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Sample records for length polymorphism pcr-rflp

  1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP for rapid diagnosis of neonatal sepsis

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    Anusha Rohit

    2016-01-01

    Full Text Available Background & objectives: The difficulties in diagnosis of neonatal sepsis are due to varied clinical presentation, low sensitivity of blood culture which is considered the gold standard and empirical antibiotic usage affecting the outcome of results. Though polymerase chain reaction (PCR based detection of bacterial 16S rRNA gene has been reported earlier, this does not provide identification of the causative agent. In this study, we used restriction fragment length polymorphism (RFLP of amplified 16S rRNA gene to identify the organisms involved in neonatal sepsis and compared the findings with blood culture. Methods: Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture and PCR-RFLP. Results: Bacterial DNA was detected by 16S rRNA gene PCR in 55 cases, while BacT/Alert culture was positive in 34 cases. Staphylococcus aureus was the most common organism detected with both methods. Klebsiella spp. was isolated from four samples by culture but was detected by PCR-RFLP in five cases while Acinetobacter spp. was isolated from one case but detected in eight cases by PCR-RFLP. The sensitivity of PCR was found to be 82.3 per cent with a negative predictive value of 85.7 per cent. Eighty of the 97 neonates had prior exposure to antibiotics. Interpretation & conclusions:The results of our study demonstrate that PCR-RFLP having a rapid turnaround time may be useful for the early diagnosis of culture negative neonatal sepsis.

  2. Use of PCR-RFLP (Polymerase Chain Reaction - Restricted Fragment Length Polymorphism in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

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    H. Tonhati

    2010-02-01

    Full Text Available The milk is an important food because it contents Conjugated Linoleic Acids (CLA. These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD´s gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism. Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z43D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren’t genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

  3. [Establishing a new genotyping method of hepatitis B virus by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) to analysis on S region and its application].

    Science.gov (United States)

    Peng, Liang; Ding, Jing-Juan; Zhang, Li-Sha

    2004-08-01

    To establish a new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method of genotyping HBV using Mbo I, BsTN I, BsmA I, Hpa II and investigate the relationship between genotype and clinical spectrum of hepatitis B. 124 full-genomic HBV sequences and 13 S-genomic sequences were analyzed, genotype specific regions were identified by the restriction enzymes Mbo I, BsTN I, BsmA I, Hpa II. And 176 samples from different kinds of hepatitis B were genotyped by this method. Five samples had been randomly selected and directly sequenced their S gene, to assess the accuracy. In 176 serum samples of patients with hepatitis B from Guizhou area, genotype B and C were found in 56.8% and 43.2% respectively. The proportions of genotype B and C in ASC were 40.0% and 15.7% (chi-square = 12.16, P < 0.005); and they were 31.6% and 14.0% in CHB (chi-square = 7.88, P < 0.005). Genotyping HBV, based on S gene RFLP seems to be highly sensitive, differential and accurate and could be used in large-scale surveys. HBV genotype B and C are existed in Guizhou area.

  4. [Application of double created restriction site PCR-RFLP to identify MGMT gene polymorphisms].

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    Wang, Wei; Miao, Wenbin; Qiu, Yulan; Xia, Zhaolin

    2008-01-01

    To develop a proper assay for identifying single nucleotide polymorphisms( SNPs) of the MGMT gene. PCR primers were designed by create restriction site (CRS) method, then polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was adopted to identify four SNPs in MGMT gene. By PCR, one primer pair yielded target products containing MGMT84 SNP site, and the other primer pair yielded target products containing MGMT143, 160, 178 SNP sites. Four restriction enzymes were adopted to identify the four SNPs, respectively. The effects of PCR and RFLP were good. The methods for four SNPs of MGMT determinated by CRS-PCR-RFLP theory could be facility, economy, and rapidness.

  5. DNA polymorphism of butyrophilin gene by PCR-RFLP technique ...

    African Journals Online (AJOL)

    We used the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique to screen for DNA polymorphism in 109 cattle. In all cattle, we amplified an 863 fragment consisting of part of exon 8. The amplified fragment digested with HaeIII restriction endonuclease and subjected to electrophoretic ...

  6. A new and improved method based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the determination of A1298C mutation in the methylenetetrahydrofolate reductase (MTHFR) gene.

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    Machnik, Grzegorz; Zapala, Malgorzata; Pelc, Ewa; Gasecka-Czapla, Monika; Kaczmarczyk, Grzegorz; Okopien, Boguslaw

    2013-01-01

    Intracellular folate homeostasis and metabolism is regulated by numerous genes. Among them, 5,10-methylenetetrahydrofolate reductase (MTHFR) is of special interest because of its involvement in regulation of the homocysteine level in the body as a result of folate metabolism. Moreover, some studies demonstrated that the homocysteine plasma level in individuals may be influenced by polymorphisms present in the MTHFR gene. Two common, clinically relevant mutations have been described: MTHFR C677T and MTHFR A1298C. Although several laboratory techniques allow genotyping of both polymorphisms, PCR-RFLP analysis is simple to perform, relatively cheap, and thus one of the most utilized. In the case of A1298C, the PCR-RFLP technique that utilizes MboII endonuclease class II requires an acrylamide gel electrophoresis, since agarose gel electrophoresis is unable to resolve short deoxyribonucleic acid (DNA) fragments after restriction digestion. Agarose gel electrophoresis is commonly preferred over that of acrylamide. To resolve this inconvenience, a novel PCR-RFLP, AjuI-based method to genotype A1298C alleles has been developed that can be performed on standard agarose gel.

  7. Polymorphism of calpastatin gene in Arabic sheep using PCR- RFLP

    African Journals Online (AJOL)

    Calpastatin has been known as candidate gene in muscle growth efficiency and meat quality. This gene has been located to chromosome 5 of sheep. In order to evaluate the calpastatin gene polymorphism, random blood sample were collected from 111 Arabic ram sheep from different regions. The DNA extraction was ...

  8. Application of Created Restriction Site PCR-RFLP to Identify POT1 Gene Polymorphism.

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    Wang, Tuanwei; Wang, Sihua; Duan, Xiaoran; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Feng, Feifei; Yu, Songcheng; Wu, Yiming; Wang, Wei

    2016-06-01

    Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3'-end of the forward primer was next to the polymorphic site, and the third base from the 3'-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.

  9. [Application of created restriction site PCR-RFLP to identify alcohol dehydrogenase 2 gene polymorphism].

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    Jiao, Jie; Wang, Wei; Liu, Jing; Xia, Zhaolin

    2009-01-01

    To develop a appropriate assay for identifying single nucleotide polymorphism (SNP) of alcohol dehydrogenase 2 (ADH2) gene. According to base substitution situation of one single base mutational site, we designed the present study primers. One of the primers was designed on the basis of neighbourhood sequence of the mutational site, that is, we made one mismatch base to let product a new enzyme site between the 3' end of the primer and the single base mutation type after the PCR amplification. Then PCR-RFLP was adopted to identify the SNP in ADH2 gene. One primer pair can get target products containing ADH2 SNP site by PCR, restriction enzymes Bsh1236I were adopted to identify the SNP site. The expected results were reached. It suggested that the method of detecting the SNP of ADH2 based on CRS-PCR-RFLP theory is facilitated, economic, and rapid.

  10. Application of CRS-PCR-RFLP to identify CYP1A1 gene polymorphism.

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    Ding, Mingcui; Duan, Xiaoran; Feng, Xiaolei; Wang, Pengpeng; Wang, Wei

    2017-11-01

    Cytochrome P4501A1 (CYP1A1) is a member of the cytochrome P450 gene family and plays an important role in the metabolism of exogenous and endogenous material. In recent research, it has been shown that its genetic polymorphisms are associated with many diseases. But the isoschizomers such as the BsrDI enzyme required for the detection of this polymorphism are expensive. The study used an improved PCR-RFLP method with mismatched base for detection of the single-nucleotide polymorphism rs1048943. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and the restriction enzyme StyI for RFLP was cheaper than other enzymes. A total of 320 samples from Han Chinese were tested to evaluate this novel method. The PCR results were confirmed by DNA sequencing. After detecting 320 Chinese Han individuals, the genotype frequencies were 63.74% for AA, 31.54% for AG, and 4.72% for GG. The allelic frequencies were 75.48% for A and 24.52% for G. The x 2 test showed the genotype and allele frequencies of CYP1A1 do not deviate from Hardy-Weinberg equilibrium, and the sequences of amplified products were consistent with the one published in GenBank with the exception of mismatched base. Based on PCR with mismatched primers we designed, the CYP1A1 polymorphism could be identified effectively with low cost. © 2017 Wiley Periodicals, Inc.

  11. IDENTIFICATION OF POLYMORPHISM OF FSH BETA-SUBUNIT GENE AS SPERM QUALITY MARKER IN BALI CATTLE USING PCR-RFLP

    OpenAIRE

    A.B.L. Ishak; C. Sumantri; R. R. Noor; I. Arifiantini

    2011-01-01

    The aim of study was to identify the association of FSH beta-subunit gene polymorphisms with sperm quality traits. A total of 470 samples of normal mature bull from several breeds were used for population study and 127 bulls from National and Regional AI centre of Indonesia for association study. To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allele frequency of the A and B in Bali cattle were (0.000) and (1.000), respectively. The absence of other allel...

  12. PCR-RFLP

    African Journals Online (AJOL)

    2012-03-30

    Mar 30, 2012 ... The. cpDNA PCR-RFLP based genetic distance (GD) among 30 tea accessions ranged from 0 to 0.071, with the mean of 0.049. This study suggests that the optimization system was suitable for PCR-RFLP analysis of. cpDNA in tea. Key words: Camellia sinensis, PCR-RFLP, chloroplast DNA, establishment.

  13. Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

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    Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

    2017-09-01

    It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

  14. IDENTIFICATION OF POLYMORPHISM OF FSH BETA-SUBUNIT GENE AS SPERM QUALITY MARKER IN BALI CATTLE USING PCR-RFLP

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    A.B.L. Ishak

    2014-10-01

    Full Text Available The aim of study was to identify the association of FSH beta-subunit gene polymorphisms withsperm quality traits. A total of 470 samples of normal mature bull from several breeds were used forpopulation study and 127 bulls from National and Regional AI centre of Indonesia for association study.To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allelefrequency of the A and B in Bali cattle were (0.000 and (1.000, respectively. The absence of otherallele A suggested that the Bali cattle was monomorphic, while Brahman, FH, Simmental and Limousinewere polymorphic. The highest observed heterozygosity were found in Limousine (0.318 and thehighest expected heterozygosity were in Simmental (0.420. The higher incident of percentage of spermabnormalities were found in Simmental, Limousin, Brahman compared to Bali and FH. Among all typesof sperm abnormalities, the abaxial and microcephalus were found in highest number.

  15. Development and Validation of a Simple, Rapid and Inexpensive PCR-RFLP Method for Genotyping of Common IL28B Polymorphisms: A Useful Pharmacogenetic Tool for Prediction of Hepatitis C Treatment Response.

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    Sharafi, Heidar; Pouryasin, Ali; Alavian, Seyed Moayed; Behnava, Bita; Keshvari, Maryam; Mehrnoush, Leila; Salimi, Shima; Kheradvar, Osveh

    2012-03-01

    In 2009, 3 genome-wide association studies implicated IL28B single-nucleotide polymorphisms (SNPs) as the strongest genetic pretreatment predictor of sustained virological response (SVR) in hepatitis C infection. Recently, the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) included IL28B testing in their guidelines. The main aim of this study was to develop and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for genotyping of common IL28B polymorphisms (rs12979860 and rs8099917). Two methods were developed to genotype common IL28B polymorphisms: 1) PCR-sequencing as a reference method and 2) PCR-RFLP as a rapid and inexpensive method. Both polymorphisms were genotyped in 104 Iranian hepatitis C patients by both methods simultaneously. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results. Genotyping of rs12979860 and rs8099917 by PCR-RFLP was concordant with PCR-sequencing in 104 (100%) individuals. The analytical sensitivity and specificity of the PCR-RFLP method for genotyping of both SNPs are 100%. Among these 104 patients with chronic hepatitis C, the frequency of the rs12979860 CC, CT and TT genotypes were 40.4%, 47.1% and 12.5% and the frequency of the rs8099917 TT, GT and GG genotypes were 59.6%, 35.6% and 4.8%, respectively. Also, three IL28B haplotypes (rs12979860-rs8099917) were found among our patients including C-T, T-G and T-T with 63.9%, 22.6% and 13.5% frequency, respectively. C-G haplotype was absent in all of our patients. We have developed a validated, fast, and simple PCR-RFLP method for genotyping of common IL28B SNPs that is more cost-effective than sequencing.

  16. IDENTIFICATION OF POLYMORPHISM OF FSH BETA-SUBUNIT GENE AS SPERM QUALITY MARKER IN BALI CATTLE USING PCR-RFLP

    Directory of Open Access Journals (Sweden)

    A.B.L. Ishak

    2011-12-01

    Full Text Available The aim of study was to identify the association of FSH beta-subunit gene polymorphisms with sperm quality traits. A total of 470 samples of normal mature bull from several breeds were used for population study and 127 bulls from National and Regional AI centre of Indonesia for association study. To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allele frequency of the A and B in Bali cattle were (0.000 and (1.000, respectively. The absence of other allele A suggested that the Bali cattle was monomorphic, while Brahman, FH, Simmental and Limousine were polymorphic. The highest observed heterozygosity were found in Limousine (0.318 and the highest expected heterozygosity were in Simmental (0.420. The higher incident of percentage of sperm abnormalities were found in Simmental, Limousin, Brahman compared to Bali and FH. Among all types of sperm abnormalities, the abaxial and microcephalus were found in highest number.

  17. Using PCR-RFLP Technology to Teach Single Nucleotide Polymorphism for Undergraduates

    Science.gov (United States)

    Zhang, Bo; Wang, Yan; Xu, Xiaofeng; Guan, Xingying; Bai, Yun

    2013-01-01

    Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a…

  18. [Analysis of polymorphism on ribosomal internal transcribed spacer 2 in four species of sandflies with PCR-RFLP].

    Science.gov (United States)

    Gu, Deng-An; Zhang, Yi; Lan, Qin-Xian

    2012-04-30

    Internal transcribed spacer 2 region in 4 species of sandflies from China (Phlebotomus chinensis, Ph. wui, Ph. longiductus and Ph. alexandri) was genotyped by PCR-RFLP. Four kinds of RFLP band patterns were created by Dra I, that were A 450 bp, B 154 bp/127 bp/93 bp/66 bp/10 hp, C 311 bp/80 bp/60 bp, and D 353 bp/300 bp/160 bp/58 bp. There were identical pattern types in various sandflies, AA in Ph. chinensis, BB in Ph. alexandri, CC in Ph. wui, and DD in Ph. longiductus. The results indicated that PCR-RFLP can be used to identify the four species of Phlebotomus in China.

  19. Analysis of Single Nucleotide Polymorphism (SNP rs22114085 Associated with Canine Atopic Dermatitis by PCR-RFLP Method

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    Martina Miluchová

    2012-05-01

    Full Text Available Canine atopic dermatitis (cAD is a common inflammatory skin disease that is considered to be a naturally occurring, spontaneous model of human atopic dermatitis (eczema. The aim of the paper was to identify of the SNP rs22114085 in different dog breeds. The material involved 52 dogs from 5 different breeds. Canine genomic DNA was isolated from saliva by modified method with using DNAzol® and linear polyacrylamide (LPA carrier and from blood by using commercial kit NucleospinBlood and used in order to estimate rs22114085 SNP genotypes by PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. The C allele was distributed in Czech Pointer, Chihuahua, German Wirehaired Pointer with an allele frequency ranging from 0.4545 to 1.00. In the population of Czech Pointer we detected all genotypes CC, CT and TT with frequency in male 0.25, 0.5833 and 0.1667, and in female 0.2728, 0.3636 and 0.3636, subsequently. In German Wirehaired Pointer was detected homozygote genotype CC in male and heterozygote genotype CT in female with frequency 1 and 1. In Chihuahua was observed homozygote genotype CC and heterozygote genotype CT with frequency 0.3333 and 0.6667, subsequently. In Golden retriever and Pincher we detected genotype TT with frequency 1.

  20. A Comprehensive Experiment for Molecular Biology: Determination of Single Nucleotide Polymorphism in Human REV3 Gene Using PCR-RFLP

    Science.gov (United States)

    Zhang, Xu; Shao, Meng; Gao, Lu; Zhao, Yuanyuan; Sun, Zixuan; Zhou, Liping; Yan, Yongmin; Shao, Qixiang; Xu, Wenrong; Qian, Hui

    2017-01-01

    Laboratory exercise is helpful for medical students to understand the basic principles of molecular biology and to learn about the practical applications of molecular biology. We have designed a lab course on molecular biology about the determination of single nucleotide polymorphism (SNP) in human REV3 gene, the product of which is a subunit of…

  1. Aplicación de PCR-RFLP para subtipificar Campylobacter jejuni PCR-RFLP for Campylobacter jejuni subtyping

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    G. Giacoboni

    2005-06-01

    Full Text Available Diez cepas de Campylobacter jejuni aisladas de fetos porcinos abortados fueron identificadas por pruebas bioquímicas: 8 como C. jejuni biotipo II de Lior, y 2 como C. jejuni biotipo I. Para poder subtipificarlas se utilizó la técnica de reacción en cadena de la polimerasa (PCR para amplificar el gen flaA y al producto obtenido se lo digirió con la enzima de restricción DdeI (RFLP. Se pudieron obtener 6 subtipos a partir de C. jejuni biotipo II, mientras que los dos aislamientos de biotipo I correspondieron a un mismo subtipo. Aunque existe una amplia variedad de técnicas de biología molecular que son aplicadas con fines epidemiológicos para Campylobacter, PCR-RFLP, demostró ser una técnica simple y accesible, capaz de subtipificar a C. jejuni.Ten Campylobacter jejuni isolates, 8 identified as C. jejuni biotype II of Lior and 2 as C. jejuni biotipe I, were recovered from aborted pig fetuses. In order to discriminate among strains, restriction fragment length polymorphism (RFLP using DdeI of polymerase chain reaction (PCR products of flaA gen was used. C. jejuni biotype II strains could be diferenciated in 6 by PCR-RFLP, and one subtype was obtained from C. jejuni biotype I. Although there is great variability of molecular techniques applied to the Campylobacter epidemiological studies, PCR-RFLP demonstrated to be a simple and accessible technique to discriminate Campylobacter jejuni isolates.

  2. Genotoxicity Evaluation of Acephate and Profenofos by the PCR-RFLP Assay

    OpenAIRE

    Bhinder, Preety; Chaudhry, Asha

    2014-01-01

    Objectives: In this study we have evaluated the genotoxic potential of pesticides acephate and profenofos by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the mosquito Culex quinquefasciatus taken as experimental model. Material and Methods: Second instar larvae were treated with LC20 of each pesticide for 24 h and induced mutations in the sequence of mitochondrial 16S rRNA gene were studied from restriction patterns generated with PacI and PsiI rest...

  3. Detection and identification of Malassezia species in domestic animals and aquatic birds by PCR-RFLP

    OpenAIRE

    Zia, M.; Mirhendi, H.; Toghyani, M.

    2015-01-01

    The present study aimed at detection and species-level identification of the Malassezia yeasts in domestic animals and aquatic birds by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Samples were collected using tape strips and swabs from 471 animals including 97 horses, 102 cattle, 105 sheep, 20 camels, 60 dogs, 30 cats, 1 hamster, 1 squirrel, 50 aquatic birds and 5 turkeys. Tape-strip samples were examined by direct microscopy. All samples were inoculated on ...

  4. Close relatedness of Acanthamoeba pustulosa with Acanthamoeba palestinensis based on isoenzyme profiles and rDNA PCR-RFLP patterns.

    Science.gov (United States)

    Kim, Y H; Ock, M S; Yun, H C; Hwang, M Y; Yu, H S; Kong, H H; Chung, D I

    1996-12-01

    The taxonomic validity of morphological group III Acanthamoeba spp. is uncertain. In the present study, six type strains of group III Acanthamoeba spp., A. culbertsoni, A. healyi, A. pustulosa, A. palestinensis, A. royreba and A. lenticulata were subjected for the evaluation of their taxonomic validity by comparison of the isoenzyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment length polymorphism (RFLP), and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between the species. The type strains of A. palestinensis and A. pustulosa showed almost identical patterns of isoenzymes and rDNA PCR RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenzymes and rDNA PCR-RFLP. It is likely that A. pustulosa is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.

  5. Genotyping species of the Sporothrix schenckii complex by PCR-RFLP of calmodulin.

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    Rodrigues, Anderson Messias; de Hoog, G Sybren; de Camargo, Zoilo Pires

    2014-04-01

    Sporotrichosis is one of the most common subcutaneous mycosis in Latin America and is caused by 4 pathogenic thermodimorphic fungi in the genus Sporothrix. From both therapeutic and epidemiological perspectives, it is essential to identify the causative agents down to the species level. Traditional parameters may overlap among closely related species, and we propose polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as an alternative approach. In the present study, the calmodulin gene was amplified and digested with HhaI to yield 5 different electrophoretic patterns representing all medically important Sporothrix species: Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa, and Sporothrix luriei. The PCR-RFLP protocol described here is a simple and inexpensive method and is highly suitable for accurate routine genotyping of relevant Sporothrix species. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Identification of five sea cucumber species through PCR-RFLP analysis

    Science.gov (United States)

    Lv, Yingchun; Zheng, Rong; Zuo, Tao; Wang, Yuming; Li, Zhaojie; Xue, Yong; Xue, Changhu; Tang, Qingjuan

    2014-10-01

    Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene ( COI) was used to identifing five sea cucumber species ( Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

  7. Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species.

    Science.gov (United States)

    Şakalar, Çağrı; Kuk, Salih; Erensoy, Ahmet; Dağli, Adile Ferda; Özercan, İbrahim Hanifi; Çetınkaya, Ülfet; Yazar, Süleyman

    2014-01-01

    To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

  8. Identification of goat cashmere and sheep wool by PCR-RFLP analysis of mitochondrial 12S rRNA gene.

    Science.gov (United States)

    Geng, Rong-Qing; Yuan, Chao; Chen, Yu-Lin

    2012-12-01

    The efficacy of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial 12S rRNA gene in identification of goat cashmere and sheep wool samples was evaluated. The specific fragments of the mitochondrial 12S rRNA gene, which were about 440 bp, were obtained using the PCR. Restriction enzyme digestion of the PCR products with endonucleases BspT I and Hinf I revealed species-specific RFLP patterns. Application of this technique on mixed samples could identify goat cashmere and sheep wool from each other within the proportion of 8:1. The technique, however, could detect only one species when the proportion of mixture was more than 9:1. The PCR-RFLP technique was demonstrated to possess potential value in precise identification of goat cashmere and sheep wool.

  9. Effective Natural PCR-RFLP Primer Design for SNP Genotyping Using Teaching-Learning-Based Optimization With Elite Strategy.

    Science.gov (United States)

    Cheng, Yu-Huei; Kuo, Che-Nan; Lai, Ching-Ming

    2016-10-01

    SNP (single nucleotide polymorphism) genotyping is the determination of genetic variations of SNPs between members of a species. In many laboratories, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) is a usually used biotechnology for SNP genotyping, especially in small-scale basic research studies of complex genetic diseases. PCR-RFLP requires an available restriction enzyme at least for identify a target SNP and an effective primer pair conforms numerous constraints. However, the lots of restriction enzymes, tedious sequence and complicated constraints make the mining of available restriction enzymes and the design of effective primer pairs become a major challenge. In the study, we propose a novel and available CI (Computation Intelligence)-based method called TLBO (teaching-learning-based optimization) and introduce the elite strategy to design effective primer pairs. Three common melting temperature computations are available in the method. REHUNT (Restriction Enzymes HUNTing) is first combined with the method to mine available restriction enzymes. Robust in silico simulations for the GA (genetic algorithm), the PSO (particle swarm optimization), and the method for natural PCR-RFLP primer design in the SLC6A4 gene with two hundred and eighty-eight SNPs had been performed and compared. These methods had been implemented in JAVA and they are freely available at https://sites.google.com/site/yhcheng1981/tlbonpd-elite for users of academic and non-commercial interests.

  10. Lab-on-a-chip-based PCR-RFLP assay for the confirmed detection of short-length feline DNA in food.

    Science.gov (United States)

    Ali, Md Eaqub; Al Amin, Md; Hamid, Sharifah Bee Abd; Hossain, M A Motalib; Mustafa, Shuhaimi

    2015-01-01

    Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.

  11. A Novel Teaching-Learning-Based Optimization for Improved Mutagenic Primer Design in Mismatch PCR-RFLP SNP Genotyping.

    Science.gov (United States)

    Cheng, Yu-Huei

    2016-01-01

    Many single nucleotide polymorphisms (SNPs) for complex genetic diseases are genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in small-scale basic research studies. It is an essential work to design feasible PCR-RFLP primer pair and find out available restriction enzymes to recognize the target SNP for PCR experiments. However, many SNPs are incapable of performing PCR-RFLP makes SNP genotyping become unpractical. A genetic algorithm (GA) had been proposed for designing mutagenic primer and get available restriction enzymes, but it gives an unrefined solution in mutagenic primers. In order to improve the mutagenic primer design, we propose TLBOMPD (TLBO-based Mutagenic Primer Design) a novel computational intelligence-based method that uses the notion of "teaching and learning" to search for more feasible mutagenic primers and provide the latest available restriction enzymes. The original Wallace's formula for the calculation of melting temperature is maintained, and more accurate calculation formulas of GC-based melting temperature and thermodynamic melting temperature are introduced into the proposed method. Mutagenic matrix is also reserved to increase the efficiency of judging a hypothetical mutagenic primer if involve available restriction enzymes for recognizing the target SNP. Furthermore, the core of SNP-RFLPing version 2 is used to enhance the mining work for restriction enzymes based on the latest REBASE. Twenty-five SNPs with mismatch PCR-RFLP screened from 288 SNPs in human SLC6A4 gene are used to appraise the TLBOMPD. Also, the computational results are compared with those of the GAMPD. In the future, the usage of the mutagenic primers in the wet lab needs to been validated carefully to increase the reliability of the method. The TLBOMPD is implemented in JAVA and it is freely available at http://tlbompd.googlecode.com/.

  12. Species determination of Malaysian Bactrocera pests using PCR-RFLP analyses (Diptera: Tephritidae).

    Science.gov (United States)

    Chua, Tock H; Chong, Yi Vern; Lim, Saw Hoon

    2010-04-01

    Identification of Bactrocera carambolae Drew and Hancock, B. papayae Drew and Hancock, B. tau Walker, B. latifrons Hendel, B. cucurbitae Coquillett, B. umbrosa Fabricius and B. caudata Fabricius would pose a problem if only a body part or an immature stage were available. Analysis of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of cytochrome oxidase I (COI) gene using primers COIR, COIF, UEA7 and UEA10 and restriction enzymes (MseI, RsaI and Alu1) was carried out. The banding profiles in the electrophoresis gel were analysed. The COI gene in six Bactrocera spp. was successfully amplified by COIR and COIF, as well as UEA7 and UEA10, while B. caudata was amplified successfully only by UEA primers. Using COI amplified PCR products and restriction enzymes, distinct banding profiles for B. tau, B. latifrons, B. cucurbitae and B. umbrosa were observed, but not for B. carambolae and B. papayae. However, using UEA7, UEA10 and RsaI, B. caudata could be identified, while B. carambolae and B. papayae might possibly be separated from one another. It was also shown that adult body parts or immature life stages of B. carambolae, B. papayae, B. latifrons and B. cucurbitae produced the same banding profiles as the adults. PCR-RFLP analyses are able to identify positively five Bactrocera species, while B. papayae and B. carambolae might possibly be separated from one another, even if immature life stages or adult body parts are used.

  13. Detection and identification of Malassezia species in domestic animals and aquatic birds by PCR-RFLP

    Science.gov (United States)

    Zia, M.; Mirhendi, H.; Toghyani, M.

    2015-01-01

    The present study aimed at detection and species-level identification of the Malassezia yeasts in domestic animals and aquatic birds by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Samples were collected using tape strips and swabs from 471 animals including 97 horses, 102 cattle, 105 sheep, 20 camels, 60 dogs, 30 cats, 1 hamster, 1 squirrel, 50 aquatic birds and 5 turkeys. Tape-strip samples were examined by direct microscopy. All samples were inoculated on modified Leeming and Notman agar medium. DNA extracted from the yeast colonies was amplified by PCR using primers specific for 26S rDNA. RFLP of the PCR products was performed using Hin6I enzyme, and PCR and RFLP products were visualized by agarose gel electrophoresis. Malassezia yeasts were detected at the following frequencies: 15.46% in horses, 12.74% in cattle, 12.38% in sheep, 28.33% in dogs, 26.66% in cats and 26% in aquatic birds. Eighty colonies of 6 species were isolated: Malassezia globosa 41.25%, Malassezia furfur 22.5%, Malassezia restricta 15%, Malassezia sympodialis 15%, Malassezia pachydermatis 5% and Malassezia slooffiae 1.25%. Therefore different lipophilic Malassezia species are found in a wide diversity of animals and aquatic birds. PCR-RFLP is a suitable technique for identification of different Malassezia species. PMID:27175148

  14. Software for optimization of SNP and PCR-RFLP genotyping to discriminate many genomes with the fewest assays

    Directory of Open Access Journals (Sweden)

    Wagner Mark C

    2005-05-01

    Full Text Available Abstract Background Microbial forensics is important in tracking the source of a pathogen, whether the disease is a naturally occurring outbreak or part of a criminal investigation. Results A method and SPR Opt (SNP and PCR-RFLP Optimization software to perform a comprehensive, whole-genome analysis to forensically discriminate multiple sequences is presented. Tools for the optimization of forensic typing using Single Nucleotide Polymorphism (SNP and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP analyses across multiple isolate sequences of a species are described. The PCR-RFLP analysis includes prediction and selection of optimal primers and restriction enzymes to enable maximum isolate discrimination based on sequence information. SPR Opt calculates all SNP or PCR-RFLP variations present in the sequences, groups them into haplotypes according to their co-segregation across those sequences, and performs combinatoric analyses to determine which sets of haplotypes provide maximal discrimination among all the input sequences. Those set combinations requiring that membership in the fewest haplotypes be queried (i.e. the fewest assays be performed are found. These analyses highlight variable regions based on existing sequence data. These markers may be heterogeneous among unsequenced isolates as well, and thus may be useful for characterizing the relationships among unsequenced as well as sequenced isolates. The predictions are multi-locus. Analyses of mumps and SARS viruses are summarized. Phylogenetic trees created based on SNPs, PCR-RFLPs, and full genomes are compared for SARS virus, illustrating that purported phylogenies based only on SNP or PCR-RFLP variations do not match those based on multiple sequence alignment of the full genomes. Conclusion This is the first software to optimize the selection of forensic markers to maximize information gained from the fewest assays, accepting whole or partial genome sequence data as input. As

  15. Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions.

    Science.gov (United States)

    Mikaeili, Fattaneh; Mathis, Alexander; Deplazes, Peter; Mirhendi, Hossein; Barazesh, Afshin; Ebrahimi, Sepideh; Kia, Eshrat Beigom

    2017-09-26

    The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment length polymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.

  16. [PCR-RFLP and sequencing studies of malaria vectors (Diptera, Culicidae, Anopheles) in Kyrgyzstan].

    Science.gov (United States)

    Goriacheva, I I; Zvantsov, A B; Gordeev, M I; Bezzhonova, O V; Usenbaev, N T; Ezhov, M N

    2011-01-01

    The results of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, obtained after treatment of PCR-products with restriction endonuclease CfoI, could identify two members of the Anopheles maculipennis complex: An. maculipennis and An. artemievi. Treatment of amplification products with restriction endonuclease BsuI gave rise to fragment lengths of 192 and 218 bp, characteristic of An. artemievi, in the populations of the Talas (settlement of Kizil-Adyr, Kara-Bura District), Dzhelalabad (towns of Tashkumyr and Kara-Kul), and Osh (town of Gulcha, Alai District; village of Langar, Kara-Suisky District) Regions. After treatment of PCR-products with restriction endonuclease BstACI, fragment lengths of 292 and 150 bp, characteristic of An. messeae, were obtained for the mosquitoes of Issyk-Kul (town of Balykchi) and Naryn (settlement of Kochkorka, Kochkor District) Regions. To identify the molecular forms of An. superpictus, the investigators sequenced the amplification products obtained by PCR with 5.8S and 28S rRNA gene-specific primers. Analysis of the primary structure of the second internal transcribed spacer, by using the international databases, has indicated that molecular form X is prevalent in the study districts of Kyrgyzstan. The COI-COII region of the mitochondrial genome of the vector also underwent PCR-RFLP analysis. Three new haplotypes with restriction patterns of about 540, 420, 200, 150, 140 bp, about 540, 360, 280, 150, 140 bp, and about 580, 540, and 150, 140 bp have been identified along with the previously described haplotype X characterized by restriction products of 540, 420, 260, 150, and 140 bp in length.

  17. A MspI PCR-RFLP within bovin growth hormone gene and its ...

    African Journals Online (AJOL)

    user

    2009-10-05

    Oct 5, 2009 ... Mixed model analyses of sperm quality traits considering genotype and environment as fixed effects and ... Key words: Iranian Holstein bull, PCR-RFLP, bGH-Msp-I, polymorphism, sperm quality trait. INTRODUCTION ..... Vincent AL, Evans G, Short TH, Southwood OI, Plastow GS, Tuggle. CK, Rothschild MF ...

  18. Glutathione S-transferase P1 gene polymorphisms and susceptibility ...

    Indian Academy of Sciences (India)

    matched and ethnicity-matched healthy controls (n = 200) were genotyped for polymorphisms in GSTP1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotype distribution of g.313A>G and ...

  19. Haplotype identification and detection of mitochondrial DNA heteroplasmy in Varroa destructor mites using ARMS and PCR-RFLP methods.

    Science.gov (United States)

    Gajić, Bojan; Stevanović, Jevrosima; Radulović, Željko; Kulišić, Zoran; Vejnović, Branislav; Glavinić, Uroš; Stanimirović, Zoran

    2016-11-01

    In the present study, amplification refractory mutation system (ARMS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were used for identification of recently described Serbia 1 (S1) and Peshter 1 (P1) mitochondrial haplotypes of Varroa destructor. Based on single nucleotide polymorphisms (SNPs) within cytochrome oxidase 1 (cox1) and cytochrome b (cytb) gene sequences, a total of 64 adult V. destructor females were analyzed from locations where the S1 and P1 haplotypes had been detected previously. Results of haplotype identification obtained by ARMS and PCR-RFLP methods were completely consistent with the sequencing data. Furthermore, in some analyzed samples the occurrence of site heteroplasmy at haplotype-defining sites was detected, as it was confirmed by double peaks in the sequence chromatograms. Neither mites with simultaneous nucleotide variability, nor those with combined SNP and heteroplasmy in cox1 and cytb were found. Given that this is the first occurrence of site heteroplasmy in V. destructor, the origin of this phenomenon and possible specific traits of heteroplasmic mites have yet to be determined.

  20. Mutagenicity Assessment of Organophosphates using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay

    OpenAIRE

    Bhinder, Preety; Chaudhry, Asha

    2013-01-01

    Objectives: In this study we have evaluated the mutagenicity of organophosphate pesticides acephate, chlorpyrifos, and profenofos using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the mosquito Culex quinquefasciatus taken as an experimental model. Materials and Methods: Second instar larvae were treated with LC20 of each pesticide for 24 h and mutations induced in the sequence of mitochondrial COII gene (690bp) were studied from restriction pattern...

  1. Prolactin-RsaI gene polymorphism in East Anatolian Red cattle in ...

    African Journals Online (AJOL)

    The aim of the study was to determine by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method the gene and genotype frequencies of PRL gene in native East Anatolian Red (EAR) cattle, which are raised as a genetic resource in Turkey. PCR-RFLP analysis involved the use of the ...

  2. Molecular Detection and Genetic Characterization of Toxoplasma gondii in Farmed Minks (Neovison vison in Northern China by PCR-RFLP.

    Directory of Open Access Journals (Sweden)

    Wen-Bin Zheng

    Full Text Available Toxoplasma gondii is a worldwide prevalent parasite, affecting a wide range of mammals and human beings. Little information is available about the distribution of genetic diversity of T. gondii infection in minks (Neovison vison. This study was conducted to estimate the prevalence and genetic characterization of T. gondii isolates from minks in China. A total of 418 minks brain tissue samples were collected from Jilin and Hebei provinces, northern China. Genomic DNA were extracted and assayed for T. gondii infection by semi-nested PCR of B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2 (5'+3' SAG2, alter.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP technology. 36 (8.6% of 418 DNA samples were overall positive for T. gondii. Among them, 5 samples were genotyped at all loci, and 1 sample was genotyped for 9 loci. In total, five samples belong to ToxoDB PCR-RFLP genotype#9, one belong to ToxoDB genotye#3. To our knowledge, this is the first report of genetic characterization of T. gondii in minks in China. Meanwhile, these results revealed a distribution of T. gondii infection in minks in China. These data provided base-line information for controlling T. gondii infection in minks.

  3. Molecular Detection and Genetic Characterization of Toxoplasma gondii in Farmed Minks (Neovison vison) in Northern China by PCR-RFLP.

    Science.gov (United States)

    Zheng, Wen-Bin; Zhang, Xiao-Xuan; Ma, Jian-Gang; Li, Fa-Cai; Zhao, Quan; Huang, Si-Yang; Zhu, Xing-Quan

    2016-01-01

    Toxoplasma gondii is a worldwide prevalent parasite, affecting a wide range of mammals and human beings. Little information is available about the distribution of genetic diversity of T. gondii infection in minks (Neovison vison). This study was conducted to estimate the prevalence and genetic characterization of T. gondii isolates from minks in China. A total of 418 minks brain tissue samples were collected from Jilin and Hebei provinces, northern China. Genomic DNA were extracted and assayed for T. gondii infection by semi-nested PCR of B1 gene. The positive DNA samples were typed at 10 genetic markers (SAG1, SAG2 (5'+3' SAG2, alter.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. 36 (8.6%) of 418 DNA samples were overall positive for T. gondii. Among them, 5 samples were genotyped at all loci, and 1 sample was genotyped for 9 loci. In total, five samples belong to ToxoDB PCR-RFLP genotype#9, one belong to ToxoDB genotye#3. To our knowledge, this is the first report of genetic characterization of T. gondii in minks in China. Meanwhile, these results revealed a distribution of T. gondii infection in minks in China. These data provided base-line information for controlling T. gondii infection in minks.

  4. Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene.

    Science.gov (United States)

    Kumar, Deepak; Singh, S P; Karabasanavar, Nagappa S; Singh, Rashmi; Umapathi, V

    2014-11-01

    Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

  5. Prognostic value of IDH1 mutations identified with PCR-RFLP assay in acute myeloid leukemia patients

    International Nuclear Information System (INIS)

    Elsayed, Gh.M.; Zaher, A.; Elnoshokaty, E.H.; Nassar, H.R.; Moneer, M.M.

    2014-01-01

    Background: Somatic mutations in isocitrate dehydrogenase 1 (1DH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique. Aim of the work: The aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML. Methods: Residual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was adapted to IDHl gene, codon 132 mutations screening. Results: The frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p = 0.03). Patients with 1DH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p = 0.08). Conclusion: IDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDF11 mutations in AML.

  6. Genetic differentiation of Colletotrichum gloeosporioides and C. truncatum associated with Anthracnose disease of papaya (Carica papaya L.) and bell pepper (Capsium annuum L.) based on ITS PCR-RFLP fingerprinting.

    Science.gov (United States)

    Maharaj, Ariana; Rampersad, Sephra N

    2012-03-01

    Members of the genus Colletotrichum include some of the most economically important fungal pathogens in the world. Accurate diagnosis is critical to devising disease management strategies. Two species, Colletotrichum gloeosporioides and C. truncatum, are responsible for anthracnose disease in papaya (Carica papaya L.) and bell pepper (Capsicum annuum L.) in Trinidad. The ITS1-5.8S-ITS2 region of 48 Colletotrichum isolates was sequenced, and the ITS PCR products were analyzed by PCR-RFLP analysis. Restriction site polymorphisms generated from 11 restriction enzymes enabled the identification of specific enzymes that were successful in distinguishing between C. gloeosporioides and C. truncatum isolates. Species-specific restriction fragment length polymorphisms generated by the enzymes AluI, HaeIII, PvuII, RsaI, and Sau3A were used to consistently resolve C. gloeosporioides and C. truncatum isolates from papaya. AluI, ApaI, PvuII, RsaI, and SmaI reliably separated isolates of C. gloeosporioides and C. truncatum from bell pepper. PvuII, RsaI, and Sau3A were also capable of distinguishing among the C. gloeosporioides isolates from papaya based on the different restriction patterns that were obtained as a result of intra-specific variation in restriction enzyme recognition sites in the ITS1-5.8S-ITS2 rDNA region. Of all the isolates tested, C. gloeosporioides from papaya also had the highest number of PCR-RFLP haplotypes. Cluster analysis of sequence and PCR-RFLP data demonstrated that all C. gloeosporioides and C. truncatum isolates clustered separately into species-specific clades regardless of host species. Phylograms also revealed consistent topologies which suggested that the genetic distances for PCR-RFLP-generated data were comparable to that of ITS sequence data. ITS PCR-RFLP fingerprinting is a rapid and reliable method to identify and differentiate between Colletotrichum species.

  7. Alkaline Extraction of DNA from Pathogenic Fungi for PCR-RFLP Analysis

    OpenAIRE

    Matsumoto, Masaru; Mishima, Shinobu; Matsuyama, Nobuaki; 松元, 賢; 松山, 宣明

    1997-01-01

    For the preparation of DNA samples from fungal mycelia alkaline extraction method was applied and assessed its usefulness for PCR-RFLP analysis. Using alkaline treatment protocols, 18S ribosomal DNAs (rDNA) derived from fungal genomic DNA of Pyricularia oryzae, P. zingiberi, Rhizoctonia solani and R. oryzae were PCR-amplified and digested with Hha I, Msp I and Hae ill. RFLP analysis with HhaI showed the divergent polymorphism between genus Pyricularia and Rhizoctonia. The alkaline DNA extract...

  8. Performance of PCR-restriction fragment length polymorphism analysis of the Helicobacter pylori ureB gene in differentiating gene variants

    DEFF Research Database (Denmark)

    Colding, H; Hartzen, S H; Mohammadi, M

    2003-01-01

    Recently, PCR-restriction fragment length polymorphism (PCR-RFLP) of the urease genes of Helicobacter pylori was evaluated in a meta-analysis; acceptable discriminatory indices of the ureAB and C genes were found. In the present investigation, we found a discriminatory index of 0.95 for 191...... unrelated clinical H. pylori isolates with PCR-RFLP typing of the ureB gene (933 bp), combining the results obtained with restriction enzymes HaeIII and Sau3A, and a mixture of the enzymes. We therefore find that PCR-RFLP typing of the ureB gene of H. pylori with restriction enzymes HaeIII and Sau3A...

  9. Performance of PCR-restriction fragment length polymorphism analysis of the Helicobacter pylori ureB gene in differentiating gene variants

    DEFF Research Database (Denmark)

    Colding, H; Hartzen, S H; Mohammadi, M

    2003-01-01

    unrelated clinical H. pylori isolates with PCR-RFLP typing of the ureB gene (933 bp), combining the results obtained with restriction enzymes HaeIII and Sau3A, and a mixture of the enzymes. We therefore find that PCR-RFLP typing of the ureB gene of H. pylori with restriction enzymes HaeIII and Sau3A......Recently, PCR-restriction fragment length polymorphism (PCR-RFLP) of the urease genes of Helicobacter pylori was evaluated in a meta-analysis; acceptable discriminatory indices of the ureAB and C genes were found. In the present investigation, we found a discriminatory index of 0.95 for 191...

  10. Intraspecific differentiation of Colletotrichum gloeosporioides sensu lato based on in silico multilocus PCR-RFLP fingerprinting.

    Science.gov (United States)

    Ramdeen, Stephen; Rampersad, Sephra N

    2013-02-01

    Colletotrichum gloeosporioides sensu lato is one of the most common and widely distributed plant pathogens in the world. Understanding fungal biodiversity is hinged on accurate identification and delimitation at the inter- and intraspecific levels. Sequences of the ITS1-5.8S-ITS2 region (ITS), β-tubulin (TUB), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of 30 C. gloeosporioides sensu lato isolates, collected from anthracnose infected papaya fruits grown in the main production areas in Trinidad, were analyzed by in silico PCR-RFLP analysis with the aim of identifying which gene region(s) had the highest level of intraspecific polymorphism. Restriction site polymorphisms generated from 13 restriction enzymes enabled the identification of specific enzymes that were successful at intraspecific discrimination of the C. gloeosporioides isolates. Genetic distance values were reflective of the level of polymorphisms obtained for the four different gene regions. In both cases (calculated genetic distance and percentage of polymorphic loci from RFLP profiles), ACT and ITS gene regions had the highest level of restriction site polymorphisms and genetic diversity, GPDH and TUB had the lowest. Cluster analysis based on PCR-RFLP genetic distance data revealed sub-specific placement of the isolates which appeared to be gene-dependent. The implications of these findings are discussed relative to biodiversity monitoring and the need for multilocus, polyphasic investigations which must take into account the possibility of exaggerated estimates of genetic diversity.

  11. Restriction fragment length polymorphism catalog for molecular identification of Japanese Tetranychus spider mites (Acari: Tetranychidae).

    Science.gov (United States)

    Osakabe, Masahiro; Kotsubo, Yu; Tajima, Ryusen; Hinomoto, Norihide

    2008-08-01

    Species identification is a basic issue in biosecurity. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) is a useful molecular diagnostic tool for species identification. However, the lack of transferability of data has been a serious shortcoming of this method. A RFLP catalog, i.e., a graph of PCR-RFLP patterns expected from sequence data, was devised as a tool to facilitate PCR-RFLP data sharing among laboratories. Twelve species of Tetranychus spider mites have been recorded in Japan to date. In this study, we analyzed DNA sequences of the internal transcribed spacer (ITS) region in nuclear ribosomal DNA of 11 Tetranychus species. For the species identification using PCR-RFLP, we chose six candidates from 131 restriction endonucleases and developed an RFLP catalog of all known Japanese Tetranychus species except Tetranychus neocaledonicus André. The RFLP catalog revealed that most Tetranychus species had diagnostic restriction fragments. The RFLP catalog is transferable and simple molecular diagnostic tool, and it has the ability to add more species and newly found intraspecific variations. Therefore, we believe that the RFLP catalog will contribute to biosecurity as a practical diagnostic tool for species identification of spider mites.

  12. Use of PCR-RFLP and PCR-HWP1 for identification of Candida species isolated from cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    Peyman Solimani

    2014-08-01

    Full Text Available Background: Due to the predisposing conditions in patients with cystic fibrosis (CF caused by defective mucociliary clearance facilitating colonization and invasion with Candida species has dramatically increased. Traditional methods for identifying problems are imminent and time-consuming. Therefore, molecular techniques utilizing amplification of target DNA provide quick and precise methods for the diagnosis and identification of Candida species. Therefore, the aim of the current study was to identify the most medically common isolated Candida species from the air way of CF patients by PCR-RFLP and amplification of HWP1 gene. Materials and Methods: A total of 42 CF patients presenting symptoms who referred to pediatric respiratory diseases research center were screened for the presence of Candida spp. The isolates initially were phenotypically identified and confirmed by molecular approaches based on restriction fragment length polymorphism ( PCR -RFLP for the discrimination of C. albicans of non-albicans and the amplification of HWP1 gene for the discrimination of C. albicans from C. dubliniensis and C. africana was conducted. Results: The results show that C. albicans was the most frequently isolated species (83.8% followed by non-albicans included C. parapsilosis (7.1%, C. glabrata (3.2%, and C. tropicalis (3.2%. The restriction patterns of each Candida species were perfectly specific. Since MspI could not discriminate between the three morphological related species, C. albicans, C. dubliniensis and C. africana, we used PCR amplification of HWP1 gene, which (7.1% species from C. albicans identified as C. dubliniensis, however C. africana strains were not found. Conclusion: The present study found that C. albicans as predominant species wereisolated from the CF patients. It could be concluded that molecular diagnostic methods are reliable and would be useful for the identification of medically important Candida species in clinical samples

  13. Identificazione rapida di mutazioni associate a farmaco-resistenza in ceppi di citomegalovirus umano mediante nPCR-RFLP

    Directory of Open Access Journals (Sweden)

    Maria Cristina Medici

    2007-03-01

    Full Text Available We developed a nested-PCR followed by restriction fragment length polymorphism (RFLP for the detection of human cytomegalovirus (HCMV UL97 M460V/I, H520Q, C592Q,A594V, L595S/F and C603W mutations associated to ganciclovir (GCV resistance.The method uses five primer pairs and seven enzymes already published and newly combined.The detection limit of nPCR was assessed in a single serial dilution assay to be about 0.13 PFU/reaction. Expected restriction fragment patterns were obtained by nPCR-RFLP on either wild-type reference strains or strains and sequences of HCMV containing mutations. Then the nPCR-RFLP was used on 24 sera/plasma belonging to 22 transplant recipients (kidney, bone marrow, or kidney-pancreas: 13 subjects never treated with GCV (control group and 9 subjects treated with GCV oral profilaxis (study group. All codons detected from the control group (six in 8 cases and four in 1 case were identified as wild-type. All codons detected from the study group (six in 6 cases, three in 2 cases, and four in the second sample of 1 case whose first sample was negative by nPCR were wild-type except one, which showed a restriction pattern referring to M460V and/or M460I ATA-codified, definitively proved to be M460V by sequence analysis.This was the case of a renal transplant recipient at the end of profilaxis. In conclusion, the procedure seems to be quite sensitive and specific as well as able to detect mixed population of mutants or mutants and wild-type. It could represent a good tool in monitoring the emergence of HCMV mutants in renal transplant recipients treated with GCV.

  14. Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species.

    Science.gov (United States)

    Van Bortel, W; Sochanta, T; Harbach, R E; Socheat, D; Roelants, P; Backeljau, T; Coosemans, M

    2002-09-01

    A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north-eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR-RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR-RFLP assay can detect species not included in the initial set-up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non-vector anophelines in South-east Asia.

  15. Discrimination of the Thai rejuvenating herbs Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna collettii (Black Kwao Khruea) using PCR-RFLP.

    Science.gov (United States)

    Wiriyakarun, Suchaya; Yodpetch, Woraluk; Komatsu, Katsuko; Zhu, Shu; Ruangrungsi, Nijsiri; Sukrong, Suchada

    2013-07-01

    The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name "Kwao Khruea" is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating "Kwao Khruea" species.

  16. Molecular identification of Candida species isolated from cases of neonatal candidemia using polymerase chain reaction-restriction fragment length polymorphism in a tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Akeela Fatima

    2017-01-01

    Full Text Available Context: Candida spp. is an emerging cause of bloodstream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs in various Candida species are responsible for the increase in morbidity and mortality due to candidemia. Hence, the rapid identification of Candida isolates is very important for the proper management of patients with candidemia. Aims: The aim was to re-evaluate the identification of various Candida spp. by polymerase chain reaction (PCR-restriction fragment length polymorphism (RFLP and to evaluate the accuracy, speed, and cost of phenotypic methodology versus PCR-RFLP. Settings and Design: Hospital-based cross-sectional study. Materials and Methods: Ninety consecutive clinical isolates of seven Candida species, isolated from blood of neonates and identified by routine phenotypic methods, were re-evaluated using universal primers internal transcribed spacer 1 (ITS1 and ITS4 for PCR amplification and Msp I restriction enzyme for RFLP. Statistical Analysis Used: Kappa test for agreement. Results: The results of PCR-RFLP were 100% in agreement with those obtained using conventional phenotypic methods. Identification could be achieved within 3 work days by both the methods. Our routine methods proved to be cost effective than PCR-RFLP. Conclusions: We can continue with our routine phenotypic methods and PCR-RFLP can be used for periodic quality control or when conventional methods fail to identify a species.

  17. Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP.

    Science.gov (United States)

    Quijada, A; Liston, A; Delgado, P; Vázquez-Lobo, A; Alvarez-Buylla, E R

    1998-03-01

    In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300-900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon's index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease.

  18. Genotyping of β-Lactoglobulin gene by PCR-RFLP in Sahiwal and Tharparkar cattle breeds

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    Gupta Neelam

    2006-05-01

    Full Text Available Abstract Background Improvement of efficiency and economic returns is an important goal in dairy farming, as in any agricultural enterprise. The primary goal of dairy industry has been to identify an efficient and economical way of increasing milk production and its constituents without increasing the size of the dairy herd. Selection of animals with desirable genotypes and mating them to produce the next generation has been the basis of livestock improvement and this would continue to remain the same in the coming years. The use of polymorphic genes as detectable molecular markers is a promising alternative to the current methods of trait selection once these genes are proven to be associated with traits of interest in animals. The point mutations in exon IV of bovine β-Lactoglobulin gene determine two allelic variants A and B. These variants were distinguished by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP analysis in two indigenous Bos indicus breeds viz. Sahiwal and Tharparkar cattle. DNA samples (228 in Sahiwal and 86 in Tharparkar were analyzed for allelic variants of β-Lactoglobulin gene. Polymorphism was detected by digestion of PCR amplified products with Hae III enzyme, and separation on 12% non-denaturing gels and resolved by silver staining. Results The allele B of β-Lactoglobulin occurred at a higher frequency than the allele A in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in Sahiwal and Tharparkar breeds were 0.031, 0.276, 0.693 and 0.023, 0.733, 0.244 respectively. Frequencies of A and B alleles were 0.17 and 0.83, and 0.39 and 0.61 in Sahiwal and Tharparkar breeds respectively. The Chi-square test results (at one degree of freedom at one per cent level revealed that the Tharparkar population was not in Hardy-Weinberg equilibrium as there was a continuous migration of animals in the herd studied, where as, the results are not significant for the Sahiwal

  19. GROWTH HORMONE GENE GENOTYPING BY Msp I RESTRICTION ENZYME AND PCR-RFLP METHODS IN ACEH CATTLE BREED AT INDRAPURI DISTRICT OF ACEH PROVINCE

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    W.P.B. Putra

    2014-10-01

    Full Text Available The objective of this research was to identify growth hormone (GH genes genotype in selectedAceh cattle at Indrapuri’s Breeding and Forage Centre (IBFC of Aceh Cattle. Fourty one cattleconsisting of 21 male and 20 female cattle were used in this study. The genomic DNA was extractedfrom blood using Sambrook et al. (1989 methods. Polymerase Chain Reaction - Restriction FragmentLength Polymorphism (PCR-RFLP and mehod of sequencing was used to detect MspI site on GH gene.Based on sequencing results, it can be concluded that all cattle were monomorphism. The frequency ofTT genotype were 1.00 and same as T allele frequency. The transition of C (cytosine into T (thymineon 1548 bp caused the lost of restriction site.

  20. Polymorphism of calpastatin gene in Arabic sheep using PCR- RFLP

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... Sci. 67: 3313-3321. Goll DE, Thompson VF, Taylor RG, Ouali A (1998). The calpain system and skeletal muscle growth. Canadian J. Animal Sci. 78: 503-512. Goll DE, Thompson VF, Taylor RG, Zaleweska T (1992). Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin . Bio.

  1. DNA polymorphism of butyrophilin gene by PCR-RFLP technique

    African Journals Online (AJOL)

    STORAGESEVER

    2008-07-18

    Jul 18, 2008 ... PME (1989). Rapid and simple method for purification of nucleic acid. J. Clin. Microbiol, 28: 495-503. Frank WW, Heid HW, Grund C, Winter S, Freudenstein C, Schmid E,. Jarasch ED, Keenan TW (1981). Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in.

  2. Identification of blood meals in field captured sand flies by a PCR-RFLP approach based on cytochrome b gene.

    Science.gov (United States)

    González, Estela; Gállego, Montserrat; Molina, Ricardo; Abras, Alba; Alcover, M Magdalena; Ballart, Cristina; Fernández, Anna; Jiménez, Maribel

    2015-12-01

    Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand flies. Information about blood meal preferences in sand flies is essential to understand the epidemiology of the disease to adopt control measures. In previous studies, a polymerase chain reaction (PCR) of 359bp fragment of the conserved gene cytochrome b (cyt b) and further sequencing were applied in the study of blood meal sources in sand flies collected in the area of a leishmaniasis outbreak in southwest Madrid, Spain, providing significant information about blood meal preferences in the focus. In this work, a PCR-restriction fragment length polymorphism (RFLP) targeting a fragment of 359bp of vertebrate cyt b gene was developed. Restriction endonucleases HaeIII and HinfI generated specific patterns consistent with the blood meal sources found in sand flies. The protocol has been validated with twenty six engorged females collected in the field with CDC traps. Blood meals from nine vertebrates were identified based on PCR-cyt b and sequencing-human, dog, cat, horse, hare, rabbit, sheep, goat and chicken - and mixed blood meals (sheep/human; sheep/goat) - and successfully distinguished by PCR-RFLP. Therefore, this approach is an efficient and reliable alternative method to be applied in entomological surveys. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Identification of Eastern United States Reticulitermes Termite Species via PCR-RFLP, Assessed Using Training and Test Data

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    Ryan C. Garrick

    2015-06-01

    Full Text Available Reticulitermes termites play key roles in dead wood decomposition and nutrient cycling in forests. They also damage man-made structures, resulting in considerable economic loss. In the eastern United States, five species (R. flavipes, R. virginicus, R. nelsonae, R. hageni and R. malletei have overlapping ranges and are difficult to distinguish morphologically. Here we present a molecular tool for species identification. It is based on polymerase chain reaction (PCR amplification of a section of the mitochondrial cytochrome oxidase subunit II gene, followed by a three-enzyme restriction fragment length polymorphism (RFLP assay, with banding patterns resolved via agarose gel electrophoresis. The assay was designed using a large set of training data obtained from a public DNA sequence database, then evaluated using an independent test panel of Reticulitermes from the Southern Appalachian Mountains, for which species assignments were determined via phylogenetic comparison to reference sequences. After refining the interpretive framework, the PCR-RFLP assay was shown to provide accurate identification of four co-occurring species (the fifth species, R. hageni, was absent from the test panel, so accuracy cannot yet be extended to training data. The assay is cost- and time-efficient, and will help improve knowledge of Reticulitermes species distributions.

  4. A new HpaII PCR-RFLP within the porcine prolactin receptor (PRLR) gene and study of its effect on litter size and number of teats

    Czech Academy of Sciences Publication Activity Database

    Putnová, L.; Knoll, A.; Dvořák, J.; Čepica, Stanislav

    2002-01-01

    Roč. 119, - (2002), s. 57-63 ISSN 0931-2668 R&D Projects: GA ČR GA523/99/0842; GA AV ČR KSK5052113 Keywords : polymorphism * PCR-RFLP * pig Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.795, year: 2002

  5. Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

    Science.gov (United States)

    Asing; Ali, Md Eaqub; Abd Hamid, Sharifah Bee; Hossain, M A Motalib; Mustafa, Shuhaimi; Kader, Md Abdul; Zaidul, I S M

    2016-01-01

    The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

  6. Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis in the Food Chain and Traditional Chinese Medicines.

    Directory of Open Access Journals (Sweden)

    Asing

    Full Text Available The Malayan box turtle (Cuora amboinensis (MBT is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP assay with a very short target length (120 bp to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

  7. Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

    Directory of Open Access Journals (Sweden)

    M.S. Santos

    2010-08-01

    Full Text Available Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP. More specifically: a to evaluate 3 different amplification regions, b to investigate 3 different restriction enzymes, and c to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2 were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

  8. Development of a polymerase chain reaction/restriction fragment length polymorphism method for Saccharomyces cerevisiae and Saccharomyces bayanus identification in enology.

    Science.gov (United States)

    Masneuf, I; Aigle, M; Dubourdieu, D

    1996-05-01

    Several yeast strains of the species Saccharomyces cerevisiae, S. bayanus and S. paradoxus, first identified by hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerevisiae and S. bayanus. Enological strains classified as S. bayanus, S. chevalieri, and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uvarum strains belong to S. bayanus. Enologists should no longer use the name of S. bayanus for S. cerevisiae Gal strains, and should consider S. bayanus as a distinct species.

  9. Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

    Directory of Open Access Journals (Sweden)

    Caldeira Roberta L

    2000-01-01

    Full Text Available Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

  10. Identification of three Iranian species of the genus Rattus (Rodentia, Muridae using a PCR-RFLP technique on mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    safieh Akbary rad

    2009-11-01

    Full Text Available Abstract Three species of the genus Rattus Fisher, 1803 have been reported from Iran: the brown rat (R. norvegicus, the black rat (R. rattus and the Himalayan rat (R. pyctoris. The first two were introduced, whilst R. pyctoris is native and lives in mountainous regions from Pakistan to north-eastern Iran. In this study, the mitochondrial DNA from twenty six rats were analysed using a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragments Length Polymorphism method to investigate inter-specific variation. Part of the 16S rRNA and cytochrome b genes were amplified and digested with three restriction enzymes: AluI, MboI and HinfI. Restriction fragments resulted in four different haplotypes and allowed to distinguish the three Rattus species. Our results suggest that the Himalayan rats are more closely related to R. rattus than to R. norvegicus and provide the basics for further phylogenetic studies. Riassunto Identificazione di tre specie iraniane del genere Rattus (Rodentia, Muridae tramite PCR-RFLP su DNA mitocondriale. Tre specie del genere Rattus risultano diffuse in Iran: il surmolotto (R. norvegicus, il ratto nero (R. rattus e il ratto himalayano (R. pyctoris. Le prime due specie sono state introdotte, mentre R. pyctoris è presente nelle aree montane che si sviluppano dal Pakistan all’Iran nordorientale. In questo studio, il DNA mitocondriale di 26 individui è stato analizzato tramite PCR-RFLP per evidenziare variazioni inter-specifiche. Parte dei geni del rRNA 16S e del citocromo b è stata amplificata e quindi sottoposta a digestione tramite tre diversi enzimi: AluI, MboI e HinfI. I frammenti di restrizione hanno permesso di individuare quattro aplotipi mitocondriali e di distinguere le tre specie. I risultati ottenuti suggeriscono che il ratto

  11. KERAGAMAN DURIAN BERDASARKAN FRAGMEN INTERNAL TRANSCRIBED SPACERS (ITS DNA RIBOSOMAL MELALUI ANALISIS PCR-RFLP

    Directory of Open Access Journals (Sweden)

    R. U. Hikmah

    2016-11-01

    Full Text Available Durian adalah salah satu jenis buah-buahan tropis yang memiliki nilai ekonomi tinggi. Durian memiliki bermacam-macam kultivar dengan morfologi yang sulit untuk membedakan. jaminan identitas penting bagi informasi mendasar dalam meningkatkan efisiensi pemuliaan dan pengembangan durian. identifikasi molekuler dianggap lebih akurat dibandingkan dengan karakter morfologi. Polymerase Chain Reaction Restriction Fragment Length Polimorfism (PCR-RFLP adalah metode untuk menganalisis hasil DNA fragmen panjang perbedaan yang mencerna menggunakan enzim restriksi dengan endonuklease. Sampel yang digunakan dalam penelitian ini adalah aksesi durian diambil secara acak di kecamatan Gunungpati, Semarang. Genomik DNA diisolasi berdasarkan protokol Kit nukleon Phytopure dengan modifikasi. ITS wilayah ribosom DNA diamplifikasi menggunakan teknik PCR-RFLP mengeksploitasi primer spesifik L ITS dan ITS 4 menghasilkan ITS panjang fragmen pada 800 bp. Amplikon yang dicerna menggunakan enam enzim restriksi AluI, Eco471, Bsp1431, BsuRI, Mph11301 dan Ade1. Hasil penelitian dari 11 aksesi durian yang diperiksa menunjukkan bahwa enzim Bsp1431 memiliki dua situs tertentu yang dipotong pada ukuran 550 bp dan 120 bp. enzim BsuR1 memiliki luka situs tertentu dalam ukuran 600 bp. Sedangkan enzim Eco471 telah mencerna situs spesifik pada ukuran 450 bp. Kesimpulan dari penelitian ini adalah durian nomor aksesi ke-12, 15, dan 17 memiliki hubungan genetik dekat dan diduga berada di salah satu spesies.Durian is one of the types of tropical fruits that has high economic value. Durian has an assortment of cultivars with morphology that are difficult to distinguish. Identity assurance is important for the fundamental information in increasing the efficiency of breeding and development of durian. Molecular identification is considered to be more accurate than the morphological characters. Polymerase Chain Reaction Restriction Fragment Length Polimorfism (PCR-RFLP is a method to analyze

  12. Identification of aphid (Hemiptera: Aphididae) species of economic importance in Kenya using DNA barcodes and PCR-RFLP-based approach.

    Science.gov (United States)

    Kinyanjui, G; Khamis, F M; Mohamed, S; Ombura, L O; Warigia, M; Ekesi, S

    2016-02-01

    Aphids are among pests of economic importance throughout the world. Together with transmitting plant viruses, aphids are capable of inflicting severe crop production losses. They also excrete honeydew that favours the growth of sooty mold which reduces the quality of vegetables and fruits and hence their market values. Rapid and accurate identification of aphids to the species level is a critical component in effective pest management and plant quarantine systems. Even though morphological taxonomy has made a tremendous impact on species-level identifications, polymorphism, morphological plasticity and immature stages are among the many challenges to accurate identification. In addition, their small size, presence of cryptic species and damaged specimens dictate the need for a strategy that will ensure timely and accurate identification. In this study, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based on mitochondrial cytochrome c oxidase subunit I gene and DNA barcoding were applied to identify different aphid species collected from different agro-ecological zones of Kenya. Three restriction enzymes RsaI, AluI and Hinf1 produced patterns that allowed unambiguous identification of the species except Aphis craccivora and Aphis fabae. Analyses of the barcode region indicated intraspecific and interspecific sequence divergences of 0.08 and 6.63%, respectively. DNA barcoding identified all species, including the morphologically indistinguishable A. craccivora and A. fabae and separated two subspecies of A. fabae. Based on these results, both PCR-RFLPs and DNA barcoding could provide quick and accurate tools for identification of aphid species within Aphididae subsequently aiding in effective pest management programmes and enhance plant quarantine systems.

  13. Application of PCR-based restriction fragment length polymorphism for the identification of mycobacterial isolates.

    Science.gov (United States)

    Deepa, P; Therese, K L; Madhavan, H N

    2005-05-01

    Conventional identification of mycobacteria is achieved by standard biochemical tests that are time consuming, laborious and is not always conclusive. This study was thus undertaken to standardize a simple, rapid and cost-effective polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) using primers coding for the 16S - 23S rRNA spacer region to identify the mycobacterial isolates to the species level. The PCR with primers targeting the 16S-23S rRNA spacer region was standardized using the standard mycobacterial strains and applied on 51 clinical isolates. The PCR amplified products were subjected to RFLP using the restriction enzymes, Hae III, MspI and BstXI. The results obtained were compared with those of conventional biochemical tests. PCR was sensitive to detect 2.5 pg of H37Rv DNA (370 bp for slow grower mycobacteria) and 1.5 pg of M. fortuitum DNA (450 bp for rapid grower mycobacteria). Based on the PCRRFLP products obtained the 51 mycobacterial isolates were classified into 41 slow growers and 10 rapid growers. Among the 41 slow growers, 40 were identified as M. tuberculosis, one as M. xenopi and 10 rapid growers as M. fortuitum. PCR using primers targeting the 16S-23S rRNA spacer region was a reliable tool for rapid identification of mycobacterial isolates into slow and rapid growers within 4 h of isolation and further speciation by PCR-RFLP within 6-8 h.

  14. Detection of the mosquitocidal toxin genes encoding Cry11 proteins from Bacillus thuringiensis using a novel PCR-RFLP method Detección de genes que codifican proteínas mosquitocidas Cry11 de Bacillus thuringiensis mediante un método de PCR-RFLP novedoso

    Directory of Open Access Journals (Sweden)

    D. H. Sauka

    2010-02-01

    Full Text Available A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method for detection of cry11 genes from Bacillus thuringiensis was established. Based on the analysis of conserved regions of the cry11 genes, 2 oligonucleotide primers were designed to amplify a 1459-bp fragment of the cry11Aa gene, and a 1471-bp of the cry11Ba and cry11Bb genes. The amplification products were digested with restriction endonuclease HinfI. Exotic B. thuringiensis strains and native isolates collected from soils, leaves and stored product dust of Argentina were analyzed to study the distribution of cry11 genes. The PCR-RFLP patterns revealed the detection of cry11 genes in 3 of 64 exotic strains and in 10 of 107 native B. thuringiensis isolates tested. Just the cry11Aa gene subclass was detected among these bacteria. Since the methodology was also developed to detect cry11Ba and cry11Bb genes, an experimental future confirmation will be required. Based on the results obtained, the PCR-RFLP method presented may be a valuable tool for specific detection of the mosquitocidal toxin genes encoding Cry11 proteins from B. thuringiensis.En el presente estudio se estableció una estrategia basada en la amplificación génica (PCR y el posterior análisis de restricción (RFLP para detectar todos los genes cry11 de Bacillus thuringiensis informados hasta ahora. De acuerdo con el análisis de las regiones conservadas en los genes cry11, se diseñaron dos cebadores para amplificar un fragmento de 1459 pb de los genes cry11Aa y un fragmento de 1471 pb de los genes cry11Ba y cry11Bb. Los productos de la amplificación fueron digeridos con la enzima de restricción HinfI. Se analizaron cepas exóticas de B. thuringiensis y aislamientos nativos de Argentina obtenidos a partir de muestras de suelos, hojas y polvillo de silos, para estudiar la distribución de los genes cry11. Los patrones de PCR-RFLP revelaron la presencia de genes cry11 en 3 de las 64 cepas ex

  15. Analysis of bovine growth hormone gene polymorphism of local and ...

    African Journals Online (AJOL)

    Analysis of bovine growth hormone gene polymorphism of local and Holstein cattle breeds in Kerman province of Iran using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) ... African Journal of Biotechnology ... RFLPs in this segment were studied using AluI restriction enzyme.

  16. Detection of MspI polymorphism and the single nucleotide ...

    African Journals Online (AJOL)

    The aim of this study was to detect the genetic polymorphism of GH gene in five camel breeds reared in Egypt which are Sudany, Somali, Mowaled, Maghrabi and Falahy, using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) technique. Also, this work aimed to identify the single nucleotide ...

  17. Association of transforming growth factor-ß3 gene polymorphism ...

    African Journals Online (AJOL)

    Genotyping for the TGF-β3 gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and BslI restriction endonuclease showed a mutation in 294-bp fragment located on the fourth intron of chromosome 5. Polymorphism in TGF-β3 gene was significantly (P < 0.1) associated with ...

  18. Keragaman Genetik Gen Hormon Pertumbuhan (GH|MboII pada Itik Sikumbang Janti Menggunakan Penciri PCR-RFLP

    Directory of Open Access Journals (Sweden)

    T.D. Nova

    2016-02-01

    Full Text Available Penelitian ini bertujuan untuk mengetahui keragaman gen hormon pertumbuhan (GH dengan enzim MboII pada itik Sikumbang Janti dengan menggunakan penciri PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism. Penelitian ini menggunakan sebanyak 50 sampel darah itik Sikumbang Janti. Sampel darah itik Sikumbang Janti diambil melalui vena achilaris sebanyak ± 1 ml. DNA sampel darah diisolasi menggunakan protocol Genomik DNA Purification Kit (Promega. DNA total diamplifikasi menggunakan sepasang primer F : 5’-CTG GAG CAG GCA GGA AAA TT-3’ dan R: 5’-TCC AGG GAC AGT GAC TCA AC-3’ yang menghasilkan fragmen exon 1 gen GH dengan panjang 801 bp. Produk amplifikasi direstriksi dengan menggunakan MboII yang mengenali situs pemotongan GAAGA (N/8↓ . Dari 46 sampel hasil restriksi diperoleh dua posisi. Pada posisi 618 bp dengan genotip yaitu genotip heterozigot (+/- yang terdiri dari 3 pita (266 bp, 535 bp dan 801 bp, genotip homozigot (+/+ yang terdiri dari 3 pita (109 bp, 266 bp, 426 bp dan genotip homozigot (-/- yang terdiri dari 1 pita ( 801 dan terdapat dua tipe alel, yaitu alel (+ dan all (-, all (+ sebesar 0,79 dan alel (- sebesar 0,21. Sedangkan pada posisi 727 bp memiliki genotip yaitu genotip heterozigot (+/- yang terdiri dari 3 pita (109 bp, 266 bp, 426 bp, dan genotip homozigot (-/- yang terdiri dari 3 pita dan terdapat dua tipe alel, yaitu frekuensi alel (+ sebesar 0,61 dan frekuensi alel (- sebesar 0,39. Dari hasil penelitian ini dapat disimpulkan bahwa gen GH-MboII memiliki keragamanan yang tinggi serta menunjukkan adanya keseimbangan atau tidak menyimpang dari keseimbangan Hardy Weinberg pada posisi keragaman 618 bp dan pada posisi 727 dalam ketidakseimbangan Hardy Weinberg.

  19. Distribution and differential diagnosis of Entamoeba Histolytica from Entamoeba Dispar by the PCR-RFLP method in central Iran

    International Nuclear Information System (INIS)

    Hooshyar, Hossein; Rezian, Mostafa; Kazemi, Bahram

    2003-01-01

    Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable human protozoan parasites that are genetically distinct species. The potential invasive pathogenic Entamoeba histolytica and non-invasive parasites Entamoeba dispar can be differentiated by molecular and other methods. We used Polymer Chain Reaction (PCR) to determine the ratio of two species in a population in central Iran.Human stool samples(n=12 148) were randomly collected in Tehran and Karaj and examined for E.histolytica/E.dispar cysts with direct and formalin-ether methods.Eighty -seven (0.7%) cases were positive, of which 49 (62.8%) isolates were successfully cultured in Robison's medium. A pair of oligonucleotide primers designed from sequence data for genomic DNA coding the 30-KD surface antigen of E.histolytica/E.dipar was used to amplify a 374 base-pair (bp) fragment. The restriction fragment length polymorphism (RFLP) pattern obtained from a standard E.histolytica isolate had had two fragments (219 bp and 155bp), but the standard isolate of E. disparshowed three fragments (155, 152 and 67bp ). Differential diagonosis of 49 isolates of E. histolytica/ E. dispar from Tehran and Karaj using PCR-RFLP revealed that 46(93.9%) were E. were E. dispar while only 2(4.1%) were E. histolytica .One person (2%) had a mixed infection and showed both patterns The differential diagonosis of the potentially pathogenic parasite E.histolytica from the non-pathogenic E. dispar is of clinical and epidemiological importance. This study demonstrated E. dispar is much more prevalent than E.histolytica among the c yst passersin Tehran and Kraj in Central Iran. (author)

  20. Candida species in cutaneous candidiasis patients in the Guilan province in Iran; identified by PCR-RFLP method.

    Directory of Open Access Journals (Sweden)

    Ali Akbar Fallahi

    2013-11-01

    Full Text Available Due to the epidemiological alteration in distribution of Candida species as well as significant increasing trend of either intrinsic or acquired in resistance of some of these fungi, the precise identification of Candida species is necessary for effective antifungal therapy and also for prevention of nosocomial infections. PCR-RFLP method is indicated to be a reliable, rapid and simple technique which is able to differentiate the Candida species. In the present study, we applied this method to evaluate the distribution of Candida species in patients affected with cutaneous candidiasis in the Guilan province. 896 clinical cutaneous samples were collected from different parts of skin and nail of suspected patients referred to clinical centers all over the Guilan province during 24 months. Samples were examined directly with 15% KOH and cultured on fungal specific media. Genomic DNA was extracted and the restriction enzyme Msp1 was applied for polymorphism analysis. Totally, 47 yeast strains were successfully isolated from different clinical samples and identified by conventional as well as PCR-RFLP methods. The results indicated that Candida albicans (36.17% was the most frequent species followed by C. parapsilosis (25.53%, C. tropicalis (19.14%, C. guilliermondii (14.89%, C. famata (2.12% and C. krusei (2.12%. Female finger nails were the most common location to be affected by Candida species. In conclusion, PCR-RFLP method was successfully used for recognition of clinical Candida species within the Guilan province and obtained results revealed C. albicans as the predominant causative agent of cutaneous candidiasis. However, distribution of other Candida species did not completely consist with the reported distribution of Candida species in other parts of Iran with different climate to the Guilan province.

  1. PCR-RFLP Method to Identify Salmonid Species of Economic Importance

    Directory of Open Access Journals (Sweden)

    Andreea Dudu

    2011-05-01

    Full Text Available The identification of different fish species by molecular methods has become necessary to avoid both the incorrect labelling of individuals involved in repopulation programmes and the commercial frauds on the fish market. Different fish species of great economical importance, like the salmonids, which are very much requested for their meat, can be identified using molecular techniques such as PCR-RFLP. The method is based on the amplification of a target region from the genome by PCR reaction followed by endonucleases digestion to detect the polymorphism of restriction fragments. In our study we analysed the following salmonid species from Romania: Salmo trutta fario, Salmo labrax, Salvelinus fontinalis, Onchorhynchus mykiss, Thymallus thymallus and Hucho hucho. In order to discriminate between the analysed species we amplified a fragment of mitochondrial genome comprising tRNAGlu/ cytochrome b/ tRNAThr/ tRNAPro/ D-loop/ tRNAPhe, followed by digestion with a specific restriction enzyme. The direct digestion of unpurified PCR products generated species-specific restriction patterns and proved to be a simple, reliable, inexpensive and fast method. Thus, it may be successfully utilized in specialized laboratories for the correct identification of the fish species for multiple purposes, including the traceability of fish food products.

  2. Association between A59V polymorphism in exon 3 of leptin gene ...

    African Journals Online (AJOL)

    ONOS

    2010-09-06

    Sep 6, 2010 ... We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to screen for DNA polymorphisms of the leptin gene in 255 cows of Iranian Holstein. Amplified region is located in exon 3 of leptin gene. The genomic bovine leptin sequences, which consist of three ...

  3. Association of Gln27Glu polymorphism of the β-2- adrenergic ...

    African Journals Online (AJOL)

    A group of 66 patients with preeclampsia and 72 control subjects were analyzed for the Arg16Gly and Gln27Glu polymorphisms of the ADRB2 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Comparisons of the ADRB2 genotypes or alleles between different groups were performed ...

  4. Benzimidazole -Resistance in Haemonchus Contortus: New PCR-RFLP Method for the Detection of Point Mutation at Codon 167 of Isotype 1 Β-Tubulin Gene

    Directory of Open Access Journals (Sweden)

    A Eslami

    2012-12-01

    Full Text Available Background: Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT-HC167 MF-primer, to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubu­lin gene of Haemonchus contortus.Methods: There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleo­tide polymorphism (SNP at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer, in which the nucleotide T at the posi­tion 443 was substituted through a nucleotide A creating a restriction site for restriction endonuc­lease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achiev­ing a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, respec­tively.Results: All worms had two alleles encoding for phenylalanine (BZss homozygote for both codons.Conclusion: Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP.

  5. Differentiation of Candida glabrata, C. nivariensis and C. bracarensis based on fragment length polymorphism of ITS1 and ITS2 and restriction fragment length polymorphism of ITS and D1/D2 regions in rDNA

    DEFF Research Database (Denmark)

    Mirhendi, H; Bruun, B; Schønheyder, H C

    2011-01-01

    Different molecular methods for the discrimination of Candida glabrata, C. bracarensis and C. nivariensis were evaluated and the prevalence of these species among Danish blood isolates investigated. Control strains were used to determine fragment length polymorphism in the ITS1, ITS2, ITS1-5.8S...... enzymes were suitable for RFLP differentiation of the species. Enzymatic digestion of the D1/D2 domain with TatI produced unique band sizes for each of the three species. PCR-RFLP and PNA-FISH were in agreement for all of the isolates tested. None of the 133 Danish blood isolates were C. nivariensis or C....... bracarensis. Fragment size polymorphism of ITS1 and RFLP of the D1/D2 domain or the ITS region are useful methods for the differentiation of the species within the C. glabrata group. C. bracarensis and C. nivariensis are rare among Danish C. glabrata blood isolates....

  6. Differentiation between Aspergillus flavus and Aspergillus parasiticus from Pure Culture and Aflatoxin-Contaminated Grapes Using PCR-RFLP Analysis of aflR-aflJ Intergenic Spacer

    International Nuclear Information System (INIS)

    El Khoury, A.; Atoui, A.; Lebrihi, A.; Rizk, T.; Lteif, R.; Kallassy, M.

    2011-01-01

    Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification. (author)

  7. ESTANDARIZACIÓN DE UNA PRUEBA DE PCR-RFLP PARA LA IDENTIFICACIÓN DE Rhodococcus rhodnii EN INSECTOS TRIATOMINOS

    Directory of Open Access Journals (Sweden)

    J. Rodríguez

    2007-12-01

    Full Text Available Triatomines are insects of great importance for public health because they transmit the parasite Trypano-soma cruzi, etiological agent of Chagas disease. These insects harbour intestinal symbiotic flora from theRhodococcus genus, which provide the nutrients lacking in the insect diet, nutrients which are essentialfor normal insect development and growth. Study of these bacteria is of special interest due to thepossibility of designing paratransgenesis control strategies as well as for implications that interactionsbetween these bacteria and parasites can develop inside triatomine insects. Taking into account thedifficulty in identifying bacteria from the Rhodococcus genus by traditional methods, in this work apolymerase chain reaction (PCR followed by a restriction fragment length polymorphism analysis wasstandardized to identify Rhodococcus rhodnii isolated from Rhodnius prolixus. Gene coding for bacterial16 S rRNA was amplified and obtained products were sequenced and digested with several restrictionenzymes. Results show a total correlation between number and size of the restriction fragments obtainedin vitro and in silico as well. Therefore, applicability of this PCR-RFLP assay for R. rhodnii identificationhas been demonstrated.

  8. Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Dinka eMandakovic

    2016-05-01

    Full Text Available The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS, a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination and fish samples (coinfection, aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants. Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

  9. Molecular Characterization of Yeast Strains Isolated from Different Sources by Restriction Fragment Length Polymorphism

    International Nuclear Information System (INIS)

    Ali, M. S.; Latif, Z.

    2016-01-01

    Various molecular techniques like analysis of the amplified rDNA internal transcribed spacers (ITS), intragenic spacers and total ITS region analysis by restriction fragment length polymorphism (RFLP) has been introduced for yeast identification but there are limited databases to identify yeast species on the basis of 5.8S rDNA. In this study, twenty nine yeast strains from various sources including spoiled fruits, vegetables, foodstuffs, and concentrated juices were characterized by PCR-RFLP. PCR-RFLP has been used to characterize yeasts present in different spoiled food samples after isolation of the yeasts. By using this technique, the isolated yeast strains were characterized by direct 5.8S-ITS rDNA region amplification. RFLP analysis was applied to each of the amplification products (varied from 400bp to 800bp) detected, and the corresponding yeast identifications were made according to each specific restriction patterns obtained after treatment with two endonucleases TaqI and HaeIII which yielded a specific banding pattern for each species. For further confirmation amplified products of eleven selected isolates were sequenced and blast on NCBI. Both RFLP and sequence analyses of the strains with accession nos. KF472163, KF472164, KF472165, KF472166, KF472167, KF472168, KF472169, KF472170, KF472171, KF472172, KF472173 gave significantly similar results. The isolates were found to belong five different yeast species including; Candida spp., Pichia spp., Kluyveromyces spp., Clavispora spp. and Hanseniaspora spp. This method provides a fast, easy, reliable and authentic way for determining yeast population present in different type of samples, as compared to traditional characterization technique. (author)

  10. Genetic Background and Population Genetics of Hungarian Brown Trout Populations Using PCR-RFLP and Microsatellite Markers

    Directory of Open Access Journals (Sweden)

    Ágnes Ősz

    2015-12-01

    4 University of West Hungary, Mosonmagyaróvár Vár 2., 9200 Mosonmagyaróvár, Hungary Based on the analyses of the mitochondrial DNA of several European brown trout populations, five evolutionary lineages of brown trout were indentified (Atlantic, Danubian, Mediterranean, Adriatic, Marble. The species is bred primarily for stock enhancement of natural waters, however the most hatchery-maintained broodstocks originate from the Atlantic lineage. Due to the hydrogeography of Hungary our stocks should theoretically belong to the Danubian lineage; however, this has not been investigated earlier by genetic studies. For our genetic analysis, 702 fin clips were collected from two brown trout broodstocks (Lillafüred and Szilvásvárad as well as populations of natural streams (Bán, Jósva, Kemence, Apátkút, Bittva and Kölöntés in Hungary. Sequencing of the control region in mitochondrial DNA, three PCR-RFLP (mitochondrial DNA control region, lactate dehydrogenase and somatolactin genes and five microsatellite markers were used to distinguish between Danubian and Atlantic lineages of brown trout. The proportion of the mitochondrial haplotype of the Danubian lineage was low, with the exception of the Apátkúti, Kölöntés streams and Szilvásvárad broodstock. Analyses of nuclear PCR-RFLP and microsatellites markers showed various distributions of alleles characteristic of the Atlantic or Danubian lineages, although the Atlantic genotype has dominated in all population. In case of the analyses of microsatellites the polymorphism varied greatly at all locations. In addition we found several alleles that were not described earlier in other populations. Those alleles probably would be typical of Hungarian brown trout populations. Overall the populations were effectively in Hardy-Weinberg equilibrium for both PCR-RFLP and microsatellite markers. The remarkably high proportion of allochthonous Atlantic alleles in the analyzed sites is a clear indicator of the import

  11. Linkage maping of a HaeIII PCR-RFLP within the porcine EXT1 gene

    Czech Academy of Sciences Publication Activity Database

    Čepica, Stanislav; Rohrer, G. A.; Masopust, Martin

    2002-01-01

    Roč. 33, - (2002), s. 81-82 ISSN 0268-9146 R&D Projects: GA ČR GA523/99/0842; GA AV ČR KSK5052113 Keywords : PCR- RFLP * mapping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.443, year: 2002

  12. Differentiation between a pathogenic and a non-pathogenic form of Gyrodactylus salaris using PCR-RFLP

    DEFF Research Database (Denmark)

    Kania, Per Walther; Jørgensen, Thomas Rohde; Buchmann, Kurt

    2007-01-01

    A new method based on PCR-RFLP is presented. It is able to differentiate between the Danish non-pathogenic form of Gyrodactylus salaris and the Norwegian pathogenic form.......A new method based on PCR-RFLP is presented. It is able to differentiate between the Danish non-pathogenic form of Gyrodactylus salaris and the Norwegian pathogenic form....

  13. Karakterisasi Molekuler Ikan Gurami Soang (Osphronemus gouramy Lac. yang Mati pada Rentang Waktu Berbeda Menggunakan PCR-RFLP Gen Major Histocompatibility Complex Kelas II B

    Directory of Open Access Journals (Sweden)

    Jaka Tri Spetiawan

    2017-08-01

    Full Text Available Gurami (Osphronemus gouramy Lac. is a popular fish species among Indonesian people. Several Gurami strains have been cultivated by fish farmer, one of which is Gurami Soang. This strain is belived to have a faster growth rate compared to other strains. However, like other strains, the fingerling of Soang strain have also a low survival and suceptible to disease, especially that caused by Aeromonas hydrophila infection. It has been proved that seeds from a single spawning event show varibale disease resistance. The difference in resistance among individuals is suggested related to the difference in their genetic component. One of the genes responsible for resistance is Major Histocompatibility Complex (MHC class II B gene. Variability in resistance can be analyzed by using PCR - RFLP technique. PCR-RFLP is a technique that can produce a specific DNA fragments by PCR, followed by cutting the PCR product using restriction enzymes to describe the presence or absence of restriction sites in DNA fragments. This research aims to determine genetic marker to differiantiate between resitant and irresistant individual of Gurami Soang infected by A. hydrophila which die at a different time priod based on PCR-RFLP MHC class IIB gene. The study used survey method with purposive random sampling. The Data of PCR-RFLP band patterns were analyzed descriptively. The result indicated that cutting of the MHC class II B gene using HinfI produce two RFLP bands with 300 bp and 100 bp length in all samples. Meanwhile, the MHC IIB gene was not cuted by PstI, HindIII, BamHI and EcoRI enzymes forall samples. These mean that MHC II gene in all individuals were monomorphic. Therefore,it can be concluded that there is no specific genetic marker to differentiate gurami soang individulas which was dying in different time periods.

  14. Analysis of mitochondrial DNA for authentication of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) by polymerase chain reaction-restriction fragment length polymorphism.

    Science.gov (United States)

    Fajardo, Violeta; González, Isabel; López-Calleja, Inés; Martin, Irene; Rojas, Maria; Pavón, Miguel Angel; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2007-01-01

    The prevention of fraudulent labeling of game meat constitutes an important part of food regulatory control and quality assurance systems. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis based on mitochondrial deoxyribonucleic acid (DNA) was developed for authentication of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon). Amplification and restriction site analysis of a DNA fragment about 720 base pairs (bp) from the mitochondrial 12S rRNA gene of all analyzed species permitted the selection of Msel and Apol endonucleases for meat speciation. The 12S rRNA restriction profiles obtained allowed the unequivocal identification of chamois, pyrenean ibex, and mouflon/sheep and their differentiation from meats of domestic species such as cattle, goat, and swine. The highly variable mitochondrial D-loop gene was also targeted to attempt discrimination between mouflon and sheep meats. A D-loop region (700-1000 bp) was amplified and sequenced in all game and domestic species analyzed, and a primer set was designed for the selective amplification of a 370 bp DNA fragment from mouflon and sheep. PCR-RFLP analysis with the selected Maell enzyme generated a single electrophoretic profile characteristic for sheep, whereas 3 different fragment patterns were obtained for mouflon meats. Consequently, the PCR-RFLP technique developed can be routinely applied in inspection programs in order to verify the correct labeling of game species.

  15. Identification of Panulirus homarus puerulus larvae by restriction fragment length polymorphism of mitochondrial cytochrome oxidase I gene.

    Science.gov (United States)

    Dharani, G; Maitrayee, G A; Karthikayalu, S; Kumar, T S; Anbarasu, M; Vijayakumaran, M

    2009-02-01

    Molecular identification of puerulus larvae of Panulirus homarus of the genus Panulirus from Indian coast was studied by employing Polymerase Chain Reaction, Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the mitochondrial DNA (mtDNA) Cytochrome Oxidase Gene (COI) by agarose gel electrophoresis and Denaturing Gradient Gel Electrophoresis (DGGE). The size of amplified fragment of COI gene was estimated to be approximately 1300 base pairs (bp). Single fragment amplification was recorded during different stages of the life cycle. The RFLP digestion was carried out using five different restriction enzymes (BsplI, HhaI, RsaI, TaqI and AluI). The RFLP profile of the different endonucleases, varied between 1-5 restriction types. RFLP analysis using endonuclease TaqI enabled identification of P. homarus during different stages of its life history.

  16. Detección de Bartonella bacilliformis usando PCR-RFLP

    Directory of Open Access Journals (Sweden)

    C Padilla

    1998-01-01

    Full Text Available Reportamos la aplicación de las técnicas moleculares PCR-RFLP para la confirmación de infecciones por Bartonella bacilliformis. El método de PCR-RFLP se basa en la amplificación in vitro de un fragmento de 380 pb correspondiente al gen citrato sintetasa a partir de sangre y cultivos in vitro. El análisis del producto de amplificación por cortes con las enzimas restricción Hinfl y Taql permite caracterizar molecularmente que el brote ocurrido en Urubamba, Cuzco fue producido por Bartonella bacilliformis. Este método es aplicado directamente a sangre y a cultivos.

  17. PHYLOGENETIC RELATIONSHIPS AMONGST 10 Durio SPECIES BASED ON PCR-RFLP ANALYSIS OF TWO CHLOROPLAST GENES

    Directory of Open Access Journals (Sweden)

    Panca J. Santoso

    2013-07-01

    Full Text Available Twenty seven species of Durio have been identified in Sabah and Sarawak, Malaysia, but their relationships have not been studied. This study was conducted to analyse phylogenetic relationships amongst 10 Durio species in Malaysia using PCR-RFLP on two chloroplast DNA genes, i.e. ndhC-trnV and rbcL. DNAs were extracted from young leaves of 11 accessions from 10 Durio species collected from the Tenom Agriculture Research Station, Sabah, and University Agriculture Park, Universiti Putra Malaysia. Two pairs of oligonucleotide primers, N1-N2 and rbcL1-rbcL2, were used to flank the target regions ndhC-trnV and rbcL. Eight restriction enzymes, HindIII, BsuRI, PstI, TaqI, MspI, SmaI, BshNI, and EcoR130I, were used to digest the amplicons. Based on the results of PCR-RFLP on ndhC-trnV gene, the 10 Durio species were grouped into five distinct clusters, and the accessions generally showed high variations. However, based on the results of PCR-RFLP on the rbcL gene, the species were grouped into three distinct clusters, and generally showed low variations. This means that ndhC-trnV gene is more reliable for phylogenetic analysis in lower taxonomic level of Durio species or for diversity analysis, while rbcL gene is reliable marker for phylogenetic analysis at higher taxonomic level. PCR-RFLP on the ndhC-trnV and rbcL genes could therefore be considered as useful markers to phylogenetic analysis amongst Durio species. These finding might be used for further molecular marker assisted in Durio breeding program.

  18. Prevalence of renal lesions in slaughtered cattle in Shiraz, Iran, and detection of Leptospira in them by nested PCR-RFLP.

    Science.gov (United States)

    Taghadosi, Vahideh; Hosseinzadeh, Saeid; Shekarforoush, Seyed Shahram; Samiei, Azadeh

    2016-12-01

    Renal diseases in cattle are frequently not recognized due to the subclinical conditions. Some species of Leptospira are the main cause of infectious agents that damage the kidneys and lead to abortion and economic losses in cattle and are also of major concern in the public health. This study was aimed to assess the prevalence of renal lesions of slaughtered cattle in the Shiraz abattoir and to determine the correlation between rejected kidneys and infection with Leptospira using nested PCR-restriction fragment length polymorphism (RFLP) techniques. Out of 1000 inspected animals, 205 (20.5 %) revealed the renal lesions. Chronic nephritis (7.5 %), white-spotted kidney (7.3 %), and petechial hemorrhage (3.5 %) were the most prevalent forms of the lesions. A direct correlation between increasing the age and significant increase in the rate of lesions was also observed (P = 0.03). Using nested PCR-RFLP assay, 40.8 % of the tested kidneys were turned to be infected to the pathogenic species of Leptospira. The risk of infection of the kidneys with white spot to pathogenic species of Leptospira (53.8 %) was more than that of the kidneys with other lesions (25.0 %) (P = 0.014). The odd ratio indicates that the kidneys with white spot lesions are likely to be infected with pathogenic species of Leptospira, five times greater than other lesions. This study showed that renal lesions especially white-spotted kidney, which were considerably associated with Leptospira in slaughtered cattle in Shiraz, were very high. This is important in terms of public health and in particular, increases the risk of transmission of disease to human specially in the high-risk careers including farmers, veterinarians, and abattoir workers.

  19. PCR-RFLP analyses for studying the diversity of GH and Pit-1 genes in Slovak Simmental cattle

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    Anna Trakovická

    2013-10-01

    Full Text Available The aim of this study was evaluation of growth hormone (GH and specific pituitary transcription factor (Pit-1 genes diversity in population of 353 Slovak Simmental cows. The analyses were based on single nucleotide polymorphisms GH/AluI and Pit-1/HinfI detections. A polymorphic site of GH gene (AluI has been linked to differences in circulating metabolites, metabolic hormones and milk yield. Bovine Pit-1 is responsible for pituitary development and hormone secreting gene expression, including GH gene. The Pit-1/HinfI locus was associated with growth, milk production and reproduction performance in cattle. Samples of genomic DNA were analyzed by PCR-RFLP method. Digestion of GH gene PCR products with restriction enzyme AluI revealed allele L and V with frequency 0.695 and 0.305, respectively. The digested Pit-1 gene PCR products with enzyme HinfI revealed alleles A (0.249 and B (0.751. Dominant genotypes were for GH gene heterozygous LV (0.47 and for Pit-1 gene homozygous BB (0.56 animals. The observed heterozygosity, effective allele numbers and polymorphism information content of GH/AluI and Pit-1/HinfI bovine loci population were 0.42/0.37, 1.73/1.59 and 0.33/0.30, respectively. The median polymorphic information content of loci was also transferred to the higher observed homozygosity in population (0.58/0.63. Keywords: cattle, growth hormone, leptin, PCR, Pit-1, polymorphism.

  20. HERITABILITIES OF BODY SIZE BY GROWTH HORMONE (GH-Msp1 GENOTYPES USING PCR-RFLP IN ONGOLE GRADE CATTLE

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    U. Paputungan

    2016-02-01

    Full Text Available Genotypic performance in term of heritability as the crucial factor of animal economical traits for body size inheritance had not been fully studied in Ongole-grade cattle. The objectives of this research were to define the heritability values of live weight, chest girth and body length in Ongole-crossbred cattle. Total of 37 blood samples were collected from parental cows and 2 blood samples from parental Ongole breed bulls. All blood samples were screened for the presence of growth hormone (GH locus using PCR-RFLP method involving restricted enzyme Msp1 on agarose-gel (1.2%. Data were analyzed using statistical program in Excel XP. Results showed that the phenotypic estimation average of Ongole grade cattle population of live weight, chest girth and body length were 445.41 ± 45.95 kg, 175.35 ± 4.11 cm, and 139.70 ± 5.73cm, respectively. The heritability values of animal live weight, chest girth and body length in this study were 0.24, 0.003, and 0.41, respectively. These heritability values of animal live weight and body length would be categorized as moderate to high genotypic performance values, while the heritability of animal chest girth was included in low heritability standard of the animal economical trait performance.

  1. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

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    N Maleki-Ravasan

    2009-07-01

    Full Text Available   Abstract Background: We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to iden­tify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method."nMethods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006-2007. For PCR-RFLP a set of conserved vertebrate prim­ers were used to amplify a part of the host mitochondrial cytochrome b (cyt b gene followed by digestion of the PCR products by Hae III enzyme."nResults: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on hu­mans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%, human (10%, dog (4%, human and cow (21%, dog and cow (14%, and human and dog (3%. "nConclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advan­tages and limitations of PCR and ELISA assays are discussed.

  2. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

    Science.gov (United States)

    Maleki-Ravasan, N; Oshaghi, MA; Javadian, E; Rassi, Y; Sadraei, J; Mohtarami, F

    2009-01-01

    Background We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method. Methods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006–2007. For PCR-RFLP a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b (cyt b) gene followed by digestion of the PCR products by Hae III enzyme. Results: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%), human (10%), dog (4%), human and cow (21%), dog and cow (14%), and human and dog (3%). Conclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advantages and limitations of PCR and ELISA assays are discussed. PMID:22808367

  3. Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays

    Directory of Open Access Journals (Sweden)

    N Maleki-Ravasan

    2009-06-01

    Full Text Available  Background: We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to iden­tify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method.Methods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006-2007. For PCR-RFLP a set of conserved vertebrate prim­ers were used to amplify a part of the host mitochondrial cytochrome b (cyt b gene followed by digestion of the PCR products by Hae III enzyme.Results: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on hu­mans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%, human (10%, dog (4%, human and cow (21%, dog and cow (14%, and human and dog (3%. Conclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advan­tages and limitations of PCR and ELISA assays are discussed.

  4. Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene.

    Science.gov (United States)

    Soares, Vítor Yamashiro Rocha; Silva, Jailthon Carlos da; Silva, Kleverton Ribeiro da; Pires e Cruz, Maria do Socorro; Santos, Marcos Pérsio Dantas; Ribolla, Paulo Eduardo Martins; Alonso, Diego Peres; Coelho, Luiz Felipe Leomil; Costa, Dorcas Lamounier; Costa, Carlos Henrique Nery

    2014-06-01

    An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

  5. Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

    Directory of Open Access Journals (Sweden)

    Vítor Yamashiro Rocha Soares

    2014-06-01

    Full Text Available An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis of the mitochondrial cytochrome B (cytb gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1, Bos taurus (1 and Equus caballus (2. Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

  6. Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism

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    R Mirnejad

    2011-01-01

    Full Text Available Objectives: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR-restriction fragment length polymorphism (RFLP. Materials and Methods : Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium and a 559 bp fragment (U. urealyticum. Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. Results: Of the 210 samples, a total of 100 (47.6% samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%, and coinfections with both species were detected in four samples (1.9%. The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. Conclusion: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.

  7. PCR-RFLP Analysis of a fliC Gene Fragment in Avian Salmonella Isolates

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    Zohreh Ebrahimvandi

    2014-07-01

    Full Text Available Background: Salmonella are a genus of zoonotic bacteria of worldwide economic and health importance. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or watercontaminated by infected feces. Objectives: The aim of this study was to apply a PCR-RFLP method based on the fliC gene to identify the serotypes of Salmonella isolates from Karaj, Iran. Materials and Methods: A total of 30 Salmonella isolates were serotyped by specific antisera. For the PCR-RFLP method based on the fliC gene, extracted DNA was used as the template for amplifying the fliC gene (1500 bp using specific primers. PCR products were subjected to digestion using HhaI restriction endonuclease. Results: This study determined 30 serotypes as Salmonella durban (56.6%, Salmonella uno (23.3%, Salmonella enteritidis (3.3%, Salmonella tinda (3.3%, Salmonella mjimweme (3.3%, Salmonella Thompson (3.3%, Salmonella sIIO8 (3.3 % and Salmonella sIIO7 (3.3%. Observations indicated that HhaI is able to discriminate Salmonella tinda and Salmonella thompson, yet Salmonella enteritidis, Salmonella durban and Salmonella mjimweme had the same pattern with this enzyme. Also Salmonella sIIO8, Salmonella sIIO7 and Salmonella uno showed the same pattern. Thus, regarding the size and the number of resulting fragments from this enzyme, four patterns were obtained for HhaI. Conclusion: A large number of Salmonella serotypes need to be analyzed by the PCR-RFLP method and different enzymes must be used to give reliable results.

  8. M protein typing of Thai group A streptococcal isolates by PCR-Restriction fragment length polymorphism analysis

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    Good Michael F

    2005-10-01

    Full Text Available Abstract Background Group A streptococcal (GAS infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF and rheumatic heart disease (RHD. RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP-based assay for molecular typing the M protein gene (emm of GAS. Results Using one pair of primers, 13 known GAS M types showed one to four bands of PCR products and after digestion with Alu I, they gave different RFLP patterns. Of 106 GAS isolates examined from the normal Thai population and from patients with GAS-associated complications including RHD, 95 isolates gave RFLP patterns that corresponded to the 13 known M types. Only 11 isolates gave RFLP patterns that differed from the 13 known M types. These were then analyzed by DNA sequencing and six additional M types were identified. In addition, we found that M93 GAS was the most common M type in the population studied, and is consistent with a previous study of Thai GAS isolates. Conclusion PCR-RFLP analysis has the potential for the rapid screening of different GAS M types and is therefore considerably advantageous as an alternative M typing approach in developing countries in which GAS is endemic.

  9. PCR-RFLP diagnostic method for identifying Globodera species in Slovenia

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    Sasa ŠIRCA

    2011-01-01

    Full Text Available Species identification within the genus Globodera is based on the morphological and morphometrical characters of the cysts and second stage juveniles, and these are included in the majority of identification keys. Morphometrical methods are fast and can be applied to most of samples but they demand a trained and experienced specialist. Furthermore, some morphometrical characters may overlap between populations and beetwen species, leading to inaccurate identification. To confirm and complement the morphometrical identification of Globodera species molecular methods have been developed. Sequences of the internal transcribed spacer regions ITS1 and ITS2 of the rDNA gene cluster proved to be useful for identifying nematode species identification. A PCR-RFLP molecular method was used to identify Globodera rostochiensis, G. pallida, G. tabacum and G. achilleae. Globodera rostochiensis, G. pallida, G. tabacum and G. achilleae can be distinguished with PCR-RFLP analysis of the rDNA ITS fragment using five restriction enzymes. The RFLP patterns of G. rostochiensis, G. tabacum and G. achilleae were species-specific, while those of G. pallida varied. South American populations of G. pallida differed from other populations as their RFLP patterns were demonstrated to be distinct by in silico restriction of the ITS sequences deposited at NCBI.

  10. GroEL PCR- RFLP - An efficient tool to discriminate closely related pathogenic Vibrio species.

    Science.gov (United States)

    Silvester, Reshma; Alexander, Deborah; Antony, Ally C; Hatha, Mohamed

    2017-04-01

    Vibrio sp. are autochthonous to marine and estuarine waters. Several species of Vibrio are pathogens. It is of utmost importance to detect and discriminate the Vibrio sp. that are often involved in food and water borne infections. Since 16S rRNA based identification has limited utility in differentiating the closely related pathogenic species from non pathogenic species, we have evaluated the discriminatory power of groEL PCR-RFLP for identification of closely related Vibrio sp. Accordingly, in the current study, the efficiency of groEL PCR- RFLP for detection and accurate differentiation of known pathogens among Vibrio sp. such as V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. fluvialis, V. alginolyticus, V. anguillarum was evaluated. PCR amplified groEL gene fragment of each Vibrio sp. was digested separately using 5 restriction enzymes viz. Hha1, Rsa1, Alu1, Dde1 and Mbo1. The accuracy of the method was further validated by insilico restriction analysis of multiple strains of each species using NEBcutter. The method proved to be efficient for detection and differentiation of Vibrio species under study. Phylogenetic analysis also revealed groEL gene to be a better phylogenetic marker for Vibrio compared to 16S rRNA. Hence, the method can be employed for accurate detection of Vibrio sp. including the closely related species. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Identification of amplified fragment length polymorphism (AFLP ...

    African Journals Online (AJOL)

    Identification of amplified fragment length polymorphism (AFLP) fragments linked to soybean mosaic virus resistance gene in Glycine soja and conversion to a sequence characterized amplified regions (SCAR) marker for rapid selection.

  12. Complementary DNA-amplified fragment length polymorphism ...

    African Journals Online (AJOL)

    Complementary DNA-amplified fragment length polymorphism (AFLP-cDNA) analysis of differential gene expression from the xerophyte Ammopiptanthus mongolicus in response to cold, drought and cold together with drought.

  13. Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene Agrupamento genético de isolados do vírus da bronquite infecciosa das aves no Brasil com base na análise do gene S1 por RT-PCR-RFLP

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    Maria de Fátima S. Montassier

    2008-03-01

    Full Text Available Twelve Brazilian isolates and one reference vaccine strain of avian infectious bronchitis virus (IBV were propagated in embryonating chicken eggs. The entire S1 glycoprotein gene of these viruses was analysed by reverse-transcriptase-polymerase chain reaction and restriction fragment length polymorphism (RT-PCR-RFLP, using the restriction enzymes HaeIII, XcmI and BstyI. The RFLP patterns led to the classification of these isolates into five distinct genotypes: A, B, C, D and Massachusetts. Five of twelve isolates were grouped in Massachusetts genotype and the remaining seven viruses were classified into four distinct genotypes: A (2, B (2, C (2 or D (1. Such genotyping classification agreed with previous immunological analysis for most of these viruses, highlighting the occurrence of a relevant variability among the IBV strains that are circulating in Brazilian commercial poultry flocks.Doze isolados de campo do Brasil e uma estirpe de referência vacinal do vírus da bronquite infecciosa das aves (VBI foram propagadas em ovos embrionados SPF. O gene S1 dessas amostras foi analisado por RT-PCR seguido de RFLP, empregando-se as enzimas de restrição HaeIII, XcmI e BstyI. Observou-se a existência de cinco genotipos diferentes: M (Massachusetts, A , B, C e D. Cinco dos doze isolados de campo do VBI foram classificados no genótipo Massachusetts e os sete vírus restantes foram classificados em quatro genotipos diferentes; A (2, B (2, C (2 ou D (1. Os resultados desta genotipagem concordam com os dados obtidos na análise imunológica previamente realizada para a maior parte destes vírus, destacando a ocorrência de uma variabilidade marcante entre os isolados do VBI que estão circulando nas granjas avícolas comerciais do Brasil.

  14. Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay.

    Science.gov (United States)

    Acardi, Soraya Alejandra; Liotta, Domingo Javier; Santini, María Soledad; Romagosa, Carlo Mariano; Salomón, Oscar Daniel

    2010-09-01

    In this study, a genotypification of Leishmania was performed using polimerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing techniques to identify species of Leishmania parasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpis were grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantum by PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpis among the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantum in the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

  15. Molecular characterization of enteroinvasive Escherichia coli ipa genes by PCR-RFLP analysis Caracterização molecular do gene ipa de Escherichia coli enteroinvasora pela análise de PCR- RFLP

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    Adriana Gibotti

    2004-06-01

    Full Text Available In this study, polymorphism in ipa genes was found in five out of nine EIEC serotypes studied. When SalI and HindII were used in RFLP-PCR assays many EIEC serotypes showed polymorphism in ipaB and ipaD. On the other hand, no polymorphism was observed in ipaA and ipaC in these strains. The polymorphism present in EIEC strains is serotype-dependent, since restriction patterns were conserved amongst strains belonging to the same serotype. When IpaB deduced amino acid sequences of S. flexneri M90T and FBC124-13 were compared, ten amino acids changes could be observed mainly in the amino-terminal region. The deduced EIEC IpaD amino-acid sequence presented 91% similarity with the Shigella strain. In this case, amino acid changes were spread out through the whole structure, except in the carboxyl-terminal region.No presente estudo, foi encontrado polimorfismo no gene ipa em cinco sorotipos de EIEC, de nove estudados. Quando enzimas de restrição SalI e HindII foram utilizadas no ensaio de PCR-RFLP, amostras de EIEC apresentaram polimorfismo em ipaB e ipaD. Por outro lado, não foram observados polimorfismos nos genes ipaA e ipaC nestas cepas, quando diversas enzimas de restrição foram utilizadas. O polimorfismo presente em cepas de EIEC é sorotipo-dependente, uma vez que os padrões de restrição foram conservados entre as cepas pertencentes ao mesmo sorotipo. Quando a seqüência deduzida de aminoácidos de IpaB de S. flexneri M90T e FBC124-13 foram comparadas, mudanças foram observadas em dez aminoácidos na região amino-terminal. A seqüência deduzida de aminoácidos de IpaD de EIEC apresentou similaridade de 91% com a cepa de Shigella. Neste caso, mudanças de aminoácidos ocorreram em toda a estrutura da molécula de IpaD, exceto na região carboxi-terminal.

  16. Bovine papillomavirus in Brazil: detection of coinfection of unusual types by a PCR-RFLP method.

    Science.gov (United States)

    Carvalho, R F; Sakata, S T; Giovanni, D N S; Mori, E; Brandão, P E; Richtzenhain, L J; Pozzi, C R; Arcaro, J R P; Miranda, M S; Mazzuchelli-de-Souza, J; Melo, T C; Comenale, G; Assaf, S L M R; Beçak, W; Stocco, R C

    2013-01-01

    Bovine papillomavirus (BPV) is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil.

  17. A PCR-RFLP method for the simultaneous differentiation of three Entamoeba species.

    Science.gov (United States)

    Fontecha, Gustavo A; García, Kimberly; Rueda, María Mercedes; Sosa-Ochoa, Wilfredo; Sánchez, Ana Lourdes; Leiva, Byron

    2015-01-01

    Amoebiasis caused by Entamoeba histolytica continues to be one of the most common parasitic diseases in the developing world. Despite its relevance, due to the lack of accurate diagnostic methods, the true clinical and public health importance of this parasite remains uncertain. The aim of this study was to develop a new diagnostic tool to differentiate E.histolytica from the morphologically undistinguishable E.dispar and E.moshkovskii. We developed a specific, fast and simple PCR-RFLP method that was able to accurately differentiate experimentally-obtained restriction patterns from the three Entamoeba species. This new method could prove useful for clinical and epidemiological purposes. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    Directory of Open Access Journals (Sweden)

    Tülin GÜVEN GÖKMEN

    Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

  19. Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment

    OpenAIRE

    Millard, Julie T.; Chuang, Edward; Lucas, James S.; Nagy, Erzsebet E.; Davis, Griffin T.

    2013-01-01

    A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electropho...

  20. Identification of Pit-1 Gen Using PCR-RFLP of Padjadjaran Sheep and Evaluate of Growth Rate

    Science.gov (United States)

    Prajoga, S. B. K.; Andriani, L.; Subhandiana, H.

    2018-02-01

    The objectives of this research were to evaluate variation of Pit-1 gen of Padjadjaran Sheep and evaluate of their growth rate. The data comprised of 15 lambs female and 15 lambs male records of 0 - 12 mouths old lambs. Variation of Padjadjaran Sheep PIT-1 gen was analyzed using PCR-RFLP and used primer by 5’-GAGGGATAATTACAAATGGTCC-3’ and 5’-TGTTAACAGCTGTGGGACACAC-3’, length fragment analyse using HinfI restriction enzyme. Presence or absence of deference bands in a PCR fragment of the result can be distinguished by differences in the electrophoretic migration of the fragment. The result showed that the variation Pit1 gene showed that there was in the position 345 bp, 137 bp and 115 bp. In this experiment the difference migration did occur in all samples (monomorphic), that was amplificated with reverse and forward primer. The average male birth weight (BW), weaning weight (WN) and Yearling Weight (YW) was 2.29 ±0.42kg 8.96 ±1,89 kg and 30.12 ±5,65 kg. The average female birth weight (BW), weaning weight (WN) and Yearling Weight (YW) was 2.33 ±0.49 kg; 8.23 ±1.99 kg and 26.11 ±5.50 kg. Correlation value between age and body weight was 0,99 for female and 0,97 for male. The highest body weight gain per month was 2.85 kg for female at the age of six months and 3.4 kg for male at the age of one year. The best equition of growth rate was Ŷ = 1,862 + 0,127X1 0,076X2

  1. Complementary DNA-amplified fragment length polymorphism ...

    African Journals Online (AJOL)

    owner

    2011-05-09

    May 9, 2011 ... Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology was used to analyze ... that 9 of the studied expressed sequence tags (ESTs) are related to protein modification, 12 ESTs are involved in the .... primers were used during the first strand synthesis of our cDNA synthesis ...

  2. Bovine Papillomavirus in Brazil: Detection of Coinfection of Unusual Types by a PCR-RFLP Method

    Science.gov (United States)

    Carvalho, R. F.; Sakata, S. T.; Giovanni, D. N. S.; Mori, E.; Brandão, P. E.; Richtzenhain, L. J.; Pozzi, C. R.; Arcaro, J. R. P.; Miranda, M. S.; Mazzuchelli-de-Souza, J.; Melo, T. C.; Comenale, G.; Assaf, S. L. M. R.; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomavirus (BPV) is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil. PMID:23865043

  3. Genotyping of the Holstein-Friesian crossbred cattle for CD18 gene using PCR-RFLP

    Directory of Open Access Journals (Sweden)

    A. S. Khade

    2014-05-01

    Full Text Available Aim: The present study was undertaken in Holstein-Friesian (HF crossbred cattle with the objective to find out genotype of HF crossbred cattle for Bovine Leucocyte Adhesion Deficiency (BLAD by using PCR-RFLP. Materials and Methods: 50 blood samples were collected from HF crossbred cattle and subjected to PCR. The amplified PCR products were digested using Taq I restriction enzyme at 65 oC overnight. After restriction digestion, the final PCR products were electrophoresed on 2.5 % agarose gel. Results: All the 50 animals under present investigation were found to be normal as the amplified PCR product upon digestion with Taq I restriction enzyme, revealed two bands of 313 bp and 54 bp for normal animals. Conclusions: In the present investigation D128G carrier frequency was found to be 0 %. However, recent reports suggest that the mutant gene has already been observed in the HF crossbred cattle population of India, which makes it necessary to screen the animals to avoid the risk of spreading BLAD in the breeding cattle population.

  4. Molecular identification of Indian crocodile species: PCR-RFLP method for forensic authentication*.

    Science.gov (United States)

    Meganathan, P R; Dubey, Bhawna; Haque, Ikramul

    2009-09-01

    South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile-based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA-based identification of species using PCR-RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species-specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.

  5. Bovine Papillomavirus in Brazil: Detection of Coinfection of Unusual Types by a PCR-RFLP Method

    Directory of Open Access Journals (Sweden)

    R. F. Carvalho

    2013-01-01

    Full Text Available Bovine papillomavirus (BPV is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil.

  6. Autoscreening of restriction endonucleases for PCR-restriction fragment length polymorphism identification of fungal species, with Pleurotus spp. as an example.

    Science.gov (United States)

    Yang, Zhi-Hui; Huang, Ji-Xiang; Yao, Yi-Jian

    2007-12-01

    A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.

  7. Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning ▿

    Science.gov (United States)

    Ruecker, Norma J.; Hoffman, Rebecca M.; Chalmers, Rachel M.; Neumann, Norman F.

    2011-01-01

    Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common. PMID:21498746

  8. Simple and rapid human papillomavirus genotyping method by restriction fragment length polymorphism analysis with two restriction enzymes.

    Science.gov (United States)

    Chen, Linghan; Watanabe, Ken; Haruyama, Takahiro; Kobayashi, Nobuyuki

    2013-07-01

    Cervical cancer, the third most common cancer that affects women worldwide, is caused by the human papillomavirus (HPV) and is treatable when detected at an early stage. To date, more than 100 different HPV types have been described, and the development of simple, low-cost, and accurate methods to distinguish HPV genotypes is highly warranted. In this study, an HPV genotyping assay based on polymerase chain reaction (PCR) was evaluated. This method involved the use of MY09/11 primers followed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes HpyCH4V and NlaIII. Cervical specimens preserved using CytoRich Blue fluid were collected from 1,134 female volunteers for HPV detection, and 1,111 valid samples were amplified using PCR. The PCR method was sensitive enough to detect 25 copies of HPV18, and three copies of HPV16. Out of 202 PCR-positive samples, HPV genotypes were determined in 189 samples (93.6%) by this RFLP method. Results were then evaluated further by capillary sequencing method. Concordant results between the two tests were as high as 96.0%. Thirteen samples, which tested negative with RFLP, were verified as non-specific amplifications with PCR. In conclusion, this PCR-RFLP method using restriction enzymes HpyCH4V and NlaIII is simple, non-labor intensive, and is applicable for the inexpensive determination of HPV genotypes in clinical samples. Copyright © 2013 Wiley Periodicals, Inc.

  9. Rapid differentiation of closely related isolates of two plant viruses by polymerase chain reaction and restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Barbara, D J; Morton, A; Spence, N J; Miller, A

    1995-09-01

    Immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the product has been shown to be an effective procedure for discriminating serologically indistinguishable isolates of two plant viruses, raspberry bushy dwarf (RBDV) and zucchini yellow mosaic (ZYMV). For both viruses, only limited sequence information was available at the time of primer design, but most of the isolates which were tested could be amplified (the one exception being a serologically quite distinct isolate of ZYMV). Restriction endonucleases revealing diagnostic RFLPs were readily identified. Each of two isolates of ZYMV could be detected in the presence of the other and the relative proportions approximately quantified by visual estimation of the relative intensity of the appropriate bands. A range of isolates of different RBDV pathotypes were compared; isolates were grouped in ways that accorded with their known history. Computer analysis of the published sequence from which the primers had been derived showed the sequenced isolate to be identical with an isolate imported from the USSR. The PCR/RFLP procedure is rapid (it can be completed in less than 2 days), effective and will probably be generally applicable to distinguishing closely related virus isolates, even where little sequence information is available.

  10. DNA Polymorphism of Insulin-like Growth Factor-binding Protein-3 Gene and Its Association with Cashmere Traits in Cashmere Goats

    OpenAIRE

    Liu, Haiying; Liu, Chao; Yang, Guiqin; Li, Hui; Dai, Jin; Cong, Yuyan; Li, Xuejian

    2012-01-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) gene is important for regulation of growth and development in mammals. The present investigation was carried out to study DNA polymorphism by PCR-RFLP of IGFBP-3 gene and its effect on fibre traits of Chinese Inner Mongolian cashmere goats. The fibre traits data investigated were cashmere fibre diameter, combed cashmere weight, cashmere fibre length and guard hair length. Four hundred and forty-four animals were used to detect polymorphis...

  11. Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment.

    Science.gov (United States)

    Millard, Julie T; Chuang, Edward; Lucas, James S; Nagy, Erzsebet E; Davis, Griffin T

    2013-11-12

    A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to three weeks. This experiment, which has an engaging and versatile scenario, provides students with exposure to key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context.

  12. Cryptosporidium hominis infection diagnosed by real-time PCR-RFLP.

    Science.gov (United States)

    Cheun, Hyeng-Il; Kim, Kyungjin; Yoon, Sejoung; Lee, Won-Ja; Park, Woo-Yoon; Sim, Seobo; Yu, Jae-Ran

    2013-06-01

    There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.

  13. PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

    Directory of Open Access Journals (Sweden)

    Diego Rayan Teixeira de Sousa

    2015-05-01

    Conclusion: The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

  14. Diversity analysis of Bemisia tabaci biotypes: RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region

    OpenAIRE

    Rabello, Aline R.; Queiroz, Paulo R.; Simões, Kenya C.C.; Hiragi, Cássia O.; Lima, Luzia H.C.; Oliveira, Maria Regina V.; Mehta, Angela

    2008-01-01

    The Bemisia tabaci complex is formed by approximately 41 biotypes, two of which (B and BR) occur in Brazil. In this work we aimed at obtaining genetic markers to assess the genetic diversity of the different biotypes. In order to do that we analyzed Bemisia tabaci biotypes B, BR, Q and Cassava using molecular techniques including RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region. The analyses revealed a high similarity between the individuals of the B and Q biotypes, which could be distin...

  15. Genetic Characterization of Campylobacter Jejuni and C. coli Isolated From Broilers Using flaA PCR-Restriction Fragment Length Polymorphism Method in Shiraz, Southern Iran.

    Science.gov (United States)

    Khoshbakht, Rahem; Tabatabaei, Mohammad; Hosseinzadeh, Saeid; Shirzad Aski, Hesamaddin; Seifi, Saeed

    2015-05-01

    Thermophilic campylobacters, particularly Campylobacter jejuni and C. coli are the main agents of human campylobacteriosis. Campylobacter contaminated chicken products is the most important source of foodborne gastroenteritis. Evaluation of genetic diversity among Campylobacter population is critical for understanding the epidemiology of this bacterium and developing effective control strategies against Campylobacter infections and other related disorders. The aim of this study was to investigate the polymorphism of thermophilic Campylobacter isolated from broiler fecal samples in Shiraz, southern Iran. Ninety Campylobacter isolates were recovered from broiler feces using enrichment process followed by cultivation method. The isolates were species typing on the basis of polymerase chain reaction (PCR) detection of 16SrRNA and multiplex PCR for determining two thermophilic species. To evaluate strain diversity of thermophilic Campylobacter isolates, flaA PCR-Restriction Fragment Length Polymorphism (RFLP) was performed using DdeI restriction enzyme. All 90 Campylobacter isolates confirmed by m-PCR were successfully typed using flaA-PCR-RFLP. Eleven different types were defined according to flaA-typing method and the RFLP patterns were located at three separate clusters in RFLP image analysis dendrogram. Campylobacter jejuni isolates significantly showed more variety than C. coli isolates. A relatively low genetic diversity existed among C. jejuni and C. coli isolated from broilers in Shiraz, southern Iran. In our knowledge, this was the first report of genetic diversity among broiler originated human pathogen thermophilic campylobacters in Shiraz, southern Iran.

  16. [Polymorphism of vitamin D receptor gene Fok I in Mongolian population of China].

    Science.gov (United States)

    Xing, Shao-ji; Zhou, Li-she; Xu, Xiu-ju

    2006-04-01

    To investigate the polymorphism distribution of vitamin D receptor (VDR) gene Fok I in Mongolian population of China. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze three genotypes FF, Ff and ff in the start codon of VDR gene (Fok I) in unrelated normal healthy Mongolian individuals of China. In the population, we obtained the allelic frequencies of 57% and 43% for (F) and (f) allele and the percentage of genotypes FF, Ff and ff to be 31%, 52%, and 17% respectively. The polymorphism frequency and distribution of this VDR gene Fok I in Mongolian population of China exhibit its own characteristics.

  17. Identification of Malassezia Species Isolated from Patients with Pityriasis Versicolor Using PCR-RFLP Method in Markazi Province, Central Iran.

    Science.gov (United States)

    Didehdar, Mojtaba; Mehbod, Amir Sayed Ali; Eslamirad, Zahra; Mosayebi, Mahdi; Hajihossein, Reza; Ghorbanzade, Behzad; Khazaei, Mahmoud Reza

    2014-05-01

    The lipophilic yeasts of Malassezia species are members of the normal skin microbial that are cause of pityriasis versicolor. Pityriasis versicolor is a common superficial fungal infection with world-wide distribution. The phenotypic methods for identification of Malassezia species usually are time consuming and unreliable to differentiate newly identified species. But DNA-based techniques rapidly and accurately identified Malassezia species. The purpose of this study was isolation and identification of Malassezia Species from patients with pityriasis versicolor by molecular methods in Markazi Province, Central Iran in 2012. Mycologic examinations including direct microscopy and culture were performed on clinical samples. DNA extraction was performed from colonies. The ITS1 region of rDNA from isolates of Malassezia species were amplified by PCR reaction. The PCR were digested by Cfo I enzyme. From 70 skin samples, were microscopically positive for Malassezia elements, 60 samples were grown on culture medium (85.7%). Using PCR-RFLP method, that was performed on 60 isolates, 37(61.6%) M. globosa, 14(23.3%) M. furfur, 5(8.4%) M. sympodialis and 4(6.7%) M. restrictawere identified. In one case was isolated M. globosa along with M. restricta. The PCR-RFLP method is a useful and reliable technique for identification of differentiation of Malas-sezia species.

  18. Analisis Variabilitas Genetik Rigidoporus microporus (Jamur Akar Putih pada Karet dengan Teknik PCR-RFLP Fragment ITS

    Directory of Open Access Journals (Sweden)

    Ria Novianti

    2011-07-01

    Full Text Available The eleven isolates studied fell into two groups, which showed that the fungus did not have high genetic variability. Isolates from diseased rubber trees in shorter vicinities, which were due to pathogen’s vegetative spread through root contact, have close genetic relationship. Meanwhile isolates from far apart trees, either in one estate or from different islands, does not necessarily have distant genetic relationship. It might be due to the fact that long distance dispersal of the disease could also due to rhizomorph contaminating rubber stumps. PCR-RFLP Fragment ITS Techniques can be applied to study genetic relationship of Rigidoporus microporus isolates. It is expected that the technique will be applied with more isolates from different provinces by using additional different restriction enzymes. Kesebelas isolat yang diteliti terbagi ke dalam dua kelompok, yang menunjukkan bahwa jamur ini tidak mempunyai variabilitas genetik yang tinggi. Isolat-isolat dari pohon karet sakit yang letaknya berdekatan, yang terjadi karena penularan vegetatif melalui kontak akar, mempunyai hubungan kekerabatan genetik yang erat. Isolat-isolat dari pohon karet sakit yang berjauhan, baik dalam satu kebun maupun dari pulau lain, tidak selalu mempunyai hubungan kekerabatan genetik yang jauh. Ini dapat disebabkan karena adanya penyebaran penyakit jarak jauh secara vegetatif dengan terbawanya rizomorf pada akar stump. Teknik PCR-RFLP Fragment ITS dapat dimanfaatkan untuk penelitian kekerabatan Rigidoporus microporus. Pemakaiannya perlu diperluas dengan pengumpulan isolat yang lebih tersebar, dengan memakai enzim restriksi yang lebih banyak.

  19. Genotyping of Pasteurella multocida ovine and bovine isolates from Iran based on PCR-RFLP of ompH gene

    Directory of Open Access Journals (Sweden)

    A. Ghanizadeh

    2015-10-01

    Full Text Available Pasteurella multocida (P. multocida, A Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. The outer membrane of Gram-negative bacteria contains of many different protein in very high copy numbers. One of the major outer membrane, the H proteins have functional as high immunogenicity and antigenicity. In this study to increase information about epidemiology of ovine and bovine P. multocida, the 24 isolates from sheep and nine isolates from cattle were investigated by PCR-RFLP analysis of the ompH gene. In all 33 isolates, digestion of the amplified fragment of ompH gene by using EcoRI, cfoI and HindIII produced 3, 5 and 3 different restriction patterns respectively. Sixteen RFLP patterns were found among 33 investigated P.multocida isolates. This study showed that, the PCR RFLP based on ompH gene is potentially a useful method for typing of P. multocida isolates from sheep and cattle. The RFLP patterns of this gene exhibited extensive restriction site heterogeneity, which may be particularly suitable for fingerprinting of P. multocida isolates.Considering ompH protein as a protective immunogenic moiety of P.ultocida, the results of this study showed a heterogenic bacteria and this means the possibility to produce a multivalent vaccine to be protective against diseases caused by this organism in sheep and cattle in Iran.

  20. A PCR-RFLP assay for the detection and differentiation of Campylobacter jejuni, C. coli, C. fetus, C. hyointestinalis, C. lari, C. helveticus and C. upsaliensis.

    Science.gov (United States)

    Kamei, Kazumasa; Asakura, Masahiro; Somroop, Srinuan; Hatanaka, Noritoshi; Hinenoya, Atsushi; Nagita, Akira; Misawa, Naoaki; Matsuda, Motoo; Nakagawa, Shinsaku; Yamasaki, Shinji

    2014-05-01

    Although Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of human gastrointestinal diseases, other Campylobacter species are also involved in human and animal infections. In this study, we developed a cytolethal distending toxin (cdt) gene-based PCR-RFLP assay for the detection and differentiation of C. jejuni, C. coli, C. fetus, C. hyointestinalis, C. lari, C. helveticus and C. upsaliensis. Previously designed common primers, which can amplify the cdtB gene of C. jejuni, C. coli and C. fetus, were used for detecting seven Campylobacter species and differentiating between them by restriction digestion. The PCR-RFLP assay was validated with 277 strains, including 35 C. jejuni, 19 C. coli, 20 C. fetus, 24 C. hyointestinalis, 13 C. lari, 2 C. helveticus, 22 C. upsaliensis, 3 other Campylobacter spp. and 17 other species associated with human diseases. Sensitivity and specificity of the PCR-RFLP assay were 100 % except for C. hyointestinalis (88 % sensitivity). Furthermore, the PCR-RFLP assay successfully detected and differentiated C. jejuni, C. coli and C. fetus in clinical and animal samples. The results indicate that the PCR-RFLP assay is useful for the detection and differentiation of seven Campylobacter species important for human and animal diseases.

  1. Molecular markers. Amplified fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    Pržulj Novo

    2005-01-01

    Full Text Available Amplified Fragment Length Polymorphism molecular markers (AFLPs has been developed combining procedures of RFLPs and RAPDs molekular markers, i.e. the first step is restriction digestion of the genomic DNA that is followed by selective amplification of the restricted fragments. The advantage of the AFLP technique is that it allows rapid generation of a large number of reproducible markers. The reproducibility of AFLPs markers is assured by the use of restriction site-specific adapters and adapter-specific primers for PCR reaction. Only fragments containing the restriction site sequence plus the additional nucleotides will be amplified and the more selected nucleotides added on the primer sequence the fewer the number of fragments amplified by PCR. The amplified products are normally separated on a sequencing gel and visualized after exposure to X-ray film or by using fluorescent labeled primers. AFLP shave proven to be extremely proficient in revealing diversity at below the species level. A disadvantage of AFLP technique is that AFLPs are essentially a dominant marker system and not able to identify heterozygotes.

  2. Discrimination of press fit candidate microorganism (Enterobacter cloacae, Bacillus licheniformis) by restriction fragment length polymorphic analysis of the 16SrRNA gene; 16S rRNA idenshi no sengen danpen kchotakei kaiseki niyoru atsunyukoho biseibutsu (Enterobacter cloacae, Bacillus licheni-formis) no shikibetsu

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Kazuhiro; Tanaka, Shinji; Otsuka, Makiko; Ichimura, Naoya; Yonebayashi, Eiji; Enomoto, Heiji

    1999-09-01

    In MeOH viewed as one of the improvement method for recovery of the petroleum with hope, the development of discrimination technique of press fit candidate microorganism and oil reservoir resident microorganism which exists in the test object oil reservoir was tried in order to monitor the survival situation of the microorganism which inserted in the oil reservoir under pressure. 16S rRNA amplified by the PCR using the universal primer The microorganism that it cut off the gene at restriction enzyme HhaI,MspI, AluI and inhabits oil reservoir water and oil reservoir rock in the object oil reservoir by ( necessarily TaqI ) and restriction fragment length polymorphic analysis was classified. As the result, the effectiveness of the this PCR-RFLP method was indicated the microorganism which showed RFLP pattern which is identical with the press fit candidate microorganism in the oil reservoir resident microorganism for the discrimination of the press fit candidate microorganism without existing. And, it was indicated that the this PCR-RFLP method was effective for the investigation of oil reservoir resident microbial community which can positively utilize source of nutrition inserted to oil reservoir with the press fit candidate microorganism under pressure, and it was possible to grasp oil reservoir resident microorganism to be especially considered in MEOR. (translated by NEDO)

  3. Development of PCR-RFLP method to distinguish between Cryptosporidium parvum and C. hominis in Taiwan water samples.

    Science.gov (United States)

    Lee, Ya-Tien; Tsaihong, John Chin; Tseng, Yu-Chuan; Tsai, Chia-Mu; Peng, Shih-Yi

    2013-01-01

    Cryptosporidium, a protozoan pathogen that causes cryptosporidiosis has emerged as an important source of diarrheal illness among humans and animals. The current routine laboratory technique used for Cryptosporidium diagnosis is light microscopy with acid-fast staining but the technique has low efficiency and sensitivity for species-specific identification. Single PCR to amplify a 220 bp fragment of 18 S ribosomal DNA of C. parvum and C. hominis was developed. The restriction enzymes, TaqI and VspI, were used to distinguish between amplicons of human and bovine C. parvum genotype. Water samples, collected from Lo-Na, Ton-Pu, Ho-Ping, and Jen-Ai, Taiwan contained only bovine C. parvum genotype whereas in the Ton-Pu and Jen-Ai samples C. hominis was also present. Thus, the used of PCR-RFLP allowed successful identification of Cryptosporidium in water samples and differentiation between human and bovine species.

  4. Genetic Diversity of African and Worldwide Strains of Ralstonia solanacearum as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the hrp Gene Region

    Science.gov (United States)

    Poussier, Stephane; Vandewalle, Peggy; Luisetti, Jacques

    1999-01-01

    The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas. PMID:10224018

  5. Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811

    Directory of Open Access Journals (Sweden)

    Bethany Richards

    2013-06-01

    Full Text Available Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2 is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

  6. Use of PCR-restriction fragment length polymorphism analysis to identify the main new world Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples.

    Science.gov (United States)

    Rotureau, Brice; Ravel, Christophe; Couppié, Pierre; Pratlong, Francine; Nacher, Mathieu; Dedet, Jean-Pierre; Carme, Bernard

    2006-02-01

    At least 13 characterized Leishmania species are known to infect humans in South America. Five of these parasites are transmitted in the sylvatic ecotopes of the whole French Guianan territory and responsible for cutaneous leishmaniasis. For the diagnosis of cutaneous leishmaniasis, restriction fragment length polymorphism (RFLP) analyses have shown promising results. Thus, the end of the small subunit and internal transcribed spacer 1 of the rRNA genes were sequenced and targeted by PCR-RFLP analysis in the 10 main New World (NW) Leishmania species from the two subgenera. Then, the procedure was tested on 40 samples from patients with cutaneous leishmaniasis, and its results were compared with those of conventional methods. (i) The results of this simple genus-specific method were in agreement with those of previous isoenzyme analyses. (ii) This method distinguished the most medically relevant Leishmania species with only one enzyme (RsaI). (iii) This method could be performed directly on human biopsy specimens (sensitivity of 85.7%). Performing NW Leishmania species typing rapidly and easily in the field constitutes a very valuable improvement for detection of Leishmania spp. Revealing great diversity with several enzymes, this method could also be useful for taxonomic, ecological, and epidemiological studies in space and time.

  7. Differentiating among horse (Equus caballus), donkey (Equus asinus) and their hybrids with combined analysis of nuclear and mitochondrial gene polymorphism.

    Science.gov (United States)

    Zhao, C J; Han, G C; Qin, Y H; Wu, Ch

    2005-08-01

    A novel and brief method of differentiating among horse (Equus caballus) and donkey (Equus asinus) and their hybrids (mule, E. asinus x E. caballus and hinny, E. caballus x E. asinus) with combined analysis of nuclear and mitochondrial gene polymorphism (CANMGP) was reported in the present report. A nuclear gene, protamine P1 gene of donkey was sequenced and compared with the known horse sequence from GenBank while a published equid mitochondrial gene, cytochrome b gene of donkey was compared with that of horse. In each of the two genes, a fixed nucleotide substitution within an exon that could be recognized by Dpn II restriction enzyme was found between the two species. Two pairs of primers were designed for amplifying the fragments within the two genes containing the informative nucleotide positions in 65 horses and 41 donkeys and 38 hybrids and conditions of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis were optimized. Horse, donkey and mule and hinny had their own specific cleavage patterns after the PCR-RFLP analysis was performed, which made it very easy to identify them from each other. As multiplex PCR can be conducted with the two pairs of primers and only one restriction enzyme is involved in PCR-RFLP analysis, the method described in the present study is a convenient way to identify horse and donkey and their hybrids. The idea involved in the method of CANMGP can be also used to differentiate other animal species or breeds and their hybrids.

  8. DETEKSI MUTASI KODON 510 dan 511 DAERAH RRDR GEN rpoB PADA ISOLAT KLINIK Mycobacterium tuberculosis MULTIDRUG RESISTANT DI BALI DENGAN PCR-RESTRICTION FRAGMENT LENGHT POLYMORPHISM

    Directory of Open Access Journals (Sweden)

    Made Rai Dwitya Wiradiputra

    2017-03-01

    Full Text Available ABSTRAK: Tujuan penelitian ini adalah untuk melakukan deteksi mutasi daerah RRDR gen rpoB Mycobacterium tuberculosis khususnya pada kodon 510 dan 511 dari isolat klinis Multidrug Resistant Tuberculosis (MDR-TB di Bali dengan metode Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP. Isolat M. tuberculosis H37Rv digunakan sebagai kontrol bakteri yang tidak mengalami mutasi dan empat isolat klinis MDR-TB digunakan sebagai sampel pada penelitian ini. Proses PCR-RFLP meliputi dua tahap, yaitu amplifikasi (PCR dan digesti. Produk PCR hasil amplifikasi didigesti dengan enzim PvuII (New England Biolabs melalui proses inkubasi pada suhu 37oC selama 3 jam diikuti dengan inaktivasi ice shock pada suhu -20oC selama 5 menit. Hasil penelitian ini menunjukan bahwa enzim restriksi PvuII dapat mendeteksi mutasi kodon 510 dan 511 daerah RRDR gen rpoB M. tuberculosis dengan teknik PCR-RFLP. Pada isolat 134 diketahui terdapat mutasi pada kodon 510 dan/atau 511 sedangkan pada isolat P10, P11, dan P16 tidak ditemukan adanya mutasi pada kodon 510 dan 511. Berdasarkan hasil penelitian sebelumnya, diketahui pula bahwa mutasi yang terjadi pada isolat 134 adalah mutasi kodon 510 (CAG?CTG.   ABSTRACT: The objective of this study is to detect mutation in the region of RRDR of rpoB gene Mycobacterium tuberculosis particularly at codon 510 and 511 from MDR-TB clinical isolates in Bali using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP method. Isolate of M. tuberculosis H37Rv was used as control of non-mutated bacteria, and four MDR-TB clinical isolates were used for sample in this study. PCR-RFLP was conducted in two steps which were amplification (PCR and digestion. PCR products were digested using PvuII restriction enzyme (New England Biolabs through incubation at 37oC for 3 hours followed by ice shock inactivation at -20oC for 5 minutes. The result of this study showed that PvuII restriction enzyme could

  9. Amplified fragment length polymorphism (AFLP) studies on Indian ...

    African Journals Online (AJOL)

    Jane

    2011-07-11

    Jul 11, 2011 ... Amplified fragment length polymorphism (AFLP) technology was used to reveal the genetic variation in six species of Cycas collected from eleven natural populations. Two sets of primer with 4-selective nucleotides were used in this study and 78% polymorphism was found. The results correlated with.

  10. An Aiw 261 PCR-RFLP in exon 1 of the porcine SKI oncogene and mapping the gene to the RYR1 (CRC) linkage group on chromosome 6

    Czech Academy of Sciences Publication Activity Database

    Stratil, Antonín; Reiner, G.; Peelman, L. J.; Davoli, R.; VanPoucke, M.; Zambonelli, P.; Geldermann, H.

    2002-01-01

    Roč. 33, - (2002), s. 377-379 ISSN 0268-9146 R&D Projects: GA ČR GA523/00/0669; GA AV ČR KSK5052113 Keywords : PCR- RFLP * mapping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.443, year: 2002

  11. Genetic variation within and among Danish brown trout ( Salmo trutta L) hatchery strains, assessed by PCR-RFLP analysis of mitochondrial DNA segments

    DEFF Research Database (Denmark)

    Hansen, Michael Møller; Mensberg, Karen-Lise Dons; Rasmussen, Gorm

    1997-01-01

    Eleven Danish brown trout hatchery strains were studied by PCR- RFLP analysis of the ND-I and ND-5/6 segments of the mitochondrial genome. For comparison, data from wild trout representing three Danish river systems also were included. Reduced variability in terms of nucleon diversity and number...

  12. Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C-coli: the CAMPYNET experience

    DEFF Research Database (Denmark)

    Harrington, Clare S.; Moran, L.; Ridley, A.M.

    2003-01-01

    Aims: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP(fla typing) methods previously described for Campylobacter. Methods and Results: The sample set(n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two...

  13. Amplified restriction fragment length polymorphism in parasite genetics.

    Science.gov (United States)

    Masiga, D K; Tait, A; Turner, C M

    2000-08-01

    The amplified restriction fragment length polymorphism (AFLP) technique is a relatively new method for the analysis of polymorphism that has not yet been widely used in parasitology. In this article, Dan Masiga, Andy Tait and Mike Turner provide a brief introduction to AFLP and illustrate how it can be used in the investigation of marker inheritance in genetic crosses and in the analysis of polymorphism of field populations. They also briefly highlight the strengths and weaknesses of AFLP in comparison with other methods for detecting polymorphism and conclude that AFLP is a very useful addition to the range of techniques available.

  14. PCR-RFLP method to distinguish Frankliniella occidentalis, Frankliniella intonsa, Frankliniella pallida and Frankliniella tenuicornis

    Directory of Open Access Journals (Sweden)

    Przybylska Arnika

    2016-01-01

    Full Text Available Thrips from the genus Frankliniella (Thysanoptera, Thripidae are phytophagous on crops and wild plants. Some of them cause slight economic damage, however, others including F. occidentalis and F. intonsa are responsible for considerable losses in crop production. Moreover, they constitute a double threat for host plants by not only feeding on them but also vectoring viruses, some of which are on the quarantined list of the European Plant Protection Organization. The rapid detection and differentiation between more and less harmful Frankliniella species is, therefore, important in order to combat the pests at the time of their appearance. In this study, we have undertaken to develop a method of detecting F. occidentalis, F. intonsa, F. pallida, and F. tenuicornis. The protocol is based on PCR amplification of ITS1 rDNA fragments of these insects using universal primers pair giving products of slightly distinct length for studied insects. Restriction enzymes digestion which is easy to interpret, allows for visible differentiation of all these Frankliniella species. The method was shown to be species-specific and sensitive. Even single specimens in either the larvae or adult stage could be distinguished.

  15. Differentiation of sheep pox and goat poxviruses by sequence analysis and PCR-RFLP of P32 gene.

    Science.gov (United States)

    Hosamani, Madhusudan; Mondal, Bimalendu; Tembhurne, Prabhakar A; Bandyopadhyay, Santanu Kumar; Singh, Raj Kumar; Rasool, Thaha Jamal

    2004-08-01

    Sheep pox and Goat pox are highly contagious viral diseases of small ruminants. These diseases were earlier thought to be caused by a single species of virus, as they are serologically indistinguishable. P32, one of the major immunogenic genes of Capripoxvirus, was isolated and Sequenced from two Indian isolates of goat poxvirus (GPV) and a vaccine strain of sheep poxvirus (SPV). The sequences were compared with other P32 sequences of capripoxviruses available in the database. Sequence analysis revealed that sheep pox and goat poxviruses share 97.5 and 94.7% homology at nucleotide and amino acid level, respectively. A major difference between them is the presence of an additional aspartic acid at 55th position of P32 of sheep poxvirus that is absent in both goat poxvirus and lumpy skin disease virus. Further, six unique neutral nucleotide substitutions were observed at positions 77, 275, 403, 552, 867 and 964 in the sequence of goat poxvirus, which can be taken as GPV signature residues. Similar unique nucleotide signatures could be identified in SPV and LSDV sequences also. Phylogenetic analysis showed that members of the Capripoxvirus could be delineated into three distinct clusters of GPV, SPV and LSDV based on the P32 genomic sequence. Using this information, a PCR-RFLP method has been developed for unequivocal genomic differentiation of SPV and GPV.

  16. Isolation and identification of cutaneous leishmaniasis species by PCR-RFLP in Ilam province, the west of Iran.

    Science.gov (United States)

    Kermanjani, Ali; Akhlaghi, Lame; Oormazdi, Hormozd; Hadighi, Ramtin

    2017-03-01

    Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases and public health problems in Iran. CL is endemic in most parts of Ilam province, in the west of Iran. The distance from the center of country, the great number of divers rural areas, and lack of specialists and laboratory facilities have been the major causes of Leishmania species remaining unknown in this region. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to identify the Leishmania species in 61 patients with cutaneous lesions. Eventually L. major was confirmed as the cause of cutaneous leishmaniasis in Ilam province, the west of Iran.

  17. Genotyping of Trypanosoma cruzi DTUs and Trypanosoma rangeli genetic groups in experimentally infected Rhodnius prolixus by PCR-RFLP.

    Science.gov (United States)

    Sá, Amanda R N; Dias, Greicy B M; Kimoto, Karen Y; Steindel, Mário; Grisard, Edmundo C; Toledo, Max Jean O; Gomes, Mônica L

    2016-04-01

    The specific detection and genetic typing of trypanosomes that infect humans, mammalian reservoirs, and vectors is crucial for diagnosis and epidemiology. We utilized a PCR-RFLP assay that targeted subunit II of cytochrome oxidase and 24Sα-rDNA to simultaneously detect and discriminate six Trypanosoma cruzi discrete typing units (DTUs) and two genetic groups of Trypanosoma rangeli (KP1+/KP1-) in intestinal contents of experimentally infected Rhodnius prolixus. The PCR assays showed that in 23 of 29 (79.4%) mixed infections with the six T. cruzi DTUs and mixed infections with individual DTUs and/or groups KP1+ and KP1-, both parasites were successfully detected. In six mixed infections that involved TcIII, the TcI, TcII, TcV, and TcVI DTUs predominated to the detriment of TcIII, indicating the selection of genetic groups. Interactions between different genetic groups and vectors may lead to genetic selection over TcIII. The elimination of this DTU by the immune system of the vector appears unlikely because TcIII was present in other mixed infections (TcIII/TcIV and TcIII/KP1+). Both molecular markers used in this study were sensitive and specific, demonstrating their usefulness in a wide geographical area where distinct genotypes of these two species are sympatric. Although the cellular and molecular mechanisms that are involved in parasite-vector interactions are still poorly understood, our results indicate a dynamic selection toward specific T. cruzi DTUs in R. prolixus during mixed genotype infections. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Karakterisasi Molekuler Ikan Gurami Soang (Osphronemus goramy Lac. berbeda uuran menggunakan Pcr-Rflp Gen Sitokrom C Oksidase 1

    Directory of Open Access Journals (Sweden)

    Siti Nur Azizah

    2017-01-01

    Full Text Available Gourami (Osprhronemus goramy Lac. is a  freshwater fish that is  widely cultivated in Indonesia, particularly by fish farmer in Java. Soang strain has a faster growth rate than other strains. However, the fry that derived from a single cohort (generated from a single spawning shows various sizes even in the same age because they have different growth rate. Differences in growth rate may occur due to differences in metabolic capabilities especially cellular respiration. Different rate of respiration can be assumed to be due to differences in the genetic component, especially on their cytochrome c oxidase 1 gene (CO1. Therefore this study aimed to determine whether there are differences in PCR-RFLP marker CO1 gene among different sizes of gourami soang strain from the same cohort so that can be used to analyze genetic diversity. This study used survey method by applying purposive random sampling. Four restriction enzymes were used  during the  research. Molecular characters were defined descriptively based on the appearance of restriction fragment on agarose gel. The result showed that soang strain which used in this study had monomorphics allele on their CO1-HindIII. PstI, BamHI, and EcoRI, could not cut the PCR products and results no RFLP markers. So that genetic variation in the used CO1 gene fragment could not be detected using HindIII, Pst1, BamH1 and EcoR1 enzymes. The three different sizes of soang strain had similar alleles or homozygous, meaning that heterozigocity value was 0 (zero. Therefore, the CO1 gene in this study could not be used as molecular character to differentiate different sizes of gourami soang strain from the same cohort.

  19. Incidence of Giardia lamblia Subspecies by PCR-RFLP in Stool Specimens of Hospitalized Children at Urmia Mutahhari Hospital, West Azerbaijan Province, Iran.

    Directory of Open Access Journals (Sweden)

    Khosro Hazrati Tappeh

    2014-12-01

    Full Text Available Giardia lamblia is one of the most prevalent intestinal flagellate protozoa that infects a wide range of vertebrate hosts causing severe intestinal disorder in children.This study was performed to determine subspecies of G.lamblia by the PCR-RFLP method, targeting the glutamate dehydrogenase(gdhlocus, in hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province,Iran and determining the infection transformational storages in this area.Overall, 720 stool specimens were collected from the hospitalized children, 34 samples were positive and Giardia cysts were detected under the microscope. Cysts were partially purified by the sucrose density gradient method and then washed with sterile distilled water to remove effectively the PCR inhibitors. Genomic DNA of G. lamblia isolates was extracted by freeze-thaw cycles followed by phenol/ chloroform/isoamyl alcohol method. The single step PCR-RFLP assay was used to differentiate the assemblages between A and B, which were found in humans. In this method, 432 bp expected size was amplified, and then for detection of subspecies, specific restriction RsaI and BspLI enzymes were used.Totally 34 samples were positive in terms of Giardia cyst out of 720 examined samples microscopically, so the parasite spread rate is reported 4.72%. Analysis PCR-RFLP on these samples revealed that 28 samples (93.3% have the genotype BIII and 2 samples (6.7% belong to the subgroup BIV.PCR-RFLP is a proper analytical method for determining the genotype among parasite types, using the glutamate dehydrogenizes zone's genes. Based on the results, an animal origin of infection cycle is suggested.

  20. Highly sensitive detection of the PIK3CAH1047R mutation in colorectal cancer using a novel PCR-RFLP method

    International Nuclear Information System (INIS)

    Li, Wan-Ming; Hu, Ting-Ting; Zhou, Lin-Lin; Feng, Yi-Ming; Wang, Yun-Yi; Fang, Jin

    2016-01-01

    The PIK3CA H1047R mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CA H1047R mutation in high effectiveness. A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CA H1047R was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method. The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA H1047R mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CA H1047R mutation and the patients’ age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods. We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CA H1047R mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy

  1. Characterization of South American Snails of the Genus Biomphalaria (Basommatophora: Planorbidae and Schistosoma mansoni (Platyhelminthes: Trematoda in Molluscs by PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Roberta Lima Caldeira

    2016-01-01

    Full Text Available The identification of snails of the genus Biomphalaria can be done using morphological characteristics which depends on the size of the snails and skill and knowledge of researcher. These methods sometimes are not adequate for identification of species. The PCR-RFLP, using the ITS region of the rDNA, has been used to identify Brazilian species of the genus Biomphalaria. Nevertheless, there is a lack of information about snails from other Latin American countries. In addition, some snails may be infected by Schistosoma mansoni and when submitted to PCR-RFLP they show molecular profiles different from those previously standardized for the other mollusc species. In this work the molecular profiles of 15 species and the subspecies were established by PCR-RFLP of ITS-rDNA with the enzyme DdeI. Moreover, the molecular profiles of host species, B. glabrata, B. straminea, B. tenagophila, and B. prona, infected by S. mansoni were also established. The molluscs were dissected to permit morphological identification. These results contribute to a correct identification of snails of the genus Biomphalaria and detection of these snails infected by S. mansoni.

  2. Population structure of Salmonella investigated by amplified fragment length polymorphism

    DEFF Research Database (Denmark)

    Torpdahl, M.; Ahrens, Peter

    2004-01-01

    Aims: This study was undertaken to investigate the usefulness of amplified fragment length polymorphism (AFLP) in determining the population structure of Salmonella. Methods and Results: A total of 89 strains were subjected to AFLP analysis using the enzymes BglII and BspDI, a combination...

  3. Full-length sequencing and identification of novel polymorphisms in ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 96; Issue 4. Full-length sequencing and identification of novel polymorphisms in the ACACA gene of Valle del Belice sheep breed. ROSALIA DI GERLANDO SALVATORE MASTRANGELO LINA TORTORICI MARCO TOLONE ANNA MARIA SUTERA MARIA TERESA SARDINA ...

  4. Full-length sequencing and identification of novel polymorphisms in ...

    Indian Academy of Sciences (India)

    Rosalia Di Gerlando

    2017-08-16

    Aug 16, 2017 ... Full-length sequencing and identification of novel polymorphisms in the ACACA gene of Valle del Belice sheep breed. ROSALIA DI GERLANDO, SALVATORE MASTRANGELO, LINA TORTORICI, MARCO TOLONE,. ANNA MARIA SUTERA, MARIA TERESA SARDINA. ∗ and BALDASSARE PORTOLANO.

  5. Methylenetetrahydrofolate reductase polymorphisms in myeloid leukemia patients from Northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Cynara Gomes Barbosa

    2008-01-01

    Full Text Available Methylenetetrahydrofolate reductase (MTHFR: EC 1.5.1.20 polymorphisms are associated to acute lymphoid leukemia in different populations. We used the polymerase chain reaction and the restriction fragment length polymorphism method (PCR-RFLP to investigate MTHFR C677T and A1298C polymorphism frequencies in 67 patients with chronic myeloid leukemia (CML, 27 with acute myeloid leukemia FAB subtype M3 (AML-M3 and 100 apparently healthy controls. The MTHFR mutant allele frequencies were as follows: CML = 17.2% for C677T, 21.6% for A1298C; AML-M3 = 22.2% for C677T, 24.1% for A1298C; and controls = 20.5% for C677T, 21% for A1298C. Taken together, our results provide evidence that MTHFR polymorphisms have no influence on the development of CML or AML-M3.

  6. Association of growth hormone (GH gene polymorphism with growth and carcass in Sumba Ongole (SO cattle

    Directory of Open Access Journals (Sweden)

    P. P. Agung

    2017-08-01

    Full Text Available A study was conducted to identify the polymorphism in the intron 3 of the Growth Hormone (GH gene and also to evaluate the association of the GH gene polymorphism with growth parameters and dressing percentage in the Sumba Ongole (SO cattle. A total of 267 individual DNA samples were used in the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP analysis. The SO cattle growth parameters data (n=44 including birth weight (BW, weaning weight at 205 days of age (WW205, yearling weight at 365 days of age (YW365 and also dressing percentage (DP (n=122 were investigated in this study. There were three genotypes (AA, AB, and BB of the GH gene based on the PCR-RFLP analysis with allele frequency was 0.87 and 0.13 for A allele and B allele respectively. The highest genotype frequency in the SO cattle is AA (0.76 and the lowest is BB (0.02. The Heterozygosity Observed (Ho value in the SO cattle population is 0.23 and Polymorphism Information Content (PIC value is 0.20. Therefore, the genetic diversity in the SO cattle based on the GH gene polymorphism is quite low. There is no association (P>0.05 in BW, WW205, YW365, and DP with genotypes of the GH gene. As the result, the GH gene in this study cannot be used as a genetic marker in the SO cattle breeding program.

  7. PCR-RFLP studies on chromosome 3p in formaldehyde-fixed, paraffin-embedded cervical cancer tissues.

    Science.gov (United States)

    Karlsen, F; Rabbitts, P H; Sundresan, V; Hagmar, B

    1994-09-15

    Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.

  8. Molecular authentication of Maytenus sp by PCR-RFLPAutenticação molecular de Maytenus sp por PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Sandra Sayuri Nakamura

    2013-05-01

    Full Text Available Maytenus aquifolia and Maytenus ilicifolia are native plants from South America and popularly known as ‘Espinheira-santa’. Both are used as tea due to their efficiency in the treatment of ulcer, gastritis and indigestion. However, adulteration of processed Maytenus genus tea with Sorocea genus may happen due to their botanical similarity, compromising the quality of the products and opening a derogatory business opportunity that may lead to the discrediting of medicinal plant products. This study aimed to distinguish Maytenus sp and Sorocea bonplandii by PCR-RFLP of a chloroplast DNA (cpDNA intergenic region. Three commercial products of processed tea leaves of Maytenus sp, and in natura leaves of Maytenus sp and S. bonplandii were analyzed. PCR detected unique fragments for all samples in natura. The trnH-psbA region amplicon of both M. ilicifolia and M. aquifolia was 660 bp, and for S. bonplandii was 565 bp. These PCR products can be used as markers to distinguish the two genera. Forty-five percent of the processed samples presented only Maytenus genus, without adulterations. However, the amplification of 38% of the samples suggests adulteration with S. bonplandii while 17% seem to be adulterated with another plant (fragment of 649 bp in brand A and 690 bp in brand B. Three out of the fifteen restriction enzymes were able to detect M. ilicifolia and M. aquifolia in natura and in processed leaf samples. It was concluded that PCR technique is efficient to distinguish Maytenus sp from S. bonplandii, and other adulterating plants in processed commercial products of ‘Espinheira-santa’ tea. The trnH-psbA spacer of cpDNA is easily amplified and has satisfactory discriminating capacity to help in authentication processes of samples of the genera in natura and in processed plants. Maytenus aquifolia e Maytenus ilicifolia são plantas nativas da América do Sul e conhecidas popularmente como “Espinheira-santa”. Ambas são usadas como ch

  9. Identification of medically important Candida species by polymerase chain reaction-restriction fragment length polymorphism analysis of the rDNA ITS1 and ITS2 regions

    Directory of Open Access Journals (Sweden)

    Suphi Bayraktar

    2017-01-01

    Full Text Available Aim: We aimed to identify the distribution of species in candidal strains isolated from clinical samples and restriction fragment length polymorphism (RFLP method based on Msp I and Bln I restrictive enzyme cuts of polymerase chain reaction (PCR products after the amplification of ITS1 and ITS2 regions of rDNA genotypically. Materials and Methods: One hundred and fifty candidal strains isolated from various clinical samples were studies/ included. Phenotypic species assessment was performed using automated VITEK-2 system and kit used with the biochemical tests. Common genomic region amplification peculiar to candidal strains was carried out using ITS1 and ITS2 primer pairs. After the amplification, PCR products were cut with Msp I and Bln I restriction enzymes for species identification. Results: The majority of Candida isolates were isolated from urine (78.6% while other isolates were composed of strains isolated from swab, wound, blood and other samples by 11.3%, 3.3%, 2% and 4.7%, respectively. The result of RFLP analysis carried out with Msp I and Bln I restriction enzymes showed that candidal strains were Candida albicans by 45.3%, Candida glabrata by 19.3%, Candida tropicalis by 14.6%, Candida parapsilosis by 5.3%, Candida krusei by 5.3%, Candida lusitaniae by 0.6% and other candidal strains by 9.3%. Conclusion: When the ability to identify Candida to species level of phenotypic and PCR-RFLP methods was assessed, a great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida species. Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains.

  10. Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure

    Directory of Open Access Journals (Sweden)

    Maria Lodovica Gullino

    2012-01-01

    Full Text Available A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP and tested for ochratoxin A (OTA production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%, while A. carbonarius was the major species during 2007 (46.6%. All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES, as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7% isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46 in both years, but also some strains of A. tubingensis (11 and A. japonicus (6 produced lower amounts of OTA.

  11. Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china

    Directory of Open Access Journals (Sweden)

    Zhao Jianjun

    2011-02-01

    Full Text Available Abstract In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV, a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR and restriction fragment length polymorphism (RFLP. We selected an 829 bp fragment of the nucleoprotein (N gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively.

  12. Evaluation of the use of amplified 16S rRNA gene-restriction fragment length polymorphism analysis to detect enterobacter cloacae and bacillus licheniformis for microbial enhanced oil recovery field pilot

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Kazuhiro; Tanaka, Shinji; Otsuka, Makiko; Ichimura, Naoya [Lansai Research Institute, Kyoto (Japan); Yonebayashi, Hideharu [Japan National Oil Corp., Chiba (Japan); Hong, Chengxie; Enomoto, Heiji [Tohoku University, Miyagi (Japan)

    1999-09-01

    Evaluation of effectiveness of restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene of microorganisms injected into an oil reservoir, for monitoring their levels over time, was conducted. Two microorganisms, enterobacter cloacae TRC-322 and Bacillus licheniformis TRC-18-2-a, were focused in this paper among the microorganisms selected for injection, and gene fragments of the 16S rRNA gene of these microorganisms were amplified by polymerase chain reaction (PCP), using one set of universal primers. Samples of the reservoir brine and reservoir rock were obtained; the microorganisms inhabiting in the reservoir were isolated from these samples, and the 16S rRNA gene of these microorganisms was amplified, condition remaining the same. RFLP analysis was performed on the 16S rRNA gene of each of these microorganisms, using restriction endonucleases HhaI, MspI, AluI and TaqI as necessary. Comparison of the resultant rRNA gene fragments, demonstrated that closely-related species displaying RFLP profile similar to that of E. cloacae TRC-322 or B. licheniformis TRC-18-2-a were not among the microorganisms isolated from the reservoir. PCR-RFLP analysis of the 16S rRNA gene, using the protocol; presented in this paper, is effective to detect the presence appropriate injecting microorganisms. This method was also effective for studying microorganisms isolated from the reservoir, which have the ability to grow on a molasses. (author)

  13. Diferenciación de especies de Rhodococcus mediante una prueba de PCR-RFLP basada en los genes codificantes para la subunidad 16S ribosomal

    OpenAIRE

    Pavía, Paula; Calderon, Camila; Puerta, Concepción

    2005-01-01

    Dada la dificultad en diferenciar las especies de Rhodococcus por pruebas bioquímicas, se desarrolló una prueba de Reacción en Cadena de la Polimerasa (PCR), seguida de un ensayo de Polimorfismo de Longitud de Fragmentos de Restricción (PCR-RFLP) para la diferenciación de las mismas. R. equi, R. rhodnii y otras bacterias fueron cultivadas en agar sangre y BHI a 37 y 26 °C. El ADN bacteriano fue extraído y amplificado con los iniciadores descritos por Hypsa y Dale. Los productos de amplificaci...

  14. Utilisation de la PCR-RFLP sur de l'ADN chloro-plastique pour l'étude des relations phylogénétiques au sein du genre Phaseolus

    Directory of Open Access Journals (Sweden)

    Baudoin JP.

    1998-01-01

    Full Text Available Phylogenetic relationships among 74 accessions belonging to six species of Phaseolus are investigated using variation in chloroplast DNA assessed according to a PCR-RFLP protocol. Three fragments of chloroplast DNA are amplified using universal primers, and then digested with 10 restriction enzymes. Thirty-six haplotypes are identified on the basis of the polymorphism in fragment number and size. Three main phylogenetic groups, strongly supported through bootstrap analysis, are identified: (1 accessions from Phaseolus lunatus and Phaseolus xolocotzii; (2 accessions from Phaseolus glabellus; (3 accessions from Phaseolus vulgaris, Phaseolus polyanthus and Phaseolus coccineus. Within the third group, accessions of Phaseolus coccineus are scattered along the phylogenetic tree, which provides some evidence that coccineus accessions are paraphyletic with respect to Phaseolus vulgaris and Phaseolus polyanthus. An analysis of molecular variance applied on four species show that they are significantly differentiated with 79% of molecular variance among species and 21% within species. The results agree with previous investigations on chloroplast DNA variation in the genus Phaseolus, and suggest that PCRRFLP methods, which are technically less labour-intensive than previous methods, are of great value for phylogenetic investigations at the generic level.

  15. New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus.

    Science.gov (United States)

    Semighini, C P; Delmas, G; Park, S; Amstrong, D; Perlin, D; Goldman, G H

    2001-07-01

    In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.

  16. Establishment of a heteroplasmic mouse strain with interspecific mitochondrial DNA haplotypes and improvement of a PCR-RFLP-based measurement system for estimation of mitochondrial DNA heteroplasmy.

    Science.gov (United States)

    Shitara, Hiroshi; Cao, Liqin; Yamaguchi, Midori; Yonekawa, Hiromichi; Taya, Choji

    2017-08-01

    Mitochondrial DNA segregation is one of the characteristic modes of mitochondrial inheritance in which the heteroplasmic state of mitochondrial DNA is transmitted to the next generation in variable proportions. To analyze mitochondrial DNA segregation, we produced a heteroplasmic mouse strain with interspecific mitochondrial DNA haplotypes, which contains two types of mitochondrial DNA derived from C57BL/6J and Mus spretus strains. The strain was produced on a C57BL/6J nuclear genomic background by microinjection of donor cytoplasm into fertilized eggs. The PCR-RFLP semi-quantitative analysis method, which was improved to reduce the effect of heteroduplex formation, was used to measure the proportion of heteroplasmic mitochondrial DNA in tissues. Founder mice contained up to approximately 14% of exogenous Mus spretus mitochondrial DNA molecules in their tails, and the detected proportions differed among tissues of the same individual. Heteroplasmic mitochondrial DNA is transmitted to the next generation in varying proportions under the maternal inheritance mode. This mitochondrial heteroplasmic mouse strain and the improved PCR-RFLP measurement system enable analysis of the transmission of heteroplasmic mitochondrial DNA variants between tissues and generations.

  17. ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and Strains from Cases of Human Cutaneous Leishmaniasis in States of the Mexican Southeast

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    Amalia Monroy-Ostria

    2014-01-01

    Full Text Available American cutaneous leishmaniasis includes a spectrum of clinical forms localized cutaneous, diffuse cutaneous, and mucocutaneous leishmaniasis which can be caused by different strains of Leishmania belonging to the L. mexicana or L. braziliensis complexes which may coexist in the same endemic area. We evaluated the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 163 clinical samples and 21 Mexican isolates of Leishmania. In relation to the Mexican isolates of Leishmania 52% displayed a pattern similar to the L. (L. mexicana, 5% showed a mixed pattern compatible with L. (L. mexicana and L. (V. braziliensis, eight with L. (L. amazonensis and L. (L. mexicana, and one to L. (V. braziliensis. Most of the clinical samples, 109/116 (94%, gave a pattern similar to that of the L. mexicana, two clinical samples gave similar patterns to that of Leishmania braziliensis, and 5 samples gave patterns that suggest a coinfection of L. (L. mexicana and L. (V. braziliensis or L. (L. mexicana and L. (L. amazonensis. The ITS1 PCR-RFLP assay is a multipurpose tool for diagnosis of Leishmania from clinical samples and enables determination of the infecting species of New World Leishmania in the field in relatively short time and low cost.

  18. A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries.

    Science.gov (United States)

    Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Amani, Hizem; Mezhoud, Habib; Babba, Hamouda

    2016-10-01

    Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1-G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6-G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.

  19. Amplified-fragment length polymorphism fingerprinting of Mycoplasma species

    DEFF Research Database (Denmark)

    Kokotovic, Branko; Friis, N.F.; Jensen, J.S.

    1999-01-01

    Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including......I restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp, The method was able...

  20. Analysis of three genetic polymorphisms in Malaysian essential ...

    African Journals Online (AJOL)

    Analysis of three genetic polymorphisms in Malaysian essential hypertensive and type 2 diabetic subjects. ... Genotyping of all the three polymorphisms was performed by PCR-RFLP method with the respective primers and restriction enzymes. The genotypic and allelic frequencies of the respective polymorphisms of the ...

  1. Length polymorphism scanning is an efficient approach for revealing chloroplast DNA variation.

    Science.gov (United States)

    Matthew E. Horning; Richard C. Cronn

    2006-01-01

    Phylogeographic and population genetic screens of chloroplast DNA (cpDNA) provide insights into seedbased gene flow in angiosperms, yet studies are frequently hampered by the low mutation rate of this genome. Detection methods for intraspecific variation can be either direct (DNA sequencing) or indirect (PCR-RFLP), although no single method incorporates the best...

  2. Characterization of a western North American carnivore community using PCR-RFLP of cytochrome b obtained from fecal samples

    NARCIS (Netherlands)

    Bidlack, Allison L.; Reed, Sarah E.; Palsboll, Per J.; Getz, Wayne M.

    2007-01-01

    We developed a simple and reliable method to identify carnivore scats to species using PCR and RFLP of a portion of the mtDNA cytochrome b gene, which works for seven of the most common carnivores in western North America. We identified a short (196 bp) polymorphic region of cytochrome b which would

  3. Detection and Molecular Characterization of Cryptosporidium Species in Recreational Waters of Chaharmahal va Bakhtiyari Province of Iran Using Nested-PCR-RFLP

    Directory of Open Access Journals (Sweden)

    K Manouchehri Naeini

    2011-03-01

    Full Text Available Background: The aim of this study was to detect and characterize Cryptosporidium spp. in water sam­ples collected from recreational ponds of Chaharmahal va Bakhtiyari Province of Iran .Meth­ods: Thirty water samples were collected from November 2009 to May 2010. Each sample con­tained 10 liters of water. We used the SSU rRNA-based PCR-RFLP technique.Results: Out of thirty samples examined, 6 (20% were positive for different Cryptosporidium spp. Restriction pattern analysis showed that C. parvum has been the most prevalent genotype, fol­lowed by C. hominis and C. canis , respectively. In this area, the higher prevalence of C. par­vum compared with other genotypes is consistent with the distribution of cattle.Conclusion: Farm animals, particularly cattle are the main source of cryptosporidial contamina­tion for recreational waters in this area.

  4. Genotypic Characterisation of Indigenous Soybean Rhizobia by PCR-RFLP of 16S rDNA, rep-PCR and RAPD Analysis

    Directory of Open Access Journals (Sweden)

    Sulejman Redžepović

    2003-01-01

    Full Text Available The taxonomy of nitrogen fixing bacteria that form symbiotic associations with leguminous plants has been deeply changed in recent years. The use of very sensitive and accurate molecular methods has enabled the detection of large rhizobial diversity, particularly among natural field populations of these soil bacteria. The aim of the present investigation was to identify and characterise the indigenous soybean rhizobia isolated from different soil types in eastern Croatia that are regularly used for agricultural production. The actual composition and genetic diversity of natural field population was studied by using different PCR fingerprinting methods such as 16S rDNA PCR-RFLP, rep-PCR and RAPD analysis. Eighteen rhizobial strains, isolated from soybean nodules, were characterised and compared with reference and/or type strains representing Bradyrhizobium japonicum, B. elkanii and Sinorhizobium fredii. Cluster analysis of combined RFLP patterns obtained with six restriction endonucleases revealed that all soybean isolates differ significantly from B. elkanii and S. fredii type strains, while they were closely related to B. japonicum type strains. However, a considerable level of genetic diversity was determined among B. japonicum isolates. PCR-RFLP of 16S rDNA clearly showed the existence of two divergent groups among indigenous bradyrhizobia. After identification at the species level, all isolates were further characterised by RAPD and rep-PCR. Both RAPD and rep-PCR generated highly specific and reproducible patterns that enabled accurate strain differentiation. Among B. japonicum strains a high level of diversity was found with these two fingerprinting methods. Dendrograms derived from RAPD, REP and ERIC profiles showed that all indigenous strains could be divided into three main groups. The grouping of strains was consistent with all methods used in this study.

  5. The -2518 A/G MCP-1 polymorphism as a risk factor of inflammatory bowel disease.

    Science.gov (United States)

    Walczak, Anna; Przybyłowska, Karolina; Sygut, Andrzej; Dziki, Lukasz; Chojnacki, Cezary; Chojnacki, Jan; Dziki, Adam; Majsterek, Ireneusz

    2012-05-01

    Inflammatory bowel diseases (IBD) are disorders originated from immune disturbances. The AIM OF THE STUDY was to evaluate the association between the -2518 A/G MCP-1 polymorphism and the risk of IBD development. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Study group consisted of 197 subjects with IBD (120 with ulcerative colitis and 77 with Crohn's disease) as well as 210 healthy controls. The presence of the -2518 G/G MCP-1 genotype in the investigated groups seems to be connected with higher risk of inflammatory bowel disease as well as Crohn's disease only (OR 2.26; 95% CI 1.44-3.54 and OR 2.08; 95% CI 1.21-3.46, respectively). Our data showed that the -2518 A/G MCP-1 polymorphism might be associated with the IBD occurrence and might be used as predictive factor of these diseases in a Polish population.

  6. Use of mgc2-polymerase chain reaction-restriction fragment length polymorphism for rapid differentiation between field isolates and vaccine strains of Mycoplasma gallisepticum in Israel.

    Science.gov (United States)

    Lysnyansky, Inna; Garcia, Maricarmen; Levisohn, Sharon

    2005-06-01

    Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment length polymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries.

  7. Association of G22A and A4223C ADA1 gene polymorphisms and ADA activity with PCOS.

    Science.gov (United States)

    Salehabadi, Mahshid; Farimani, Marzieh; Tavilani, Heidar; Ghorbani, Marzieh; Poormonsefi, Faranak; Poorolajal, Jalal; Shafiei, Gholamreza; Ghasemkhani, Neda; Khodadadi, Iraj

    2016-06-01

    Adenosine deaminase-1 (ADA1) regulates the concentration of adenosine as the main modulator of oocyte maturation. There is compelling evidence for the association of ADA1 gene polymorphisms with many diseases but the importance of ADA1 polymorphisms in polycystic ovary syndrome (PCOS) has not been studied before. This study investigates serum total ADA activity (tADA), ADA1 and ADA2 isoenzyme activities, and genotype and allele frequencies of G22A and A4223C polymorphisms in healthy and PCOS women. In this case-control study 200 PCOS patients and 200 healthy women were enrolled. Genomic DNA was extracted from whole blood and the PCR-RFLP technique was used to determine the G22A and A4223C variants. The genotype frequencies were calculated and the association between polymorphic genotypes and enzyme activities were determined. tADA activity was significantly lower in the PCOS group compared with the control group (27.76±6.0 vs. 39.63±7.48, respectively). PCOS patients also showed reduced activity of ADA1 and ADA2. PCOS was not associated with G22A polymorphism whereas AA, AC, and CC genotypes of A4223C polymorphism were found distributed differently between the control and the PCOS women where the C allele showed a strong protective role for PCOS (odds ratio=1.876, p=0.033). The present study for the first time showed that lower ADA activity may be involved in pathogenesis of PCOS by maintaining a higher concentration of adenosine affecting follicular growth. As a novel finding, we also showed great differences in genotype distribution and allele frequencies of A4223C polymorphism between groups indicating a protective role for C allele against PCOS. AbbreviationsADA: adenosine deaminase PCOS: polycystic ovary syndrome PCR-RFLP: polymerase chain reaction-restriction fragment length polymorphism tADA: total adenosine deaminase.

  8. Frequencies of glutathione s-transferase (GSTM1, GSTM3 AND GSTT1) polymorphisms in a Malaysian population.

    Science.gov (United States)

    Alshagga, Mustafa A; Mohamed, Norazlina; Nazrun Suhid, Ahmad; Abdel Aziz Ibrahim, Ibrahim; Zulkifli Syed Zakaria, Syed

    2011-08-01

    Glutathione S-transferase (GST) is a xenobiotic metabolising enzyme (XME), which may modify susceptibility in certain ethnic groups, showing ethnic dependent polymorphism. The aim of this study was to determine GSTM1, GSTM3 and GSTT1 gene polymorphisms in a Malaysian population in Kuala Lumpur. Blood or buccal swab samples were collected from 137 Form II students from three schools in Wilayah Persekutuan Kuala Lumpur. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Glutathione-S-transferase GSTM3 gene frequencies were 89% for AA, 10% for AB and 1% for BB. The gene frequencies for deleted GSTM1 and GSTT1 were 66% and 18% respectively. This study suggested that the Malay population is at risk for environmental diseases and provides the basis for gene-environment association studies to be carried out.

  9. PCR-restriction fragment length polymorphism analysis of indigenous nitrogen-fixing micro organisms lineages

    International Nuclear Information System (INIS)

    Liew Woan Ying Pauline; Jong Bor Chyan; Khairuddin Abdul Rahim

    2006-01-01

    The use of PCR-RFLP analysis as a useful microbial identification tool has been evaluated for years. This approach was verified effective worldwide, where differential DNA bands and sequence markers distinctive to specific microbes or microbial groups have been identified. In our study, PCR-RFLP technique has been adopted in the identification of our indigenous N 2 -fixing isolates obtained from several local environments. RFLP was carried out with suitable restriction enzymes and the patterns were documented. Representatives of the different patterns were selected and analysed with the 16S ribosomal DNA sequencing method. The results demonstrated correlation between the differential RFLP patterns and the 16S rDNA identities. (Author)

  10. Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

    Science.gov (United States)

    Oh, Byung Ho; Choe, Yong Beom; Ahn, Kyu Joong

    2010-01-01

    Background This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. Objective The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. Methods 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. Results The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. Conclusion According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups. PMID:20548904

  11. Species authentication of commercial beef jerky based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene.

    Science.gov (United States)

    Chen, Shi-Yi; Liu, Yi-Ping; Yao, Yong-Gang

    2010-11-01

    In this study, we determined species-specific variations by analyzing the mitochondrial 12S rRNA gene sequence variation (∼440 bp) in 17 newly obtained sequences and 90 published cattle, yak, buffalo, goat, and pig sequences, which represent 62 breeds and 17 geographic regions. Based on the defined species-specific variations, two endonucleases, Alu I and Bfa I, were selected for species authentication using raw meat/tissue samples and the PCR-RFLP method. Goat and pig were identified using the Alu I enzyme, while cattle, yak, and buffalo were identified by digestion with Bfa I. Our approach had relatively high detection sensitivity of cattle DNA in mixed cattle and yak products, with the lowest detectable threshold equaling 20% of cattle DNA in a mixed cattle/yak sample. This method was successfully used to type commercial beef jerky products, which were produced by different companies utilizing various processing technologies. Our results show that several yak jerky products might be implicated in commercial fraud by using cattle meat instead of yak meat. Copyright © 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  12. [Correlation analysis of G870A CCND1 gene polymorphism with digestive system tumors].

    Science.gov (United States)

    Yang, Shu-Min; Shi, Ya-Lin

    2016-11-20

    To study the correlation of G870A CCND1 gene polymorphism and digestive system tumors. From August 2010 to August 2014, 164 digestive system cancer patients (including 82 patients with gastric cancer and 82 with colorectal cancer) and 82 healthy subjects (control group) were examined with PCR-restriction fragment length polymorphism (PCR-RFLP). The distribution of CCND1 gene G870A frequency in the 3 groups and its association with tumor staging and grading were analyzed. The frequencies of the GG, GA and AA genotypes in G870A CCND1 gene loci in patients with gastric cancer and colorectal cancer differed significantly from those in the control group (Pdigestive system tumors (Pdigestive system cancer risk than the GG genotype (Pdigestive system tumors. The allele A is associated with an increased risk of digestive system tumors and correlated with the tumor differentiation and staging of the tumor.

  13. A novel single-nucleotide polymorphism in the 5' upstream region of the prolactin receptor gene is associated with fiber traits in Liaoning cashmere goats.

    Science.gov (United States)

    Zhou, J P; Zhu, X P; Zhang, W; Qin, F; Zhang, S W; Jia, Z H

    2011-10-13

    The most important traits of Chinese Liaoning cashmere goat fiber are fiber diameter, weight, and length. We looked for polymorphisms and their possible association with cashmere fiber traits in the 5' upstream region (5' UTR) of the prolactin receptor gene (PRLR), which encodes an anterior pituitary peptide hormone involved in different physiological activities; it is the principal endocrine regulator in pelage replacement in mammals. A novel single-nucleotide polymorphism (SNP) was found in the 5' UTR of PRLR by PCR-RFLP in an analysis of 590 goats. Two genotypes (CC and CT) were observed. The frequencies of allele C and T were 0.93 and 0.07, respectively. Association analysis revealed that the PRLR 5' UTR polymorphism (SNP5) was significantly associated with cashmere fiber weight and diameter. This novel SNP in hircine PRLR has potential as a molecular marker for cashmere fiber weight and diameter in Liaoning cashmere goats.

  14. Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

    Science.gov (United States)

    Daniel, Hubert D-J; David, Joel; Raghuraman, Sukanya; Gnanamony, Manu; Chandy, George M; Sridharan, Gopalan; Abraham, Priya

    2017-05-01

    Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P < 0.00) and 96% (κ = 0.794, P < 0.00) correlated with NS5b sequencing, respectively. Sixty percent and 75% of discordant samples by 5'NCR PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing. © 2016 Wiley Periodicals, Inc.

  15. Association of polymorphisms in avian apoVLDL-II gene with body ...

    African Journals Online (AJOL)

    Association of polymorphisms in avian apoVLDL-II gene with body weight and abdominal fat weight. HH Musa, GH Chen ... Blood samples from the respective populations were taken for DNA extraction, and then slaughter for fat determination. Polymorphism was detected by PCR-RFLP and PCR-SSCP techniques.

  16. Polymorphisms and haplotypes in methylenetetrahydrofolate reductase gene and head and neck squamous cell carcinoma risk.

    Science.gov (United States)

    Galbiatti, Ana Lívia Silva; Ruiz, Mariangela Torreglosa; Rodrigues, Juliana Olsen; Raposo, Luiz Sérgio; Maníglia, José Victor; Pavarino, Érika Cristina; Goloni-Bertollo, Eny Maria

    2012-01-01

    Functional polymorphisms in genes encoding enzymes involved in folate metabolism might modulate head and neck carcinoma risk because folate participates in DNA methylation and synthesis. We therefore conducted a case-control study of 853 individuals (322 head and neck cancer cases and 531 non-cancer controls) to investigate associations among MTHFR C677T and MTHFR A1298C polymorphisms and head and neck squamous cell carcinoma risk. Interactions between these two polymorphisms and risk factors and clinical histopathological parameters were also evaluated. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to genotype the polymorphisms and Chi-square test and multiple logistic regression were used for statistical analyses. The variables age≥49 years, male gender, tobacco habits and alcohol consumption, MTHFR 1298 AC or CC genotypes, combined genotypes with two or more polymorphic alleles and 677T and 1298C polymorphic alleles were associated with increased risk for this disease (PA1298C polymorphism was more frequent in patients with oral cavity as primary site (PA1298C polymorphism has higher risk for this disease.

  17. Restriction fragment length polymorphism of two HLA-B-associated transcripts genes in five autoimmune diseases

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1991-01-01

    The restriction fragment length polymorphism of the two human HLA-B-associated transcripts (BATs) genes, BAT1 and BAT2, identifying polymorphic bands of 12, 8, 2.5, and 1.1 kb, and at 3.3, 2.7, 2.3, and 0.9 kb, respectively, was investigated in patients with primary biliary cirrhosis (PBC...

  18. Amplified fragment length polymorphism and pulsed field gel electrophoresis for subspecies differentiation of Serpulina pilosicoli

    DEFF Research Database (Denmark)

    Møller, Kristian; Jensen, Tim Kåre; Boye, Mette

    1999-01-01

    Pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) were compared for their ability to differentiate between 50 porcine Serpulina pilosicoli isolates. Both techniques were highly sensitive, dividing the isolates into 36 and 38 groups, respectively. Due...

  19. Full-length sequencing and identification of novel polymorphisms in ...

    Indian Academy of Sciences (India)

    The aim of this work was to sequence the entirecoding region of ACACA gene in Valle del Belice sheep breed to identify polymorphic sites. A total of 51 coding exons of ACACA gene were sequenced in 32 individuals of Valle del Belice sheep breed. Sequencing analysis and alignment of obtained sequences showed the ...

  20. MTHFR Gene C677T Polymorphism in Autism Spectrum Disorders

    Directory of Open Access Journals (Sweden)

    Elif Funda Sener

    2014-01-01

    Full Text Available Aim. Autism is a subgroup of autism spectrum disorders, classified as a heterogeneous neurodevelopmental disorder and symptoms occur in the first three years of life. The etiology of autism is largely unknown, but it has been accepted that genetic and environmental factors may both be responsible for the disease. Recent studies have revealed that the genes involved in the folate/homocysteine pathway may be risk factors for autistic children. In particular, C677T polymorphism in the MTHFR gene as a possible risk factor for autism is still controversial. We aimed to investigate the possible effect of C677T polymorphism in a Turkish cohort. Methods. Autism patients were diagnosed by child psychiatrists according to DSM-IV and DSM-V criteria. A total of 98 children diagnosed as autistic and 70 age and sex-matched children who are nonautistic were tested for C677T polymorphism. This polymorphism was studied by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP methods. Results. MTHFR 677T-allele frequency was found to be higher in autistic children compared with nonautistic children (29% versus 24%, but it was not found statistically significant. Conclusions. We conclude that other MTHFR polymorphisms such as A1298C or other folate/homocysteine pathway genes may be studied to show their possible role in autism.

  1. XRCC1 gene polymorphisms and risk of ameloblastoma.

    Science.gov (United States)

    Yanatatsaneejit, Pattamawadee; Boonsuwan, Titiporn; Mutirangura, Apiwat; Kitkumthorn, Nakarin

    2013-06-01

    Ameloblastoma is a common benign odontogenic tumour with inherently aggressive behaviour. Genetic susceptibility of single nucleotide polymorphism (SNP) can likely predict ameloblastoma at risk patients but this data remains limited. Here, we studied XRCC1 polymorphism as a risk factor for ameloblastoma. Eighty-two ameloblastoma samples and blood from 140 healthy controls were used to perform polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for XRCC1 at codons 194, 280 and 399, and confirmed by sequence analysis. Compare to healthy control, a significant increase was noted in the occurrence of polymorphism at codon 194 and 399 in ameloblastoma patients. At codon 194, tryptophan encoded by T, was the susceptibility allele showed an ODD ratio of (95% CI)=1.62 (1.05-2.48), p=0.027. At codon 399, glycine encoded by A was the susceptibility allele showing ODD ratio of (95% CI)=1.83 (1.19-2.84), p=0.005. Moreover at codon 399, we found AG as the susceptibility genotype (2.06 (1.14-3.72), p=0.015). However, we did not find any significant increase in polymorphic occurrence in ameloblastoma patients at codon 280. For haplotype analysis of 3 codons, we found GGC as protective haplotype, and AGT as the risk haplotype. Our data suggest that polymorphism at codons 194 and 399, likely contributes to the risk of developing ameloblastoma. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Detection of VDR gene ApaI and TaqI polymorphisms in patients with type 2 diabetes mellitus using PCR-RFLP method in a Turkish population

    NARCIS (Netherlands)

    Dilmec, Fuat; Uzer, Elmas; Akkafa, Feridun; Kose, Elif; van Kuilenburg, André B. P.

    2010-01-01

    Type 2 diabetes mellitus (T2DM) is by far the most common type of diabetes and is characterized by insulin resistance and altered insulin secretion. Some genes, such as the vitamin D receptor gene (VDR, NM_001017535; GI: 7421), involved in its metabolic pathway have been regarded as good candidates

  3. GGC and StuI polymorphism on the androgen receptor gene in endometrial cancer patients

    International Nuclear Information System (INIS)

    Sasaki, Masahiro; Karube, Akihiro; Karube, Yuko; Watari, Michiko; Sakuragi, Noriaki; Fujimoto, Seiichiro; Dahiya, Rajvir

    2005-01-01

    Androgens have an anti-proliferative effect on endometrial cells. Human androgen receptor (AR) gene contains two polymorphic short tandem repeats of GGC and CAG, and a single-nucleotide polymorphism on exon 1 that is recognized by the restriction enzyme, StuI. Prior studies have shown that the lengths of the CAG repeat are inversely and linearly related to AR activity and associated with endometrial cancer. However, little is known about the GGC repeat and the StuI polymorphism of the AR gene. Thus, we investigated whether these AR polymorphisms are risk factors for endometrial cancer. To test this hypothesis, the genetic distributions of these polymorphisms were investigated in blood samples from endometrial cancer patients and healthy controls. The allelic and genotyping profiles were analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and direct DNA sequencing, and analyzed statistically. The GGC repeat was significantly longer in endometrial cancer patients as compared to normal healthy controls. In general, an increased risk of endometrial cancer was found with increasing GGC repeat. The relative risk for the 17 GGC repeat was greater than 4, as compared to controls. However, the StuI polymorphism was not significantly different between patients and controls. The findings suggest that increased numbers of GGC repeat on the AR gene may be a risk factor for endometrial cancer

  4. Restriction fragment length polymorphism in calpain (CAPN2 gene in crossbred cattle

    Directory of Open Access Journals (Sweden)

    Maria Aparecida Cassiano Lara

    2012-12-01

    Full Text Available With advances in molecular genetics have been possible to predict the genetic value of the animal, in particular its potential to transmit desired characters to their offspring, including characters difficult to evaluate or with low heritability, as is the case of the meat tenderization. It is known that Bos taurus indicus features differences in meat tenderization, being assigned this variability to their lowest proteolysis post-mortem, as result of high activity of calpastatin. This inhibitor decreases the activity of calpain, which are the enzymes responsible for the degradation of muscle fibers during the maturation of the meat. Moreover, there were previously observed differences in the frequencies of allele A of calpain among European breeds (Hereford, Aberdeen Angus and Holstein and Bos taurus indicus (Gir, Guzerá and Nelore. This variability has been related to tenderness of meat, as cattle with Bos taurus taurus origin have more tender meat than Bos taurus indicus, showing small values of shear force. One explanation is that the Capn2A product could confer greater proteolytic activity than the encoded by the allele Capn2B. If allele A is associated with tender meat, it will be possible the early identification of the animals that have the potential to produce meat with qualities that attend the needs of the consumer market, in order to add economic value to the final product of the animal production chain. For this reason, biochemical and genetic studies related to calpain and calpastatin systems have been considered promising for the clarification of the physiological changes that occur in muscle structure during the period post-mortem, whose results have contributed to the improvement of meat quality. The objectives of this study were to investigate the RFLP in calpain (Capn2 gene and its relation with meat tenderization in 252 crossbred (Bos taurus taurus x Bos taurus indicus. The analyses were carried through by PCR-RFLP technique

  5. Multi-drug resistance 1 genetic polymorphism and prediction of chemotherapy response in Hodgkin's Lymphoma

    Directory of Open Access Journals (Sweden)

    Haddadin William J

    2011-07-01

    Full Text Available Abstract Background The human multi-drug resistance gene (MDR1, which encodes the major trans-membrane transporter P-glycoprotein (P-gp, was found to be associated with susceptibility to cancer and response to chemotherapy. The C3435T Polymorphism of MDR1 gene was correlated with expression levels and functions of P-gp. Here, we studied the association between MDR1 C3435T polymorphism and susceptibility to Hodgkin lymphoma (HL and patient's response to ABVD chemotherapy regimen. Methods a total of 130 paraffin embedded tissue samples collected from HL patients were analyzed to identify the C3435T polymorphism. As a control group, 120 healthy subjects were enrolled in the study. The C3435T Polymorphism was genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP method. Data analysis was carried out using the statistical package SPSS version 17 to compute all descriptive statistics. Chi-square and Fisher exact tests were used to evaluate the genotype distribution and allele frequencies of the studied polymorphism. Results these studies revealed that the frequency of T allele was significantly higher in HL patients compared to the controls (P 0.05. Conclusions these results suggest that MDR1 C3435T polymorphism might play a role in HL occurrence; however this polymorphism is not correlated with the clinical response to ABVD.

  6. Rapid detection of dihydropteroate polymorphism in AIDS-related Pneumocystis carinii pneumonia by restriction fragment length polymorphism

    DEFF Research Database (Denmark)

    Helweg-Larsen, J; Eugen-Olsen, J; Lundgren, B

    2000-01-01

    are associated with failure of sulpha prophylaxis and increased mortality in HIV-1 positive patients with PCP, suggesting that DHPS mutations may cause sulpha resistance. To facilitate detection of DHPS mutations we developed a restriction fragment length polymorphism (RFLP) assay, detecting mutations at codon...

  7. PLA2 polymorphism of platelet glycoprotein IIb/IIIa but not Factor V Leiden and prothrombin G20210A polymorphisms is associated with venous thromboembolism and more recurrent events in central Iran.

    Science.gov (United States)

    Pourgheysari, Batoul; Boroujeni, Hamid Rouhi; Hasheminia, Ali Mohammad; Drees, Fatima

    2013-07-01

    Inherited thrombophilic gene polymorphisms have been linked to the pathogenesis of venous thromboembolism (VTE). As there are very limited data of these polymorphisms in the Iranian population, we aimed to investigate the correlation between them and VTE in central Iran. Seventy-two unrelated VTE patients and 306 healthy control individuals were recruited for the study. Genotyping from venous blood with EDTA for the factor V Leiden (FVL), prothrombin (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and PLA2 polymorphism of platelet glycoprotein IIb/IIIa were undertaken by PCR-restriction fragment length polymorphism (PCR-RFLP). A total of 57 investigated polymorphisms with a mean of 0.79 per individual and 151 with a mean of 0.49 were found in patients and controls, respectively (Pgenetic risk factor (P=0.007) and more recurrent events occurred in such patients. Patients with PLA2 polymorphism had more recurrent events than the other patients (P=0.02). Patients with more than one genetic risk factor and recurrent events were younger. The prevalence of these polymorphisms is different from some previously published data in other populations, but is consistent with some others. Higher prevalence of PLA2 polymorphism of GPIIa/IIIb in VTE patients is indicative of the impact of this polymorphism in the pathogenesis of VTE in this population. Because of the impact of coinheritance on the recurrence and the age of occurrence, such patients may need to be managed differently.

  8. Association of Interleukin-4 Receptor Gene Polymorphism with Chronic Periodontitis

    Directory of Open Access Journals (Sweden)

    M. Khoshhal

    2011-10-01

    Full Text Available Introduction & Objective: Periodontitis is a multifactorial disease in which host immune system and genetic factors have an important role in its pathogenesis. Genetic polymorphisms in cytokines and their receptors have been proposed as potential markers for periodontal diseases. The aim of the present study was to evaluate whether IL-4R gene polymorphism is associated with chronic periodontitis (CP or not? Materials & Methods: In this cross sectional study ninety non smoker patients (61 women and 29 men with chronic periodontitis were selected according to established criteria. They were categorized into three groups according to their clinical attachment level (CAL. Mutation at position 375(alanine/glutamine, 411(leucine/serine, 478(serine/proline, 406 (arginine/ cysteine in the IL-4R gene was detected by a polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP method.Results: The distribution of mutations for IL-4 polymorphism at amino acids 375 (P=0.41, 411(P=0.22, 478(P=0.17, 406(P=0.77 were not significantly different among mild, moderate and sever chronic periodontitis patients. Conclusion: This study suggests that there is no correlation between IL-4R polymorphism of chronic periodontitis.(Sci J Hamadan Univ Med Sci 2011;18(3:63-69

  9. Physical localization of NORs and ITS length variants in old ...

    Indian Academy of Sciences (India)

    No variation at the number of Ag-NORs per metaphase was found among the 51 durum wheat cultivars, but the PCR-RFLP technique carried out with the restriction enzyme HpaII, allowed the detection of ITS length variants among them. The molecular data was used in order to establish the genetic relationships among ...

  10. Genetic Diversity and Sectional Relationships from an Amplified Fragment Length Polymorphism Analysis of Taiwan Bananas

    OpenAIRE

    Chang, Shu-Fen; Chang, Yueh-Long; Yen, Yung-Fu; Miyajima, Ikuo; Huang, Kuang-Liang

    2017-01-01

    Phylogenetic relationships among 19 Musa species or cultivars were examined through DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis. The AFLP analysis was performed on the Musa species or cultivars with 21 primer combinations, yielding a total of 6,348 DNA bands, among which 6,113 (96.3%) were polymorphic. M. itinerans var. formosana demonstrated 133 monomorphic bands, which is the most among all samples. Unweighted pair–group method with arithmetic averages was...

  11. [Recent advances of amplified fragment length polymorphism and its applications in forensic botany].

    Science.gov (United States)

    Li, Cheng-Tao; Li, Li

    2008-10-01

    Amplified fragment length polymorphism (AFLP) is a new molecular marker to detect genomic polymorphism. This new technology has advantages of high resolution, good stability, and reproducibility. Great achievements have been derived in recent years in AFLP related technologies with several AFLP expanded methodologies available. AFLP technology has been widely used in the fields of plant, animal, and microbes. It has become one of the hotspots in Forensic Botany. This review focuses on the recent advances of AFLP and its applications in forensic biology.

  12. Use of pcr-rflp of thefla a gene for detection and subtyping of Campylobacter jejuni strains Potentially related to Guillain-barré syndrome, isolated from humans and animals

    Directory of Open Access Journals (Sweden)

    E. Scarcelli

    2009-12-01

    Full Text Available The objectives of the present study were the subtyping of Campylobacter jejuni subsp. jejuni strains obtained from humans and different animal species using PCR-RFLP, and the detection, by means of the same technique, of strains related to serotype PEN O19:LIO 7, the main C. jejuni serotype linked to Guillain-Barré Syndrome (GBS. Seventy C. jejuni strains isolated from human feces (n=33, primates (n=15, dogs (n=5, swine (n=2, bovines (n=1, abortion material from goats (n=2 and poultry carcasses (n=12, all collected in the state of São Paulo, were subtyped by means of PCR-RFLP of fla A gene, using restriction endonucleases Hae III, Afa I and Mbo I. Seven subtypes were observed when using the enzyme Hae III; eight when using Mbo I; and seven when using Afa I. The combination of the three endonucleases led to 16 fla-RFLP subtypes, from which ten subtypes shared strains of human and animal origin. From these, seven subtypes were observed in human and broiler strains. In eight subtypes, the other animal species shared patterns with human strains. It was inferred that, besides broilers, swine, goats, dogs and primates may be sources of infection for human in São Paulo. PCR-RFLP is a highly discriminatory technique that may be applied to molecular epidemiology studies of samples from different origins. Besides, the study also enabled the detection of two human strains and two primate strains related to serotype PEN O19: LIO 7.

  13. CAG-encoded polyglutamine length polymorphism in the human genome

    Directory of Open Access Journals (Sweden)

    Hayden Michael R

    2007-05-01

    Full Text Available Abstract Background Expansion of polyglutamine-encoding CAG trinucleotide repeats has been identified as the pathogenic mutation in nine different genes associated with neurodegenerative disorders. The majority of individuals clinically diagnosed with spinocerebellar ataxia do not have mutations within known disease genes, and it is likely that additional ataxias or Huntington disease-like disorders will be found to be caused by this common mutational mechanism. We set out to determine the length distributions of CAG-polyglutamine tracts for the entire human genome in a set of healthy individuals in order to characterize the nature of polyglutamine repeat length variation across the human genome, to establish the background against which pathogenic repeat expansions can be detected, and to prioritize candidate genes for repeat expansion disorders. Results We found that repeats, including those in known disease genes, have unique distributions of glutamine tract lengths, as measured by fragment analysis of PCR-amplified repeat regions. This emphasizes the need to characterize each distribution and avoid making generalizations between loci. The best predictors of known disease genes were occurrence of a long CAG-tract uninterrupted by CAA codons in their reference genome sequence, and high glutamine tract length variance in the normal population. We used these parameters to identify eight priority candidate genes for polyglutamine expansion disorders. Twelve CAG-polyglutamine repeats were invariant and these can likely be excluded as candidates. We outline some confusion in the literature about this type of data, difficulties in comparing such data between publications, and its application to studies of disease prevalence in different populations. Analysis of Gene Ontology-based functions of CAG-polyglutamine-containing genes provided a visual framework for interpretation of these genes' functions. All nine known disease genes were involved in DNA

  14. R497K polymorphism in epidermal growth factor receptor gene is associated with the risk of acute coronary syndrome

    Directory of Open Access Journals (Sweden)

    Pan Xin-Min

    2008-07-01

    Full Text Available Abstract Background Previous studies suggested that genetic polymorphisms in the epidermal growth factor receptor (EGFR gene had been implicated in the susceptibility to some tumors and inflammatory diseases. EGFR has been recently implicated in vascular pathophysiological processes associated with excessive remodeling and atherosclerosis. Acute coronary syndrome (ACS is a clinical manifestation of preceding atherosclerosis. Our purpose was to investigate the association of the EGFR polymorphism with the risk of ACS. In this context, we analyzed the HER-1 R497K and EGFR intron 1 (CAn repeat polymorphisms in 191 patients with ACS and 210 age- and sex-matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP strategy and direct sequencing. Results There were significant differences in the genotype and allele distribution of R497K polymorphism of the EGFR gene between cases and controls. The Lys allele had a significantly increased risk of ACS compared with the Arg allele (adjusted OR = 1.49, 95% CI: 1.12–1.98, adjusted P = 0.006. However, no significant relationship between the number of (CAn repeats of EGFR intron 1 (both alleles P = 0.911. Considering these two polymorphisms together, there was no statistically significant difference between the two groups. Conclusion R497K polymorphism of the EGFR gene is significantly associated with the risk of ACS. Our data suggests that R497K polymorphism may be used as a genetic susceptibility marker of the ACS.

  15. Exon 3-deleted and full-length growth hormone receptor polymorphism frequencies in an Iranian population

    OpenAIRE

    Palizban, A.A.; Radmansorry, M.; Bozorgzad, M.

    2014-01-01

    The functional role of the exon 3 growth hormone receptor (d3GHR) polymorphism in human and its distributions in different populations is not clearly understood. The presence of full length growth hormone (flGHR) is the most important in metabolic risk factors. The aim of this study was to define the frequency distribution of d3GHR/full-length GHR in an Iranian population. The presence of the d3GHR polymorphism in healthy volunteers blood DNA (n=80, male=30 and female=50) was assessed by PCR ...

  16. Androgen Receptor Repeat Length Polymorphism Associated with Male-to-Female Transsexualism

    Science.gov (United States)

    Hare, Lauren; Bernard, Pascal; Sánchez, Francisco J.; Baird, Paul N.; Vilain, Eric; Kennedy, Trudy; Harley, Vincent R.

    2012-01-01

    Background There is a likely genetic component to transsexualism, and genes involved in sex steroidogenesis are good candidates. We explored the specific hypothesis that male-to-female transsexualism is associated with gene variants responsible for undermasculinization and/or feminization. Specifically, we assessed the role of disease-associated repeat length polymorphisms in the androgen receptor (AR), estrogen receptor β (ERβ), and aromatase (CYP19) genes. Methods Subject-control analysis included 112 male-to-female transsexuals and 258 non-transsexual males. Associations and interactions were investigated between CAG repeat length in the AR gene, CA repeat length in the ERβ gene, and TTTA repeat length in the CYP19 gene and male-to-female transsexualism. Results A significant association was identified between transsexualism and the AR allele, with transsexuals having longer AR repeat lengths than non-transsexual male control subjects (p = .04). No associations for transsexualism were evident in repeat lengths for CYP19 or ERβ genes. Individuals were then classified as short or long for each gene polymorphism on the basis of control median polymorphism lengths in order to further elucidate possible combined effects. No interaction associations between the three genes and transsexualism were identified. Conclusions This study provides evidence that male gender identity might be partly mediated through the androgen receptor. PMID:18962445

  17. Association of the methylenetetrahydrofolate reductase polymorphism in Korean patients with childhood acute lymphoblastic leukemia.

    Science.gov (United States)

    Kim, Nam Keun; Chong, So Young; Jang, Moon Ju; Hong, Seung Ho; Kim, Heung Sik; Cho, Eun Kyung; Lee, Jung Ae; Ahn, Myung Ju; Kim, Chul Soo; Oh, Doyeun

    2006-01-01

    Methylenetetrahydrofolate reductase plays a central role in converting folate to methyl donor for DNA methylation. Recently, methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) mutations were discovered to be associated with childhood acute lymphoblastic leukemia (ALL), as well as colon cancer, lymphoma, esophageal and stomach cancer. Therefore, it was hypothesized that the MTHFR polymorphisms are associated with the risk of childhood ALL in the Korean population. DNA samples taken from 66 patients with ALL and 100 age-matched controls were analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for detection of MTHFR C677T and A1298C mutations. The frequency of the AC genotype for MTHFR A1298C polymorphism was significantly different between the controls and the cases (OR, 2.22; CI, 95% 1.09-4.51, p=0.03). The 1298AC+CC genotype was also significantly different (OR, 2.11; 95% CI, 1.06-4.22; p=0.049). There was, however, no significant difference for MTHFR C677T polymorphism and combined genotype frequencies between the two groups. Although no consistent results on associations between MTHFR A 1298C polymorphism and ALL in the populations studied were obtained, the A1298C polymorphism, at least in Koreans, may be a genetic determinant among childhood ALL patients.

  18. Risk conferred by FokI polymorphism of vitamin D receptor (VDR) gene for essential hypertension.

    Science.gov (United States)

    Swapna, N; Vamsi, U Mohana; Usha, G; Padma, T

    2011-09-01

    The vitamin D receptor (VDR) gene serves as a good candidate gene for susceptibility to several diseases. The gene has a critical role in regulating the renin-angiotensin system (RAS) influencing the regulation of blood pressure. Hence determining the association of VDR polymorphisms with essential hypertension is expected to help in the evaluation of risk for the condition. The aim of this study was to evaluate association between VDRFok I polymorphism and genetic susceptibility to essential hypertension. Two hundred and eighty clinically diagnosed hypertensive patients and 200 normotensive healthy controls were analyzed for Fok I (T/C) [rs2228570] polymorphism by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Genotype distribution and allele frequencies in patients and controls, and odds ratios (ORs) were calculated to predict the risk for developing hypertension by the individuals of different genotypes. The genotype distribution and allele frequencies of Fok I (T/C) [rs2228570] VDR polymorphism differed significantly between patients and controls (χ(2) of 18.0; 2 degrees of freedom; P = 0.000). FF genotype and allele F were at significantly greater risk for developing hypertension and the risk was elevated for both the sexes, cases with positive family history and habit of smoking. Our data suggest that VDR gene Fok I polymorphism is associated with the risk of developing essential hypertension.

  19. Investigation on estrogen receptor alpha gene polymorphisms in Iranian women with recurrent pregnancy loss

    Science.gov (United States)

    Mahdavipour, Marzieh; Idali, Farah; Zarei, Saeed; Talebi, Saeed; Fatemi, Ramina; Jeddi-Tehrani, Mahmood; Pahlavan, Somayeh; Rajaei, Farzad

    2014-01-01

    Background: Recurrent pregnancy loss (RPL) is a multifactorial disorder. Environmental factors and genetics can affect pregnancy outcomes. Objective: Conflicting data suggest an association between estrogen receptor alpha (ESR1) gene polymorphisms and RPL. In this study, such association was investigated in Iranian women with RPL. Materials and Methods: In this case control study, blood samples were collected from 244 women with a history of three or more consecutive pregnancy losses and 104 healthy women with at least two live births. Using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP), we studied -397C/T and -351A/G polymorphisms on ESR1 gene in case and control subjects. Results: The genotypic frequencies of -397C/T and -351A/G polymorphisms on ESR1were not significantly different between RPL and control groups (p=0.20 and p=0.09, respectively). A significantly negative correlation was observed between -397C/T and -351A/G (r=-0.852, p<0.001) in RPL women and complete linkage disequilibrium between the investigated polymorphisms was found (D’: 0.959; r-square= 0.758, p<0.001). Conclusion: This investigation suggests that the analyzed polymorphisms on ESR1gene are not associated with an increased risk of RPL in the studied population. PMID:25071847

  20. SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination.

    Directory of Open Access Journals (Sweden)

    Weiping Xu

    Full Text Available A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100% and a specificity of 99.9% (2910/2912; 95%CI 99.7-100% when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.

  1. SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination.

    Science.gov (United States)

    Xu, Weiping; Morris, Ulrika; Aydin-Schmidt, Berit; Msellem, Mwinyi I; Shakely, Delér; Petzold, Max; Björkman, Anders; Mårtensson, Andreas

    2015-01-01

    A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7-100%) when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.

  2. SYBR Green Real-Time PCR-RFLP Assay Targeting the Plasmodium Cytochrome B Gene – A Highly Sensitive Molecular Tool for Malaria Parasite Detection and Species Determination

    Science.gov (United States)

    Xu, Weiping; Morris, Ulrika; Aydin-Schmidt, Berit; Msellem, Mwinyi I.; Shakely, Delér; Petzold, Max; Björkman, Anders; Mårtensson, Andreas

    2015-01-01

    A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5–10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as ‘final positive’ if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5–100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7–100%) when compared against ‘final positive’ samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings. PMID:25774805

  3. Correlation between single nucleotide polymorphisms of NADPH oxidase p22phox gene and ischemic stroke in Shanghai Han population

    Directory of Open Access Journals (Sweden)

    Wei XU

    2015-09-01

    Full Text Available Objective This paper aims to investigate the distribution of genotypes and alleles of nicotinamide adenine dinucleotide phosphate (NADPH oxidase p22phox -930A/G, 242C/T and -675A/T, so as to evaluate the association between three single-nucleotide polymorphisms (SNPs and risk of atherosclerotic ischemic stroke in permanent resident population of Han nationality living in Shanghai area. Methods The genotypes and allele frequencies of NADPH oxidase p22phox subunit -930A/G, 242C/T and -675A/T were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis in 205 patients with ischemic stroke and 136 healthy controls. Results In patients with ischemic stroke, the results of PCR-RFLP in variant genetic loci were different. For -930A/G, one band appeared at 268 bp of genotype AA; 2 bands appeared at 197 and 71 bp of genotype GG; 3 bands appeared at 268, 197 and 71 bp of genotype AG. For 242C/T, one band appeared at 348 bp of genotype CC; 2 bands appeared at 188 and 160 bp of genotype TT; 3 bands appeared at 348, 188 and 160 bp of genotype CT. For -675A/T, 2 bands appeared at 158 and 54 bp of genotype TT; 3 bands appeared at 212, 158 and 54 bp of genotype AT. The genotypes and allele frequency of all three SNPs of NADPH oxidase p22phox gene had no significant difference between ischemic stroke patients and healthy controls (P > 0.05. Conclusions The genetic polymorphism of NADPH oxidase p22phox gene -930A/G, 242C/T and -675A/T might have no association with ischemic stroke. DOI: 10.3969/j.issn.1672-6731.2015.09.011

  4. Transposition rates of Mycobacterium tuberculosis IS6110 restriction fragment length polymorphism patterns

    NARCIS (Netherlands)

    Eilers, Paul H. C.; van Soolingen, Dick; Thi Ngoc Lan, Nguyen; Warren, Rob M.; Borgdorff, Martien W.

    2004-01-01

    To determine the rate at which IS6110 restriction fragment length polymorphism (RFLP) patterns in Mycobacterium tuberculosis change over time, we applied a smooth nonparametric survival model to several data sets, including data from previous publications on the rate of change. The results strongly

  5. Use of Restriction Fragment Length Polymorphism of 18S rRNA ...

    African Journals Online (AJOL)

    The restriction fragment length polymorphism (RFLP) pattern of PCR products obtained was the same for T. brucei subspecies: T.b. brucei and T.b. gambiense but different for other trypanosome species and L. donovani. RFLP analysis was also done with genomic DNA from different trypanosome species, subspecies and ...

  6. Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis

    DEFF Research Database (Denmark)

    On, Stephen L.W.; Atabay, H.I.; Amisu, K.O.

    2004-01-01

    Aims: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. Methods and Results: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and Nor...

  7. Differential diagnosis of genetic disease by DNA restriction fragment length polymorphisms

    NARCIS (Netherlands)

    Bolhuis, P. A.; Defesche, J. C.; van der Helm, H. J.

    1987-01-01

    DNA restriction fragment length polymorphisms (RFLPs) are used for diagnosis of genetic disease in families known to be affected by specific disorders, but RFLPs can be also useful for the differential diagnosis of hereditary disease. An RFLP pattern represents the inheritance of chromosomal markers

  8. Association between the catechol-o-methyltransferase val158met polymorphism with susceptibility and severity of carpal tunnel syndrome

    Directory of Open Access Journals (Sweden)

    Erkol İnal E

    2015-12-01

    Full Text Available Carpal tunnel syndrome (CTS is the most common entrapment neuropathy of the upper extremity. In this study, we aimed to clarify the relationships between the catechol-O-methyltransferase (COMT gene Val158Met (rs4680 polymorphism and development, functional and clinical status of CTS. Ninety-five women with electro diagnostically confirmed CTS and 95 healthy controls were enrolled in the study. The functional and clinical status of the patients was measured by the Turkish version of the Boston Questionnaire and intensity of pain related to the past 2 weeks was evaluated on a visual analog scale (VAS. The Val158Met polymorphism was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP, method. We divided patients according to the genotypes of the Val158Met polymorphism as Val/Val, Val/Met and Met/Met. There were not any significant differences in terms of Val158Met polymorphisms between patients and healthy controls (p >0.05. We also did not find any relationships between the Val158Met polymorphism and CTS (p >0.05. In conclusion, although we did not find any relationships between CTS and the Val158Met polymorphism, we could not generalize this result to the general population. Future studies are warranted to conclude precise associations.

  9. Glutathione S-Transferase P1 (GSTP1 gene polymorphism increases age-related susceptibility to hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Kuo Wu-Hsien

    2010-03-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is one of the most frequent malignant neoplasms in the world. Genetic polymorphism has been reported to be a factor increasing the risk of HCC. Phase II enzymes such as glutathione s-transferases (GSTP1, GSTA1 play important roles in protecting cells against damage induced by carcinogens. The aim of this study was to estimate the relationship of the GSTP1 and GSTA1 gene polymorphisms to HCC risk and clinico-pathological status. Methods Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP was used to measure GSTP1 (A→G and GSTA1 (C→T gene polymorphisms in 386 healthy controls and 177 patients with HCC. Results Neither gene polymorphism was associated with the clinico-pathological status of HCC and serum expression of liver-related clinico-pathological markers. No association between the GSTA1 gene polymorphism and HCC susceptibility was found. However, in the younger group, aged ≤ 57 years, individuals with AG or GG alleles of GSTP1 had a 2.18-fold (95%CI = 1.09-4.36; p = 0.02 and 5.64-fold (95%CI = 1.02-31.18; p = 0.04 risk, respectively, of developing HCC compared to individuals with AA alleles, after adjusting for other confounders. Conclusion AG and GG alleles of GSTP1 gene polymorphisms may be considered as factors increasing the susceptibility to and risk of HCC in Taiwanese aged ≤ 57 years.

  10. Genetic polymorphisms in Plasmodium falciparum chloroquine resistance genes, pfcrt and pfmdr1, in North Sulawesi, Indonesia.

    Science.gov (United States)

    Reteng, Patrick; Vrisca, Visia; Sukarno, Inka; Djarkoni, Ilham Habib; Kalangi, Jane Angela; Jacobs, George Eduardo; Runtuwene, Lucky Ronald; Eshita, Yuki; Maeda, Ryuichiro; Suzuki, Yutaka; Mongan, Arthur Elia; Warouw, Sarah Maria; Yamagishi, Junya; Tuda, Josef

    2017-04-04

    Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.

  11. Analysis of genetic diversity identified by amplified fragment length polymorphism marker in hybrid wheat.

    Science.gov (United States)

    Ejaz, M; Qidi, Z; Gaisheng, Z; Na, N; Huiyan, Z; Qunzhu, W

    2015-08-07

    Amplified fragment length polymorphism markers were used to assess genetic diversity in 10 male sterile wheat crop lines (hetero-cytoplasm with the same nucleus) in relation to a restorer wheat line. These male sterile lines were evaluated using 64 amplified fragment length polymorphism primer combinations, and 13 primers produced polymorphic bands, generating a total 682 fragments. Of the 682 fragments, 113 were polymorphic. The polymorphic information content and marker index values demonstrated the utility of the primer combinations used in the present study. Unweighted pair group method with arithmetic mean and principal coordinate analysis of the genotypic data revealed clustering of accessions based on genetic relationships, and accessions were separated into 2 groups with their restorer line. Jaccard's similarity coefficient values suggested good variability among the male sterile lines, indicating their utility in breeding programs. The fallouts of analysis of molecular variance showed large within-group population variation, accounting for 77% of variation, while among-group comparison accounted for 23% of the total molecular variation, which was statistically significant. The molecular diversity observed in this study will be useful for selecting appropriate accessions for plant improvement and hybridization through molecular-breeding approaches and for developing suitable conservation strategies.

  12. High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Fussing, V.; Ojeniyi, B.

    2004-01-01

    The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non- L. monocytogenes strains representing six other Listeria species...... of different origin. The AFLP technique was compared with three other molecular typing methods - ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE) - in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included....... Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable...

  13. Restriction fragment length polymorphisms of the DNA of selected Naegleria and Acanthamoeba amebae.

    Science.gov (United States)

    McLaughlin, G L; Brandt, F H; Visvesvara, G S

    1988-09-01

    Fourteen strains of Naegleria fowleri, two strains of N. gruberi, and one strain each of N. australiensis, N. jadini, N. lovaniensis, Acanthamoeba sp., A. castellanii, A. polyphaga, and A. comandoni isolated from patients, soil, or water were characterized by restriction fragment length polymorphisms. Total cellular DNA (1 microgram) was digested with either HindIII, BglII, or EcoRI; separated on agarose gels; and stained with ethidium bromide. From 2 to 15 unusually prominent repetitive restriction fragment bands, totaling 15 to 50 kilobases in length and constituting probably more than 30% of the total DNA, were detected for all ameba strains. Each species displayed a characteristic pattern of repetitive restriction fragments. Digests of the four Acanthamoeba spp. displayed fewer, less intensely staining repetitive fragments than those of the Naegleria spp. All N. fowleri strains, whether isolated from the cerebrospinal fluid of patients from different parts of the world or from hot springs, had repetitive restriction fragment bands of similar total lengths (ca. 45 kilobases), and most repetitive bands displayed identical mobilities. However, polymorphic bands were useful in identifying particular isolates. Restriction fragment length polymorphism analysis generally was consistent with taxonomy based on studies of infectivity, morphology, isoenzyme patterns, and antibody reactivity and suggests that this technique may help classify amebae isolated from clinical specimens or from the environment.

  14. Characterization of six rat strains (Rattus norvegicus by mitochondrial DNA restriction fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    Hilsdorf A.W.

    1999-01-01

    Full Text Available Restriction fragment length polymorphism (RFLP was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

  15. TC2 C776G polymorphism studies in patients with oral cancer in the Polish population

    Directory of Open Access Journals (Sweden)

    Katarzyna Malinowska

    2016-11-01

    Full Text Available The first signs of oral cancer may resemble developing infections in the mucous membranes, with throat cancer symptoms being similar to those of upper respiratory tract infections. This greatly hinders rapid diagnosis and treatment. Better knowledge of the changes occurring in the metabolism of folic acid can help in understanding the carcinogenesis affecting DNA methylation and genome stability. Polymorphisms in genes encoding enzymes involved in this pathway may influence enzyme activity and thereby interfere with the concentrations of homocysteine and S-adenosylmethionine, which are important for DNA synthesis and cellular methylation reactions. The aim of the study was to determine the risk of oral cancer associated with the TC2 C776G polymorphism, as determined in 119 patients. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The test genotype was found to correspond to the Hardy-Weinberg (HW equilibrium (p > 0.05. In our population G/G homozygosity of C776G TC2 gene polymorphism increases the risk of oral cancer; OR (odds ratio: 4.3875; 95% CI (confidence interval: 2.0518-9.319; p = 0.001. Regarding C/G genotype of the C776G TC2 gene, polymorphism also increases the risk of developing this cancer; OR 2.4146 95% CI: 1.2803-4.5541; p = 0.01.

  16. Identification of polymorphisms in the RNase3 gene and the association with allergic rhinitis.

    Science.gov (United States)

    Kang, Inhong; An, Xue-hua; Oh, Yeon-Kyun; Lee, Sang Heon; Jung, Ha Min; Chae, Soo-Cheon; Lee, Jae Hoon

    2010-03-01

    Eosinophil cationic protein (ECP), a potent cytotoxic molecule, is released by activated eosinophils. ECP has been suggested to be involved in tissue remodeling of allergic diseases. The ECP (RNase3) gene is a candidate gene in atopic diseases. RNase3 polymorphisms have been reported to have an association with atopy. We determined whether polymorphisms in the RNase3 gene are associated with allergic rhinitis in a Korean population. The Taqman assay, restriction fragment length polymorphism (PCR-RFLP), and high-resolution melt (HRM) were used for genotyping. Three single nucleotide polymorphisms (SNPs; g.-550A>G, g.371G>C, and g.499G>C) were identified. The genotype of the SNPs was analyzed in patients with allergic rhinitis and controls without allergic rhinitis. The genotype and allele frequencies were compared between both groups. The genotype frequencies of the g.-550A>G and g.371G>C SNPs were not significantly different between patients with allergic rhinitis and controls (P > 0.05). However, in patients with allergic rhinitis, the genotype and allele frequencies of the g.499G>C SNP of RNase 3 were significantly different from those of the control group (P associated with allergic rhinitis (P = 0.048), while the G-G-G haplotype was negatively associated with allergic rhinitis (P = 0.004). Our study suggests that RNase3 polymorphisms are potentially associated with susceptibility to allergic rhinitis.

  17. Association of reduced folate carrier-1 (RFC-1) polymorphisms with ischemic stroke and silent brain infarction.

    Science.gov (United States)

    Cho, Yunkyung; Kim, Jung O; Lee, Jeong Han; Park, Hye Mi; Jeon, Young Joo; Oh, Seung Hun; Bae, Jinkun; Park, Young Seok; Kim, Ok Joon; Kim, Nam Keun

    2015-01-01

    Stroke is the second leading cause of death in the world and in South Korea. Ischemic stroke and silent brain infarction (SBI) are complex, multifactorial diseases influenced by multiple genetic and environmental factors. Moderately elevated plasma homocysteine levels are a major risk factor for vascular diseases, including stroke and SBI. Folate and vitamin B12 are important regulators of homocysteine metabolism. Reduced folate carrier (RFC), a bidirectional anion exchanger, mediates folate delivery to a variety of cells. We selected three known RFC-1 polymorphisms (-43C>T, 80A>G, 696T>C) and investigated their relationship to cerebral infarction in the Korean population. We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to analyze associations between the three RFC-1 polymorphisms, disease status, and folate and homocysteine levels in 584 ischemic stroke patients, 353 SBI patients, and 505 control subjects. The frequencies of the RFC-1 -43TT, 80GG, and 696CC genotypes differed significantly between the stroke and control groups. The RFC-1 80A>G substitution was also associated with small artery occlusion and SBI. In a gene-environment analysis, the RFC-1 -43C>T, 80A>G, and 696T>C polymorphisms in the ischemic stroke group had combined effects with all environmental factors. In summary, we found that the RFC-1 -43C>T, 80A>G, and 696T>C polymorphisms may be risk factors for ischemic stroke.

  18. Prognostic Value of a CYP2B6 Gene Polymorphism in Patients with Acute Myeloid Leukemia.

    Science.gov (United States)

    Alazhary, Nevin M; Shafik, Roxan E; Shafik, Hanan E; Kamel, Mahmoud M

    2015-01-01

    The objectives of this study aimed to detect a CYP2B6 polymorphism in de novo cases of acute myeloid leukemia patients and identify any role in disease progression and outcome. DNA was isolated from peripheral blood of 82 newly diagnosed acute myeloid leukemia cases and the CYP2B6 G15631T gene polymorphism was assayed by PCR restriction fragment length polymorphism (PCR-RFLP). The frequency of the GG genotype (wild type) was 48 (58.5%) and that of the mutant type T allele was 34 (41.9%). GT genotype heterozygous variants were found in 28 (34%), and TT genotype homozygous variants in 6 (7.3%) cases. We found no significant association between the CYP2B6 G15631T polymorphism and complete response (CR) (p-value=0.768), FAB classification (p-value=0.51), cytogenetic analysis (p-value=0.673), and overall survival (p-value=0.325). Also, there were no significant links with early toxic death (p-value=0.92) or progression- free survival (PFS) (p-value=0.245). Our results suggest that the CYP2B6 polymorphism has no role in disease progression, therapeutic outcome, patient free survival, early toxic death and overall survival in acute myeloid leukemia patients.

  19. Methylenetetrahydrofolate reductase gene polymorphisms in Egyptian Turner Syndrome patients.

    Science.gov (United States)

    Ismail, Manal F; Zarouk, Waheba A; Ruby, Mona O; Mahmoud, Wael M; Gad, Randa S

    2015-01-01

    Folate metabolism dysfunctions can result in DNA hypomethylation and abnormal chromosome segregation. Two common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) encoding gene (C677T and A1298C) reduce MTHFR activity, but when associated with aneuploidy, the results are conflicting. Turner Syndrome (TS) is an interesting model for investigating the association between MTHFR gene polymorphisms and nondisjunction because of the high frequency of chromosomal mosaicism in this syndrome. To investigate the association of MTHFR gene C677T and A1298C polymorphisms in TS patients and their mothers and to correlate these polymorphisms with maternal risk of TS offspring. MTHFR C677T and A1298C polymorphisms were genotyped in 33 TS patients, their mothers and 15 healthy females with their mothers as controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing technique. Genotype and allele frequencies of both C677T and A1298C were not significantly different between TS cases and controls. There were no significant differences in C677T genotype distribution between the TS mothers and controls (p=1). The MTHFR 1298AA and 1298AC genotypes were significantly increased in TS mothers Vs. control mothers (p=0.002). The C allele frequency of the A1298C polymorphism was significantly different between the TS mothers and controls (p=0.02). The association of A1298C gene polymorphism in TS patients was found to increase with increasing age of both mothers (p=0.026) and fathers (p=0.044) of TS cases. Our findings suggest a strong association between maternal MTHFR A1298C and risk of TS in Egypt.

  20. Lack of association between urotensin-II (UTS2 gene polymorphisms (Thr21Met and Ser89Asn and migraine

    Directory of Open Access Journals (Sweden)

    Betül Ozan

    2017-08-01

    Full Text Available Migraine is a common neurovascular brain disorder with heterogeneous clinical presentation, including recurrent headache attacks. The pathophysiology of migraine is complex, and a number of genomic regions have been associated with the development of migraine. In this study, we analyzed the allele and genotype frequencies of the urotensin-II gene (UTS2 polymorphisms, Thr21Met and Ser89Asn, among Turkish patients with migraine. A total of 146 patients with migraine (14 with aura [MA group] and 132 without aura [MO group] were genotyped for Thr21Met and Ser89Asn polymorphisms and compared with 154 age- and sex-matched healthy controls. The UTS2 gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. No significant differences were observed in allele and genotype frequencies for Thr21Met and Ser89Asn polymorphisms between the patients with migraine and control group. Similarly, we did not observe significant differences in allele and genotype frequencies between MA and MO and control group. Moreover, the haplotype analysis showed no association between UTS2 gene haplotypes (MN, MS, TN, and TS and migraine. In summary, Thr21Met and Ser89Asn polymorphisms of the UTS2 gene are not risk factors for migraine in our sample of Turkish migraine patients.

  1. Polymorphisms in methylenetetrahydrofolate reductase and cystathionine beta-synthase in oral cancer - a case-control study in southeastern Brazilians

    Directory of Open Access Journals (Sweden)

    Andressa Barbosa

    Full Text Available ABSTRACT INTRODUCTION: Oral squamous cell carcinoma (OSCC is a serious public health problem, due to its high mortality rate and worldwide rising incidence. OSCC susceptibility is mediated by interactions between genetic and environmental factors. Studies suggest that genetic variants encoding enzymes involved in folate metabolism may modulate OSCC risk by altering DNA synthesis/repair and methylation process. OBJECTIVE: The goals of this study were to evaluate the association of three genotypic polymorphism (MTHFR C677T, MTHFR A1298C and CBS 844ins68 and oral cancer risk in southeastern Brazilians and evaluate the interactions between polymorphisms and clinical histopathological parameters. METHODS: This case-control study included 101 cases and 102 controls in the state of Espírito Santo, Brazil. MTHFR genotyping was done by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism and CBS genotyping by PCR (polymerase chain reaction analysis. RESULTS: MTHFR C677T polymorphism was associated with lymph node involvement. Genotype CT + TT acted as a protective factor. MTHFR A1298C AC + CC genotype was associated with tumor differentiation, and possibly with a better prognosis. In risk analysis, no correlation was observed between genotypes and OSCC. CONCLUSION: We concluded that MTHFR C677T, MTHFR A1298C and CBS 844ins68 polymorphisms were not associated with OSCC risk in southeastern Brazilians; however, we suggest a prognosis effect associated with MTHFR C677T and A1298C polymorphisms in OSCC.

  2. Genomic variations of Mycoplasma capricolum subsp capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

    DEFF Research Database (Denmark)

    Kokotovic, Branko; Bolske, G.; Ahrens, Peter

    2000-01-01

    The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size...... found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes....

  3. Association of restriction fragment length polymorphism in alcohol dehydrogenase 2 gene with alcohol induced liver damage.

    OpenAIRE

    Sherman, D I; Ward, R J; Warren-Perry, M; Williams, R; Peters, T J

    1993-01-01

    OBJECTIVE--To investigate the role of genetically determined differences in the enzymes of alcohol metabolism in susceptibility to liver damage from misusing alcohol. DESIGN--Use of pADH36 probe to study PVU II restriction length fragment polymorphism in alcohol dehydrogenase 2 gene in white alcohol misusers and controls. SETTING--Teaching hospital referral centres for liver disease and alcohol misuse. SUBJECTS--45 white alcohol misusers (38 with alcoholic liver disease) and 23 healthy contro...

  4. DNA Polymorphism of Insulin-like Growth Factor-binding Protein-3 Gene and Its Association with Cashmere Traits in Cashmere Goats

    Directory of Open Access Journals (Sweden)

    Haiying Liu

    2012-11-01

    Full Text Available Insulin-like growth factor binding protein-3 (IGFBP-3 gene is important for regulation of growth and development in mammals. The present investigation was carried out to study DNA polymorphism by PCR-RFLP of IGFBP-3 gene and its effect on fibre traits of Chinese Inner Mongolian cashmere goats. The fibre traits data investigated were cashmere fibre diameter, combed cashmere weight, cashmere fibre length and guard hair length. Four hundred and forty-four animals were used to detect polymorphisms in the hircine IGFBP-3 gene. A 316-bp fragment of the IGFBP-3 gene in exon 2 was amplified and digested with HaeIII restriction enzyme. Three patterns of restriction fragments were observed in the populations. The frequency of AA, AB and BB genotypes was 0.58, 0.33 and 0.09 respectively. The allelic frequency of the A and B allele was 0.75 and 0.25 respectively. Nucleotide sequencing revealed a C>G transition in the exon 2 region of the IGFBP-3 gene resulting in R158G change which caused the polymorphism. Least squares analysis revealed a significant effect of genotypes on cashmere weight (p0.05. The animals of AB and BB genotypes showed higher cashmere weight, cashmere fibre length and hair length than the animals possessing AA genotype. These results suggested that polymorphisms in the hircine IGFBP-3 gene might be a potential molecular marker for cashmere weight in cashmere goats.

  5. ANÁLISIS GENÓMICO DE PARVOVIRUS CANINO POR PCR - RFLP A PARTIR DE AISLAMIENTOS DE CASOS CLÍNICOS SINTOMÁTICOS TOMADOS EN BOGOTÁ - COLOMBIA -ESTUDIO PRELIMINAR-

    Directory of Open Access Journals (Sweden)

    Ángela Castillo

    2001-12-01

    Full Text Available Con el objetivo de detemúnar el genotipo del parvovirus canino (CPV circulante en Bogotá, se analizaron 56 muestras tomadas a conveniencia, de materia fecal y sangre de perros menores a un año con gastroenteritis hemorrágica presuntiva de parvovirosis, por reacción en cadena de la polirnerasa - huella genórnica (PCR - RFLP. Se obtuvieron fragmentos amplificados de DNA de 2.2 Kb por PCR de la cepa vacunal de CPV-2 y de cuatro muestras de heces, no se obtuvieron amplificados de las muestras de sangre. Los productos amplificados fueron digeridos por la enzima de restricción Rsa I pemútiendo la identificación de los genotipos CPV-2a y CPV-2b así como su diferenciación con el parvovirus tipo - 2 (CPV-2.

  6. High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Fussing, V.; Ojeniyi, B.

    2004-01-01

    of different origin. The AFLP technique was compared with three other molecular typing methods - ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE) - in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included...... for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains...

  7. A simple PCR-RFLP test for direct identification of Melanocortin Receptor 1 (MC1R alleles causing red coat colour in Holstein cattle

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    Alessio Valentini

    2010-01-01

    Full Text Available A direct test to determine the presence of the recessive alleles causing red colour in Holstein cattle at DNA level is proposed.Digestions with two restriction enzymes were used to detect individuals carrying recessive alleles of MelanocortinReceptor 1 (MC1R gene, responsible for coat coloration. Direct sequencing of the PCR products confirmed the identifiedgenotypes. Compared to previously described methods this is an effective, relatively economic and quick method. Thistest could be employed not only to facilitate the detection of polymorphisms in populations but also to exclude animalscarrying alleles resulting in an undesired coat colour from breeding schemes.

  8. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in chronic myeloid leukemia: an Egyptian study.

    Science.gov (United States)

    Khorshied, Mervat Mamdooh; Shaheen, Iman Abdel Mohsen; Abu Khalil, Reham E; Sheir, Rania Elsayed

    2014-01-01

    Methylenetetrahydrofolate reductase (MTHFR) gene plays a pivotal role in folate metabolism. Several genetic variations in MTHFR gene as MTHFR-C677T and MTHFR-A1298C result in decreased MTHFR activity, which could influence efficient DNA methylation and explain susceptibility to different cancers. The etiology of chronic myeloid leukemia (CML) is obscure and little is known about individual's susceptibility to CML. In order to assess the influence of these genetic polymorphisms on the susceptibility to CML and its effect on the course of the disease among Egyptians, we performed an age-gender-ethnic matched case-control study. The study included 97 CML patients and 130 healthy controls. Genotyping of MTHFR-C677T and -A1298C was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The results showed no statistical difference in the distribution of MTHFR-C677T and -A1298C polymorphic genotypes between CML patients and controls. The frequency of MTHFR 677-TT homozygous variant was significantly higher in patients with accelerated/blastic transformation phase when compared to those in the chronic phase of the disease. In conclusion, our study revealed that MTHFR-C677T and -A1298C polymorphisms could not be considered as genetic risk factors for CML in Egyptians. However, MTHFR 677-TT homozygous variant might be considered as a molecular predictor for disease progression.

  9. Evaluation of Apolipoprotein A5 Polymorphism in Coronary- Heart Disease Patients

    Directory of Open Access Journals (Sweden)

    Somayeh Haqparast

    2012-02-01

    Full Text Available Background and Objectives: Apolipoprotein A5 (APOA5 gene is important in determining plasma triglyceride levels, a major cardiovascular disease risk factor. Mutation in this gene affected plasma triglyceride level. We looked for possible associations of the APOA5 gene polymorphism S19W with coronary heart disease (CHD in a sample of Iranian population. Materials and Methods: A total of 73 CHD patients and 55 controls were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP for this single nucleotide polymorphism. Serum lipids and Fast Blood Sugar concentrations were measured in all subjects with enzymatic method. Results: Allele frequencies observed in our population were 0.041 for the W allele and 0.959 for the S allele which are similar to other populations (p>0.05. There is no evidence that APOA5 S19W, is a risk factor of CHD in our sample (p>0.05. In addition, we observed no association between the APOA5 W allele and elevated plasma TG levels (p>0.05 in the CHD group. This result was also present in the control group (p>0.05. Conclusion: The APO A5 gene polymorphism in S19W gene has no association with the high susceptibility to CHD.

  10. Association of TRB3 Q84R polymorphism with polycystic ovary syndrome in Chinese women

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    Tu Binbin

    2011-04-01

    Full Text Available Abstract Background Tribbles 3 (TRB3 affects insulin signalling by inhibiting insulin-stimulated Akt phosphorylation and subsequent activation. A single nucleotide polymorphism located in the second extron of the human TRB3 gene is thought to be associated with insulin resistance. The latter is a core abnormality in PCOS independent of obesity. The present study was designed to clarify the relationships of TRB3 Q84R polymorphism with PCOS in a Chinese women group. Methods A case-control study with two groups: PCOS group (n = 336 and control group of infertility women for tubal and/or male factor (n = 116 was performed. Genotyping of the TRB3 R84 variant was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. Results The frequency of genotype QQ in PCOS women was significantly lower, while genotype QR and RR were significantly higher than that in control group (p Conclusions TRB3 Q84R polymorphism is associated with obesity and especially glucose metabolism and not associated with polycystic ovary syndrome because of compositional characteristics of phenotype in Chinese PCOS women.

  11. IDENTIFICATION OF GH|ALUI AND GHR|ALUI GENES POLYMORPHISMS IN INDONESIAN BUFFALO

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    E. Andreas

    2014-10-01

    Full Text Available Growth hormone (GH is an anabolic hormone which sintesized and secreted by somatrotop cell inpituitary anterior lobe. GH exert its effect on growth and metabolism by interacting with a specificreceptor on the surface of the target cells. Growth hormone receptor (GHR has been suggested ascandidate gene for traits related to meat production in Bovidae. The objectives of this study were toidentify polymorphism of GH and GHR genes in buffalo. The 452 DNA samples buffalo were collectedfrom five populations in Indonesia (Siborong-Borong-Medan (65, Lebak-Banten (29, Pandeglang-Banten (180, Semarang-Central Java, and Mataram-West Nusa Tenggara (103. A gene fragment of theGH|AluI gene at 432 bp located on exon 3 and GHR|AluI gene at 298 bp on exon 10 were successfullyamplified by using the techniques of a PCR (polymerase chain reaction and genotyped by PCR-RFLP(restriction fragment length polymorphism then -SSCP (single strand conformation polymorphism. Theresults showed no polymorphisms were detected in these genes. All buffaloes tested had LL genotype forlocus GH|AluI and AA genotype for locus GHR|AluI.

  12. Association of PDCD1 polymorphism to systemic lupus erythematosus and rheumatoid arthritis susceptibility.

    Science.gov (United States)

    do Canto, Luisa Matos; Farias, Ticiana Della Justina; Medeiros, Mayara Delagnelo; Coêlho, Cíntia Callegari; Sereia, Aline Fernanda Rodrigues; de Carlos Back, Lia Kubelka Fernandes; de Mello, Filipe Martins; Zimmermann, Adriana Fontes; Pereira, Ivânio Alves; de Souza, Ilíada Rainha

    This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

  13. [The role of smoking and metabolic enzyme polymorphisms in the organic solvent induced chronic encephalopathy].

    Science.gov (United States)

    Wang, H Y; Ma, Y M; Cui, J Z; Zhao, D; Liu, A J; Hong, J; Lu, Y G; Wang, K J

    2016-03-20

    The organic solvents and other exogenous compounds of metabolic enzymes genetic variation may affect the risk of the toxic effect of organic solvents exposure. Therefore, this research we observed the glutathione transferase M1 and T1 (GSTM1, GSTT1) deletion mutation genotype, two kinds of microsomal epoxide hydrolase (mEPHX) genetic polymorphism, organic solvents exposure and smoking effection in chronic cases of toxic encephalopathy (CTE) correlation. The object was 115 patients who had a long history of organic solvents exposure, were divieded into two groups: CTE (n=83) , no CET (n=32) according to clinical diagnosis. DNA was isolated from patients in white blood cells through the multiple-polymerase chain reaction to determine the loss of GSTM1 and GSTT1 genotype. two kinds of mEPHX polymorphism were analysised through the PCR-RFLP (restriction fragment length polymorphism). The relative risk has obviously improved when lack of GSTM1 genotypes to CTE (RR=2.35, 95% CI 2.35 0.96). in according to the patient's Smoking condition and classify genotype, patients lack of GSTM1 genotypes had a significantly higher risk CTE than GSTM1+genotype patients (RR=3.13, 95% CI 3.13 1.2) , both mEPHX polymorphisms had nothing to do with an increased risk of CTE. The GSTM1 genotypes played an important role in the organic solvent induced the CTE of susceptibility.it was Influenced by the interaction between smoking at the same time.

  14. Association of ADAM33 gene polymorphisms with adult allergic asthma and rhinitis in a Chinese Han population

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    Jin Lianhong

    2008-09-01

    Full Text Available Abstract Background Rhinitis and asthma are very common diseases involving genetic and environmental factors. Most patients with asthma also have rhinitis, which suggests the concept of 'one airway, one disease.' A disintegrin and metalloproteinase 33 (ADAM33 is the first asthma-susceptible gene to be discovered by positional cloning. To evaluate the potential influence of ADAM33 gene polymorphisms on allergic rhinitis (AR and allergic asthma (AS, a case-control study was conducted on the Han population of northeast China. Methods Six polymorphic sites (V4, T+1, T2, T1, S1, and Q-1 were genotyped in 128 patients with AR, 181 patients with AS, and 151 healthy controls (CTR. Genotypes were determined by the polymerase chain restriction fragment length polymorphism (PCR-RFLP method. Data were analyzed using the chi-square test with Haploview software. Results The single nucleotide polymorphisms (SNPs, V4 G/C, T+1 A/G, and T1 G/A, of the ADAM33 gene may be the causal variants in AR, whereas ADAM33 V4 G/C, T2 A/G, T1 G/A, and Q-1A/G may participate in the susceptibility of AS. Conclusion These results suggest that polymorphisms of the ADAM33 gene may modify individual susceptibility to AR and AS in a Chinese Han population.

  15. Matrix Metalloproteinase-9 rs17576 Gene Polymorphism and Behçet's Disease: Is There an Association?

    Science.gov (United States)

    Kamal, Asmaa; Elgengehy, Fatema T; Abd Elaziz, Mohamed Momtaz; Gamal, Sherif M; Sobhy, Nesreen; Medhat, Amira; El Dakrony, Al Hussein M

    2017-07-01

    Clinical studies have reported a significant association between matrix metalloproteinases (MMP), particularly (MMP-9), and inflammatory diseases including Behçet's disease (BD). To study the relationship between MMP-9 rs17576 gene polymorphism and the development of BD, and its relation to disease activity among Egyptian patients. A total of 100 BD patients and 100 healthy control volunteers were genotyped for MMP-9 rs17576 polymorphism with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), followed by the confirmation of our results in random subgroups using direct DNA sequencing technique. The frequency of the GG genotype and G allele was significantly higher in BD patients as compared to the normal controls (p = 0.011, OR 8.61; p = 0.03, OR 1.65, respectively). There was no significant association between the MMP-9 rs17576 polymorphism and the clinical outcomes of BD. our study suggests a significant association of the MMP-9 rs17576 A/G polymorphism with increased risk of BD development in Egyptian patients.

  16. HLA-DPB1 typing with polymerase chain reaction and restriction fragment length polymorphism technique in Danes

    DEFF Research Database (Denmark)

    Hviid, T V; Madsen, H O; Morling, N

    1992-01-01

    We have used the polymerase chain reaction (PCR) in combination with the restriction fragment length polymorphism (RFLP) technique for HLA-DBP1 typing. After PCR amplification of the polymorphic second exon of the HLA-DPB1 locus, the PCR product was digested with seven allele-specific restriction...

  17. Restriction fragment length polymorphism (RFLP) of two HLA-B-associated transcripts (BATs) genes in healthy Danes

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1990-01-01

    The restriction fragment length polymorphism (RFLP) of the two human HLA-B-associated transcripts (BATs) genes, BAT1 and BAT2, was investigated using 5 different restriction enzymes and two human BAT1 and BAT2 cDNA probes. Two of the enzymes, NcoI and RsaI, revealed polymorphic patterns which were...

  18. Application of fluorescent amplified fragment length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica

    DEFF Research Database (Denmark)

    Fearnley, C.; On, S.L.W.; Kokotovic, Branko

    2005-01-01

    An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y enterocolitica strains according to th...

  19. Association of CYP1A1 and CYP1B1 polymorphisms with bone mineral density variations in postmenopausal Mexican-Mestizo women.

    Science.gov (United States)

    Chávez, Bertha; Vilchis, Felipe; Rojano-Mejía, David; Coral Vázquez, Ramón Mauricio; Aguirre-García, María Del Carmen; Canto, Patricia

    2017-08-01

    Herein, we investigated potential associations between polymorphisms of genes related to estrogen metabolism and bone mineral density (BMD) in postmenopausal women. This was a cross-sectional study, in which two hundred and ninety postmenopausal Mexican-Mestizo women were studied. The BMD of the lumbar spine (LS), total hip (TH), and femoral neck (FN) was measured. The distribution of the genetic polymorphisms, including rs1799814 and rs1048943 at CYP1A1 as well as rs1056836 at CYP1B1, were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-stranded conformational polymorphism (SSCP), and DNA sequencing. Deviations from Hardy-Weinberg equilibrium (HWE) were tested, and linkage disequilibrium (LD) was calculated by direct correlation (r 2 ). Moreover, haplotype analysis was performed. All polymorphisms were in HWE. The genotype and allele distributions of the three single nucleotide polymorphisms (SNPs) studied showed no significant differences. However, statistical significance was reached when constructing haplotypes. The CG haplotype in CYP1A1 was associated with variations in LS and FN BMD after adjustment for covariates (p = 0.021 and 0.045, respectively), but the association with TH BMD was not significant. These results suggested that the CG haplotype in CYP1A1 may play an important role in the mechanism of osteoporosis and may be useful as a genetic marker.

  20. MTHFR C677T and A1298C gene polymorphisms and their relation to homocysteine level in Egyptian children with congenital heart diseases.

    Science.gov (United States)

    Zidan, Haidy E; Rezk, Noha A; Mohammed, Doaa

    2013-10-15

    To investigate the association of combined MTHFR C677T and A1298C gene polymorphisms with congenital heart diseases (CHD) in Egyptian children and their mothers and to determine their effect on homocysteine level in these children. MTHFR C677T and A1298C polymorphisms were genotyped in 160 Egyptian children (80 patients with CHD and 80 healthy controls) and their mothers using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while, homocysteine (Hcy) level was measured optically by enzymatic method. We found that MTHFR 677TT genotype, T allele, 1298CC genotype, and C allele were associated with 2.61, 2.0, 2.91 and 1.99 fold increased risk of CHD in Egyptian children respectively. Furthermore, the frequencies of MTHFR 1298AC and CC genotypes and C allele significantly increased in mothers with CHD affected children. The homocysteine levels were significantly increased in MTHFR 677TT and 1298CC genotypes in children with CHD. Our study demonstrated an association of MTHFR A1298C polymorphisms with CHD in Egyptian children and their mothers, while, MTHFR C677T polymorphisms were significantly associated with the risk of CHD in the children only. An association between combined MTHFR A1298C and C677T polymorphisms and CHD was recorded in the children and their mothers. Also, homocysteine levels were significantly increased with both MTHFR 677TT and 1298CC genotypes in Egyptian children with CHD. © 2013.

  1. The interleukin-18 gene promoter -607 A/C polymorphism contributes to non-small-cell lung cancer risk in a Chinese population

    Directory of Open Access Journals (Sweden)

    Jia YC

    2016-03-01

    Full Text Available Youchao Jia,1,2 Aimin Zang,2 Shunchang Jiao,1 Sumei Chen,1 Fu Yan1 1Department of Medical Oncology, General Hospital of Chinese PLA, Beijing, 2Department of Oncology, Affiliated Hospital of Hebei University, Hebei, People’s Republic of China Abstract: The purpose of the present study was to determine the relationship between interleukin-18 (IL-18 -607 A/C polymorphism and the risk of non-small-cell lung cancer (NSCLC and its impact on the serum IL-18 level. The genotyping of IL-18 -607 A/C polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The results showed that the AA/AC genotype distribution in NSCLC patients was significantly higher than that of healthy controls (P=0.02. However, no significant differences were found between the two subgroups when stratified by clinical characteristics. Furthermore, serum IL-18 levels were found to be significantly higher in the NSCLC patients than in the controls (P=0.01 as detected by enzyme-linked immunosorbent assay analysis. There was no correlation between serum IL-18 levels and different genotypes. In conclusion, these findings suggest that IL-18 -607 A/C polymorphism increases the risk of NSCLC in the Chinese population, and this polymorphism could not functionally affect the IL-18 levels. Keywords: IL-18, polymorphism, NSCLC

  2. Vascular endothelial growth factor (-460 C/T, +405 G/C, and +936 C/T) polymorphisms and endometriosis risk in Tunisian population.

    Science.gov (United States)

    Henidi, Belkis; Kaabachi, Wajih; Naouali, Abir; Kaabachi, Safa; Zhioua, Amel; Haj Sassi, Faycal; Hamzaoui, Kamel

    2015-01-01

    The vascular endothelial growth factor (VEGF), a major angiogenic factor, is known to play an important role in the development of endometriosis. The aim of this study was to investigate the association of three VEGF (-460 C/T, +405 G/C, and +936 C/T) polymorphisms with the risk of endometriosis in the Tunisian population. This study includes 105 women with endometriosis and 150 women with no laparoscopic evidence of disease. Genotyping of the VEGF -460 C/T, +405 G/C, and +936 C/T polymorphisms were performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The distribution of genotypes (P = 0.006) and allele (P = 0.0009) frequencies of the +936 C/T polymorphism was significantly different between patients and controls. Patients with stages III-IV endometriosis showed a higher VEGF + 936T allele frequency than controls (P = 0.0001). However, the distribution of genotypes and allele frequencies of the VEGF -460 C/T and +405 G/C polymorphisms did not differ significantly between endometriosis patients and controls. These findings suggest that the VEGF +936 C/T polymorphism may be a risk factor for endometriosis development and the VEGF +936 T allele is associated with an increased risk of stages III-IV endometriosis in the Tunisian population.

  3. Restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene.

    Science.gov (United States)

    Masina, P; Rando, A; Cocozza, S

    1984-10-01

    By Southern blot analysis, a restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene was detected. Two alleles, characterized by 9.7- and 12.4-kb BamHI fragments and by 15.3- and 18.0-kb HindIII fragments, have been detected in a small population of White New Zealand rabbits. The long allele is the most frequent (about 70%). The simultaneous changes in the restriction patterns of the two endonucleases and the constant distance between BamHI and HindIII sites in short and long fragments suggest the possibility that the two alleles arise from a rearrangement phenomenon involving a DNA segment 2.7 kb long. In addition, the presence of the two alleles in individuals genetically unrelated to the White New Zealand breed suggests that this polymorphism is widespread.

  4. Genetic variation in hemp and marijuana (Cannabis sativa L.) according to amplified fragment length polymorphisms.

    Science.gov (United States)

    Datwyler, Shannon L; Weiblen, George D

    2006-03-01

    Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.

  5. The isolation and localization of arbitrary restriction fragment length polymorphisms in Southern African populations

    International Nuclear Information System (INIS)

    Conn, V.

    1987-01-01

    The main aim of this study was to contribute to the mapping of the human genome by searching for and characterizing a number of RFLPs (restriction fragment length polymorphisms) in the human genome. The more specific aims of this study were: 1. To isolate single-copy human DNA sequences from a human genomic library. 2. To use these single-copy sequences as DNA probes to search for polymorphic variation among Caucasoid individuals. 3. To show by means of family studies that the RFLPs were inherited in a co-dominant Mendelian fashion. 4. To determine the population frequencies of these RFLPs in Southern African Populations, namely the Bantu-speaking Negroids and the San. 5. To assign these RFLP-detecting DNA sequences to human chromosomes using somatic cell hybrid lines. In this study DNA was labelled with Phosphorus 32

  6. Exon 3-deleted and full-length growth hormone receptor polymorphism frequencies in an Iranian population.

    Science.gov (United States)

    Palizban, A A; Radmansorry, M; Bozorgzad, M

    2014-01-01

    The functional role of the exon 3 growth hormone receptor (d3GHR) polymorphism in human and its distributions in different populations is not clearly understood. The presence of full length growth hormone (flGHR) is the most important in metabolic risk factors. The aim of this study was to define the frequency distribution of d3GHR/full-length GHR in an Iranian population. The presence of the d3GHR polymorphism in healthy volunteers blood DNA (n=80, male=30 and female=50) was assessed by PCR using specific primers. The 935-bp and 592-bp fragments indicate the presence of the flGHR and the exon3 deletion of GHR, respectively. The distribution of the GHR genotypes in this study were 31.4% (n=24) for fl/flGHR, 49.7 % (n=41) for fl/d3GHR, and 19.0 % (n=15) for d3/d3GHR. Frequencies of fl allele and d3 allele were 55.4% and 44.4% within whole population, respectively. There was no difference in allels frequencies of GHR in male (fl=0.583, d3=0.417) and female (fl=0.540, d3=0.460) when compared with whole population. The results showed that the frequency of d3/d3GHR isoform was significantly lower than that of the fl/flGHR and d3/flGHR. The frequencies of GHR polymorphisms were likely consistent with previous reports. Our finding is also consistant with Mexican population. The advantage of existence of the d3/d3 rather than fl/flGHR polymorphisms in individuals and in correlation with diseases opens new insights for GH and insilin-like-growth factor-1 (IGF-I) axis.

  7. New insights in HLA-E polymorphism by refined analysis of the full-length gene.

    Science.gov (United States)

    Olieslagers, T I; Voorter, C E M; Groeneweg, M; Xu, Y; Wieten, L; Tilanus, M G J

    2017-03-01

    Human leukocyte antigen (HLA)-E is a non-classical HLA class I molecule that plays a role in both the innate and the adaptive immune response through interaction with receptors on natural killer- and T-cells. The HLA-E gene is characterized by limited polymorphism compared with the classical HLA loci on chromosome 6. At the start of this study, only 13 variable sites had been identified (IPD-IMGT/HLA Database v3.18.0). While most previous studies focused on polymorphism in exons 2 and 3 or specific gene regions, polymorphism in the other exons and introns could influence protein expression and function as well. Studies that investigate extended HLA-E polymorphism are therefore needed to better understand the functional relevance of HLA-E in health and disease. The aim of this study was to examine the variability of the full-length HLA-E gene region in individuals originating from different populations. A total of 7 new HLA-E alleles were identified using full-length HLA-E sequencing of 123 individuals from Asian, Dutch or Hunan Han origin. Furthermore, genome variation analysis of the third phase of the 1000 genomes database showed 107 new variable sites in 2504 individuals originating from 26 different populations. Our study demonstrates that the nucleotide variability of the HLA-E gene is much higher than previously known, albeit in only a limited number of individuals. Overall only 2 variants, HLA-E*01:01 and *01:03, are frequently present worldwide, suggesting that balancing selection is acting on HLA-E. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

    DEFF Research Database (Denmark)

    Kokotovic, Branko; On, Stephen L.W.

    1999-01-01

    A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species...... to available epidemiological data. We conclude that this amplified fragment length polymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp....

  9. Genetic variation in an endemic salamander, Salamandra atra, using amplified fragment length polymorphism.

    Science.gov (United States)

    Riberon, Alexandre; Miaud, Claude; Guyetant, R; Taberlet, P

    2004-06-01

    The pattern of genetic differentiation of the endemic alpine salamander, Salamandra atra, has been studied using amplified fragment length polymorphism (AFLP) from 11 populations throughout the range of the two currently recognized subspecies, atra and aurorae. Five different primer combinations produced 706 bands and were analyzed by constructing a phylogenetic tree using NJ and principal component analysis. Significant genetic variation was revealed by AFLP between and within populations but, our results show a lack of genetic structure. AFLP markers seems to be unsuitable to investigate complex and recent diversification.

  10. Genomic diversity among Danish field strains of Mycoplasma hyosynoviae assessed by amplified fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Kokotovic, Branko; Friis, Niels F.; Nielsen, Elisabeth O.

    2002-01-01

    Genomic diversity among strains of Mycoplasma hyosynoviae isolated in Denmark was assessed by using amplified fragment length polymorphism (AFLP) analysis. Ninety-six strains, obtained from different specimens and geographical locations during 30 years and the type strain of M. hyosynoviae S16(T......) were concurrently examined for variance in BglII-MfeI and EcoRI-Csp6I-A AFLP markers. A total of 56 different genomic fingerprints having an overall similarity between 77 and 96% were detected. No correlation between AFLP variability and period of isolation or anatomical site of isolation could...

  11. Direct endonuclease digestion and multi-analysis of restriction fragment length polymorphisms by microchip electrophoresis.

    Science.gov (United States)

    Akamine, Rie; Yatsushiro, Shouki; Yamamura, Shouhei; Kido, Jun-ichi; Shinohara, Yasuo; Baba, Yoshinobu; Kataoka, Masatoshi

    2009-12-05

    A high-performance multi-analysis system for genotypic mutation by means of restriction fragment length polymorphisms (RFLP) involving endonuclease treatment of PCR-amplified DNA on a microchip and subsequent analysis by microchip electrophoresis for DNA sizing was developed. A Hitachi SV1210 system, with which 12 samples can be analyzed on a plastic chip with good accuracy as to DNA sizing between 25 and 300 bp, was employed for RFLP analysis. We performed RFLP analysis of the ABO genotypes of blood donors for whom the ABO type was known. Six blood samples were analyzed by PCR to amplify two different regions of the genomic DNA, each of the amplified DNAs containing a different nucleotide polymorphism. To analyze the genes at polymorphic sites 261 and 526, restriction endonucleases Kpn I and Ban I were employed, respectively. When an amplified DNA was digested with each endonuclease on a microchip for 20 min, sequential analysis revealed the presence or absence of the respective restriction site. This analysis was performed within 7 min using a 1/10 volume of a DNA sample in comparison with the conventional method, and the estimated DNA size differed from the predicted size by less than 10 bp. The results indicate the potential of microchip electrophoresis for RFLP with on-chip direct endonuclease digestion and sequential analysis, offering high resolution in a short time.

  12. Identification of polymorphisms of XRCC1 gene in patients with cancer in a city of northern Brazil

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    Artemis Socorro N. Rodrigues

    2015-06-01

    Full Text Available ABSTRACT Introduction: Cancer is considered a genetic disease. For this reason, identification and characterization of the genes involved in its origin and progression are of fundamental importance in understanding its molecular basis. Objective: Our objective was to determine whether people from Macapá with a diagnosis of cancer have genetic polymorphisms related to the XRCC1 gene. Materials and methods: We analyzed 30 samples of deoxyribonucleic acid (DNA of cases with cancer and 30 control samples. All samples were amplified and analyzed by the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP method, with the use of restriction enzyme MspI. Results: Regarding the 194T polymorphism, we found that all samples of the cases presented the polymorphic allele Trp (Arg/Trp. In control samples, 96.6% also identify the polymorphic allele Trp and, among these, one was homozygous for the same allele (Trp/Trp. Regarding the 399A polymorphism, 83.3% of the cases and 23.3% of the controls had the Arg/ Gln genotype, respectively. We found that 73.3% of controls and 16.6% of cases had the Arg/Arg genotype. Among the controls, we found only a sample that was homozygous for the polymorphic allele Trp/Trp. Conclusion: Our results demonstrated the allele frequency of 194Trp polymorphism in both sample groups analyzed. We also found a significant number of polymorphic allele 399A in people with cancer. Thus, we can highlight 399Gln polymorphism as a genetic marker of cancer risk in this population.

  13. Glutathione-S-transferase and microsomal epoxide hydrolase polymorphism and viral-related hepatocellular carcinoma risk in India.

    Science.gov (United States)

    Kiran, Manjula; Chawla, Yogesh Kumar; Kaur, Jyotdeep

    2008-12-01

    Hepatocellular carcinoma (HCC) is the fourth most common cancer worldwide, the main etiological factors being chronic infections with hepatitis B and C viruses. Genetic polymorphic forms of glutathione-S-transferase (GST) and microsomal epoxide hydrolase (mEPHX) have been associated with risk for various malignancies. The present study was undertaken to evaluate the association of GSTT1 and GSTM1 null genotypes and mEPHX polymorphisms with hepatitis virus-related HCC risk in an Indian population. Three groups of subjects were considered, control (n = 169), chronic viral hepatitis (n = 174), and HCC (n = 63). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for this polymorphic study. Genotype distributions between categories were compared using the chi2 test; odds ratios (ORs) and 95% confidence interval were calculated to express the relative risk. GSTT1 null genotype was associated with 2.23-fold (p GST and mEPHX variants share a positive association with viral-related HCC risk in Indian population, although a larger sample size is still required to confirm the results.

  14. [Correlation Study on Chinese Medical Syndrome Types of Chronic Atrophic Gastritis Patients, Hp, and IL-1β Polymorphism].

    Science.gov (United States)

    Zhang, Jian-zhi; Feng, Quan-lin; Hu, Yi-lu; Yang, Ti; Zhang, Ying

    2015-12-01

    To explore the correlation between Chinese medical (CM) syndrome types of chronic atrophic gastritis (CAG) patients and Helicobacter pylori (Hp) infection, polymorphisms of IL-1B, and IL-1β. Totally 192 CAG patients and 202 healthy subjects (as the healthy control group) were recruited in this case-control study. The Hp infection was tested by 13C-urea breath test and colloidal gold-labeled assay (GICA). The concentration of peripheral blood IL-1β was measured by ELISA. The polymorphisms of IL-1B gene in the promoter region were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Pi-Wei weakness syndrome (PWWS) was dominant in CAG patients (31.77%, 61/192 cases). The Hp infection ratio in CAG patients was 53.65% (103/192 cases), of which, Pi-Wei damp-heat syndrome(PWDHS, 64.86%, 24/37 cases) and Gan-Wei disharmony syndrome (GWDS, 66.67%, 24/36 cases) were dominant. Compared with the health control group, the plasma concentration of IL-1β was obviously elevated in CAG patients with PWDHS, GWDS, and static blood obstructing collaterals syndrome (SBOCS) (all P 0.05). The incidence risk of CAG was not associated with IL-1B polymorphism. But CM syndrome types of CAG patients was associated with Hp infection and peripheral blood IL-1β levels.

  15. Association of the 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) polymorphisms in Korean patients with adult acute lymphoblastic leukemia.

    Science.gov (United States)

    Oh, Doyeun; Kim, Nam Keun; Jang, Moon Ju; Kim, Hugh Chul; Lee, Jae Hoon; Lee, Jung Ae; Ahn, Myung Ju; Kim, Chul Soo; Kim, Heung Sik; Park, Seonyang; Chio, Hyun Sook; Min, Yoo Hong

    2007-01-01

    Methylenetetrahydrofolate reductase (MTHFR) plays a central role in converting folate to methyl donor for DNA methylation. Because MTHFR is a key enzyme in folate metabolism, changes in its activity resulting from polymorphisms in the MTHFR gene could modify the susceptibility to cancer. Recently, the C677T and A1298C mutations of MTHFR were discovered to be associated with susceptibility in acute lymphoblastic leukemia (ALL). The association between MTHFR polymorphisms and susceptibility and clinical outcome in ALL was studied in 118 adult ALL patients and matched healthy controls (n =427). DNA samples taken from patients with ALL and controls were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays to detect the MTHFR C677T and A1298C mutations. No significant difference was found in the development of adult ALL among those with different MTHFR genotypes of the C677T or A1298C polymorphisms. However, the MTHFR 677CT+TT genotype showed a tendency to be associated with adult ALL [crude odds ratio (OR), 0.67; 95% confidence interval (CI), 0.44-1.02; adjusted OR, 0.74 95% CI, 0.47-1.14]. The MTHFR C677T and A1298C polymorphisms are not significant risk factors in adult acute leukemia in the Korean population.

  16. Association of methylenetetrahytrofolate reductase (MTHFR) C677T and A1298C polymorphisms with the susceptibility of childhood acute lymphoblastic leukaemia (ALL) in Chinese population.

    Science.gov (United States)

    Li, Xiaolei; Liao, Qingchuan; Zhang, Shunguo; Chen, Minling

    2014-01-29

    The aim of this study was to investigate the relationship between the polymorphisms of the methylenetetrahytrofolate reductase (MTHFR) gene and susceptibility to childhood acute lymphoblastic leukemia (ALL). A case-control study was conducted among 98 children with ALL and 93 age- and sex- matched non-ALL controls. Genotyping of MTHFR C677T and A1298C polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The odds ratios (ORs) of MTHFR genotypes were used to assess the associations of these polymorphisms with childhood ALL susceptibility. No significant differences were observed for frequencies of the 677CC, 677CT and 677TT genotypes between patients and controls. Frequencies of the 1298AA, 1298 AC and 1298CC genotypes between the two groups were significantly different. The risk of ALL with the 1298C allele carriers (AC + CC) was elevated by 1.1 times compared with the AA genotype [OR = 2.100; 95% CI (1.149; 3.837); P = 0.015]. The MTHFR A1298C polymorphism is associated with susceptibility to childhood ALL in the Chinese population.

  17. Simple, specific molecular typing of dengue virus isolates using one-step RT-PCR and restriction fragment length polymorphism.

    Science.gov (United States)

    Ortiz, Alma; Capitan, Zeuz; Mendoza, Yaxelis; Cisneros, Julio; Moreno, Brechla; Zaldivar, Yamitzel; Garcia, Mariana; Smith, Rebecca E; Motta, Jorge; Pascale, Juan Miguel

    2012-10-01

    A one-step RT-PCR and one-enzyme RFLP was used to detect and distinguish among flaviviruses, including the four serotypes of dengue and the St. Louis Encephalitis, West Nile and Yellow Fever viruses in cultured virus samples or acute-phase human serum. Using a previously described RT-PCR, but novel RFLP procedure, results are obtained in 24 h with basic PCR and electrophoresis equipment. There is 95% agreement between RT-PCR/RFLP results and those achieved by indirect immunofluorescence assays, and 100% agreement between RT-PCR/RFLP results and gene sequencing. This method is more rapid than tests of cytopathic effect based on virus isolation in tissue culture, and simpler than real-time PCR. It does not require specialized equipment, radioisotopes or computer analysis and is a method that can be applied widely in the developing world. It allows for prompt determination of whether a flavivirus is the cause of illness in a febrile patient, rapid identification of dengue serotypes in circulation, and improved patient management in cases where prior dengue exposure make dengue hemorrhagic fever or dengue shock syndrome a risk. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Study of Polymorphism of the DRD2 Gene (-141C Ins/Del, rs1799732 with Attention Deficit Hyperactivity Disorder a Population Sample of Children in Iranian-Azeri

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    Leila Mehdizadeh Fanid

    2017-03-01

    Full Text Available BackgroundAttention deficit hyperactivity disorder (ADHD, is a multifactorial disorder and converging evidence has implicated abnormalities of dopamine neurotransmission. The aim of this study was to examine the association of -141 polymorphisms in DRD2 gene with ADHA among Iranian-Azeri population.Materials and Methods A case–control association study included 153 patients with attention deficit hyper activity disorder (case group, and 133 healthy subjects (control group. Genomic DNA was extracted peripheral blood samples by salting-out method. Single nucleotide polymorphism (SNP genotyping was performed by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP technique. The data analysis was performed through Chi-square, with a significance level of 0.05.Results: There was not significant difference in the allele and genotype frequencies between ADHD and -141C Ins/Del polymorphism in cases and controls (P>0.05. Ins/Ins homozygous dominants were more frequent in control group than the case group, but there was not significant difference observed (P>0.05. Del/Del homozygous dominants were not observed. No significant difference was detected in the allele and genotype frequencies between ADHD and -141 Insertion/Deletion polymorphism in cases and control groups (P>0.05.ConclusionOur results do not detected association between the -141C Ins/Del, rs1799732, polymorphism and ADHD disorder in population of Children in Iranian-Azeri.

  19. Polymorphism of the SCNN1g Gene and its Association with Eggshell Quality

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    Kheirkhah Z

    2017-06-01

    Full Text Available Eggshell quality is the main trait to assess egg quality. Marker assisted selection can be used to improve this trait. During eggshell formation, a mass of inorganic minerals is deposited. The Sodium Channel (SCNN1 gene family plays an essential role in cation transportation and SCNN1g is a member of this gene family. The objective of this study was to estimate the frequency of SCNN1g gene variants and to find its associations with eggshell quality in Hy-Line breed. 100 hens were randomly selected and their eggs and blood samples were collected. DNA was extracted and purified using the phenol-chloroform method and genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis. GLM procedure of SAS software was used to evaluate the association of SCNN1g gene polymorphism with egg weight, specific gravity, eggshell strength, eggshell weight, and eggshell thickness. Based on the polymorphism of SCNN1g gene, three genotypes were observed including AA, AG, and GG with frequencies of 0.26, 0.57, and 0.17, respectively. Genotype only had a significant effect on eggshell strength (P < 0.05. Other traits were not significantly influenced by genotypes of this gene. Therefore, introducing this gene in marker-assisted selection programs may improve eggshell strength of Hy-Line breed.

  20. Hyperhomocysteinemia, enzyme polymorphism and thiobarbituric Acid reactive system in children with high coronary risk family history.

    Science.gov (United States)

    Szamosi, Tamas; Roth, Erzsebet; Szamosi, Tamas; Tomsits, Erika; Tordai, Attila; Szabo, Terez

    2004-10-01

    To investigate both the frequency and the genetic background of hyperhomocysteinemia and the frequency of increased plasma thiobarbituric acid reactive system (TBARS) levels in children and adolescents whose parents had premature coronary heart disease (CHD). The study was performed on children and adolescents aged 4-18 years (105 offspring of parents with CHD before age 45 and 74 referents from families without any evidence of premature atherosclerosis). Fasting serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and total triglyceride (TG) levels were measured by enzymatic methods. Low density lipoprotein cholesterol (LDL-C) level was calculated by the Friedewald formula. Plasma total homocysteine (THCy) level was measured by fluorescence polarisation immunoassay. Plasma TBARS level was determined by fluorimetric method. 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme polymorphism was analyzed by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP). Hyperhomocysteinemia was found in 32 cases and in 4 controls. Increased plasma THCy level was found in 10 children and adolescents from 12 cases homozygous for the C677T polymorphism of the MTHFR gene. No similar high frequency was observed in heterozygous subjects. Elevated fasting plasma TBARS levels were found in 38 cases and in 8 controls. The frequency differences were significant (p history.

  1. High levels of genetic variability and differentiation in hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes populations revealed by PCR-RFLP analysis of the mitochondrial DNA D-loop region

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    Sabuj Kanti Mazumder

    2009-01-01

    Full Text Available The hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes is an important anadromous clupeid species from the Western division of the Indo-Pacific region. It constitutes the largest single fishable species in Bangladesh. Information on genetic variability and population structure is very important for both management and conservation purposes. Past reports on the population structure of T. ilisha involving morphometric, allozyme and RAPD analyses are contradictory. We examined genetic variability and divergence in two riverine (the Jamuna and the Meghna, two estuarine (Kuakata and Sundarbans and one marine (Cox's Bazar populations of T. ilisha by applying PCR-RFLP analysis of the mtDNA D-loop region. The amplified PCR products were restricted with four restriction enzymes namely, XbaI, EcoRI, EcoRV, and HaeIII. High levels of haplotype and gene diversity within and significant differentiations among, populations of T. ilisha were observed in this study. Significant F ST values indicated differentiation among the river, estuary and marine populations. The UPGMA dendrogram based on genetic distance resulted in two major clusters, although, these were subsequently divided into three, corresponding to the riverine, estuarine and marine populations. The study underlines the usefulness of RFLP of mtDNA D-loop region as molecular markers, and detected at least two differentiated populations of T. ilisha in Bangladesh waters.

  2. Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis.

    Science.gov (United States)

    Hsieh, Ming-Kun; Shih, Pei-Yin; Wei, Chia-Fong; Vickroy, Thomas W; Chou, Chi-Chung

    2016-03-30

    The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols. © 2015 Society of Chemical Industry.

  3. PPARα gene variants as predicted performance-enhancing polymorphisms in professional Italian soccer players

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    Proia P

    2014-12-01

    Full Text Available Patrizia Proia,1 Antonino Bianco,1 Gabriella Schiera,2 Patrizia Saladino,2 Valentina Contrò,1 Giovanni Caramazza,3 Marcello Traina,1 Keith A Grimaldi,4 Antonio Palma,1 Antonio Paoli5 1Sport and Exercise Sciences Research Unit, 2Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy; 3Regional Sports School of CONI Sicilia, Sicily, Italy; 4Biomedical Engineering Laboratory, Institute of Communication and Computer Systems, National Technical University of Athens, Athens, Greece; 5Department of Biomedical Sciences, University of Padova, Padua, Italy Background: The PPARα gene encodes the peroxisome proliferator-activator receptor alpha, a central regulator of expression of other genes involved in fatty acid metabolism. The purpose of this study was to determine the prevalence of G allele of the PPARα intron 7 G/C polymorphism (rs4253778 in professional Italian soccer players. Methods: Sixty professional soccer players and 30 sedentary volunteers were enrolled in the study. Samples of venous blood were obtained at rest, in the morning, by conventional clinical procedures; blood serum was collected and total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides were measured. An aliquot of anticoagulant-treated blood was used to prepare genomic DNA from whole blood. The G/C polymorphic site in PPARα intron 7 was scanned by using the PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism protocol with TaqI enzyme. Results: We found variations in genotype distribution of PPARα polymorphism between professional soccer players and sedentary volunteers. Particularly, G alleles and the GG genotype were significantly more frequent in soccer players compared with healthy controls (64% versus 48%. No significant correlations were found between lipid profile and genotype background. Conclusion: Previous results

  4. Endothelin receptor A -231 G>A polymorphism: no linkage to primary pediatric headache.

    Science.gov (United States)

    Lisi, Veronica; Garbo, Greta; Battistella, PierAntonio; Miccichè, Flavia; Stecca, Anna; Terrazzino, Salvatore; Franzoi, Malida; Tripoli, Elisa; Leon, Alberta; Clementi, Maurizio

    2006-03-01

    To assess whether the biallelic -231 G>A polymorphism of the endothelin type A receptor (EDNRA) gene, previously shown to be a marker of increased risk for developing migraine, has a role in the susceptibility to primary pediatric headache. Several studies suggest that endothelin has a role in migraine. A recent association study has shown that the biallelic -231 G>A polymorphism of the EDNRA gene is associated to migraine in an elderly population. A total of 126 consecutive unrelated pediatric patients affected by primary headache, classified according to the International Headache Society criteria in migraine (migraine with aura, n = 3; migraine without aura, n = 80), and tension-type headache (episodic tension-type headache, n = 36; chronic tension-type headache, n = 7) patients, were recruited to the study. Sixty-seven healthy blood donors were used as a control group. Genomic DNA was extracted from buccal swabs or blood samples and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the above-mentioned polymorphism. Allele and genotype frequencies for primary headache patients were analyzed in comparison with the control group. No significant differences were found in the distribution of the EDNRA -231 G>A polymorphic variant when considering both genotype (migraine chi2 = 2.78, P = .25; tension-type headache chi2 = 3.58, P = .17) and allelic frequencies (migraine chi2 = 1.48, P = .22; tension-type headache chi2 = 0.39, P = .56). Furthermore, no significant genotype-related difference was found in relation to clinical features, such as age at onset, frequency, and length of the attacks. Our study shows that the -231 G>A polymorphism in the EDNRA gene is neither associated with primary juvenile headache nor significantly correlated with main clinical features characteristic of the headache pathology in pediatric settings.

  5. Coccidioides species determination: does sequence analysis agree with restriction fragment length polymorphism?

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    Johnson, Suzanne M; Carlson, Erin L; Pappagianis, Demosthenes

    2015-06-01

    Fifteen Coccidioides isolates were previously examined for genetic diversity using restriction fragment length polymorphism (RFLP); two fragment patterns were observed. Two isolates demonstrated one banding pattern (designated RFLP group I), while the remaining 13 isolates demonstrated a second pattern (designated RFLP group II). Recently, molecular studies supported the division of the genera Coccidioides into two species: Coccidioides posadasii and Coccidioides immitis. It has been assumed that the species division corresponds to the RFLP grouping. We tested this hypothesis by amplifying the ribosomal DNA internal transcribed spacer region as well as the dioxygenase, serine proteinase, and urease genes from 13 isolates previously examined by RFLP and then sequencing the PCR products. The appropriate species for each isolate was assigned using phylogenetically informative sites. The RFLP grouping agreed with the Coccidioides species assignment for all but one isolate, which may represent a hybrid. In addition, polymorphic sites among the four genes examined were in agreement for species assignment such that analysis of a single gene may be sufficient for species assignment.

  6. ALIS-FLP: Amplified ligation selected fragment-length polymorphism method for microbial genotyping

    DEFF Research Database (Denmark)

    Brillowska-Dabrowska, A.; Wianecka, M.; Dabrowski, Slawomir

    2008-01-01

    A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs......RI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding...... for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA-PFGE. Versatility of the method is highlighted, i.e. its...

  7. From the chromosome to DNA: Restriction fragment length polymorphism analysis and its clinical application.

    Science.gov (United States)

    Todd, R; Donoff, R B; Kim, Y; Wong, D T

    2001-06-01

    Understanding how chromosomal alterations contribute to acquired and inherited human disease requires the ability to manage the enormous physical and informational complexity of the deoxyribonucleic acid (DNA) packaged within. Important concepts and techniques involved in the analysis of DNA include restriction enzymes, Southern blotting, and restriction fragment length polymorphism/linkage analysis. These techniques have been essential in the understanding and diagnosis of several syndromes associated with the head and neck. The purpose of this article is to introduce DNA structure, describe some techniques fundamental to DNA analysis, and provide a brief overview of the clinical applications of this technology with respect to dentinogenesis imperfecta and oral field cancerization. Copyright 2001 American Association of Oral and Maxillofacial Surgeons.

  8. Restriction fragment length polymorphism typing of infectious bursal disease virus field strains in Turkey.

    Science.gov (United States)

    Sareyyüpoğlu, B; Akan, M

    2006-12-01

    Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of immature chickens. It is caused by IBD virus (IBDV) and is responsible for major economic losses in the poultry industry worldwide. In this study, 280 bursa samples from 56 commercially reared chicken flocks in Turkey with clinical symptoms of IBD were examined for IBDVs using the reverse transcription (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) assay. The assay was conducted on a 743-bp fragment of the VP2 gene with the restriction enzymes BstNI, MboI, and SspI. The results indicate the existence of field isolates with new molecular patterns different from those previously published that may well be unique and specific to geographical regions.

  9. Investigating of yeast species in wine fermentation using terminal restriction fragment length polymorphism method.

    Science.gov (United States)

    Sun, Yue; Liu, Yanlin

    2014-04-01

    The objective of this study was to examine the potential of terminal restriction fragment length polymorphism (T-RFLP) in monitoring yeast communities during wine fermentation and to reveal new information on yeast community of Chinese enology. Firstly, terminal restriction fragment (TRF) lengths database was constructed using 32 pure yeast species. Ten of these species were firstly documented. The species except for Candida vini, Issatchenkia orientalis/Candida krusei, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces cerevisiae, Saccharomyces kudriarzevii and Zygosaccharomyces bisporus could be distinguished by the T-RFLP targeting 5.8S-ITS rDNA. Moreover, the yeast communities in spontaneous fermentation of Chardonnay and Riesling were identified by T-RFLP and traditional methods, including colony morphology on Wallerstein Nutrient (WLN) medium and 5.8S-ITS-RFLP analysis. The result showed that T-RFLP profiles of the yeast community correlated well with that of the results identified by the traditional methods. The TRFs with the highest intensity and present in all the samples corresponded to Saccharomyces sp. Other species detected by both approaches were Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia minuta var. minuta, Saccharomycodes ludwigii/Torulaspora delbrueckii and Candida zemplinina. This study revealed that T-RFLP technique is a rapid and useful tool for monitoring the composition of yeast species during wine fermentation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Analysis of polymorphisms of tumor necrosis factor-alpha and polymorphic xenobiotic metabolizing enzymes in inflammatory bowel disease: study from northern India.

    Science.gov (United States)

    Mittal, Rama D; Manchanda, Parmeet K; Bid, Hemant K; Ghoshal, Uday C

    2007-06-01

    Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine associated with inflammatory diseases, while GSTM1 and T1 enzymes catalyze detoxification of products of oxidative stress and hence reduce inflammation. Thus, both may play important roles in the pathogenesis of inflammatory bowel disease (IBD). The present study aimed to evaluate the effect of polymorphism of the TNF-alpha promoter at the -308 site, GSTM1 and GSTT1 in patients with IBD and healthy controls from northern India. Genotyping was performed in 114 patients with IBD (22 Crohn's disease [CD] and 92 ulcerative colitis [UC]) in TNF-alpha and 105 (20 CD and 85 UC) in GSTM1 and T1 and 164 healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR methods. Patients with IBD were comparable to healthy controls in relation to age and gender. Genotypic and allelic frequencies of TNF-alpha were comparable among patients with IBD and healthy controls. GSTM1 null genotype was more frequent in UC than in healthy controls (52/85 vs 49/164; P GST genotypes further increases the risk, possibly due to gene-gene interaction. TNF-alpha is unlikely to be an important determinant of susceptibility to IBD in the Indian population.

  11. NRAMP1 and VDR gene polymorphisms in susceptibility to pulmonary tuberculosis among Andhra Pradesh population in India: a case-control study.

    Science.gov (United States)

    Medapati, Rooth Vasantha; Suvvari, Sridevi; Godi, Sudhakar; Gangisetti, Paddaiah

    2017-06-05

    The aim of the present study was to evaluate the association of NRAMP1 -3'UTR, 274-CT,VDR- Fok1 VDR-Taq1 Polymorphisms with the risk of pulmonary tuberculosis. A case -control study was conducted on Andhra Pradesh Population of India. Analysis of gene polymorphisms of NRAMP1 gene (3'UTR, 274CT) and VDR gene (Fok1 and Taq1) was done by using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in Tuberculosis (TB) patients and healthy controls. The obtained results were observed using 2% Agarose Gel electrophoresis and analysed statistically using Chi-square test and Odds Ratio. Statistical significance was observed between the patients and the controls in the NRAMP1-3'UTR (P = 0.005; OR = 2.997; 95% CI = 1.019-8.813) and VDR-Taq1 (P  0.05). 3'UTR-NRAMP1 gene and VDR-Taq1 gene Polymorphisms are statistically associated with the susceptibility of TB in Andhra Pradesh Population in India.

  12. Study of the Relation between E23K Single Nucleotide Polymorphism of KCNJ11 Gene and Probability of Coronary Heart Disease in Iran

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    M Fasihi Ramandi

    2010-10-01

    Full Text Available Introduction: The G to A mutation in KCNJ11 the ATP-sensitive potassium channel subunit, results in glutamate (E to lysine (K substitution at codon 23, and the A allele is shown to have a relationship with type II diabetes in our previous study. Their role in coronary heart disease (CHD is not exactly obvious. We hypothesized that the polymorphism would be associated with increased susceptibility to CHD. Methods: The E23K gene polymorphism of KCNJ11 gene was analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP methods in 55 controls and 73CHD patients. Serum lipids and Fasting Blood Sugar concentrations were measured in all subjects. Results: Among the CHD patients, the frequency of the A allele was higher (34.9% vs. 26.4%, P0.05 than among controls. No significant differences were found in allele frequencies between CHD and controls (P>0.05. Also, there were no significant differences in GG and combined (GA+AA genotypes frequencies (42.5% vs. 56.4%, and 57.5% vs. 43.6%, P>0.05. Conclusion: The E23K gene polymorphism in KCNJ11 gene has no association with the high susceptibility to CHD.

  13. Toll-like receptors-2 and -9 (TLR2 and TLR9) gene polymorphism in patients with type 2 diabetes and diabetic foot.

    Science.gov (United States)

    Wifi, Mohamed-Naguib Abdalla; Assem, Maha; Elsherif, Rasha Hamed; El-Azab, Hameda Abdel-Fattah; Saif, Aasem

    2017-04-01

    Toll-like receptors (TLRs) are innate immune receptors that mediate the inflammatory response in diabetes mellitus (DM). The aim of this study is to evaluate the association of TLR2 and TLR9 gene polymorphism in patients with type 2 DM (T2DM) and diabetic foot (DF).The study included 90 subjects divided into group I (30 patients with T2DM and DF), group II (30 patients with T2DM and no evidence of DF), and group III (normal control subjects). TLR2 (1350 T/C, rs3804100) and TLR9 (1237 T/C, rs5743836) genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for all subjects.There was a statistically significant difference in the distribution of TLR9-1237 T/C genotypes between groups I and II (P < .029) as well as between groups I and III (P < .001). Calculated risk estimation revealed that TLR9-1237 polymorphism conferred almost 20 times increased risk of DF disorders in T2DM (OR = 20, 95% CI = 5.38-74.30). There was no statistical difference in the distribution of TLR2-1350T/C genotypes between the 3 groups.TLR9-1237 T/C gene polymorphism may be considered as a molecular risk for DF among patients with T2DM.

  14. Impact of the Ile105Val Polymorphism of the Glutathione S-transferase P1 (GSTP1) Gene on Obesity and Markers of Cardiometabolic Risk in Young Adult Population.

    Science.gov (United States)

    Chielle, E O; Trott, A; da Silva Rosa, B; Casarin, J N; Fortuna, P C; da Cruz, I B M; Moretto, M B; Moresco, R N

    2017-05-01

    The aim of the study was to investigate the association between Glutathione S-transferase P1 (GSTP1) gene polymorphism with obesity and markers of cardiometabolic risk. A cross-sectional study was carried out in individuals aged≥18 and ≤30 years. The study included 54 normal weight, 27 overweight and 68 obese volunteers. Anthropometric measurements and biochemical parameters were evaluated, the DNA was extracted from blood samples and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to measure GSTP1 Ile 105 Val gene polymorphism of the study participants. Also, biochemical analysis and hormone assays were carried out. A positive association between GSTP1 polymorphism and obesity was observed on subjects carrying at least one G allele (AG and GG). GG genotype was found only in the obese group. The G allele carriers presented 2.4 times higher chance of obesity when compared to those with the AA genotype. These results were independent of sex and age. We suggest that despite a study in population regional (south of Brazil), the GSTP1 gene polymorphism may play a significant role in the increase of susceptibility of obesity and contribute to identify the cardiovascular risk in young adults. © Georg Thieme Verlag KG Stuttgart · New York.

  15. Association of polymorphisms in the leptin and thyroglobulin genes with meat quality and carcass traits in beef cattle

    Directory of Open Access Journals (Sweden)

    Thiago Dutra de Carvalho

    2012-10-01

    Full Text Available The objective of the present study was to estimate the allelic and genotypic frequencies of the polymorphisms E2FB (AY138588.1: c.305C> T, located in the leptin gene (LEP, and TG5 (X05380.1:g.-422C>T, located in the thyroglobulin gene (TG, and evaluate the association of these polymorphisms in crossbred cattle of seven distinct genetic groups with the following traits: slaughter weight (SW, hot carcass weight (HCW, hot carcass yield (HCY, carcass fat thickness (CFT, ribeye area (REA, marbling (MARM and shear force (SF. The animals were genotyped using the PCR-RFLP (Polymorphism Chain Reaction-Restriction Fragment Length Polymorphism technique, using 201 products obtained from F1 Caracu × Nellore, Angus × Nellore and Valdostana × Nellore cows, mated to Canchim, Caracu and Red Angus bulls (only Caracu × Nellore cows were used with Red Angus bulls. The allelic and genotypic frequencies were compared using the Chi-squared test. Associations between the genotype of each polymorphism and the traits were analyzed using the General Linear Model (GLM of statistical software SAS. The least squares means of genotypes of the polymorphisms were compared using Student's t test. The E2FB polymorphism in the LEP gene was associated with CFT, showing the potential for use in national programs for genetic improvement of beef cattle, through the inclusion of SNP in genotyping commercial tests. The TG5 polymorphism in the TG gene was not associated with any of the evaluated traits and was considered ineffective for selection of beef cattle in Brazilian herds.

  16. Polymorphism in the internal transcribed spacer (ITS of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

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    Gomes Eliane A.

    2002-01-01

    Full Text Available Inter- and intraspecific variation among 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 species were evaluated by analysis of the internal transcribed sequence (ITS of the rDNA region using restriction fragment length polymorphism (RFLP. The ITS region was first amplified by polymerase chain reaction (PCR with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 560 and 750 base pairs (bp, were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the isolates. Cleavage of amplified fragments with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II revealed extensive polymorphism. All eight genera and most species presented specific restriction patterns. Species not identifiable by a specific pattern belonged to two genera: Rhizopogon (R. nigrescens, R. reaii, R. roseolus, R. rubescens and Rhizopogon sp., and Laccaria (L. bicolor and L. amethystea. Our data confirm the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.

  17. IL-6 and IL-10 gene polymorphisms in patients with aggressive periodontitis: effects on GCF, serum and clinic parameters.

    Science.gov (United States)

    Pirim Gorgun, Emine; Toker, Hulya; Korkmaz, Ertan Mahir; Poyraz, Omer

    2017-01-16

    Genetic variations observed in cytokines affect periodontitis susceptibility. The aim of this study was to investigate interleukin(IL)-6(-174) and IL-10(-597) gene polymorphisms in generalized aggressive periodontitis (GAgP) patients. Also, we aimed to evaluate the effects of IL-6 and IL-10 gene polymorphisms on the clinical outcomes of non-surgical periodontal therapy and cytokine levels in gingival crevicular fluid(GCF) and serum. Fifty-three patients with GAgP and 50 periodontally healthy individuals were included in this study. Clinical parameters, GCF and blood samples were collected at baseline and at 6-week. Non-surgical periodontal therapy was performed in patients with GAgP. Gene analysis were determined by PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism) and cytokine levels were determined by enzyme-linked immunosorbent assay(ELISA).GAgP patients showed significant improvement on clinical parameters after periodontal therapy(pperiodontal therapy, while patients with IL-6(-174) GG genotype had higher levels of GCF IL-10 levels.

  18. Association of TP53 codon 72 and CDH1 genetic polymorphisms with colorectal cancer risk in Bangladeshi population.

    Science.gov (United States)

    Rivu, Sanzana Fareen; Apu, Mohd Nazmul Hasan; Shabnaz, Samia; Nahid, Noor Ahmed; Islam, Md Reazul; Al-Mamun, Mir Md Abdullah; Nahar, Zabun; Rabbi, Sikder Nahidul Islam; Ahmed, Maizbha Uddin; Islam, Mohammad Safiqul; Hasnat, Abul

    2017-08-01

    Till now no pharmacogenetic study of TP53 codon 72 (Arg72Pro) and CDH1 rs16260 (-160Ccolorectal cancer. So the aim of the study is to determine whether there is an elevated risk of colorectal cancer development with TP53 codon 72 and CDH1 rs16260 genetic polymorphism in Bangladeshi population for the first time. To investigate the association of these two SNPs, we conducted a case-control study with 288 colorectal cancer patients and 295 healthy volunteers by using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. We found an increased risk of association between Arg/Pro heterozygosity (adjusted OR=2.58, 95% CI=1.77-3.77, pcolorectal cancer predisposition. In case of CDH1 rs16260 polymorphism, C/A heterozygous and A/A mutant homozygous are significantly (pcolorectal cancer risk with adjusted OR of 1.94 and 2.63, respectively. The combined genotype of C/A and A/A was also found to be strongly associated with colorectal cancer risk compared to C/C genotype (adjusted OR=2.02, 95% CI=1.42-2.87, pcolorectal cancer development in Bangladeshi population. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. A Restriction Fragment Length Polymorphism Map and Electrophoretic Karyotype of the Fungal Maize Pathogen Cochliobolus Heterostrophus

    Science.gov (United States)

    Tzeng, T. H.; Lyngholm, L. K.; Ford, C. F.; Bronson, C. R.

    1992-01-01

    A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical to map distance should make this RFLP map a useful tool for cloning genes. PMID:1346261

  20. Identification and characterization of some aromatic rice mutants using amplified fragment length polymorphism (AFLP) technique

    International Nuclear Information System (INIS)

    Fahmy, E.M.; Sobieh, S. E. S.; Ayaad, M. H.; El-Gohary, A. A.; Rownak, A.

    2012-12-01

    Accurate identifying of the genotypes is considered one of the most important mechanisms used in the recording or the protection of plant varieties. The investigation was conducted at the experimental form belonging to the egyptian Atomic Energy Authority, Inshas. The aim was to evaluate grain quality characteristics and molecular genetic variation using Amplified Fragment Length Polymorphism (AFLP) technique among six rice genotypes, Egyptian Jasmine aromatic rice cultivar and five aromatic rice mutants in (M3 mutagenic generation). Two mutation (Egy22 and Egy24) were selected from irradiated Sakha 102 population with 200 and 400Gy of gamma rays in the M2 generation, respectively, and three mutations ( Egy32, Egy33, and Egy34) were selected from irradiated Sakha 103 population with 200, 300, 400Gy of gamma rays in the M2 generation, respectively. The obtained results showed that the strong aroma was obtained for mutant Egy22 as compared with Egyptian Jasmine rice cultivar (moderate aroma). Seven primer combinations were used through six rice genotypes on the molecular level using AFLP marker. The size of AFLP Fragments Were Ranged from 51- 494bp. The total number of amplified bands was 997 band among them 919 polymorphic bans representing 92.2%. The highest similarity index (89%) was observed between Egyptian Jasmine and Egy32 followed by (82%) observed between Egyptian Jasmine and Egy34. On the other hand, the lowest similarity index was (48%) between Egyptian Jasmine and Egy24. In six rice genotypes, Egy24 produced the highest number of the AFLP makers giving 49 unique markers (23 positive and 26 negative), then Egy22 showed 23 unique markers (27 positive and 6 negative) while Egy33 was characterized by 17 unique markers (12 positive and 5 negative). At last Egyptian Jasmine was discriminated by the lowest number of markets, 10 (6 positive and 4 negative). The study further confirmed that AFLP technique was able to differentiate rice genotypes by a higher number

  1. Restriction fragment length polymorphism (RFLP) of two HLA-B-associated transcripts (BATs) genes in healthy Danes

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1990-01-01

    The restriction fragment length polymorphism (RFLP) of the two human HLA-B-associated transcripts (BATs) genes, BAT1 and BAT2, was investigated using 5 different restriction enzymes and two human BAT1 and BAT2 cDNA probes. Two of the enzymes, NcoI and RsaI, revealed polymorphic patterns which were...... investigated in healthy Danes. The cDNA/restriction enzyme combination BAT1/NcoI identifies polymorphic bands at 12 kb, 8 kb, 2.5 kb, and 1.1 kb, while the BAT2/RsaI combination identifies polymorphic bands at 3.3 kb, 2.7 kb, 2.3 kb, and 0.9 kb. The frequencies of these markers were determined in 90 unrelated...

  2. Mapped DNA probes from Ioblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

    Science.gov (United States)

    M.R. Ahuja; M.E. Devey; A.T. Groover; K.D. Jermstad; D.B Neale

    1994-01-01

    A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm....

  3. Enterocytozoon bieneusi Identification Using Real-Time Polymerase Chain Reaction and Restriction Fragment Length Polymorphism in HIV-Infected Humans from Kinshasa Province of the Democratic Republic of Congo

    Directory of Open Access Journals (Sweden)

    Roger Wumba

    2012-01-01

    in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n=19 among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3% and 5 type IV strains of E. bieneusi (26.3% were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo, and the concurrence of type I and type IV strains.

  4. Enterocytozoon bieneusi Identification Using Real-Time Polymerase Chain Reaction and Restriction Fragment Length Polymorphism in HIV-Infected Humans from Kinshasa Province of the Democratic Republic of Congo

    Science.gov (United States)

    Wumba, Roger; Jean, Menotti; Benjamin, Longo-Mbenza; Madone, Mandina; Fabien, Kintoki; Josué, Zanga; Jean, Sala; Eric, Kendjo; AC, Guillo-Olczyk; Marc, Thellier

    2012-01-01

    Objective. To determine the prevalence and the genotypes of Enterocytozoon bieneusi in stool specimens from HIV patients. Methods. This cross-sectional study was carried out in Kinshasa hospitals between 2009 and 2012. Detection of microsporidia including E. bieneusi and E. intestinalis was performed in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI) in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM) was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n = 19) among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3%) and 5 type IV strains of E. bieneusi (26.3%) were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo), and the concurrence of type I and type IV strains. PMID:22811884

  5. Characterization of mitochondrial DNA polymorphisms in the Han population in Liaoning Province, Northeast China.

    Science.gov (United States)

    Xu, Feng-Ling; Yao, Jun; Ding, Mei; Shi, Zhang-Sen; Wu, Xue; Zhang, Jing-Jing; Wang, Bao-Jie

    2018-03-01

    This study characterized the genetic variations of mitochondrial DNA (mtDNA) to elucidate the maternal genetic structure of Liaoning Han Chinese. A total of 317 blood samples of unrelated individuals were collected for analysis in Liaoning Province. The mtDNA samples were analyzed using two distinct methods: sequencing of the hypervariable sequences I and II (HVSI and HVSII), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the coding region. The results indicated a high gene diversity value (0.9997 ± 0.0003), a high polymorphism information content (0.99668) and a random match probability (0.00332). These samples were classified into 305 haplotypes, with 9 shared haplotypes. The most common haplogroup was D4 (12.93%). The principal component analysis map, the phylogenetic tree map, and the genetic distance matrix all indicated that the genetic distance of the Liaoning Han population from the Tibetan group was distant, whereas that from the Miao group was relatively close.

  6. Significant Association of Interleukin-10 Polymorphisms with Childhood Leukemia Susceptibility in Taiwan.

    Science.gov (United States)

    Lo, Wen-Jyi; Chang, Wen-Shin; Hsu, Han-Fang; Ji, Hong-Xue; Hsiao, Chieh-Lun; Tsai, Chia-Wen; Yeh, Su-Peng; Chen, Chuan-Mu; Bau, DA-Tian

    2016-01-01

    Mounting evidence supports the notion that inflammatory processes play a role in carcinogenesis, and interleukin-10 (IL10) is an important inflammatory cytokine. This study aimed to evaluate the contribution of IL10 A-1082G (rs1800896), T-819C (rs3021097) and A-592C (rs1800872) genotypes to the risk of childhood acute lymphoblastic leukemia (ALL) in Taiwan. Associations of these IL10 polymorphic genotypes with ALL risk were analyzed in 266 patients with childhood ALL patients and 266 non-cancer healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. The results showed that CC genotype carriers at IL10 T-819C were at lower risk for childhood ALL (odds ratio=0.33, 95% confidence interval=0.16-0.68). On the contrary, AC and CC genotype carriers at IL10 A-592C were at higher risk for childhood ALL (odds ratio=1.73 and 6.34, 95% confidence interval=1.19-2.51 and 3.16-12.72, respectively). There was no difference in the distribution of A-1082G genotypes between childhood ALL and control groups. The genotypes at IL10 T-819C and A-592C may serve as predictive biomarkers for childhood ALL in Taiwan. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Polymorphisms in the Estrogen Receptor Beta Gene and the Risk of Unexplained Recurrent Spontaneous Abortion

    Science.gov (United States)

    Mahdavipour, Marzieh; Zarei, Saeed; Fatemi, Ramina; Edalatkhah, Haleh; Heidari-Vala, Hamed; Jeddi-Tehrani, Mahmood; Idali, Farah

    2017-01-01

    Background: Recurrent Spontaneous Abortion (RSA) is caused by multiple genetic and non-genetic factors. Around 50% of the RSA cases have no known etiology and are considered as Unexplained RSA (URSA). Estrogens, via binding to their receptors, play an important role in female reproduction. This study aimed to investigate whether single nucleotide polymorphisms (SNPs; +1082G/A, +1730G/A and rs1256030 C/T) in the estrogen receptor beta (ESR2) gene are associated with susceptibility to URSA in a population of Iranian women. Methods: In this case-control study, the study groups consisted of 240 subjects with a history of URSA and 102 fertile women as controls. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were measured on day 2–3 of menstrual cycle. Two functional SNPs, +1082G/A (a silent mutation in exon 5) and +1730G/A (3′ untranslated region of the exon 8), and one intron, rs1256030C/T, in the ESR2 gene were genotyped, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results: Serum levels of LH were significantly increased in URSA women. No significant differences in distribution of +1082G/A, +1730G/A and rs1256030C/T between URSA and control groups were observed. Conclusion: Our findings suggest that the studied SNPs on ESR2 gene may not be associated with URSA. PMID:28706612

  8. Association of VDR polymorphisms with type 2 diabetes mellitus in Chinese Han and Hui populations.

    Science.gov (United States)

    Xu, J R; Yang, Y; Liu, X M; Wang, Y J

    2014-11-14

    We investigated the association between vitamin D receptor (VDR) and susceptibility to type 2 diabetes mellitus (T2DM). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was conducted to examine single nucleotide polymorphisms (SNPs) of the rs1544410 (BsmI, G>A), rs757343 (Tru9I, G>A), rs731236 (TaqI, T>C), and rs739837 (BglI, G>T) loci of the VDR gene in 334 healthy individuals (Hui 115, Han 219) and 355 T2DM patients (Hui 154, Han 201) living in the Ningxia Hui Autonomous Region of China. The genotypic frequency and allelic frequency distributions in the VDR gene showed no significant difference between T2DM patients and controls in the Chinese Hui population. However, statistical differences in the genotypic frequency at rs739837 and in the genotypic and allelic frequencies at rs1544410 were observed between T2DM patients and controls in the Chinese Han population (P Hui [OR (95%CI) = 4.714 (1.04-21.36)] and Han populations [OR (95%CI) = 1.723 (1.03-2.883)] (P < 0.05), implying that the haplotype GGCT of the VDR gene is associated with susceptibility to T2DM in these ethnicities.

  9. The effect of methylenetetrahydrofolate reductase polymorphisms on susceptibility to human papilloma virus infection and cervical cancer.

    Science.gov (United States)

    Hajiesmaeil, Mogge; Tafvizi, Farzaneh; Sarmadi, Soheila

    2016-12-01

    Cervical cancer is the third most common cancer among women worldwide. Several factors lead to cervical cancer, among which human papilloma virus (HPV) infection has a prominent role. Methylenetetrahydrofolate reductase (MTHFR) is crucial in folate metabolic pathway and plays an important role in DNA synthesis and DNA methylation. MTHFR gene polymorphisms, including C677T and A1298C, lead to reduced enzyme activity. This case-control study aims to illustrate the association between MTHFR gene polymorphisms and the risk of cervical cancer. This study was conducted on 196 samples, which included 96 cervical biopsy samples compared to 100 Pap smear samples of normal healthy women without HPV infection. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for the MTHFR polymorphism detection, followed by fluorescent amplification-based specific hybridization PCR method to detect HPV16 and HPV18. The results show that the MTHFR 677TT genotype plays a protective role in cervical cancer (P=0.0030) (OR=0.21, 95% confidence interval [CI]: 0.07-0.59). Furthermore, there was a strong significant association between MTHFR 1298CC genotype and the risk of cervical cancer (OR=10.69; 95% CI: 4.28-26.71, P=0.0001). It can be concluded that A1298C polymorphism is a genetic risk factor for cervical cancer in the assessed Iranian population group. It seems that MTHFR 1298CC genotype is more susceptible to HPV 16 infection. Combination analysis of MTHFR C677T and A1298C polymorphisms revealed that combined MTHFR 677CC and 1298CC are strongly associated with a risk of cervical cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Impact of MCP-1 and CCR-2 gene polymorphisms on coronary artery disease susceptibility.

    Science.gov (United States)

    Lin, Hsiu-Ling; Ueng, Kwo-Chang; Hsieh, Yih-Shou; Chiang, Whei-Ling; Yang, Shun-Fa; Chu, Shu-Chen

    2012-09-01

    Coronary artery disease (CAD) was the second leading cause of death during the last 3 years in Taiwan. Smooth muscle cells, monocytes/macrophages, and endothelial cells produce monocyte chemoattractant protein-1 (MCP-1) within atherosclerotic plaques following binding to the chemokine receptor-2 (CCR-2). Previous studies have well-documented the association between MCP-1 expression and susceptibility to, or clinicopathological features, of CAD. This study investigated the relationships between MCP-1-2518A/G and CCR-2-V64I genetic polymorphisms and CAD in the Taiwanese population. A total of 608 subjects, including 392 non-CAD controls and 216 patients with CAD, were recruited and subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to evaluate the effects of these two polymorphic variants on CAD. Results indicated a significant association between MCP-1 -2548 gene polymorphism and susceptibility to CAD. GG genotypes (OR = 1.629; 95 % CI = 1.003-2.644), or individuals with at least one G allele (OR = 1.511; 95 % CI = 1.006-2.270), had a higher risk of CAD as compared with AA genotypes. Results also revealed that subjects with at least one A allele of the V64I CCR2 gene polymorphism had significantly increased risk of CAD. G allele in MCP-1-2518 might contribute to higher prevalence of atrial fibrillation in CAD patients (OR = 4.254; p CCR-2 64I gene polymorphisms represent important factors in determining susceptibility to CAD, and the contribution of MCP-1-2518G could be through effects on atrial fibrillation in CAD patients.

  11. Mitochondrial DNA single nucleotide polymorphism associated with weight estimated breeding values in Nelore cattle (Bos indicus

    Directory of Open Access Journals (Sweden)

    Fernando Henrique Biase

    2007-01-01

    Full Text Available We sampled 119 Nelore cattle (Bos indicus, 69 harboring B. indicus mtDNA plus 50 carrying Bos taurus mtDNA, to estimate the frequencies of putative mtDNA single nucleotide polymorphisms (SNPs and investigate their association with Nelore weight and scrotal circumference estimated breeding values (EBVs. The PCR restriction fragment length polymorphism (PCR-RFLP method was used to detect polymorphisms in the mitochondrial asparagine, cysteine, glycine, leucine and proline transporter RNA (tRNA genes (tRNAasn, tRNAcys, tRNAgly, tRNAleu and tRNApro. The 50 cattle carrying B. taurus mtDNA were monomorphic for all the tRNA gene SNPs analyzed, suggesting that they are specific to mtDNA from B. indicus cattle. No tRNAcys or tRNAgly polymorphisms were detected in any of the cattle but we did detect polymorphic SNPs in the tRNAasn, tRNAleu and tRNApro genes in the cattle harboring B. indicus mtDNA, with the same allele observed in the B. taurus sequence being present in the following percentage of cattle harboring B. indicus mtDNA: 72.46% for tRNAasn, 95.23% for tRNAleu and 90.62% for tRNApro. Analyses of variance using the tRNAasn SNP as the independent variable and EBVs as the dependent variable showed that the G -> T SNP was significantly associated (p < 0.05 with maternal EBVs for weight at 120 and 210 days (p < 0.05 and animal's EBVs for weight at 210, 365 and 455 days. There was no association of the tRNAasn SNP with the scrotal circumference EBVs. These results confirm that mtDNA can affect weight and that mtDNA polymorphisms can be a source of genetic variation for quantitative traits.

  12. Association of vitamin D receptor gene polymorphisms with polycystic ovary syndrome among Indian women

    Directory of Open Access Journals (Sweden)

    Shilpi Dasgupta

    2015-01-01

    Full Text Available Background & objectives: The Vitamin-D receptor (VDR regulates vitamin D levels and calcium metabolism in the body and these are known to be associated with endocrine dysfunctions, insulin resistance and type-2 diabetes in polycystic ovarian syndrome (PCOS. Studies on VDR polymorphisms among PCOS women are sparse. We undertook this study to investigate the association pattern of VDR polymorphisms (Cdx2, Fok1, Apa1 and Taq1 with PCOS among Indian women. Methods: For the present study, 250 women with PCOS and 250 normal healthy control women were selected from Hyderabad city, Telangana, India. The four VDR polymorphisms were genotyped and analysed using ASM-PCR (allele specific multiple PCR and PCR-RFLP (restriction fragment length polymorphism. Results: The genotype and allele frequency distributions of only Cdx2 showed significant difference between the PCOS cases and control women, indicating protective role of this SNP against PCOS phenotype. However, significant association was observed between VDR genotypes and some of the PCOS specific clinical/biochemical traits. For example, Fok1 showed a significant genotypic difference for the presence of infertility and Cdx2 genotpes showed association with testosterone levels. Further, the two haplotypes, ACCA and ACTA, were found to be significantly associated with PCOS indicating haplotype specific risk. Interpretation & conclusions: Although VDR polymorphisms have not shown significant association with PCOS, in view of functional significance of the SNPs considered, one cannot yet rule out the possibility of their association with PCOS. Further, specifically designed studies on large cohorts are required to conclusively establish the role of VDR polymorphisms in PCOS, particularly including data on vitamin D levels.

  13. Heterogeneity among Dermatophilus congolensis isolates demonstrated by restriction fragment length polymorphisms.

    Science.gov (United States)

    Faibra, D T

    1993-01-01

    There is evidence of antigenic diversity and of differences in virulence in Dermatophilus congolensis. For the understanding of the epidemiology of dermatophilosis it is important to distinguish between strains of the organism. Twenty field isolates from cattle in Chad and Cameroon, and an American reference strain, have been examined on restriction fragment length polymorphisms. After restriction enzyme digestion of DNA by BamHI and Southern blotting, a rDNA probe consisting of plasmid pMC5 carrying a 4.8 kb insert of Mycoplasma capricolum DNA coding for the 5S, 23S and part of 16S rRNA allowed to distinguish 6 ribotypes of D. congolensis, based on their hybridized rDNA patterns. Particular ribotypes may be distributed over a wide geographical area. On the other hand, strains belonging to at least 5 different ribotypes may be found in one herd; this may partly explain the lack of success in immunization against dermatophilosis in the field.

  14. Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

    Science.gov (United States)

    Verrier, Julie; Pronina, Marina; Peter, Corinne; Bontems, Olympia; Fratti, Marina; Salamin, Karine; Schürch, Stéphanie; Gindro, Katia; Wolfender, Jean-Luc; Harshman, Keith; Monod, Michel

    2012-03-01

    A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

  15. Using amplified fragment length polymorphism analysis to differentiate isolates of Pasteurella multocida serotype 1

    Science.gov (United States)

    Blehert, D.S.; Jefferson, K.L.; Heisey, D.M.; Samuel, M.D.; Berlowski, B.M.; Shadduck, D.J.

    2008-01-01

    Avian cholera, an infectious disease caused by the bacterium Pasteurella multocida, kills thousands of North American wild waterfowl annually. Pasteurella multocida serotype 1 isolates cultured during a laboratory challenge study of Mallards (Anas platyrhynchos) and collected from wild birds and environmental samples during avian cholera outbreaks were characterized using amplified fragment length polymorphism (AFLP) analysis, a whole-genome DNA fingerprinting technique. Comparison of the AFLP profiles of 53 isolates from the laboratory challenge demonstrated that P. multocida underwent genetic changes during a 3-mo period. Analysis of 120 P. multocida serotype 1 isolates collected from wild birds and environmental samples revealed that isolates were distinguishable from one another based on regional and temporal genetic characteristics. Thus, AFLP analysis had the ability to distinguish P. multocida isolates of the same serotype by detecting spatiotemporal genetic changes and provides a tool to advance the study of avian cholera epidemiology. Further application of AFLP technology to the examination of wild bird avian cholera outbreaks may facilitate more effective management of this disease by providing the potential to investigate correlations between virulence and P. multocida genotypes, to identify affiliations between bird species and bacterial genotypes, and to elucidate the role of specific bird species in disease transmission. ?? Wildlife Disease Association 2008.

  16. Genetic variability of the stable fly assessed on a global scale using amplified fragment length polymorphism.

    Science.gov (United States)

    Kneeland, Kathleen M; Skoda, Steven R; Foster, John E

    2016-10-01

    The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is a blood-feeding, economically important pest of animals and humans worldwide. Improved management strategies are essential and their development would benefit from studies on genetic diversity of stable flies. Especially if done on a global scale, such research could generate information necessary for the development and application of more efficient control methods. Herein we report on a genetic study of stable flies using amplified fragment length polymorphism, with samples of 10-40 individuals acquired from a total of 25 locations in the Nearctic, Neotropic, Palearctic, Afrotropic and Australasian biogeographical regions. We hypothesized that genetic differentiation would exist across geographical barriers. Although FST (0.33) was moderately high, the GST (0.05; representing genetic diversity between individuals) was very low; Nm values (representing gene flow) were high (9.36). The mismatch distribution and tests of neutrality suggested population expansion, with no genetic differentiation between locations. The analysis of molecular variance (AMOVA) results showed the majority of genetic diversity was within groups. The mantel test showed no correlation between geographic and genetic distance; this strongly supports the AMOVA results. These results suggest that stable flies did not show genetic differentiation but are panmictic, with no evidence of isolation by distance or across geographical barriers. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  17. Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    Richard Atherton

    2013-06-01

    Full Text Available The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61% were classified as assemblage B (26 as BIII and 16 as BIV, 22 (32% as assemblage A (3 as AI and 19 as AII and five (7% as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

  18. Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review.

    Science.gov (United States)

    Dickie, I A; FitzJohn, R G

    2007-06-01

    Terminal restriction fragment length polymorphism (T-RFLP) is an increasingly widely used technique in mycorrhizal ecology. In this paper, we review the technique as it is used to identify species of mycorrhizal fungi and distinguish two different versions of the technique: peak-profile T-RFLP (the original version) and database T-RFLP. We define database T-RFLP as the use of T-RFLP to identify individual species within samples by comparison of unknown data with a database of known T-RFLP patterns. This application of T-RFLP avoids some of the pitfalls of peak-profile T-RFLP and allows T-RFLP to be applied to polyphyletic functional groups such as ectomycorrhizal fungi. The identification of species using database T-RFLP is subject to several sources of potential error, including (1) random erroneous matches of peaks to species, (2) shared T-RFLP profiles across species, and (3) multiple T-RFLP profiles within a species. A mathematical approximation of the risk of the first type of error as a function of experimental parameters is discussed. Although potentially less accurate than some other methods such as clone libraries, the high throughput of database T-RFLP permits much greater replication and may, therefore, be preferable for many ecological questions, particularly when combined with other techniques such as cloning.

  19. Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

    Directory of Open Access Journals (Sweden)

    Erica Chimara

    2004-11-01

    Full Text Available Mycobacterium tuberculosis complex (MTBC members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species - M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti" - were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, São Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306, M. bovis (3, and M. bovis BCG (2, were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.

  20. Association between matrix metalloproteinase 2 (MMP2) promoter polymorphisms and the susceptibility to non-Hodgkin's lymphoma in Egyptians.

    Science.gov (United States)

    Gouda, Heba Mahmoud; Khorshied, Mervat Mamdooh; El Sissy, Maha Hamdi; Shaheen, Iman Abdel Mohsen; Mohsen, Mohsen Mokhtar Abdel

    2014-08-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of extracellular matrix degradation. MMP2 is the key molecule that control invasion, tumor growth, and metastasis, and has been associated with poor prognosis in several tumors. Several epidemiological studies have focused on the associations between MMP2 promoter polymorphisms and cancer susceptibility; however, little is known about their role in hematological malignancies. The present study aimed to investigate the association of MMP2 -735C/T and -1306C/T promoter polymorphisms with B-NHL susceptibility and their clinicopathological characteristics. The study included 100 B-NHL patients and 100 healthy controls. Genotyping of MMP2 -735C/T and MMP2 -1306C/T was done by polymerase chain reaction restricted fragment length polymorphism (PCR-RFLP) technique. MMP2 -735C/T heteromutant genotype (CT) was detected in 23 % of patients, and the homomutant genotype (TT) was detected in 7 % of patients. The polymorphic allele, T allele, was associated with susceptibility to B-NHL (OR = 2.8:95 %CI = 1.48-5.28). For MMP2 -1306C/T, the frequencies of the polymorphic variants were 5 % for the heteromutant genotype (CT) and 3 % for the homomutant genotype (TT). The polymorphic allele, T allele, conferred almost fourfold increased risk of B-NHL (OR = 3.8, 95 %CI = 1.05-13.9), and the risk elevated to be almost eight folds when confined to diffuse large B-cell lymphoma (DLBCL) (OR = 7.9, 95 %CI = 1.67-32.27). MMP2 -735C/T polymorphic genotypes were correlated with advanced clinical stages of the disease (stages III and IV). In conclusion, the study revealed that the variant alleles of MMP2 -735C/T and MMP2 -1306C/T can be considered as molecular risk factors for B-NHL among Egyptians.

  1. Genetic diversity for restriction fragment length polymorphisms and heterosis for two diallel sets of maize inbreds.

    Science.gov (United States)

    Melchinger, A E; Lee, M; Lamkey, K R; Hallauer, A R; Woodman, W L

    1990-10-01

    Changes that may have occurred over the past 50 years of hybrid breeding in maize (Zea maize L.) with respect to heterosis for yield and heterozygosity at the molecular level are of interest to both maize breeders and quantitative geneticists. The objectives of this study were twofold: The first, to compare two diallels produced from six older maize inbreds released in the 1950's and earlier and six newer inbreds released during the 1970's with respect to (a) genetic variation for restriction fragment length polymorphisms (RFLPs) and (b) the size of heterosis and epistatic effects, and the second, to evaluate the usefulness of RFLP-based genetic distance measures in predicting heterosis and performance of single-cross hybrids. Five generations (parents, F1; F2, and backcrosses) from the 15 crosses in each diallel were evaluated for grain yield and yield components in four Iowa environments. Genetic effects were estimated from generation means by ordinary diallel analyses and by the Eberhart-Gardner model. Newer lines showed significantly greater yield for inbred generations than did older lines but smaller heterosis estimates. In most cases, estimates of additive x additive epistatic effects for yield and yield components were significantly positive for both groups of lines. RFLP analyses of inbred lines included two restriction enzymes and 82 genomic DNA clones distributed over the maize genome. Eighty-one clones revealed polymorphisms with at least one enzyme. In each set, about three different RFLP variants were typically found per RFLP locus. Genetic distances between inbred lines were estimated from RFLP data as Rogers' distance (RD), which was subdivided into general (GRD) and specific (SRD) Rogers' distances within each diallel. The mean and range of RDs were similar for the older and newer lines, suggesting that the level of heterozygosity at the molecular level had not changed. GRD explained about 50% of the variation among RD values in both sets. Cluster

  2. Restriction fragment length polymorphism pattern of Mycobacterium isolates from rodents in infected cattle farms.

    Science.gov (United States)

    Alizadeh, Khatereh; Mosavari, Nader; Nazari, Razieh

    2016-12-01

    Mycobacterium tuberculosis, the etiologic agent of tuberculosis, causes large-scale morbidity and mortality, particularly in developing countries. In recent years, there has been a significant increase in the drug-resistant ability of M. tuberculosis, triggering a major public health crisis. A detailed analysis of the evolution of the mycobacterial genome helps to better understand the genotype-phenotype relationship in this bacterium. Different strain typing methods have already revealed the worldwide diversity of mycobacterial isolates. Therefore, DNA-fingerprinting tools have been developed to improve tuberculosis case detection and control. Molecular typing techniques allow to detect and follow the spread of individual strains of the M. tuberculosis complex (MTC), complementing conventional epidemiological methods. Among these techniques, restriction fragment length polymorphism (RFLP) has been considered the standard method for genotyping of MTC. The aim of this work was to isolate M. tuberculosis from rodents in cattle farms contaminated with MTC located in the city of Booin-Zahra, Iran. A total of 100 samples were collected from the rodents in the contaminated farms and analyzed for the presence of Mycobacterium by growing the samples on Lowenstein-Jensen medium. All isolates were further identified by RFLP and DNA hybridization studies. As much as five samples showed the presence of Mycobacterium and these were subjected to PCR-16SrRNA, PCR-IS6110, and RD Typing (RD1, RD4, RD9, and RD12) methods. Further differentiation was performed with PvuII digestion (RFLP) and DNA hybridization using the polymorphic guanine/cytosine-rich repetitive sequences (PGRS) probe. The PGRS probe results classified two of the isolates as belonging to one cluster, whereas the remaining isolates were classified as belonging to different clusters. An analysis of the obtained genetic pattern and a comparison of these patterns with the genetic pattern of other infected farms allowed

  3. MT1A (A>G, MT1A (C>G, MT1M (A>C and MT4 (G>A single nucleotide polymorphism allele frequencies in Iranian populations

    Directory of Open Access Journals (Sweden)

    Javad Babaei

    2016-09-01

    Full Text Available It has been widely reported that metallothioneins (MTs play pivotal roles in the metabolism of the relatively nontoxic essential metals, as well as of toxic heavy metals. MT gene polymorphisms in an individual may increase or decrease the expression efficiency of the gene. The present study was aimed to investigate the frequency of genetic variation of four MTs in the Iranian population. Whole blood samples were collected from 300 Iranian healthy individuals. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP and automated Sanger sequencing were used for DNA genotype determination. The frequencies of major homozygous, heterozygous and minor homozygous genotypes in MT1 (A>G were, respectively, 76.0%, 24.0% and 0.0%; in MT1A (C>G, 88.0%, 12.0% and 0.0%; in MT1M (A>C, 86.0%, 14.0% and 0.0%; in MT4 (G>A, 80.0%, 18.7% and 1.3%. The present study showed for the first time that the polymorphism frequency distribution of these four MTs significantly differed between the studied Iranian population and other populations around the world, except for the MT4 polymorphism in the Iranian population and populations from the USA and New Zealand, which were similar. In conclusion, it could be suggested that regional and ethnic differences are the main reasons for this varied prevalence.

  4. Genetic diversity of Iranian potato soft rot bacteria based on PCR ...

    African Journals Online (AJOL)

    TENEBRA

    2012-01-19

    Jan 19, 2012 ... Key words: Genetic diversity, polymerase chain reaction-restriction fragment length polymorphism (PCR-. RFLP), recA gene, Pectobacterium spp. .... each restriction enzyme was used for digestions, which were incubated overnight at the .... (2006) with application of the molecular marker of PCR-RFLP, ...

  5. No association of NAD(P)H: quinone oxidoreductase 1 (NQO1) C609T polymorphism and risk of hepatocellular carcinoma development in Turkish subjects.

    Science.gov (United States)

    Akkiz, Hikmet; Bayram, Süleyman; Bekar, Aynur; Akgöllü, Ersin; Ülger, Yakup; Kaya, Berrin Yalinbaş; Sandikçi, Macit; Özdil, Burhan

    2010-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic enzyme that catalyzes the two-electron reduction of numerous quinoid compounds into their less toxic form, thus NQO1 protecting cells against oxidative stress. The gene coding for NQO1 has a single nucleotide polymorphism (C-->T) at nucleotide position 609 (proline to serine substitution at position 187 in amino acid sequence (P187S)) (rs1800566) of the NQO1 cDNA which results in very low enzimatic activity, so it would be expected that individuals with the homologous NQO1 C609T polymorphism would have a susceptibility developing cancer. Previous studies of the association between functional NQO1 C609T polymorphism and several human cancers have had mixed findings but association of NQO1 C609T polymorphism with hepatocellular carcinoma (HCC) development has yet to be investigated. In this study, we aim to evaluate the the association of NQO1 C609T with the risk of hepatocellular carcinoma (HCC) development among Turkish population. NQO1 C609T polymorphism was investigated in 167 confirmed subjects with HCC and 167 cancer-free control subjects matched on age, gender, smoking and alcohol consumption by using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. There is no association between the allel or genotype of NQO1 C609T polymorphism and HCC development risk in the Turkish subjects examined (p>0.05). Our result demonstrate for the first time that the NQO1 C609T polymorphism is not a genetic susceptibility factor for HCC in the Turkish population. Independent studies are need to validate our findings in a larger series, as well as in patients of different ethnic origins.

  6. Análisis de PCR-RFLP del gen de leptina y su asociación con características de la leche en ganado nativo (Bos indicus de Irán

    Directory of Open Access Journals (Sweden)

    Mojtaba Rezaei

    2015-01-01

    Full Text Available Se utilizó una muestra ganadera experimental para la caracterización de los genes y las frecuencias genotípicas y evaluar la asociación entre los polimorfismos de longitud del fragmento de restricción BsaAI en el gen de leptina y las características lácteas. Se recolectaron muestras de sangre de 390 vacas proporcionadas por el censo ganadero de la provincia de Guilán en Irán. La extracción de ADN genómico se basó en el método modificado de precipitación salina. El fragmento de 522 bp que comprende la región parcial del exón 3 e intrón 2 del locus del gen de leptina se amplificó mediante PCR-RFLP e iniciadores específicos. La digestión del fragmento amplificado con la enzima de restricción BsaAI reveló dos alelos de A y B con frecuencias de 0.447 y 0.553, y tres genotipos de AA, AB, BB con frecuencias de 0.185, 0.526 y 0.289 en la población estudiada, respectivamente. El resultado del contenido de polimorfismo fue 0.372 y para heterocigosis 0.526. Los resultados demostraron que la población estudiada estaba en equilibrio Hardy-Weinberg. El análisis estadístico indicó que el polimorfismo LEP- BsaAI tiene un efecto significativo en la producción de leche, porcentaje de grasa y proteína de la leche ( P <0.01. Animales portadores del genotipo heterocigoto (AB produjeron 1.17 y 0.7 kg/d más leche que los animales homocigotos AA y BB, respectivamente. Los animales con el genotipo AA tuvieron mayor grasa (0.28 % y porcentaje de proteína (0.17 % que el genotipo AB. Tales resultados pueden utilizarse como criterios para facilitar la selección genética en los programas de cría animal.

  7. Genetic polymorphisms in DNA base excision repair gene XRCC1 and the risk of squamous cell carcinoma of the head and neck

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    Pietruszewska Wioletta

    2009-03-01

    Full Text Available Abstract Background The genes of base excision repair (BER pathway have been extensively studied in the association with various human cancers. We performed a case-control study to test the association between two common single nucleotide polymorphisms (SNPs of XRCC1 gene with human head and neck squamous cell carcinoma (HNSCC. Methods The genotype analysis of Arg194Trp and Arg399Gln gene polymorphisms for 92 HNSCC patients and 124 controls of cancer free subjects, in Polish population were performed using the PCR-based restriction fragment length polymorphism (PCR-RFLP with endonuclease MspI. Results No altered risk has been found individually for these SNPs, however haplotypes analysis showed high association with head and neck cancer. The highest frequency, according to wild-type of Arg194Arg and Arg399Arg genotypes, was identified for Arg194Trp-Arg399Arg haplotype (OR, 2.96; 95% CI, 1.01–8.80. Conclusion Finally, we identified the combined Arg194Trp-Arg399Arg genotype of base excision repair gene XRCC1 that was associated with HNSCC and may have an impact on identification of a high-risk cancer population.

  8. Association Analysis between g.18873C>T and g.27522G>A Genetic Polymorphisms of OPG and Bone Mineral Density in Chinese Postmenopausal Women

    Directory of Open Access Journals (Sweden)

    Fei Wang

    2014-01-01

    Full Text Available Several studies report that the OPG is an important candidate gene in the pathogenesis of osteoporosis. This study aimed to detect the potential association of OPG gene polymorphisms with osteoporosis in postmenopausal women. We recruited 928 subjects containing 463 with primary postmenopausal osteoporosis and 465 healthy volunteers as controls. The BMD of neck hip, lumbar spine (L2–4, and total hip were assessed by dual-energy X-ray absorptiometry (DEXA. Through the created restriction site-polymerase chain reaction (CRS-PCR, PCR-restriction fragment length polymorphism (PCR-RFLP, and DNA sequencing methods, the g.18873C>T and g.27522G>A have been investigated. As for g.18873C>T, our data indicated that subjects with CC genotype have significantly higher BMD value than those of CT and TT genotypes (all P values A, the BMD values of subjects with GG genotype were significantly higher than those of GA and AA genotypes (all P values T and g.27522G>A genetic polymorphisms are associated with the decreased risk for osteoporosis in Chinese postmenopausal women.

  9. Osteoprotegerin Gene (OPG) Polymorphisms Associated with Peri-implantitis Susceptibility in a Chinese Han Population.

    Science.gov (United States)

    Zhou, Jian; Zhao, Yimin

    2016-11-09

    BACKGROUND The aim of this study was to investigate the association between T950C (rs2073617) and G1181C (rs2073618) polymorphisms of the osteoprotegerin gene (OPG) and the susceptibility of peri-implantitis in the Chinese Han population.  MATERIAL AND METHODS 110 patients with peri-implantitis and 116 healthy persons from the Chinese Han population were included in this study using a case-control design; rs2073617 and rs2073618 in OPG were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The linkage disequilibrium (LD) and haplotype analysis were performed with Haploview software. Hardy-Weinberg equilibrium (HWE) was assessed in the control group based on the genotype distributions of OPG polymorphisms. The genotype, allele, and haplotype distribution differences between the case and control groups were analyzed by chi-square test, and the relative risk of PD was expressed by odds ratio (OR) and 95% confidence interval (CI).  RESULTS The study results showed that people carrying the CC genotype of rs2073618 were more likely to have peri-implantitis than GG genotype carriers (OR=2.18, 95% CI=1.03-4.62, p=0.04). In addition, patients with the C allele had 1.47 times the risk of suffering from peri-implantitis (OR=1.47, 95% CI=1.01-2.13, p=0.04), but not rs2073617 polymorphism. The G-C haplotype frequency of rs2073618-rs2073617 in OPG was significantly correlated to the increased susceptibility of peri-implantitis (OR=2.27, 95% CI=1.20-4.30).  CONCLUSIONS OPG rs2073618 polymorphism may be related to the risk of peri-implantitis, but not rs2073617. Moreover, haplotype is also a non-ignorable risk factor.

  10. Genetic polymorphisms in MMP 2, 9 and 3 genes modify lung cancer risk and survival

    Directory of Open Access Journals (Sweden)

    González-Arriaga Patricia

    2012-03-01

    Full Text Available Abstract Background Matrix metalloproteases (MMPs are proteolytic enzymes that contribute to all stages of tumour progression, including the later stages of invasion and metastasis. Genetic variants in the MMP genes may influence the biological function of these enzymes and change their role in carcinogenesis and progression. We have investigated the association between the -735 C/T, the -1171 5A/6A, and the -1562 C/T polymorphisms in the MMP2, MMP3 and MMP9 genes, respectively, and the risk and survival of lung cancer. Methods The case-control study includes 879 lung cancer patients and 803 controls from a Caucasian population in Spain (CAPUA study. Genotypes were determined by PCR-RFLP. Odds ratios (OR and 95% confidence intervals (CI were calculated using unconditional logistic regression. The Kaplan-Meier method, long-rank test and Cox's were used for the survival analysis. Results The MMP9 -1562 T/T genotype was associated with a statistically significant decreased risk of developing lung cancer (OR = 0.23; 95% CI: 0.06-0.85, whereas no association was found for the MMP2 -735 C/T and MMP3 -1171 5A/6A polymorphisms. The MMP2 -735 T/T genotype was statistically significantly associated with a decreased survival in non-small cell lung cancer (NSCLC patients, identified as an independent prognosis factor of survival (hazard ratio (HR = 1.79; 95% CI: 1.00-3.20. In contrast, no association was found between the MMP3 -1171 5A/6A and the MMP9 -1562 C/T polymorphisms and survival. Conclusions These findings support the hypothesis that the MMP9 -1562 C/T polymorphism is associated with a protective effect against the development of lung cancer and suggest that the MMP2 -735 C/T polymorphism modify the length of survival in NSCLC patients.

  11. Association between ADH1C and ALDH2 polymorphisms and alcoholism in a Turkish sample.

    Science.gov (United States)

    Ayhan, Yavuz; Gürel, Şeref Can; Karaca, Özgür; Zoto, Teuta; Hayran, Mutlu; Babaoğlu, Melih; Yaşar, Ümit; Bozkurt, Atilla; Dilbaz, Nesrin; Uluğ, Berna Diclenur; Demir, Başaran

    2015-04-01

    Polymorphisms in the genes encoding alcohol metabolizing enzymes are associated with alcohol dependence. To evaluate the association between the alcohol dehydrogenase 1C (ADH1C) Ile350Val and aldehyde dehydrogenase 2 (ALDH2) Glu504Lys polymorphisms and alcohol dependence in a Turkish sample. 235 individuals (115 alcohol-dependent patients and 120 controls) were genotyped for ADH1C and ALDH2 with PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Association between the polymorphisms and family history, daily and maximum amount of alcohol consumed was investigated. The associations between alcohol dependence, severity of consumption and family history and the polymorphisms were analyzed by chi-square or Fisher's exact test where necessary. Relationship between genotypes and dependence related features was evaluated using analysis of variance (ANOVA). The -350Val allele for ADH1C (ADH1C*2) was increased in alcohol-dependent patients (P = 0.05). In individuals with a positive family history, the genotype distribution differed significantly (P = 0.031) and more patients carried the Val allele compared with controls (P = 0.025). Genotyping of 162 participants did not reveal the -504Lys allele in ALDH2. These findings suggest that ADH1C*2 is associated with alcohol dependence in the Turkish population displaying a dominant inheritance model. ADH1C*2 allele may contribute to the variance in heritability of alcohol dependence. The ALDH2 -504Lys/Lys or Glu/Lys genotypes were not present in alcohol-dependent patients, similar to that seen in European populations and in contrast to the findings in the Asian populations.

  12. Genotypic lineages and restriction fragment length polymorphism of canine distemper virus isolates in Thailand.

    Science.gov (United States)

    Radtanakatikanon, Araya; Keawcharoen, Juthatip; Charoenvisal, Na Taya; Poovorawan, Yong; Prompetchara, Eakachai; Yamaguchi, Ryoji; Techangamsuwan, Somporn

    2013-09-27

    Canine distemper virus (CDV) is known to cause multisystemic disease in all families of terrestrial carnivores. Attenuated live vaccines have been used to control CDV in a variety of species for many decades, yet a number of CDV infections in vaccinated dogs are still observed. The aims of this study were to investigate the genetic diversity of CDV lineages based on phosphoprotein (P), hemagglutinin (H) and fusion protein (F) genes and to develop the restriction fragment length polymorphism (RFLP) technique for effective differentiation among individual wild-type and vaccine lineages in Thailand. Four commercial vaccine products, thirteen conjunctival swabs and various tissues from 9 necropsied dogs suspected of having CDV infections were included. Virus isolation was performed using Vero cell expressing canine signaling lymphocyte activation molecules (Vero-DST cells). Reverse-transcription polymerase chain reaction (RT-PCR) on 3 gene regions from the dog derived specimens and the vaccines were carried out, then RFLP analysis upon F-gene amplified fragments was developed. Nucleotide sequence and phylogenetic analysis were compared with other CDV lineages in Genbank. Phylogenetic relationships revealed that CDV field isolates were separated from the vaccine lineage and could be divided into two clusters; one of which belonged to the Asia-1 lineage and another, not related to any previous recognized lineages was proposed as 'Asia-4'. RFLP patterns demonstrating concordance with phylogenetic trees of the distemper virus allowed for differentiation between the Asia-1, Asia-4 and vaccine lineages. Thus, RFLP technique is able to effectively distinguish individual wild-type canine distemper virus from vaccine lineages in Thailand. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Use of restriction fragment length polymorphisms to investigate strain variation within Neisseria meningitidis

    International Nuclear Information System (INIS)

    Williams, S.D.

    1989-01-01

    Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P 32 labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population

  14. Use of restriction fragment length polymorphisms to investigate strain variation within Neisseria meningitidis

    Energy Technology Data Exchange (ETDEWEB)

    Williams, S.D.

    1989-01-01

    Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P{sup 32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population.

  15. Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence.

    Science.gov (United States)

    Nishi, Eiji; Tashiro, Yukihiro; Sakai, Kenji

    2015-05-01

    DNA typing from forensic evidence is commonly used to identify individuals. However, when the quantity of the forensic evidence is insufficient, successful identification using DNA typing is impossible. Such evidence may also contain DNA from bacteria that occur naturally on the skin. In this study, we aimed to establish a profiling method using terminal restriction fragment length polymorphisms (T-RFLPs) of the amplified bacterial 16S ribosomal RNA (rRNA) gene. First, the extraction and digestion processes were investigated, and the T-RFLP profiling method using the 16S rRNA gene amplicon was optimized. We then used this method to compare the profiles of bacterial flora from the hands of 12 different individuals. We found that the T-RFLP profiles from one person on different days displayed higher similarity than those between individuals. In a principal component analysis (PCA), T-RFLPs from each individual were closely clustered in 11 out of 12 cases. The clusters could be distinguished from each other, even when the samples were collected from different conditions. No major change of the profile was observed after six months except in two cases. When handprints on glass plates were compared, 11 of 12 individuals were assigned to a few clusters including the cluster corresponding to the correct individual. In conclusion, a method for reproducible T-RFLP profiling of bacteria from trace amounts of handprints was established. The profiles were obtained for particular individuals clustered in PCA and were experimentally separable from other individuals in most cases. This technique could provide useful information for narrowing down a suspect in a criminal investigation.

  16. Use of Restriction Fragment Length Polymorphisms to Investigate Strain Variation Within Neisseria Meningitidis.

    Science.gov (United States)

    Williams, Shelley Diane

    Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty -six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P ^{32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analysed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population. This analysis demonstrates the lack of structure within Neisseria meningitidis due primarily to a heterogenous population and the lack of geographic segregation. The potential utility of this technique as a

  17. DNA Polymorphism of Insulin-like Growth Factor-binding Protein-3 Gene and Its Association with Cashmere Traits in Cashmere Goats.

    Science.gov (United States)

    Liu, Haiying; Liu, Chao; Yang, Guiqin; Li, Hui; Dai, Jin; Cong, Yuyan; Li, Xuejian

    2012-11-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) gene is important for regulation of growth and development in mammals. The present investigation was carried out to study DNA polymorphism by PCR-RFLP of IGFBP-3 gene and its effect on fibre traits of Chinese Inner Mongolian cashmere goats. The fibre traits data investigated were cashmere fibre diameter, combed cashmere weight, cashmere fibre length and guard hair length. Four hundred and forty-four animals were used to detect polymorphisms in the hircine IGFBP-3 gene. A 316-bp fragment of the IGFBP-3 gene in exon 2 was amplified and digested with HaeIII restriction enzyme. Three patterns of restriction fragments were observed in the populations. The frequency of AA, AB and BB genotypes was 0.58, 0.33 and 0.09 respectively. The allelic frequency of the A and B allele was 0.75 and 0.25 respectively. Nucleotide sequencing revealed a C>G transition in the exon 2 region of the IGFBP-3 gene resulting in R158G change which caused the polymorphism. Least squares analysis revealed a significant effect of genotypes on cashmere weight (pcashmere fibre length (pcashmere fibre diameter was not statistically significant (p>0.05). The animals of AB and BB genotypes showed higher cashmere weight, cashmere fibre length and hair length than the animals possessing AA genotype. These results suggested that polymorphisms in the hircine IGFBP-3 gene might be a potential molecular marker for cashmere weight in cashmere goats.

  18. The haplotype of two FSHR polymorphisms in ovarian cancer--a potential role of ethnology in risk modification.

    Science.gov (United States)

    Heubner, Martin; Riemann, Kathrin; Otterbach, Friedrich; Kimmig, Rainer; Kasimir-Bauer, Sabine; Siffert, Winfried; Wimberger, Pauline

    2009-03-01

    The precise role of gonadotropins in the carcinogenesis of epithelial ovarian cancer remains uncertain. Recently, the haplotype of two single nucleotide polymorphisms, Thr307Ala (rs6165) and Asn680Ser (rs6166), has been described as a risk factor for ovarian cancer in Chinese women. In this study we investigated the impact of this haplotype regarding the risk to develop ovarian cancer as well as possible effects upon the clinical course in a Caucasian patient sample. Determination of genotypes in 115 patients with primary epithelial ovarian cancer and 115 age-matched controls was performed by Pyrosequencing for Thr307Ala and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for Asn680Ser. Analysis of the genotypes revealed almost complete linkage disequilibrium of both SNPs. The distribution of genotypes was not statistically significant different between ovarian cancer patients and age-matched controls. Clinical parameters such as overall survival, CA12-5 elevation at primary diagnosis, age at diagnosis, FIGO stage, grading, and platinum resistance were not statistically significantly different regarding genotypes. We could not confirm the FSHR Ala307-Ser680 haplotype as a risk factor for epithelial ovarian cancer in Caucasian women. Hence, the modification of tumor risk may be affected by the ethnology of the patient collective. We could not find any associations of clinical parameters or course of the disease with the different genotypes.

  19. Association between STK11 Gene Polymorphisms and Coronary Artery Disease in Type 2 Diabetes in Han Population in China

    Directory of Open Access Journals (Sweden)

    Xiaowei Ma

    2017-01-01

    Full Text Available Background. Recent studies indicated that the Serine threonine kinase 11 (STK11, which is a key regulator of the AMP-activated protein kinase (AMPK, plays a crucial role in cardiovascular system. This study aimed to investigate whether genetic variations in the STK11 gene affect the risk of coronary artery disease (CAD in Chinese type 2 diabetics. Methods. 5 haplotype-tagging single nucleotide polymorphisms (SNPs were selected, and 288 CAD-positive cases and 159 CAD-negative controls with type 2 diabetes were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP assay. Results. The carriers of minor allele A at rs12977689 had a higher risk of CAD compared to the homozygotes of CC (OR = 1.572, 95% CI = 1.039–2.376, p=0.035, and the difference was still significant after adjustment for the other known CAD risk factors (OR′ = 1.184, 95%  CI′ = 1.036–1.353, p′=0.013. Conclusion. Genetic variability at STK11 locus is associated with CAD risk in type 2 diabetes in the Chinese population.

  20. Polymorphisms in methylenetetrahydrofolate reductase and cystathionine beta-synthase in oral cancer - a case-control study in southeastern Brazilians.

    Science.gov (United States)

    Barbosa, Andressa; Dos Santos, Marcelo; de Podestá, José Roberto Vasconcelos; Gouvêa, Sônia Alves; Von Zeidler, Sandra Ventorin; Louro, Iúri Drumond; Cordeiro-Silva, Melissa de Freitas

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is a serious public health problem, due to its high mortality rate and worldwide rising incidence. OSCC susceptibility is mediated by interactions between genetic and environmental factors. Studies suggest that genetic variants encoding enzymes involved in folate metabolism may modulate OSCC risk by altering DNA synthesis/repair and methylation process. The goals of this study were to evaluate the association of three genotypic polymorphism (MTHFR C677T, MTHFR A1298C and CBS 844ins68) and oral cancer risk in southeastern Brazilians and evaluate the interactions between polymorphisms and clinical histopathological parameters. This case-control study included 101 cases and 102 controls in the state of Espírito Santo, Brazil. MTHFR genotyping was done by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) and CBS genotyping by PCR (polymerase chain reaction) analysis. MTHFR C677T polymorphism was associated with lymph node involvement. Genotype CT+TT acted as a protective factor. MTHFR A1298C AC+CC genotype was associated with tumor differentiation, and possibly with a better prognosis. In risk analysis, no correlation was observed between genotypes and OSCC. We concluded that MTHFR C677T, MTHFR A1298C and CBS 844ins68 polymorphisms were not associated with OSCC risk in southeastern Brazilians; however, we suggest a prognosis effect associated with MTHFR C677T and A1298C polymorphisms in OSCC. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  1. Association analysis between the -2518MCP-1(A/G) polymorphism and generalized aggressive periodontitis in a Chinese population.

    Science.gov (United States)

    Zhu, X L; Meng, H X; Zhang, L; Xu, L; Chen, Z B; Shi, D; Feng, X H; Zhang, X

    2012-06-01

    It has been suggested that aggressive periodontitis has a genetic basis. Monocyte chemoattractant protein 1 (MCP-1) plays a critical role in the recruitment of monocytes and the development of periodontitis. The -2518MCP-1(A/G) polymorphism has been implicated as a risk or susceptibility factor for a variety of autoimmune conditions and inflammatory diseases. The intent of this investigation was to study whether the -2518MCP-1(A/G) polymorphism is associated with generalized aggressive periodontitis in the Chinese population. One hundred and twenty-four patients with generalized aggressive periodontitis and 94 healthy subjects were included in this case-control study. Genomic DNA was isolated from a peripheral blood sample obtained from each subject. Gene polymorphisms of -2518MCP-1(A/G) were analyzed by a standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. A logistic regression analysis was performed to test the association between the -2518MCP-1(A/G) genotype (alleles) and generalized aggressive periodontitis with adjustment of the major covariates (gender, age and smoking status). There was no significant association of the -2518MCP-1(A/G) polymorphism with generalized aggressive periodontitis in the unstratified subjects. However, when patients were stratified by gender, the frequency of the G(+) genotype was significantly lower in female patients with generalized aggressive periodontitis compared with female controls (p = 0.036, adjusted odds ratio = 0.3, 95% CI: 0.1-0.9). In female patients with generalized aggressive periodontitis, the probing pocket depth was larger in subjects with the AA genotype than in subjects with the G(+) genotype (5.07 mm vs. 4.30 mm; Z = -2.470, p = 0.014). The polymorphisms of -2518MCP-1 may play an important role in determining generalized aggressive periodontitis susceptibility in this cohort of Chinese women. © 2011 John Wiley & Sons A/S.

  2. Multi-drug resistance-1 gene polymorphisms in nephrotic syndrome: impact on susceptibility and response to steroids.

    Science.gov (United States)

    Youssef, Doaa M; Attia, Tarek A; El-Shal, Amal S; Abduelometty, Fawzya A

    2013-11-10

    Role of multidrug resistance-1 (MDR-1) gene polymorphisms has not been clarified in nephrotic syndrome (NS). Additionally, researchers studied several genetic polymorphisms to explain their influence on different patients' responses to steroid; however the data were inconsistent. Therefore, we aimed to investigate the association of MDR-1 gene polymorphisms [C1236T, G2677T/A, C3435T] and haplotypes with susceptibility to childhood nephrotic syndrome, and whether they influence steroid response. We detected MDR-1 gene polymorphisms using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 138 NS patients and 140 age and sex matched healthy children. The frequencies of MDR1 G2677T/A GT, GA, TT+AA genotypes or T allele, MDR1 C3435T TT genotype, and T allele genotype frequencies were significantly increased in NS group. While no significant differences were observed in distributions of C1236T genotypes or allele between NS patients and healthy children. Moreover, steroid non-responder NS patients had significantly higher frequencies of MDR1 G2677T/A GT, GA, and TT+AA genotypes than steroid responsive NS patients. We observed also that NS patients with age less than 6 years old had increased frequencies of MDR1 G2677T/A GT, GA, TT+AA genotypes or T allele MDR1 C3435T CT, TT genotypes and T allele. Interestingly the frequency of the TGC haplotype of MDR1 was lower in the initial steroid responders than in non-responders NS patients. On the contrary, there were no any association between the MDR1 haplotypes with NS susceptibility and they did not influence renal pathological findings. Our data suggested that MDR1 C3435T or G2677T/A gene polymorphisms are risk factors of increased susceptibility, earlier onset of NS, and steroid resistance. © 2013 Elsevier B.V. All rights reserved.

  3. Interleukin-10 -1082A/G polymorphism is associated with the development of acute pancreatitis in a Chinese population.

    Science.gov (United States)

    Cai, Feng; Cui, Ning; Ma, Hongyan; Wang, Xueli; Qiao, Guihong; Liu, Danping

    2015-01-01

    We conducted a case-control study to investigate the association between IL-10 gene polymorphism (-1082A/G, -819T/C, and -592A/C) and risk of acute pancreatitis in a Chinese population. A total of 240 patients with proven acute pancreatitis and 240 control subjects were collected between May 2012 and January 2015. Genotyping of the IL-10-1082A/G, -819T/C, and -592A/C gene polymorphisms was conducted by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. By univariate logistic regression analysis, patients with acute pancreatitis were more likely to have higher BMI (OR=2.12, 95% CI=1.45-3.12; Pacute pancreatitis and control subjects (χ(2)=9.97, P=0.007). By multiple logistic regression analysis, we found that individuals with the GG genotype of IL-10-1082A/G were associated with an increased risk of acute pancreatitis when compared with the AA genotype (OR=2.32, 95% CI=1.20-4.59; P=0.007). In dominant and recessive models, the IL-10-1082A/G gene polymorphism was significantly correlated with an elevated risk of acute pancreatitis, and the adjusted Ors (95% CI) were 1.50 (1.03-2.20) and 1.99 (1.06-3.79), respectively. However, no significant different was found between IL-10-819T/C and -592A/C gene polymorphisms and susceptibility to acute pancreatitis. In conclusion, we suggest that IL-10-1082A/G gene polymorphisms contribute to the development of acute pancreatitis in codominant, dominant and recessive models.

  4. Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis

    Science.gov (United States)

    Grant T. Kirker; M. Lynn Prewitt; Walter J. Diehl; Susan V. Diehl

    2012-01-01

    The effects of wood preservatives on the bacterial community in southern yellow pine were assessed by the molecular method ‘terminal restriction fragment length polymorphism’ (T-RFLP). Stakes, treated with 0.25 % and 0.37 % ammoniacal copper quat (ACQ-C), 0.1 % and 0.25 % chlorothalonil (CTN), 0.1 % and 0.25 % CTN with 2 % butylated hydroxytoluene (BHT), and 2 % BHT...

  5. Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

    Science.gov (United States)

    Fadda, M E; Pisano, M B; Scaccabarozzi, L; Mossa, V; Deplano, M; Moroni, P; Liciardi, M; Cosentino, S

    2013-01-01

    This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of

  6. Polymorphisms of transforming growth factor beta 1 (RS#1800468 and RS#1800471) and esophageal squamous cell carcinoma among Zhuangese population, China.

    Science.gov (United States)

    Tang, Ren-Guang; Huang, Yong-Zhi; Yao, Li-Min; Xiao, Jian; Lu, Chuan; Yu, Qian

    2013-01-01

    Epidemiological evidence has shown two polymorphisms (namely RS#1800468G>A and RS#1800471G>C) of transforming growth factor-beta 1 (TGF-β1) gene may be involved in the cancer development. However, their role in the carcinogenic process of esophageal squamous cell carcinoma (ESCC) has been less well elaborated. We conducted a hospital-based case-control study including 391 ESCC cases and 508 controls without any evidence of tumors to evaluate the association between these two polymorphisms and ESCC risk and prognosis for Zhuangese population by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS)-PCR techniques. We found that individuals with the genotypes with RS#1800471 C allele (namely RS#1800471-GC or -CC) had an increased risk of ESCC than those without above genotypes (namely RS#1800471-GG, adjusted odds ratio 3.26 and 5.65, respectively). Further stratification analysis showed that this polymorphism was correlated with tumor histological grades and TNM (tumor, node, and metastasis) stage, and modified the serum levels of TGF-β1. Additionally, RS#1800471 polymorphism affected ESCC prognosis (hazard ratio, 3.40), especially under high serum levels of TGF-β1 conditions. However, RS#1800468 polymorphism was not significantly related to ESCC risk. These findings indicated that TGF-β1 RS#1800471G>C polymorphism may be a genetic modifier for developing ESCC in Zhuangese population. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Associations of TGFBR1 and TGFBR2 gene polymorphisms with the risk of hypospadias: a case-control study in a Chinese population.

    Science.gov (United States)

    Han, Xin-Rui; Wen, Xin; Wang, Shan; Hong, Xiao-Wu; Fan, Shao-Hua; Zhuang, Juan; Wang, Yong-Jian; Zhang, Zi-Feng; Li, Meng-Qiu; Hu, Bin; Shan, Qun; Sun, Chun-Hui; Bao, Ya-Xing; Lin, Meng; He, Tan; Wu, Dong-Mei; Lu, Jun; Zheng, Yuan-Lin

    2017-10-31

    This case-control study investigated the association of transforming growth factor-β (TGF-β) receptor type I and II ( TGFBR1 and TGFBR2 ) gene polymorphisms with the risk of hypospadias in a Chinese population. One hundred and sixty two patients suffering from hypospadias were enrolled as case group and 165 children who underwent circumcision were recruited as control group. Single nucleotide polymorphisms (SNPs) in TGFBR1 and TGFBR2 genes were selected on the basis of genetic data obtained from HapMap. PCR-restriction fragment length polymorphism (PCR-RFLP) was performed to identify TGFBR1 and TGFBR2 gene polymorphisms and analyze genotype distribution and allele frequency. Logistic regression analysis was conducted to estimate the risk factors for hypospadias. No significant difference was found concerning the genotype and allele frequencies of TGFBR1 rs4743325 polymorphism between the case and control groups. However, genotype and allele frequencies of TGFBR2 rs6785358 in the case group were significantly different in contrast with those in the control group. Patients carrying the G allele of TGFBR2 rs6785358 polymorphism exhibited a higher risk of hypospadias compared with the patients carrying the A allele ( P <0.05). The TGFBR2 rs6785358 genotype was found to be significantly related to abnormal pregnancy and preterm birth (both P <0.05). The frequency of TGFBR2 rs6785358 GG genotype exhibited significant differences amongst patients suffering from four different pathological types of hypospadias. Logistic regression analysis revealed that preterm birth, abnormal pregnancy, and TGFBR2 rs6785358 were the independent risk factors for hypospadias. Our study provides evidence that TGFBR2 rs6785358 polymorphism might be associated with the risk of hypospadias. © 2017 The Author(s).

  8. Matrix-Gla Protein rs4236 [A/G] gene polymorphism and serum and GCF levels of MGP in patients with subgingival dental calculus.

    Science.gov (United States)

    Doğan, Gülnihal Emrem; Demir, Turgut; Aksoy, Hülya; Sağlam, Ebru; Laloğlu, Esra; Yildirim, Abdulkadir

    2016-10-01

    Matrix-Gla Protein (MGP) is one of the major Gla-containing protein associated with calcification process. It also has a high affinity for Ca 2+ and hydroxyapatite. In this study we aimed to evaluate the MGP rs4236 [A/G] gene polymorphism in association with subgingival dental calculus. Also a possible relationship between MGP gene polymorphism and serum and GCF levels of MGP were examined. MGP rs4236 [A/G] gene polymorphism was investigated in 110 patients with or without subgingival dental calculus, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Additionally, serum and GCF levels of MGP of the patients were compared according to subgingival dental calculus. Comparison of patients with and without subgingival dental calculus showed no statistically significant difference in MGP rs4236 [A/G] gene polymorphism (p=0.368). MGP concentrations in GCF of patients with subgingival dental calculus were statistically higher than those without subgingival dental calculus (p=0.032). However, a significant association was not observed between the genotypes of AA, AG and GG of the MGP rs4236 gene and the serum and GCF concentrations of MGP in subjects. In this study, it was found that MGP rs4236 [A/G] gene polymorphism was not to be associated with subgingival dental calculus. Also, that GCF MGP levels were detected higher in patients with subgingival dental calculus than those without subgingival dental calculus independently of polymorphism, may be the effect of adaptive mechanism to inhibit calculus formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Gender Differences in the VDR-FokI Polymorphism and Conventional Non-Genetic Risk Factors in Association with Lumbar Spine Pathologies in an Italian Case-Control Study

    Science.gov (United States)

    Colombini, Alessandra; Brayda-Bruno, Marco; Ferino, Lucia; Lombardi, Giovanni; Maione, Vincenzo; Banfi, Giuseppe; Cauci, Sabina

    2015-01-01

    Recently, the FokI polymorphism (rs2228570) in the vitamin D receptor gene (VDR) and conventional risk factors were associated with spine disorders in the Italian population, but without gender analysis. Two-hundred and sixty-seven patients (149 males, 118 females) with lumbar spine disorders were assessed by magnetic resonance imaging (MRI) and 254 (127 males, 127 females) asymptomatic controls were enrolled. The exposure to putative risk factors was evaluated and FokI polymorphism was detected by PCR-restriction fragment length polymorphism (PCR-RFLP). An association between lumbar spine pathologies and higher than average age; overweight; family history; lower leisure physical activity; smoking habit; higher number of hours/day exposure to vibration and more sedentary or intense physical job demand was observed in male patients. In contrast, in females, only higher age, overweight, family history and lower leisure physical activity were risk factors. FF genotype was a 2-fold risk factor to develop discopathies and/or osteochondrosis concomitant with disc herniation for both gender patients, while heterozygous Ff was protective for females only. In males only ff genotype was protective for discopathies and/or osteochondrosis and F allele was a 2-fold risk factor for hernia; discopathies; discopathies and/or osteochondrosis. Sex-related differences in voluntary behaviors, exposure to environmental risks and genetic background could be crucial for a gender-differentiated management of patients with spine disorders. PMID:25671813

  10. Gender Differences in the VDR-FokI Polymorphism and Conventional Non-Genetic Risk Factors in Association with Lumbar Spine Pathologies in an Italian Case-Control Study

    Directory of Open Access Journals (Sweden)

    Alessandra Colombini

    2015-02-01

    Full Text Available Recently, the FokI polymorphism (rs2228570 in the vitamin D receptor gene (VDR and conventional risk factors were associated with spine disorders in the Italian population, but without gender analysis. Two-hundred and sixty-seven patients (149 males, 118 females with lumbar spine disorders were assessed by magnetic resonance imaging (MRI and 254 (127 males, 127 females asymptomatic controls were enrolled. The exposure to putative risk factors was evaluated and FokI polymorphism was detected by PCR-restriction fragment length polymorphism (PCR-RFLP. An association between lumbar spine pathologies and higher than average age; overweight; family history; lower leisure physical activity; smoking habit; higher number of hours/day exposure to vibration and more sedentary or intense physical job demand was observed in male patients. In contrast, in females, only higher age, overweight, family history and lower leisure physical activity were risk factors. FF genotype was a 2-fold risk factor to develop discopathies and/or osteochondrosis concomitant with disc herniation for both gender patients, while heterozygous Ff was protective for females only. In males only ff genotype was protective for discopathies and/or osteochondrosis and F allele was a 2-fold risk factor for hernia; discopathies; discopathies and/or osteochondrosis. Sex-related differences in voluntary behaviors, exposure to environmental risks and genetic background could be crucial for a gender-differentiated management of patients with spine disorders.

  11. Promoter polymorphisms of the HLA-G gene, but not the HLA-E and HLA-F genes, is associated with non-segmental vitiligo patients in the Korean population.

    Science.gov (United States)

    Kim, Su Kang; Hong, Mi Sook; Shin, Min Kyung; Uhm, Yoon Kyung; Chung, Joo-Ho; Lee, Mu-Hyoung

    2011-11-01

    The aim of this study was to determine whether or not promoter polymorphisms of the class I major histocompatibility complex (HLA-E, HLA-F, and HLA-G) are associated with susceptibility to vitiligo. To identify a possible association with vitiligo, 241 patients with non-segmental vitiligo (NSV) and 395 healthy controls were recruited in this study. Three promoter single nucleotide polymorphisms (SNPs; rs1264459 of HLA-E, rs9258170 of HLA-F, and rs1736936 of HLA-G) were analyzed using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and direct sequencing. Multiple logistic regression models (co-dominant 1, co-dominant 2, dominant, recessive, and log-additive models) were applied for odds ratios (ORs), 95% confidence intervals (CIs), and P values. To obtain the defined results, P values were recalculated by a Bonferroni correction. After the Bonferroni correction, the genotype of the SNP (rs1736936) of HLA-G was shown to have significant association with NSV (P = 0.045 in the recessive model). The genotype frequencies of the HLA-G SNP (rs1736936) had a significant correlation with the age of onset of NSV (P = 0.016 in the co-dominant 1 model and P = 0.027 in the dominant model). Our results suggest that HLA-G, but not HLA-E and HLA-F, may be associated with susceptibility to NSV in the Korean population.

  12. Uteroglobin gene polymorphism (G38A) may be a risk factor in childhood idiopathic nephrotic syndrome.

    Science.gov (United States)

    Demircioglu Kılıc, Beltinge; Buyukcelik, Mithat; Oguzkan Balcı, Sibel; Pehlivan, Sacide; Kul, Seval; Col, Nilgun; Balat, Ayse

    2018-02-01

    Uteroglobin (UG) is a multifunctional protein with anti-inflammatory properties. The aim of this study was to first evaluate the role of UG gene G38A polymorphism in childhood idiopathic nephrotic syndrome (INS), and determine whether this variation may be related to the occurrence of INS or a steroid response. One hundred and thirty-six children diagnosed with INS in Gaziantep University, Department of Pediatric Nephrology, and 70 healthy volunteers were included. Children with INS were divided into two groups: steroid-sensitive (n = 84), and steroid-resistant (n = 52). Samples were examined using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) enzyme method. The distributions of AA, GG, and AG genotypes of UG gene G38A (G/A) were 16.9%, 44.9%, and 38.2% in the all-INS group, whereas they were 14.3%, 48.8%, and 36.9% in the steroid-sensitive INS (SSINS) group compared with 21.1%, 38.5%, and 40.4% in steroid-resistant INS (SRINS), and 5.7%, 41.4%, and 52.9% in controls. The risk of INS was increased almost 4-fold in children with the AA genotype (p = 0.016). The risk of having SSINS was increased 3.5-fold (p = 0.042) whereas the risk of SRINS was increased 4.8-fold in the same genotype (p = 0.014). The uteroglobin gene may play an important role in the development of INS, and the AA genotype of UG gene G38A polymorphism was found more frequently in those children. Further studies evaluating all polymorphisms in larger patient groups are needed to exactly determine the effect of UG gene on the development of INS and steroid response in children.

  13. Relation of atrophic gastritis with Helicobacter pylori-CagA(+) and interleukin-1 gene polymorphisms.

    Science.gov (United States)

    Sierra, Rafaela; Une, Clas; Ramirez, Vanessa; Alpizar-Alpizar, Warner; Gonzalez, Maria-I; Ramirez, Jose-A; De Mascarel, Antoine; Cuenca, Patricia; Perez-Perez, Guillermo; Megraud, Francis

    2008-11-14

    To determine the association of Helicobacter pylori (H pylori) CagA(+) infection and pro-inflammatory polymorphisms of the genes interleukin (IL)-1RN and IL-1B with the risk of gastric atrophy and peptic ulcers in a dyspeptic population in Costa Rica, a country with high incidence and mortality of gastric cancer. Seven biopsy specimens, a fasting blood sample and a questionnaire concerning nutritional and sociodemographic factors were obtained from 501 consecutive patients who had undergone endoscopy for dyspeptic symptoms. A histopathological diagnosis was made. Pepsinogen concentrations were analyzed by enzyme linked immunosorbent assay (ELISA). Infection with H pylori CagA(+) was determined by serology and polymerase chain reaction (PCR). IL-1B and IL-1RN polymorphisms genotyping was performed by PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR respectively. In this dyspeptic population, 86% were H pylori positive and of these, 67.8% were positive for CagA. Atrophic antral gastritis (AAG) was associated with CagA(+) status [odd ratio (OR) = 4.1; P gastritis (ABG) was associated with pepsinogen PGI/PGII < 3.4 (OR = 4.9; P < 0.04) and alcohol consumption (OR = 7.3; P < 0.02). Duodenal ulcer was associated with CagA(+) (OR = 2.9; P < 0.04) and smoking (OR = 2.4; P < 0.04). PGI < 60 microg/L as well as PGI/PGII < 3.4 were associated with CagA(+). In a dyspeptic population in Costa Rica, H pylori CagA(+) is not associated with ABG, but it is a risk factor for AAG. The pro-inflammatory cytokine polymorphisms IL-1B + 3945 and IL-1RN are not associated with the atrophic lesions of this dyspeptic population.

  14. Association between polymorphisms of the IKZF3 gene and systemic lupus erythematosus in a Chinese Han population.

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    Xinze Cai

    Full Text Available OBJECTIVE: It has been reported that IKAROS family of zinc finger 3 (IKZF3-deficient mice spontaneously develop human systemic lupus erythematosus (SLE-like phenotypes and produce anti-dsDNA Ab leading to immune complex-mediated glomerulonephritis. Polymorphism of the IKZF3 gene corresponds with the susceptibility to several immune-related diseases. Our intention was to establish an association between polymorphisms in the IKZF3 gene and SLE in the Chinese Han population. METHODS: The study involved obtaining blood samples for DNA extraction and genotyping the 4 selected single-nucleotide polymorphisms (SNPs in IKZF3, including rs12150079, rs9909593, rs907091, and rs2872507, by performing PCR restriction fragment length polymorphism analysis (PCR-RFLP. A group of 366 SLE patients were compared to 455 healthy controls. RESULTS: A significant decrease in frequencies of the rs907091 CC genotype and C allele appeared in the SLE patients unlike that observed in the controls (p = 0.001 and 0.015, respectively. The frequencies of the rs12150079 genotype and allele were different between the SLE patients and the control individuals, although the significance was only marginal (p = 0.046 and 0.049, respectively. In addition, a significantly low frequency of the GGCG haplotype was observed in the SLE patients, suggesting that it may provide protection against SLE (p = 0.011. CONCLUSION: To the best of our knowledge, this is the first study to demonstrate an important association between polymorphisms in IKZF3 and SLE in the Chinese Han population. A strong association between rs907091 in the IKZF3 gene and SLE was identified.

  15. Analysis of polymorphism in the survivin gene promoter as a potential risk factor for head and neck cancers development

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    Kostić Marija

    2013-01-01

    Full Text Available Introduction. Association studies have shown that gene polymorphisms in various classes of genes can modulate cancer risk. The -31G/C polymorphism in the promoter of survivin gene, affects the expression of the anti-apoptotic protein survivin which in turn may predispose an individual to some types of cancer. Objective. The aim of the study was to determine whether the survivin promoter -31G/C polymorphism could be a susceptibility factor for squamous cell carcinoma (SCC of the oral cavity and basal cell carcinoma (BCC of the skin. Methods. The DNA obtained from 88 patients with SCC, 60 patients with BCC and 111 healthy individuals was subjected to polymerase chain reaction-restriction fragment length polymorphism analysis (PCR- RFLP in order to determine genotype and allele frequencies in patients and control groups. Logistic regression was used for cancer risk assessment. Results. The following distribution of genotypes was obtained: CC genotype 15% in the SCC group, 13% in the BCC group and 12% in controls; CG genotype 41% in SCCs, 35% in BCCs, 48% in controls; GG genotype 44% in SCCs, 52% in BCCs and 40% in controls. Allelic frequencies were as follows: G allele 0.65 in SCCs, 0.69 in BCCs and 0.64 in the control group; C allele 0.35 in SCCs, 0.31 in BCCs and 0.36 in the control group. There was no statistically significant difference in allele or genotype frequencies between the patients and controls (p>0.05. Conclusion. In Serbian population, -31G/C polymorphism in the promoter of the survivin gene cannot be considered as a risk factor for oral squamous cell carcinoma and skin basal cell carcinoma. [Projekat Ministarstva nauke Republike Srbije, br. 175075

  16. MMP1-1607(1G>2G polymorphism and the risk of lung cancer in Lebanon

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    Hana Fakhoury

    2012-01-01

    Full Text Available Context: Matrix metalloproteinases (MMPs are a family of enzymes that degrade various components of the extracellular matrix and are involved in the development and progression of cancer. Lung cancer is the most commonly diagnosed cancer in Lebanon. MMP1 is responsible for degrading stromal collagens, which enhance the ability of neoplastic cells to cross basal membrane of both the endothelium and the vascular endothelium. A recent meta-analysis has suggested that the MMP1-1607 2G allele may be associated with an increased risk for certain types of cancers. Aim: This study was undertaken to investigate the association between guanine insertion polymorphism in the MMP1 promoter and the susceptibility to lung cancer in the Lebanese population. Settings and Design: This case-control study was conducted on 41 patients with lung cancer and 51 age-matched healthy controls, recruited from different regions of Lebanon. Methods: Cases were histologically confirmed lung cancer patients obtained from different hospitals in Lebanon. Controls were healthy unrelated individuals with no history of cancer or genetic diseases. All subjects were genotyped for MMP1 -1607(1G>2G polymorphism using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP. Results: No statistically significant differences were found when genotype and allele distribution of MMP1 -1607(1G>2G polymorphism were compared between patients with lung cancer and controls [P= 0.6 by chi-squared test on a 3x2 contingency table; allelic P=0.61, OR (95% CI = 1.18 (0.60-2.31]. Conclusion: Our data shows that MMP1 promoter polymorphism is not associated with lung cancer susceptibility in the Lebanese population.

  17. NcoI restriction fragment length polymorphism (RFLP) of the tumour necrosis factor (TNF alpha) region in primary biliary cirrhosis and in healthy Danes

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1989-01-01

    The restriction fragment length polymorphism of the human tumour necrosis factor (TNF alpha) region was investigated by means of 20 different restriction enzymes and a human TNF alpha cDNA probe. Only one of the enzymes, NcoI, revealed a polymorphic pattern consisting of fragments of 10.5 and 5...

  18. NcoI restriction fragment length polymorphism (RFLP) of the tumour necrosis factor (TNF alpha) region in primary biliary cirrhosis and in healthy Danes

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1989-01-01

    The restriction fragment length polymorphism of the human tumour necrosis factor (TNF alpha) region was investigated by means of 20 different restriction enzymes and a human TNF alpha cDNA probe. Only one of the enzymes, NcoI, revealed a polymorphic pattern consisting of fragments of 10.5 and 5.5...

  19. MTHFR A1298C polymorphism and idiopathic male infertility

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    Singh K

    2010-01-01

    Full Text Available Background : DNA methylation is an important epigenetic feature of DNA that plays a pivotal role in gene expression regulation during spermatogenesis. The enzyme methylenetetrahydrofolate reductase (MTHFR catalyses the formation of folate intermediates that are vital for DNA synthesis and methylation reactions. C677T and A1298C variants of MTHFR result in reduced plasma folate and increase the susceptibility to various multifactorial disorders. We have already shown that homozygosity for 677 (C ®T mutation in the MTHFR gene, is a risk factor for idiopathic male infertility in an Indian population. Aim: Recently, we showed that homozygosity for the 677(C;T mutation in the MTHFR gene is a risk factor for idiopathic male infertility and now we aim to assess whether the A1298C mutation in the same gene is an additional risk factor for idiopathic male infertility in an Indian population. Setting and Designs : In a case-control study 151 idiopathic male infertile patients and 140 healthy fertile control individuals were recruited from the University hospital and infertility clinics in Varanasi city, India. Materials and Methods: Genotyping for A1298C change of the MTHFR gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method. Statistical Analysis : Allele frequencies were calculated using Fisher′s exact test. Odds ratio was calculated as the measure of the association between the MTHFR genotype and idiopathic male infertility. Results : The homozygous (C/C A1298C polymorphism of the MTHFR gene was present at a statistically high significance in idiopathic azoospermic infertile men (OR=3.4494, CI: 1.0092 to 11.7899, P<0.05. Conclusion : The MTHFR 1298CC genotype is an additional genetic risk factor for idiopathic male infertility in an Indian population.

  20. MTHFR A1298C polymorphism and idiopathic male infertility.

    Science.gov (United States)

    Singh, K; Singh, S K; Raman, R

    2010-01-01

    DNA methylation is an important epigenetic feature of DNA that plays a pivotal role in gene expression regulation during spermatogenesis. The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyses the formation of folate intermediates that are vital for DNA synthesis and methylation reactions. C677T and A1298C variants of MTHFR result in reduced plasma folate and increase the susceptibility to various multifactorial disorders. We have already shown that homozygosity for 677 (C ®T) mutation in the MTHFR gene, is a risk factor for idiopathic male infertility in an Indian population. Recently, we showed that homozygosity for the 677(C;T) mutation in the MTHFR gene is a risk factor for idiopathic male infertility and now we aim to assess whether the A1298C mutation in the same gene is an additional risk factor for idiopathic male infertility in an Indian population. In a case-control study 151 idiopathic male infertile patients and 140 healthy fertile control individuals were recruited from the University hospital and infertility clinics in Varanasi city, India. Genotyping for A1298C change of the MTHFR gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Statistical Analysis : Allele frequencies were calculated using Fisher's exact test. Odds ratio was calculated as the measure of the association between the MTHFR genotype and idiopathic male infertility. The homozygous (C/C) A1298C polymorphism of the MTHFR gene was present at a statistically high significance in idiopathic azoospermic infertile men (OR=3.4494, CI: 1.0092 to 11.7899, P<0.05). The MTHFR 1298CC genotype is an additional genetic risk factor for idiopathic male infertility in an Indian population.

  1. Restriction fragment length polymorphisms of mitochondrial DNA among five freshwater fish species of the genus Astyanax (Pisces, Characidae

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    Cinthia Bachir Moysés

    2002-01-01

    Full Text Available Restriction fragment length polymorphism (RFLP analysis of mitochondrial DNA (mtDNA was employed to characterize species and populations of Astyanax, a Neotropical freshwater fish genus. Samples of five species, A. altiparanae, A. fasciatus, A. lacustris, A. scabripinnis paranae and A. schubarti, from the Upper Paraná and São Francisco river basins were analyzed. Two out of the ten restriction enzymes employed generated species-specific mtDNA patterns for each of the five species. MtDNA exhibited considerable polymorphism within and among populations. All populations sampled showed relatively high values of haplotype diversity. Geographically localized haplotypes were detected for A. altiparanae and A. fasciatus from the Upper Paraná and São Francisco basins. The relationships between populations are discussed.

  2. Species determination within Staphylococcus genus by extended PCR-restriction fragment length polymorphism of saoC gene.

    Science.gov (United States)

    Bukowski, Michal; Polakowska, Klaudia; Ilczyszyn, Weronika M; Sitarska, Agnieszka; Nytko, Kinga; Kosecka, Maja; Miedzobrodzki, Jacek; Dubin, Adam; Wladyka, Benedykt

    2015-01-01

    Genetic methods based on PCR-restriction fragment length polymorphism (RFLP) are widely used for microbial species determination. In this study, we present the application of saoC gene as an effective tool for species determination and within-species diversity analysis for Staphylococcus genus. The unique sequence diversity of saoC allows us to apply four restriction enzymes to obtain RFLP patterns, which appear highly distinctive even among closely related species as well as atypical isolates of environmental origin. Such patterns were successfully obtained for 26 species belonging to Staphylococcus genus. What is more, tracing polymorphisms detected by different restriction enzymes allowed for basic phylogeny analysis for Staphylococcus aureus, which is potentially applicable for other staphylococcal species. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Genetic analysis of polymorphisms in the kalirin gene for association with age-at-onset in European Huntington disease patients

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    Tsai Yu-Chun

    2012-06-01

    Full Text Available Abstract Background Huntington disease (HD is caused by an expanded CAG repeat in the HD gene. Although the length of the CAG repeat strongly correlates with the age-at-onset (AAO, AAO in HD individuals may differ dramatically in spite of similar expanded CAG repeat lengths. Additional genetic or environmental factors are thought to influence the disease onset. Several modifier genes have been discovered so far but they do not fully explain the variability of AAO in HD. To potentially identify a novel genetic modifier, we analyzed single nucleotide polymorphisms (SNPs in the kalirin (KALRN gene. Kalirin is a protein crucially involved in spine plasticity and its interaction with huntingtin-associated protein-1 (HAP-1 and a potential protein dysfunction might contribute to spine pathogenesis in HD. Methods The selected SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and association of SNPs with AAO was investigated with the framework of linear models in an analysis of variance and covariance. Results Eleven SNPs in the kalirin gene were examined in an association study in European HD patients. The ten coding SNPs under investigation were monomorphic, whereas SNP rs10934657 in the promoter region showed a minor allele frequency >1%. An analysis of covariance together with the influence of the expanded HD allele was applied in 680 HD patients. SNP rs10934657 did not affect the AAO of the examined HD population. Conclusions The results did not reveal an association between the analyzed kalirin polymorphisms and the AAO in HD. However, it does not exclude other SNPs of the kalirin gene as susceptible genetic modifiers.

  4. Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Samah, S; Valadez-Moctezuma, E; Peláez-Luna, K S; Morales-Manzano, S; Meza-Carrera, P; Cid-Contreras, R C

    2016-06-03

    Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.

  5. Mannose binding lectin (MBL) 2 gene polymorphism & its association with clinical manifestations in systemic lupus erythematosus (SLE) patients from western India

    Science.gov (United States)

    Pradhan, Vandana; Surve, Prathamesh; Rajadhyaksha, Anjali; Rajendran, Vinod; Patwardhan, Manisha; Umare, Vinod; Ghosh, Kanjaksha; Nadkarni, Anita

    2015-01-01

    Background & objectives: Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by production of autoantibodies. Mannose binding lectin (MBL) is an important element of the innate defense system. The present study was undertaken to determine whether variant alleles in MBL2 gene were associated with disease severity in SLE patients. Methods: The MBL alleles [-550, -221, +4, Codon 52, Codon 54 and Codon 57] were studied by PCR- RFLP (restriction fragment length polymorphism) method in 100 SLE patients fulfilling ACR (American College of Rheumatology) criteria along with 100 healthy controls. SLE disease activity was evaluated using SLE Disease Activity Index (SLEDAI) score. Results: Homozygosity for MBL variant allele (O/O) was observed in 24 per cent of the SLE patients compared to 16 per cent of the normal controls, while no difference was found for heterozygosity (A/O) (37 vs 35%). A significant difference was reported in incidence of double heterozygosity for mutant allele B and D (B/D) among SLE patients as against control group (P = 0.015). MBL genotypes did not show any association with renal involvement. Interpretation & conclusions: In this study from western India, MBL gene polymorphism showed an influence as a possible risk factor for susceptibility to SLE, but had no direct effect on disease characteristics. Further studies need to be done on a larger number of SLE patients in different regions of the country. PMID:25900955

  6. Prevalence of coagulation factor II G20210A and factor V G1691A Leiden polymorphisms in Chechans, a genetically isolated population in Jordan.

    Science.gov (United States)

    Dajani, Rana; Fatahallah, Raja; Dajani, Abdelrahman; Al-Shboul, Mohammad; Khader, Yousef

    2012-09-01

    Coagulation factor II G20210A and coagulation factor V (Leiden) G1691A single nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufficient information about their distribution among Chechans, we addressed the prevalence of these SNPs in the Chechan population in Jordan, a genetically isolated population. Factor II G20210A and factor V Leiden SNPs were analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method and Amplification refractory mutation detection system (ARMS) respectively in 120 random unrelated subjects from the Chechan population in Jordan. Among the subjects studied for factor II G20210A mutation there were three individuals carrying this mutation as heterozygous (one female and two male), giving a prevalence of 2.5 % and an allele frequency of 1.25 %. No homozygous factor II allele was found. Factor V Leiden G1691A mutation was detected as heterozygous in 22 of 120 of individuals (17 female and five male) indicating a prevalence of 18.3 % and allele frequency of 9.2 %. No homozygous allele was found. Our results indicated that prevalence of factor II G20210A mutation in the Chechan population is similar to prevalence in Jordan and Caucasian populations (1-6 %) while the prevalence of factor V Leiden was higher in the Chechan population compared to Jordan and Caucasian populations (2-15 %).

  7. [Association between interleukin-6 polymorphism in the -174 G/C region and hearing loss in the elderly with a history of occupational noise exposure].

    Science.gov (United States)

    Braga, Miula Portelinha; Maciel, Sandra Mara; Marchiori, Luciana Lozza de Moraes; Poli-Frederico, Regina Célia

    2014-01-01

    The biological processes involved in noise-induced hearing loss (NIHL) are still unclear. The involvement of inflammation in this condition has been suggested. To investigate the association between interleukin - 6 (IL-6) polymorphism and susceptibility to NIHL. This was a cross-sectional study with a sample of 191 independent elderly individuals aged >60 years of age. Information on exposure to occupational noise was obtained by interviews. Audiological evaluation was performed using pure tone audiometry and genotyped through PCR by restriction fragment length polymorphism - PCR-RFLP. Data were analyzed using the chi-square test and the odds ratio (OR), with the significance level set at 5%. Among elderly with hearing loss (78.0%), 18.8% had a history of exposure to occupational noise. There was a statistically significant association between the genotype frequencies of the IL-6 -174 and NIHL. The elderly with the CC genotype were less likely to have hearing loss due to occupational noise exposure when compared to those carrying the GG genotype (OR=0.0124; 95% CI 0.0023-0.0671; phearing loss. Copyright © 2014 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  8. [Association between single nucleotide polymorphism in Wnt3 and nonsyndromic cleft lip with or without cleft palate in Hui and Han population of Ningxia Autonomous Region].

    Science.gov (United States)

    Xin, Yanhua; Ma, Lijuan; Zhai, Kun; Zhou, Zhongwei; Yang, Xiong; Ma, Jian; Wang, Yirui; Zhu, Jinfang; Jiang, Min; Huang, Yongqing

    2013-08-01

    To investigate the association between rs142167, rs7216231 single nucleotide polymorphism (SNP) in Wnt3 and nonsyndromic cleft lip and palate (NSCL/P) in Hui and Han population of Ningxia Autonomous Region. The study consisted of 371 NSCL/P patients from Ningxia Hui and Han population (Han population 166, Hui population 205), their parents (196 fathers, 224 mothers, 150 trios) and 258 normal controls (Han population 190, Hui population 68). Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) was used to identify rs142167, rs7216231 genotypes of the samples. The data was analyzed by case-control analysis, transmission disequilibrium test (TDT) and family based associated test (FBAT). Case-control study showed that no differences in cleft lip, cleft palate, cleft lip and palate, and the total case group compared with the control group at rs142167 and rs7216231 (P > 0.05) in Hui and Han population and in stratified comparison. TDT test showed that rs142167 and rs7216231's allele had not over-transmitted (P > 0.05) in NSCL/P. FBAT test showed that G-G specific haplotypes showed statistically significant (P Hui and Han population.

  9. Do Biochemical Markers and Apa I Polymorphism in IGF-II Gene Play a Role in the Association of Birth Weight and Later BMI?

    Science.gov (United States)

    Wu, Junqing; Ren, Jingchao; Li, Yuyan; Wu, Yinjie; Gao, Ersheng

    2013-01-01

    The aim of the study was to explore the mechanisms underlying the association of birth weight with later body mass index (BMI) from the biochemical markers related to metabolism and the Apa I polymorphism in IGF-II gene. A total of 300 children were selected randomly from the Macrosomia Birth Cohort in Wuxi, China. The height and weight were measured and blood samples were collected. Plasma concentrations of 8 biochemical markers were detected. Apa I polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Biochemical markers were detected for 296 subjects and 271 subjects were genotyped for the Apa I polymorphism. No association was found between birth weight and 8 biochemical markers. In boys, the BMIs of AA, AG and GG genotypes were 16.10 ± 2.24 kg/m(2), 17.40 ± 3.20 kg/m(2), 17.65 ± 2.66 kg/m(2). And there was statistical difference among the three genotypes. But in girls, there was no statistical difference. The birth weights of AA, AG and GG genotypes were 3751.13 ± 492.43 g, 3734.00 ± 456.88 g, 3782.00 ± 461.78 g. And there was no statistical difference among the three genotypes. Biochemical markers are not associated with birth weight. Apa I polymorphism may be related to childhood BMI, but it may be not associated with birth weight. Therefore, biochemical markers and Apa I polymorphism might not play a role in the association of birth weight and BMI.

  10. Significant genotype difference in the CYP2E1 PstI polymorphism of indigenous groups in Sabah, Malaysia with Asian and non-Asian populations.

    Science.gov (United States)

    Goh, Lucky Poh Wah; Chong, Eric Tzyy Jiann; Chua, Kek Heng; Chuah, Jitt Aun; Lee, Ping-Chin

    2014-01-01

    CYP2E1 PstI polymorphism G-1259C (rs3813867) genotype distributions vary significantly among different populations and are associated with both diseases, like cancer, and adverse drug effects. To date, there have been limited genotype distributions and allele frequencies of this polymorphism reported in the three major indigenous ethnic groups (KadazanDusun, Bajau, and Rungus) in Sabah, also known as North Borneo. The aim of this study was to investigate the genotype distributions and allele frequencies of the CYP2E1 PstI polymorphism G-1259C in these three major indigenous peoples in Sabah. A total of 640 healthy individuals from the three dominant indigenous groups were recruited for this study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) at G-1259C polymorphic site of CYP2E1 gene was performed using the Pst I restriction enzyme. Fragments were analyzed using agarose gel electrophoresis and confirmed by direct sequencing. Overall, the allele frequencies were 90.3% for c1 allele and 9.7% for c2 allele. The genotype frequencies for c1/c1, c1/c2 and c2/c2 were observed as 80.9%, 18.8%, and 0.3%, respectively. A highly statistical significant difference (ppopulations. However, among these three indigenous groups, there was no statistical significant difference (p>0.001) in their genotype distributions. The three major indigenous ethnic groups in Sabah show unique genotype distributions when compared with other populations. This finding indicates the importance of establishing the genotype distributions of CYP2E1 PstI polymorphism in the indigenous populations.

  11. Influence of vitamin D receptor polymorphisms on biochemical markers of mineral bone disorders in South African patients with chronic kidney disease.

    Science.gov (United States)

    Waziri, Bala; Dix-Peek, Therese; Dickens, Caroline; Duarte, Raquel; Naicker, Saraladevi

    2018-02-07

    It remains unclear whether genetic factors may explain the reported variation in the levels of biochemical markers of chronic kidney disease mineral and bone disorders (CKD- MBD) across ethnic groups. Therefore, the aim of this study was to examine the influence of vitamin D receptor (VDR) polymorphisms on secondary hyperparathyroidism and its association with vitamin D levels in black and white South African study participants. This was a cross sectional study involving 272 CKD stage 3- 5D patients and 90 healthy controls. The four major VDR polymorphisms (Bsm 1, Fok 1, Taq 1, and Apa1) were genotyped using the polymerase chain reaction- restriction fragment length polymorphism (PCR -RFLP) method. In addition, biochemical markers of CKD-MBD were measured to determine their associations with the four VDR polymorphisms. With the exception of Taq I polymorphism, the distribution of the VDR polymorphisms differed significantly between blacks and whites. In hemodialysis patients, the Bb genotype was significantly associated with moderate secondary hyperparathyroidism (OR, 3.88; 95 CI 1.13-13.25, p = 0.03) and severe hyperparathyroidism (OR, 2.54; 95 CI 1.08-5.96, p = 0.03). This was consistent with the observed higher levels of median parathyroid hormone, fibroblast growth factor 23 and mean phosphate in patients with Bb genotype. This candidate risk genotype (Bb) was over represented in blacks compared to whites (71.0% versus 55.6%, p kidney disease. In addition, study participants with FokFf genotype are at increased of developing severe 25 -hydroxyvitamin D [25(OH)D] deficiency.

  12. Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism

    DEFF Research Database (Denmark)

    Torpdahl, Mia; Skov, Marianne N.; Sandvang, Dorthe

    2005-01-01

    subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length...... polymorphism (AFLP). When making genetic trees, all three methods resulted in similar clustering that often corresponded with serotype, although some serotypes displayed more diversity than others. Of the three techniques, MLST was the easiest to interpret and compare between laboratories. Unfortunately...

  13. Involvement of different mechanisms for the association of CAG repeat length polymorphism in androgen receptor gene with prostate cancer

    Science.gov (United States)

    Mao, Xueying; Li, Jie; Xu, Xingxing; Boyd, Lara K; He, Weiyang; Stankiewicz, Elzbieta; Kudahetti, Sakunthala C; Cao, Guangwen; Berney, Daniel; Ren, Guosheng; Gou, Xin; Zhang, Hongwei; Lu, Yong-Jie

    2014-01-01

    While androgen and androgen receptor (AR) activity have been strongly implicated in prostate cancer development and therapy, the influence of the CAG repeat, which is found within the first exon of the AR gene, on prostate carcinogenesis is still unclear. We investigated the differences in the length of the CAG repeat between prostate cancer patients and controls in the Chinese population as well as between TMPRSS2:ERG fusion positive and negative samples. A general association between prostate cancer and either longer or shorter AR CAG repeat length was not observed in the Chinese population. However, our data suggest that certain CAG repeat lengths may increase or decrease prostate cancer risk. Shorter CAG repeat length was also not shown to be associated with a higher induction rate of TMPRSS2 and ERG proximity, an essential step for TMPRSS2:ERG fusion formation. However, samples with a CAG repeat of 17 were found more frequently in the TMPRSS2:ERG fusion positive than negative prostate cancer cases and mediated a higher rate of androgen-induced TMPRSS2 and ERG co-localisation than AR with longer (24) and shorter (15) CAG repeats. This suggests that 17 CAG repeats may be associated with TMPRSS2:ERG fusion positive prostate cancer, but may have a preventive role for prostate cancer in the Chinese population, which has a low TMPRSS2:ERG fusion frequency. This study suggests that different mechanisms for the association of CAG repeat length polymorphism and prostate cancer exist in different ethnic populations. PMID:25520876

  14. Amyloid structure exhibits polymorphism on multiple length scales in human brain tissue

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jiliang; Costantino, Isabel; Venugopalan, Nagarajan; Fischetti, Robert F.; Hyman, Bradley; Frosch, Matthew; Gomez-Isla, Teresa; Makowski, Lee

    2016-09-15

    Although aggregation of Aβ amyloid fibrils into plaques in the brain is a hallmark of Alzheimer's Disease (AD), the correlation between amyloid burden and severity of symptoms is weak. One possible reason is that amyloid fibrils are structurally polymorphic and different polymorphs may contribute differentially to disease. However, the occurrence and distribution of amyloid polymorphisms in human brain is poorly documented. Here we seek to fill this knowledge gap by using X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid within individual plaques; among proximal plaques and in subjects with distinct clinical histories. A 5 µ x-ray beam was used to generate diffraction data with each pattern arising from a scattering volume of only ~ 450 µ3 , making possible collection of dozens to hundreds of diffraction patterns from a single amyloid plaque. X-ray scattering from these samples exhibited all the properties expected for scattering from amyloid. Amyloid distribution was mapped using the intensity of its signature 4.7 Å reflection which also provided information on the orientation of amyloid fibrils across plaques. Margins of plaques exhibited a greater degree of orientation than cores and orientation around blood vessels frequently appeared tangential. Variation in the structure of Aβ fibrils is reflected in the shape of the 4.7 Å peak which usually appears as a doublet. Variations in this peak correspond to differences between the structure of amyloid within cores of plaques and at their periphery. Examination of tissue from a mismatch case - an individual with high plaque burden but no overt signs of dementia at time of death - revealed a diversity of structure and spatial distribution of amyloid that is distinct from typical AD cases. We demonstrate the existence of structural polymorphisms among amyloid within and among plaques of a single individual and suggest

  15. Restriction fragment length polymorphism within the class I gene loci of the equine major histocompatibility complex

    International Nuclear Information System (INIS)

    Alexander, A.J.; Bailey, E.; Woodward, J.G.

    1986-01-01

    Fourteen standard bred horses were serotyped as homozygous for 1 of 6 Equine Leukocyte Antigen (ELA) specificities. DNA was purified from peripheral leukocytes and digested with Hind III or Pvu II. Southern blot hybridization analysis was carried out using a 32 P-labeled mouse cDNA probe (PH2IIa) specific for class I MHC genes. Both enzymes generated blots that contained a large number of bands (23 to 30) per horse. Significant polymorphism existed among most fragment sizes, while a dozen highly conserved band sizes suggested the presence of Qa/tla - like genes. Only 2 animals (both W6's) showed identical band patterns. Polymorphism was greatest between horses of different serotypes and was significantly decreased within serotypes. Unique bands were present on both blots for both W1's and W6's and may account for the serologic specificity seen in ELA W1 and W6 horses. This study is consistent with the findings in other higher vertebrates and implies that the MHC of the horse includes a highly polymorphic class I multigene family

  16. Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using restriction fragment length polymorphism.

    Science.gov (United States)

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

  17. Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQwl and DQw3 alleles of the HLA-D region

    International Nuclear Information System (INIS)

    Szafer, F.; Brautbar, C.; Tzfoni, E.

    1987-01-01

    Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQβ, DQα, and DRβ cDNA probes. Hybridization with the DQβ probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQα and DRβ probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease

  18. Expression and Association of SCD Gene Polymorphisms and Fatty Acid Compositions in Chicken Cross

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    A. Furqon

    2017-12-01

    Full Text Available Stearoyl-CoA desaturase (SCD is an integral membrane protein of endoplasmic reticulum (ER that catalyzes the rate limiting step in the monounsaturated fatty acids from saturated fatty acids. Selection for fatty acids traits based on molecular marker assisted selection is needed to increase a value of chicken meat. This study was designed to analyze expression and associations of SCD gene polymorphisms with fatty acid traits in F2 kampung-broiler chicken cross. A total of 62 F2 kampung-broiler chicken cross (29 males and 33 females were used in this study. Fatty acid traits were measured at 26 weeks of age. Samples were divided into two groups based on fatty acid traits (the highest and the lowest. Primers in exon 2 region were designed from the genomic chicken sequence. The SNP g.37284A>G was detected and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method was then used to genotype. The expression of SCD gene was analyzed using quantitative real time PCR (qRT-PCR. The result showed that there were three genotypes (AA, AG, and GG found in this study. The SCD|AciI polymorphism was significantly associated with palmitoleic acid (C16:1, fatty acids total and saturated fatty acid in 26 weeks old of F2 kampung-broiler chicken cross (P<0.05. The SCD gene was expressed for polyunsaturated fatty acids in liver tissue in two groups of chickens. In conclusion, the SCD gene could be a candidate gene that affects fatty acids traits in F2 kampung-broiler chicken cross.

  19. Screening of polymorphisms for MTHFR and DHFR genes in spina bifida children and their mothers

    Science.gov (United States)

    Husna, M. Z.; Endom, I.; Ibrahim, S.; Selvi, N. Amaramalar; Fakhrurazi, H.; Htwe, R. Ohnmar; Kanehaswari, Y.; Halim, A. R. Abdul; Wong, S. W.; Subashini, K.; Syahira, O. Nur; Aishah, S.

    2013-11-01

    Mechanism underlying the beneficial effect of folic acid supplementation in reducing the risk of neural tube defect is still not well understood. Current evidences show the involvement of folic acid metabolic gene's polymorphism as contributing factors that regulate this pathway. Therefore, the objective of this research was to determine the presence of C677T polymorphism for methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR-19 bp deletion) genes between mother-children pairs of case and control. With the approval of UKMMC ethic committee, genomic DNA was extracted from one hundred and forty consented bloods. Polymerase chain reaction (PCR), PCR-RFLP (Restriction Fragment Length Polymorphism) and sequencing were employed to verify each nucleotide change. Our result shows that mutant MTHFR and DHFR alleles are present in all Malaysian sub-ethnic groups, case and control. Even though mutant MTHFR are found to be slightly higher in the case groups, 75% of the affected child is a non carrier for this allele and 62.5% of the mothers with an affected child are genotypically normal. For DHFR, almost all (87.5-100%) investigated samples are a carrier or having a double DHFR deletion be it a case or control pairs. However, strong maternal inheritance shown by the deleted allele might be due to a cascade effect of lacks of folate consumption or maternal uniparental disomy. In conclusion, the use of MTHFR and DHFR as markers in determining the risk of having spina bifida baby is uninformative and plays a small indirect role as the genetic causes of spina bifida. Therefore, spina bifida remains etiologically unknown polygenic and quantitative developmental trait whereby the searches for positive genetic marker need to be continued.

  20. Analysis of the population structure of Uruguayan Creole cattle as inferred from milk major gene polymorphisms

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    Gonzalo Rincón

    2006-01-01

    Full Text Available The ancestors of Uruguayan Creole cattle were introduced by the Spanish conquerors in the XVII century, following which the population grew extensively and became semi-feral before the introduction of selected breeds. Today the Uruguayan Creole cattle genetic reserve consists of 575 animals. We used the tetra primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR to analyze the kappa-casein, beta-casein, alphaS1-casein and alpha-lactoalbumin gene polymorphisms and restriction fragment length polymorphism PCR (RFLP-PCR for the beta-lactoglobulin and the acylCoA:diacyl glycerol acyltransferase 1 (DGAT1 genes. The kappa-casein and beta-lactoglobulin genes presented very similar A and B allele frequencies, while the alphas1-casein and alpha-lactoalbumin gene B alleles showed much higher frequencies than the corresponding A alleles. The beta-casein B allele was not found in the population sampled. There was a very high frequency of the DGAT1 gene A allele which is associated with low milk fat content and high milk yield. All loci were in Hardy-Weinberg equilibrium and the level of heterozygosity agreed with the high genetic diversity observed in a previous analysis of this population. Preservation of the allelic richness observed in the Uruguayan Creole cattle should be considered for future dairy management and livestock genetic improvement. The results also emphasize the value of the tetra primers ARMS-PCR technique as a rapid, easy and economical way of genotyping cattle breeds for milk gene single nucleotide polymorphisms.

  1. Interaction effect between the BDNF Val66Met polymorphism and parental rearing for interpersonal sensitivity in healthy subjects.

    Science.gov (United States)

    Suzuki, Akihito; Matsumoto, Yoshihiko; Shibuya, Naoshi; Ryoichi, Sadahiro; Kamata, Mitsuhiro; Enokido, Masanori; Goto, Kaoru; Otani, Koichi

    2012-12-30

    Interpersonal sensitivity is defined as undue and excessive awareness of, and sensitivity to, the behaviour and feelings of others and is one of the vulnerable factors to depression. In a twin study, it was suggested that this personality trait was characterised by both genetic and environmental factors. In the present study, we examined the effects of the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism and parental rearing on interpersonal sensitivity in 725 healthy Japanese subjects. Assessment of interpersonal sensitivity was performed by the Japanese version of the Interpersonal Sensitivity Measure (IPSM). Perceived parental rearing was assessed by the Parental Bonding Instrument (PBI), which consists of the care and protection factors. The BDNF polymorphism was detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. There was no main effect of the BDNF genotype on the IPSM score, while the PBI factors except maternal care had significant main effect on the IPSM score. There was significant interaction effect between the BDNF genotype and maternal care of the PBI on the IPSM score. Post-hoc analysis of simple slopes showed that the negative relationship between the IPSM score and maternal care was strongest and significant in the Met/Met genotype group, intermediate in the Val/Met genotype group and weakest in the Val/Val genotype group. The present study suggests that the interaction between the BDNF Val66Met polymorphism and parental rearing, especially maternal care, influences interpersonal sensitivity in healthy subjects. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. Vascular endothelial growth factor polymorphisms and a synchronized examination of plasma and tissue expression in epithelial ovarian cancers.

    Science.gov (United States)

    Bhaskari, J; Premalata, C S; Shilpa, V; Rahul, B; Pallavi, V R; Ramesh, G; Krishnamoorthy, Lakshmi

    2016-01-01

    In this study, we have analyzed six genetic polymorphisms of the VEGF-A gene and correlated the genetic data with plasma and tissue expression of VEGF-A in epithelial ovarian carcinomas. A total of 130 cases including 95 malignant carcinomas, 17 low malignant potential and 18 benign tumours were studied. rs699947, rs833061, rs1570360, rs2010963, rs1413711 and rs3025039 were studied by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma levels of VEGF-A were estimated by enzyme-linked immunosorbent assay (ELISA) and tissue expression of VEGF-A by immunohistochemistry (IHC). Four polymorphisms of the above excluding rs699947 and rs3025039 showed significant association with malignancy, and we observed the presence of positive correlation between haplotype CCGGCC and increased expression of VEGF-A in both plasma and tissues which also correlated with poor prognosis and recurrence suggesting a probable increase in resistance to treatment in such carriers. Highly upregulated tissue expression of VEGF-A was seen in all epithelial ovarian carcinomas with intensity of expression increasing from benign to malignant cases. ELISA data from our study showed an increase in circulating levels of VEGF-A in malignancies. VEGF-A plasma levels can be employed as a biomarker for high-grade malignancy in epithelial ovarian cancers alongside tissue expression and CA-125 levels. This study is unique due to the fact that a simultaneous analysis of plasma and tissue expression has been demonstrated and is a first such study in epithelial ovarian cancers and representing the Indian population (South-east Asian) synchronized with genetic polymorphism data as well.

  3. Effect of Cytochrome P450 2C19 681G>A Polymorphism on Premature Coronary Heart Disease

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    Zhong-hai Chi

    2016-03-01

    Full Text Available Objective: The aim of our study was to evaluate the association between cytochrome P450 2C19 (CYP2C19 681G>A polymorphisms and the age of development of coronary heart disease. Additionally, the study might find some biological indicators at the gene level that could help to predict and evaluate the risk of coronary heart disease in time. Methods: This study included 352 individuals with coronary heart disease. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP was used to identify CYP2C19 681G>A. The objects were divided into wild type (GG or homozygous CYP2C19*1 wild-type and mutant type (GA/AA or the mutant CYP2C19*2 allele on the basis of genotype. The association between CYP2C19 gene polymorphism and the age of onset of coronary heart disease was assessed by multivariate linear regression analysis. Results: There was a significant association in the age of onset between the two groups (t=3.398, P=0.001, which was 58.51+12.72 years in the wild type and 53.95+11.63 years in the mutant group. The frequency of CYP2C19 681A (CYP2C19*2 allele was 3.32, 0.268, 0.227 in different age groups, which was significantly different (χ2=10.745, P=0.005 in different groups, as well as in the genotype. The result, assessed by multiple linear regression, showed that genotype, smoking, obesity, and hyperlipidemia affect the age of onset of coronary heart disease (β was -0.167, 0.156, 0.155, and 0.112, PA gene polymorphism may be one of the risk factors in susceptibility to early onset of coronary heart disease, but not an independent factor because other factors may play a synergistic role.

  4. Association of HSP70-2 Gene 1267A/G Polymorphism With Cataract Incidence Among Guilan Population

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    Zivar Salehi

    2017-01-01

    Full Text Available Background: Cataract is a visible opacity of the eye lens and it is the main reason of reversible blindness in the world. Oxidative stress is known as a major factor in cataract formation HSP70-2 protein is a molecular chaperone which is essential for cell survival in stress conditions. HSP70-2 gene is located in the human leukocyte antigen class ΙΙΙ region. This gene encodes an inducible protein. One of the common polymorphism of HSP70-2 is 1267A/G which is located in coding region. The aim of this study was to analysis of 1267A/G polymorphism of hsp70-2 gene in cataract patients. Material and Methods: This case-control study included 118 cataract patients and 122 healthy people as a control groups. Genomic DNA was extracted from peripheral blood leukocytes and genotyping determination was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method. Statistical analysis was performed by using the MedCalc software (Version 12.1. Results: The frequency of G allele was significantly higher in patients than the controls, (0.36 and 0.24, respectively. A higher frequency of the GG genotype of the HSP70-2 1267A/G polymorphism was found in the patients compared with controls (21.19% and 8.20%, respectively. The patients carrying the GG genotype were 3.2-fold at a higher risk of cataract compared with AA genotype (P=0.005. Conclusion: The finding of this study suggested that the HSP70-2 1267A/G may affect the susceptibility to cataract in the studied population. Anyway the randomized multicenter studies with greater sample size still need to confirmed our results.

  5. Genetic Polymorphism of the Lactoferrin Gene in Dairy and Beef Cattles at National Artificial Insemination and Embryo Transfer Stations

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    Anneke Anggraeni

    2016-12-01

    Full Text Available Lactoferrin (LTF adalah gen pengontrol komponen protein susu dan memiliki karakteristik sebagai antimikrobial. LTF pada susu berfungsi untuk mencegah diare, sedangkan pada sapi laktasi untuk mencegah mastitis pada ambing. Mempertimbangkan peran penting dari gen LTF, maka perlu dilakukan peningkatan kadar LTF dalam susu melalui seleksi pada taraf DNA. Polymorfisme genetik dari gen LTF diidentifikasi pada sapi perah dan potong dengan metoda Polymerase Chain Reaction - Restricsion Fragment Length Polymorphism (PCR-RFLP, dengan enzim restriksi EcoRI. Genotyping dilakukan pada sapi perah Friesian Holstein (FH total sejumlah 89 ekor, meliputi dari Balai Inseminasi Buatan Lembang (BIB Lembang untuk 17 pejantan, Balai Besar IB Singosari (BBIB Singosari untuk 32 pejantan, dan Balai embrio Transfer Cipelang (BET Cipelang pada 40 dara. Genotyping dilakukan pula pada sapi potong dara berasal dari empat bangsa, meliputi Limousin (14 ekor, Angus (5 ekor, Simmental (13 ekor dan Brahman (5 ekor dari BET Cipelang. Gen LTF|EcoRI pada sapi perah dan potong pengamatan menghasilkan dua tipe alel, yaitu alel A dan B. Kedua jenis sapi tersebut menghasilkan hanya dua genotipe, yaitu genotipe AA dan AB, tanpa genotipe BB. Ini dapat menjadi hal yang baik karena genotipe AA dan AB dipertimbangkan berasosiasi dengan ketahan pada mastitis. Nilai-nilai dari heterozygositas observasi (Ho dari gen ini lebih tinggi dibandingkan heterozigositas ekspektasi (He. Disimpulkan bahwa gen LTF|EcoRI memiliki variasi yang baik pada sapi perah dan sapi potong dari ketiga balai bibit nasional tersebut.

  6. GENETIC POLYMORPHISM AT THE K-CASEIN LOCUS IN A DAIRY HERD OF ROMANIAN SPOTTED AND BROWN OF MARAMURES BREEDS

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    ILIE DANIELA

    2007-01-01

    Full Text Available Caseins are a family of milk proteins that exist in several molecular forms and arethe main proteins present in the bovine milk. Genetic variation of these proteins hasbeen associated with the quality and quantity of cheese derived from milk.This study was focused on possibilities to evaluate the frequency of the K-casein Ballele in dairy herds from the Research and Development Station for Bovine RaisingArad in order to have breeding programs that target an increase in the frequency ofthe B allele in the dairy cattle population.In order to differentiate the favorable genotype for superior composition and highercheese yield, we used simple DNA extraction method from fresh blood andtechniques based on DNA analysis, which include polymerase chain reaction andrestriction fragment length polymorphisms (PCR-RFLP methods. Employing thesetechniques we were able to determine the k-casein genotype of all individuals in agiven population under selection, regardless of sex, age or physiological stage.As a result, it is now possible to include information on milk protein genotypes intomarker assisted selection programs and consequently improve response to selection.

  7. GENETIC POLYMORPHISM AT THE K-CASEIN LOCUS IN A DAIRY HERD OF ROMANIAN SPOTTED AND BROWN OF MARAMURES BREEDS

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    DANIELA ILIE

    2007-05-01

    Full Text Available Caseins are a family of milk proteins that exist in several molecular forms and arethe main proteins present in the bovine milk. Genetic variation of these proteins hasbeen associated with the quality and quantity of cheese derived from milk.This study was focused on possibilities to evaluate the frequency of the K-casein Ballele in dairy herds from the Research and Development Station for Bovine RaisingArad in order to have breeding programs that target an increase in the frequency ofthe B allele in the dairy cattle population.In order to differentiate the favorable genotype for superior composition and highercheese yield, we used simple DNA extraction method from fresh blood andtechniques based on DNA analysis, which include polymerase chain reaction andrestriction fragment length polymorphisms (PCR-RFLP methods. Employing thesetechniques we were able to determine the k-casein genotype of all individuals in agiven population under selection, regardless of sex, age or physiological stage.As a result, it is now possible to include information on milk protein genotypes intomarker assisted selection programs and consequently improve response to selection.

  8. Cytotoxic T-lymphocyte antigen-4 gene polymorphisms and human T-cell lymphotrophic virus-1 infection: their associations with Hashimoto's thyroiditis in Japanese patients.

    Science.gov (United States)

    Tomoyose, Takeaki; Komiya, Ichiro; Takara, Masaki; Yabiku, Kouichi; Kinjo, Yoshino; Shimajiri, Yoshinori; Yogi, Hiroyuki; Kouki, Tsuyoshi; Masuda, Masato; Takasu, Nobuyuki

    2002-08-01

    Cytotoxic T-lymphocyte antigen-4 (CTLA-4) decreases the immune response of T cells by inactivating the signal that occurs with interaction between CD28 on T cells and B7 on antigen-presenting cells. Gene polymorphisms involving CTLA-4 promoter (-318 C/T), exon 1 (49 A/G), and exon 4 (microsatellite (AT)n) have been linked to Hashimoto's thyroiditis (HT) and other autoimmune diseases. HT also has a reported association with human T-cell lymphotrophic virus-1 (HTLV-1) infection. We investigated the occurrence of CTLA-4 polymorphisms in Japanese patients with HT with and without anti-HTLV-1 antibodies (HTLV-1 Ab). DNA samples from 143 patients with HT and 199 controls were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using the restriction enzymes, Bbv 1, Tse 1, and Mse 1. In the HTLV-1 Ab-positive group the exon 1 G allele was more frequent in patients with HT than in controls (67% vs. 53%, p = 0.0377), and in HTLV-1 Ab-negative group it was also frequent in patients with HT than in controls (68% vs. 53%, p = 0.0041). Frequency of the G allele in HT with HTLV-1 Ab was comparable to those without HTLV-1 Ab. Frequency of polymorphism in the promoter did not differ between patients with HT and controls, nor between controls with and without HTLV-1 Ab. HTLV-1 infection is not associated with CTLA-4 polymorphisms in either HT or controls. HTLV-1 infection is not regulated by genetic factor such as CTLA-4, and may affect occurrence of HT as an independent purely environmental factor.

  9. Analysis of the Relationship between CGB5 155G/C Polymorphism and in vitro Fertilization-embryo Transfer Outcome (IVF-ET in the Iranian Population

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    Bahareh Babaei Houlari

    2018-01-01

    Full Text Available Abstract Background: Successful pregnancy depends on the ability of the embryo to achieve appropriate extent of trophoblastic proliferation and invasion into maternal endometrium as well as, once implanted, to induce its own blood supply. Beta Human chorionic gonadotropin (β-hCG, enhances blastocyst implantation, uterine vascularization, and angiogenesis, as well as regulates maintenance of uterine quiescence and immunological adaptation during pregnancy. The β-subunit of hCG is encoded by CGB3, CGB6, CGB5, CGB7 and CGB8 genes. The aim of this study was to evaluate the association of CGB5-G/C polymorphism and the clinical outcomes in women who underwent IVF-ET procedures. Materials and Methods: A total of 200 patients undergoing IVF-ET (100 patients with positive and 100 patients with negative IVF-ET outcome were included in this study. Genotyping of CGB5 at -155G/C polymorphic site was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. Statistical analysis was performed using the MedCalc software. Results: Our findings show that the CC genotype of the CGB5 -155G/C polymorphism is associated with decreased risk of IVF-ET failure (OR=0.29; 95%CI=0.1-0.85; p=0.02. However, the allelic distribution of the CGB5 -155G/C is not significantly different between two groups (χ2=1.46; p=0.22. Conclusion: The results of this study suggested that CGB5 (-155G/C CC genotype has a protective effect on IVF-ET outcome. More studies with larger sample sizes on different populations are necessary to elucidate the underlying mechanisms which can explain the associations found between the GGB5 gene polymorphisms and IVF-ET outcome.

  10. The Role of Genetic Polymorphisms as Related to One-Carbon Metabolism, Vitamin B6, and Gene–Nutrient Interactions in Maintaining Genomic Stability and Cell Viability in Chinese Breast Cancer Patients

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    Xiayu Wu

    2016-06-01

    Full Text Available Folate-mediated one-carbon metabolism (FMOCM is linked to DNA synthesis, methylation, and cell proliferation. Vitamin B6 (B6 is a cofactor, and genetic polymorphisms of related key enzymes, such as serine hydroxymethyltransferase (SHMT, methionine synthase reductase (MTRR, and methionine synthase (MS, in FMOCM may govern the bioavailability of metabolites and play important roles in the maintenance of genomic stability and cell viability (GSACV. To evaluate the influences of B6, genetic polymorphisms of these enzymes, and gene–nutrient interactions on GSACV, we utilized the cytokinesis-block micronucleus assay (CBMN and PCR-restriction fragment length polymorphism (PCR-RFLP techniques in the lymphocytes from female breast cancer cases and controls. GSACV showed a significantly positive correlation with B6 concentration, and 48 nmol/L of B6 was the most suitable concentration for maintaining GSACV in vitro. The GSACV indexes showed significantly different sensitivity to B6 deficiency between cases and controls; the B6 effect on the GSACV variance contribution of each index was significantly higher than that of genetic polymorphisms and the sample state (tumor state. SHMT C1420T mutations may reduce breast cancer susceptibility, whereas MTRR A66G and MS A2756G mutations may increase breast cancer susceptibility. The role of SHMT, MS, and MTRR genotype polymorphisms in GSACV is reduced compared with that of B6. The results appear to suggest that the long-term lack of B6 under these conditions may increase genetic damage and cell injury and that individuals with various genotypes have different sensitivities to B6 deficiency. FMOCM metabolic enzyme gene polymorphism may be related to breast cancer susceptibility to a certain extent due to the effect of other factors such as stress, hormones, cancer therapies, psychological conditions, and diet. Adequate B6 intake may be good for maintaining genome health and preventing breast cancer.

  11. Single Cystosorus Isolate Production and Restriction Fragment Length Polymorphism Characterization of the Obligate Biotroph Spongospora subterranea f. sp. subterranea.

    Science.gov (United States)

    Qu, Xinshun; Christ, Barbara J

    2006-10-01

    ABSTRACT Spongospora subterranea f. sp. subterranea causes powdery scab in potatoes and is distributed worldwide. Genetic studies of this pathogen have been hampered due, in part, to its obligate parasitism and the lack of molecular markers for this pathogen. In this investigation, a single cystosorus inoculation technique was developed to produce large amounts of S. subterranea f. sp. subterranea plasmodia or zoosporangia in eastern black nightshade (Solanum ptycanthum) roots from which DNA was extracted. Cryopreservation of zoosporangia was used for long-term storage of the isolates. S. subterranea f. sp. subterranea-specific restriction fragment length polymorphism (RFLP) markers were developed from randomly amplified polymorphic DNA (RAPD) fragments. Cystosori of S. subterranea f. sp. subterranea were used for RAPD assays and putative pathogen-specific RAPD fragments were cloned and sequenced. The fragments were screened for specificity by Southern hybridization and subsequent DNA sequence BLAST search. Four polymorphic S. subterranea f. sp. subterranea-specific probes containing repetitive elements, and one containing single copy DNA were identified. These RFLP probes were then used to analyze 24 single cystosorus isolates derived from eight geographic locations in the United States and Canada. Genetic variation was recorded among, but not within, geographic locations. Cluster analysis separated the isolates into two major groups: group I included isolates originating from western North America, with the exception of those from Colorado, and group II included isolates originating from eastern North America and from Colorado. The techniques developed in this study, i.e., production of single cystosorus isolates of S. subterranea f. sp. subterranea and development of RFLP markers for this pathogen, provide methods to further study the genetic structure of S. subterranea f. sp. subterranea.

  12. Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors

    International Nuclear Information System (INIS)

    Tsavaler, L.; Penhallow, R.C.; Kam, W.; Sussman, H.H.

    1987-01-01

    The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I Band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, the authors examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia

  13. Genetic differentiation of Octopus minor (Mollusca, Cephalopoda) off the northern coast of China as revealed by amplified fragment length polymorphisms.

    Science.gov (United States)

    Yang, J M; Sun, G H; Zheng, X D; Ren, L H; Wang, W J; Li, G R; Sun, B C

    2015-12-02

    Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment length polymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P octopus fisheries, so that this marine resource can be conserved for its long-term utilization.

  14. Validation of Simple Sequence Length Polymorphism Regions of Commonly Used Mouse Strains for Marker Assisted Speed Congenics Screening

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    Channabasavaiah B. Gurumurthy

    2015-01-01

    Full Text Available Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence length polymorphism (SSLP markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

  15. Validation of simple sequence length polymorphism regions of commonly used mouse strains for marker assisted speed congenics screening.

    Science.gov (United States)

    Gurumurthy, Channabasavaiah B; Joshi, Poonam S; Kurz, Scott G; Ohtsuka, Masato; Quadros, Rolen M; Harms, Donald W; Lloyd, K C Kent

    2015-01-01

    Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence length polymorphism (SSLP) markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP) array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

  16. Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from 18S ribosomal RNA gene of Trypanosoma congolense

    International Nuclear Information System (INIS)

    Osanyo, A.; Majiwa, P.W.

    2006-01-01

    Oligonucleotide primers were designed from the conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of protozoans: Trypanosoma brucei, Leishmania donovani, Triponema aequale and Lagenidium gigantum. The primers were used in polymerace chain reaction (PCR) to generate PCR products of approximately 1 Kb using genomic DNA from T. brucei and the four genotypic groups of T. congolense as template. The five PCR products so produced were digested with several restriction enzymes and hybridized to a DNA probe made from T. brucei PCR product of the same 18S rRNA gene region. Most restriction enzyme digests revealed polymorphism with respect to the location of their recognition sites on the five PCR products. The restriction fragment length polymorphism (RFLP) pattern observed indicate that the 18S rRNA gene sequences of trypanosomes: T. brucei and the four genotypes of T.congolence group are heterogeneous. The results further demonstrate that the region that was amplified can be used in specific identification of trypanosomes species and subspecies.(author)

  17. Haplotyping the human T-cell receptor β-chain gene complex by use of restriction fragment length polymorphisms

    International Nuclear Information System (INIS)

    Charmley, P.; Chao, A.; Gatti, R.A.; Concannon, P.; Hood, L.

    1990-01-01

    The authors have studied the genetic segregation of human T-cell receptor β-chain (TCRβ) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). They constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCRβ gene complex. Analysis of allele distributions between TCRβ genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequilibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCRβ gene complex. The results should provide new insight into recent reports of disease associations with the TCRβ gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome

  18. A set of highly informative rat simple sequence length polymorphism (SSLP markers and genetically defined rat strains

    Directory of Open Access Journals (Sweden)

    Yamasaki Ken-ichi

    2006-04-01

    Full Text Available Abstract Background The National Bio Resource Project for the Rat in Japan (NBRP-Rat is focusing on collecting, preserving and distributing various rat strains, including spontaneous mutant, transgenic, congenic, and recombinant inbred (RI strains. To evaluate their value as models of human diseases, we are characterizing them using 109 phenotypic parameters, such as clinical measurements, internal anatomy, metabolic parameters, and behavioral tests, as part of the Rat Phenome Project. Here, we report on a set of 357 simple sequence length polymorphism (SSLP markers and 122 rat strains, which were genotyped by the marker set. Results The SSLP markers were selected according to their distribution patterns throughout the whole rat genome with an average spacing of 7.59 Mb. The average number of informative markers between all possible pairs of strains was 259 (72.5% of 357 markers, showing their high degree of polymorphism. From the genetic profile of these rat inbred strains, we constructed a rat family tree to clarify their genetic background. Conclusion These highly informative SSLP markers as well as genetically and phenotypically defined rat strains are useful for designing experiments for quantitative trait loci (QTL analysis and to choose strategies for developing new genetic resources. The data and resources are freely available at the NBRP-Rat web site 1.

  19. Association of Per3 length polymorphism with bipolar I disorder and schizophrenia

    Directory of Open Access Journals (Sweden)

    Karthikeyan R

    2014-12-01

    Full Text Available Ramanujam Karthikeyan,1 Ganapathy Marimuthu,1 Chellamuthu Ramasubramanian,2 Gautham Arunachal,2 Ahmed S BaHammam,3 David Warren Spence,4 Daniel P Cardinali,5 Gregory M Brown,6 Seithikurippu R Pandi-Perumal7 1Department of Animal Behaviour and Physiology, School of Biological Sciences, Madurai Kamaraj University, Madurai, India; 2MS Chellamuthu Trust and Research Foundation, KK Nagar, Madurai, India; 3University Sleep Disorders Center, College of Medicine, National Plan for Science and Technology, King Saud University, Riyadh, Saudi Arabia; 4Independent researcher, Toronto, Ontario, Canada; 5Department of Teaching and Research, Faculty of Medical Sciences, Pontificia Universidad Católica Argentina, Buenos Aires, Argentina; 6Centre for Addiction and Mental Health, University of Toronto, Toronto, Ontario, Canada; 7Center for Healthful Behavior Change (CHBC, Division of Health and Behavior, Department of Population Health, NYU Langone Medical Center, Clinical and Translational Research Institute, New York, New York, USA Background: Sleep–wake disturbances have frequently been reported in bipolar disorder and schizophrenia, and are considered to be caused by an underlying circadian rhythm disorder. The study presented here was designed to investigate the existence of Per3 polymorphism in bipolar disorder type I (BD-I and schizophrenic patients in South India.Methods: Blood samples were collected from 311 BD-I patients, 293 schizophrenia patients, and 346 age- and sex-matched normal controls. Per3 genotyping was performed on DNA by polymerase chain reaction using specific primers.Results: An increased prevalence of five repeat homozygotes was seen in BD-I patients as compared with healthy controls (odds ratio =1.72 [95% confidence interval: 1.08–2.76, P=0.02]. In BD-I patients, the frequency of the five repeat allele was higher (allele frequency =0.41, and that of the four repeat allele lower (allele frequency =0.36 (χ2=4.634; P<0.03 than in

  20. A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification.

    Science.gov (United States)

    Szemiako, Kasjan; Śledzińska, Anna; Krawczyk, Beata

    2017-08-01

    Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.

  1. Differentiation of mixed lactic acid bacteria communities in beverage fermentations using targeted terminal restriction fragment length polymorphism.

    Science.gov (United States)

    Bokulich, Nicholas A; Mills, David A

    2012-08-01

    Lactic acid bacteria (LAB) are an important group of bacteria in beer and wine fermentations both as beneficial organisms and as spoilage agents. However, sensitive, rapid, culture-independent methods for identification and community analyses of LAB in mixed-culture fermentations are limited. We developed a terminal restriction fragment length polymorphism (TRFLP)-based assay for the detection and identification of lactic acid bacteria and Bacilli during wine, beer, and food fermentations. This technique can sensitively discriminate most species of Lactobacillales, and most genera of Bacillales, in mixed culture, as indicated by both bioinformatic predictions and empirical observations. This method was tested on a range of beer and wine fermentations containing mixed LAB communities, demonstrating the efficacy of this technique for discriminating LAB in mixed culture. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis to Assess Microbial Community Structure in Compost Systems

    Science.gov (United States)

    Tiquia, Sonia M.

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in composting systems. This analysis is based on the restriction endonuclease digestion of fluorescently end-labeled PCR products. The digested product is mixed with a DNA size standard, itself labeled with a distinct fluorescent dye, and the fragments are then separated by capillary or gel electrophoresis using an automated sequencer. Upon analysis, only the terminal end-labeled restriction fragments are detected. An electropherogram is produced, which shows a profile of compost microbial community as a series of peaks of varying height. This technique has also been effectively used in the exploration of complex microbial environments and in the study of bacterial, archaeal, and eukaryal populations in natural habitats.

  3. Characterization of European Yersinia enterocolitica 1A strains using restriction fragment length polymorphism and multilocus sequence analysis.

    Science.gov (United States)

    Murros, A; Säde, E; Johansson, P; Korkeala, H; Fredriksson-Ahomaa, M; Björkroth, J

    2016-10-01

    Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2-5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2-4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2-4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2-5. Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica. © 2016 The Society for Applied Microbiology.

  4. Genotyping of the fish rhabdovirus, viral haemorrhagic septicaemia virus, by restriction fragment length polymorphisms

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Winton, J.; Lorenzen, Niels

    2005-01-01

    The aim of this study was to develop a standardized molecular assay that used limited resources and equipment for routine genotyping of isolates of the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Computer generated restriction maps, based on 62 unique full-length (1524 nt......-gene by a set of three restriction enzymes was predicted to accurately enable the assignment of the VHSV isolates into the four major genotypes discovered to date. Further sub-typing of the isolates into the recently described sub-lineages of genotype I was possible by applying three additional enzymes...

  5. Phylogenetic analysis of Gossypium L. using restriction fragment length polymorphism of repeated sequences.

    Science.gov (United States)

    Zhang, Meiping; Rong, Ying; Lee, Mi-Kyung; Zhang, Yang; Stelly, David M; Zhang, Hong-Bin

    2015-10-01

    Cotton is the world's leading textile fiber crop and is also grown as a bioenergy and food crop. Knowledge of the phylogeny of closely related species and the genome origin and evolution of polyploid species is significant for advanced genomics research and breeding. We have reconstructed the phylogeny of the cotton genus, Gossypium L., and deciphered the genome origin and evolution of its five polyploid species by restriction fragment analysis of repeated sequences. Nuclear DNA of 84 accessions representing 35 species and all eight genomes of the genus were analyzed. The phylogenetic tree of the genus was reconstructed using the parsimony method on 1033 polymorphic repeated sequence restriction fragments. The genome origin of its polyploids was determined by calculating the diploid-polyploid restriction fragment correspondence (RFC). The tree is consistent with the morphological classification, genome designation and geographic distribution of the species at subgenus, section and subsection levels. Gossypium lobatum (D7) was unambiguously shown to have the highest RFC with the D-subgenomes of all five polyploids of the genus, while the common ancestor of Gossypium herbaceum (A1) and Gossypium arboreum (A2) likely contributed to the A-subgenomes of the polyploids. These results provide a comprehensive phylogenetic tree of the cotton genus and new insights into the genome origin and evolution of its polyploid species. The results also further demonstrate a simple, rapid and inexpensive method suitable for phylogenetic analysis of closely related species, especially congeneric species, and the inference of genome origin of polyploids that constitute over 70 % of flowering plants.

  6. MTHFR A1298C and C677T gene polymorphisms and susceptibility to chronic myeloid leukemia in Egypt.

    Science.gov (United States)

    Aly, Rabab M; Taalab, Mona M; Ghazy, Hayam F

    2014-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the intracellular folate metabolism which plays an important role in carcinogenesis through DNA methylation. We aimed to evaluate the association between MTHFR A1298C and C677T polymorphisms and the risks of chronic myeloid leukemia (CML). Eighty-five patients with CML and a control group containing 100 healthy, age and sex matched individuals were examined for MTHFR C677T and A1298C polymorphisms using polymerase chain reaction-restriction fragment-length (PCR-RFLP) method. The frequency of 677TT genotype in patients with CML was significantly higher compared to controls (OR=2.513, 95% CI: 0.722-4.086, P=0.025). No such association was shown for heterozygous 677CT (OR=1.010, 95% CI: 0.460-2.218, P=0.981). Moreover, for A1298C genotype, a statistically significant higher frequency of 1298CC was also detected in CML patients compared to control group (OR=1.1816, 95% CI: 0.952-3.573, P=0.036), 0.036). No such statistical significance was demonstrable for heterozygote 1298AC (OR=1.046, 95% CI: 0.740-1.759, P=0.092). In addition, patients with joint 677CT/1298AC or 677TT/1298CC genotypes showed an association with increased risk of CML (OR=1.849, 95% CI: 0.935-2.540, P=0.024; OR=1.915, 95% CI: 1.202-3.845, P=0.020 respectively). .A statistically significant increased risk of resistant to therapy was observed with 677CT and 1298AC genotypes (P=0.001, P=0.002 respectively). We conclude that both MTHFR 677TT and 1298CC polymorphisms have been associated with risk of CML and both 677CT and 1298AC genotypes are associated with higher risk of resistant to therapy.

  7. Risk of macular degeneration affected by polymorphisms in Matrix metalloproteinase-2: A case-control study in Chinese Han population.

    Science.gov (United States)

    Cheng, Jie; Hao, Xiaolin; Zhang, Zhongchen

    2017-11-01

    The purpose of this study was to investigate the correlation of single nucleotide polymorphisms (SNPs) in Matrix metalloproteinase -2 (MMP-2) gene and the risk of age-related macular degeneration (AMD) in Chinese Han population.A total of 126 AMD patients and 141 healthy controls participated in this study. Genotypes of MMP-2 gene polymorphisms were identified by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). χtest was used to detect the differences of genotypes and alleles frequencies between case and control groups. Relative risk of AMD was evaluated by odds ratios (ORs) with 95% confidence intervals (CIs).Distribution of variant allele carriers (computed tomography + TT genotypes) of MMP-2 gene rs243865 SNP was significantly different between case and control groups, and might act as protective factors for the onset of AMD (P = .044, OR = 0.583, 95% CI = 0.344-0.987). Nevertheless, the T allele might reduce the AMD risk (P = .030, OR = 0.611, 95% CI = 0.390-0.956). However, no significant association existed between rs243865 and AMD risk in the subgroup analysis based on age. GA + AA genotypes of rs243866 SNP may associate with a decreased risk of AMD in the age≤65 years subgroup (P = .028, OR = 0.399, 95% CI = 0.174-0.915).MMP-2 gene rs243865 and rs243866 SNPs associated with the risk of AMD. Further studies should be performed to confirm the results. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

  8. Polymorphisms on IFNG, IL12B and IL12RB1 genes and paracoccidioidomycosis in the Brazilian population.

    Science.gov (United States)

    Carvalho, F M C; Busser, F D; Freitas, V L T; Furucho, C R; Sadahiro, A; Kono, A S G; Criado, P R; Moretti, M L; Sato, P K; Shikanai-Yasuda, M A

    2016-09-01

    Paracoccidioidomycosis (PCM) is a systemic chronic mycosis, endemic in Latin America, especially Brazil, and is the eighth leading cause of death among chronic and recurrent infectious diseases. PCM infection is characterized by the presence of Th1 immune response; the acute form, by a mixed Th2/Th9, while the chronic form is characterized by Th17/Th22 profiles. The occurrence and severity of human PCM may also be associated with genetic factors such as single nucleotide polymorphisms (SNP) on cytokines encoding genes. We investigated the association between these polymorphisms and the different clinical forms of PCM. We included 156 patients with PCM (40 with the acute form, 99 with the chronic multifocal and 17 with the chronic unifocal form) and assayed their DNA samples for IFNG +874 T/A SNP by PCR-ARMS (Amplification Refractory Mutational System), IL12B +1188 A/C SNP on 3' UTR and IL12RB1 641 A/G SNP on exon 7 by PCR-RFLP (Restriction Fragment Length Polymorphism). We found similar genotypic and allelic frequencies of the investigated SNPs among the clinical forms of PCM. Considering male patients, the IL12RB1 641 AA genotype was more frequent in the chronic multifocal form while heterozygosis was in the chronic unifocal form of PCM (p=0.048). Although our data suggest that the AA genotype (IL12RB1) may be associated with the more disseminated chronic disease, more patients of the chronic unifocal PCM group need to be analyzed as well as the secretion patterns of IFN-γ combined with the IL-12Rβ1 expression for a better comprehension of this association. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Analysis of IL-6, IL-10 and NF-κB Gene Polymorphisms in Aggressive and Chronic Periodontitis.

    Science.gov (United States)

    Toker, Hülya; Görgün, Emine Pirim; Korkmaz, Ertan Mahir

    2017-06-01

    Pro-inflammatory cytokines, interleukin-6 (IL-6), demonstrated to be suppressed by interleukin-10 (IL-10) are known to be regulated by the transcription factor nuclear factor-κB(NF-κB). The aim of this study was to ascertain the association between genetic polymorphism of these genes (IL-6(-174), IL-10(-597) and NF-κB1-94ins/del)) and chronic/aggressive periodontitis. Forty-five patients with chronic periodontitis (CP), 58 patients with aggressive periodontitis (AP) and 38 periodontally healthy subjects were included in this study. Genomic DNA was isolated from whole blood samples. The NF-κB, IL-6, and IL-10 polymorphisms were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Among subjects for the ins/ins genotypes of NF-κB1 gene, the AA genotypes of IL-10 presented a higher frequency in chronic periodontitis group than in healthy controls (p=0.023). A statistically significant difference in genotyping frequencies between AP group and healthy controls was observed for the IL-6 gene. The AA genotype of IL-10 was overrepresented in CP and AP groups compared to healthy controls (OR=9.93, 95% CI: 2.11-46.7, OR=5.7, 95% CI: 1.22-26.89, respectively). Within the limits of this study, it can be concluded that the IL-10 (-597) AA genotype is associated with susceptibility to chronic/aggressive periodontitis and IL-6 (-174) GG genotypes and G allele seems to be associated with aggressive periodontitis. Clinical relevance: The results of the current study indicate that IL-6 and IL-10 genotypes seem to be associated with aggressive periodontitis. Also, the AA genotypes of IL-10 presented a higher frequency in chronic periodontitis subjects with carrying NF-κB1 ins/ins genotypes. Copyright© by the National Institute of Public Health, Prague 2017

  10. Interethnic diversity of the CD209 (rs4804803 gene promoter polymorphism in African but not American sickle cell disease

    Directory of Open Access Journals (Sweden)

    Jenelle A. Noble

    2015-02-01

    Full Text Available Elucidating the genomic diversity of CD209 gene promoter polymorphism could assist in clarifying disease pathophysiology as well as contribution to co-morbidities. CD209 gene promoter polymorphism has been shown to be associated with susceptibility to infection. We hypothesize that CD209 mutant variants occur at a higher frequency among Africans and in sickle cell disease. We analyzed the frequency of the CD209 gene (rs4804803 in healthy control and sickle cell disease (SCD populations and determined association with disease. Genomic DNA was extracted from blood samples collected from 145 SCD and 231 control Africans (from Mali, 331 SCD and 379 control African Americans and 159 Caucasians. Comparative analysis among and between groups was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. Per ethnic diversification, we found significant disparity in genotypic (23.4% versus 16.9% versus 3.2% and allelic frequencies (48.7% versus 42.1% versus 19.8% of the homozygote mutant variant of the CD209 (snp 309A/G gene promoter between Africans, African Americans and Caucasians respectively. Comparative evaluation between disease and control groups reveal a significant difference in genotypic (10.4% versus 23.4%; p = 0.002 and allelic frequencies (39.7% versus 48.7%; p = 0.02 of the homozygote mutant variant in African SCD and healthy controls respectively, an observation that is completely absent among Americans. Comparing disease groups, we found no difference in the genotypic (p = 0.19 or allelic (p = 0.72 frequencies of CD209 homozygote mutant variant between Africans and Americans with sickle cell disease. The higher frequency of CD209 homozygote mutant variants in the African control group reveals a potential impairment of the capacity to mount an immune response to infectious diseases, and possibly delineate susceptibility to or severity of infectious co-morbidities within and between groups.

  11. Association of kinase insert domain-containing receptor (KDR gene polymorphism/ haplotypes with recurrent spontaneous abortion and genetic structure

    Directory of Open Access Journals (Sweden)

    Shiva Shahsavari

    2015-12-01

    Full Text Available Background: Recurrent spontaneous abortion is one of the diseases that can lead to physical, psychological, and, economical problems for both individuals and society. Recently a few numbers of genetic polymorphisms in kinase insert domain-containing receptor (KDR gene are examined that can endanger the life of the fetus in pregnant women. Objective: The risk of KDR gene polymorphisms was investigated in Iranian women with idiopathic recurrent spontaneous abortion (RSA. Materials and Methods: A case controlled study was performed. One hundred idiopathic recurrent spontaneous abortion patients with at least two consecutive pregnancy losses before 20 weeks of gestational age with normal karyotypes were included in the study. Also, 100 healthy women with at least one natural pregnancy were studied as control group. Two functional SNPs located in KDR gene; rs1870377 (Q472H, and rs2305948 (V297I as well as one tag SNP in the intron region (rs6838752 were genotyped by using PCR based restriction fragment length polymorphism (PCR-RFLP technique. Haplotype frequency was determined for these three SNPs’ genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to study genetic variation. Results: Functional SNP (rs1870377 was highly linked to tag SNP (rs6838752 (D´ value=0. 214; χ2 = 16.44, p<0. 001. K means clustering showed that k = 8 as the best fit for the optimal number of genetic subgroups in our studied materials. This result was in agreement with Neighbor Joining cluster analysis. Conclusion: In our study, the allele and genotype frequencies were not associated with RSA between patient and control individuals. Inconsistent results in different populations with different allele frequencies among RSA patients and controls may be due to ethnic variation and used sample size.

  12. MTHFR gene C677T and A1298C polymorphisms and homocysteine levels in primary open angle and primary closed angle glaucoma

    Science.gov (United States)

    Micheal, Shazia; Qamar, Raheel; Akhtar, Farah; Khan, Muhammad Imran; Khan, Wajid Ali

    2009-01-01

    Purpose To investigate the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genotypes and plasma concentrations of total homocysteine (tHcy) in Pakistani patients with primary open angle glaucoma (POAG) and primary closed angle glaucoma (PCAG). Methods This was a prospective case-control study. A total of 295 patients (173 POAG, 122 PCAG) and 143 age- and sex-matched controls were subdivided into two ethnic groups, Punjabis (Punjab province, central Pakistan) and Pathans (North-West Frontier Province, northern Pakistan). Genotypes of the MTHFR C677T and A1298C polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). An enzyme-linked immunosorbent assay was used to determine the total serum homocysteine (tHcy) levels. Associations were determined by logistic regression analysis. Results Frequency distributions of genotypes and combined genotypes as well as homocysteine levels were obtained. The overall distribution of the C677T genotype was found to be significantly associated with PCAG (CC 69%, CT 21%, TT 10%; p=0.001, χ2=12.6), but not with POAG (CC 71%, CT 28%, TT 1%; p=0.98, χ2=0.02) as compared to the controls (CC 71%, CT 29%, TT 1%). The Pathan cohorts revealed no association with the disease; however, the Punjabis demonstrated a significant association with PCAG (CC 75%, CT 11%, TT 13%; pA1298C polymorphism (AA 43%, AC 54%, CC 3%; pMTHFR C677T and A1298C polymorphisms and the combined genotype TTAC were associated with PCAG in Punjabi subjects of Pakistani origin and correlated with the high serum tHcy levels seen in these patients. PMID:19936026

  13. Association of vitamin D receptor gene BsmI (A>G) and FokI (C>T) polymorphism in gestational diabetes among Saudi Women

    Science.gov (United States)

    El-Beshbishy, Hesham A.; Tawfeek, Manal A.; Taha, Inass M.; FadulElahi, Thoraya; Shaheen, Amal Y.; Bardi, Fouad A.; Sultan, Intessar I.

    2015-01-01

    Objective: Vitamin D receptor (VDR) gene polymorphism have a role in diabetes mellitus pathogenesis. Present study was conducted to determine VDR gene variants among Saudi gestational diabetics (GDM) in Madina, KSA. Methods: This cross sectional study was conducted on 112 GDM patients and 218 normal healthy control. Age, body mass index and blood pressure levels were recorded. Serum triglycerides (mg/dl), total cholesterol, HDL-cholesterol, LDL-cholesterol, fasting blood glucose FBG and post-prandial blood glucose PPBG were estimated. Extracted DNA template was amplified by PCR reaction and genotyped for single nucleotide polymorphism of BsmI and FokI by restriction fragment length polymorphism-PCR (RFLP-PCR) analysis. Results: FBG and PPBG levels in GDM patients were significantly elevated by +48.6% and +50%, respectively (P=0.005). Serum triglycerides, total cholesterol and LDL-cholesterol (mg/dl) levels in GDM patients were elevated significantly by +40.5% (P=0.005), +16% (P=0.01) and +30.8% (P=0.005), respectively. Serum HDL-cholesterol (mg/dl) showed significant decline by -10.5%. FokI VDR genotypes showed association with PPBG (P=0.05) among GDM patients. The Ff, FF and ff genotype percentage among GDM patients was 48.2%, 30.4% and 21.4%, respectively. FokI (F and f) and BsmI (B and b) alleles frequency showed no significant difference between GDM patients and control. Percentage BsmI and FokI total homozygous and heterozygous variants among GDM was 45.5% and 81.4%, respectively. Conclusion: VDR BsmI and FokI polymorphic marker not associated with Saudi GDM. PMID:26870091

  14. A brain-derived neurotrophic factor polymorphism Val66Met identifies fibromyalgia syndrome subgroup with higher body mass index and C-reactive protein.

    Science.gov (United States)

    Xiao, Yangming; Russell, I Jon; Liu, Ya-Guang

    2012-08-01

    A common single nucleotide polymorphism (SNP) in the gene of brain-derived neurotrophic factor (BDNF) results from a substitution at position 66 from valine (Val) to methionine (Met) and may predispose to human neuropsychiatric disorders. We proposed to determine whether these BDNF gene SNPs were associated with fibromyalgia syndrome (FMS) and/or any of its typical phenotypes. Patients with FMS (N = 95) and healthy normal controls (HNC, N = 58) were studied. Serum high-sensitivity C-reactive protein (hsCRP) levels were measured using an enzyme-linked immunosorbent assay (ELISA). The BDNF SNPs were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).The BDNF SNP distribution was 65 (68%) Val/Val, 28 (30%) Val/Met, and 2 (2%) Met/Met for FMS and 40 (69%), 17(29%), and 1 (2%) for HNC, respectively. The serum high-sensitivity C-reactive protein (hsCRP)and body mass index (BMI) in FMS were higher than in HNC. The FMS with BDNF Val66Val had significantly higher mean BMI (P = 0.0001) and hsCRP (P = 0.02) than did FMS carrying the Val66Met genotype. This pattern was not found in HNC. Phenotypic measures of subjective pain, pain threshold, depression, or insomnia did not relate to either of the BDNF SNPs in FMS. The relative distribution BDNF SNPs did not differ between FMS and HNC. The BDNF Val66Met polymorphism is not selective for FMS. The BDNF Val66Val SNP identifies a subgroup of FMS with elevated hsCRP and higher BMI. This is the first study to associate a BDNF polymorphism with a FMS subgroup phenotype.

  15. Molecular taxonomy of cupped oysters (Crassostrea, Saccostrea, and Striostrea) in Thailand based on COI, 16S, and 18S rDNA polymorphism.

    Science.gov (United States)

    Klinbunga, S; Khamnamtong, B; Puanglarp, N; Jarayabhand, P; Yoosukh, W; Menasveta, P

    2005-01-01

    Genetic diversity of oysters Crassostrea belcheri (Sowerby, 1871), C. iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S. forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819) (Ostreoida, Mollusca) was analyzed by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 16S ribosomal DNA with AcsI, AluI, DdeI, DraI, RsaI, and TaqI, 18S ribosomal DNA with HinfI, and cytochrome oxidase subunit I with AcsI, DdeI and MboI. A total of 54 composite haplotypes were observed. Species-diagnostic markers were specifically found in C. belcheri, C. iredalei, and S. cucullata, but not in S. forskali and Striostrea mytiloides, which shared common composite haplotypes. Neighbor-joining trees constructed from genetic distances between pairs of composite haplotypes and species indicated large genetic differences between Crassostrea and Saccostrea (including Striostrea mytiloides), but closer relationships were observed within each genus. Four groups of unidentified oysters (Crassostrea sp. and Saccostrea sp. groups 1, 2, and 3) were also genetically analyzed. Fixed RFLP markers were found in Crassostrea sp. and Saccostrea sp. group 2, but not in Saccostrea sp. groups 1 and 3. Phylogenetic and genetic heterogeneity analyses indicated that Crassostrea sp. and Saccostrea sp. group 2 should be considered as newly unidentified oyster species in Thailand.

  16. Polymorphisms at +49A/G and CT60 sites in the 3' UTR of the CTLA-4 gene and APECED-related AIRE gene mutations analysis in sporadic idiopathic hypoparathyroidism.

    Science.gov (United States)

    Goswami, R; Gupta, N; Ray, D; Rani, R; Tomar, N; Sarin, R; Vupputuri, M R

    2005-12-01

    Autoimmune diseases such as Graves' disease and type 1 diabetes have been linked with +49A/G and CT60 single nucleotide polymorphisms (SNPs) in the 3' UTR of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene. Both these SNPs are functionally relevant and linked with T-lymphocyte activation. Hypoparathyroidism is seen in 70% of patients with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome (APECED). Although calcium sensing receptor autoantibodies (CaSRAb) and generalized activation of T lymphocytes are reported among patients with sporadic idiopathic hypoparathyroidism (SIH), CTLA-4 gene SNPs and APECED-related autoimmune regulator (AIRE) gene mutations have not been assessed in them. We studied lead CTLA-4 gene SNPs and APECED-related AIRE gene mutations in 73 patients with SIH and 114 healthy subjects. The CTLA-4 gene SNPs +49A/G in exon 1, CT60A/G in 3' UTR and -318C/T in the promoter region were genotyped by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) using BstEII, NcoI and MseI endonucleases, respectively. The APECED-related AIRE gene mutations, which is R257X (Finn-major) in exon 6, 4-bp insertion and 13-bp deletion in exon 8, and Iranian Jews population 'Y85C' mutation in exon 2, were studied by PCR-RFLP (Taq-I), PCR and nucleotide sequencing, respectively. CaSRAb were studied by immunoblotting. The frequencies of CTLA-4 A/A(49), A/G(49) and G/G(49) genotypes in the patients (47.9%, 38.4% and 13.7%) and controls (45.6%, 39.5% and 14.9%, respectively) and the frequencies of CT60 A/A, A/G, and G/G genotypes in the patient (42.4%, 37.0% and 20.6%) and the control (38.6%, 40.4% and 21.0%, respectively) groups were not significantly different. The frequencies of various haplotypes including genetic loci +49A/G and CT60 and frequencies of G alleles at these positions were comparable between patient and the control groups and its presence did not correlate with clinical and biochemical indices of the disease

  17. κ-CASEIN GENE POLYMORPHISMS IN RIVERINE AND SWAMP BUFFALO IN INDONESIA

    Directory of Open Access Journals (Sweden)

    A.O. Rini

    2014-09-01

    Full Text Available Kappa-casein (κ-casein gene is known as a gene that plays a role in controlling milk protein andalso play a crucial role in the coagulation and curdling of milk. This study was aimed to identifypolymorphisms of the κ-Casein gene of local buffaloes in Indonesia. A total number of 40 heads ofriverine buffalo and 250 heads of swamp buffalo. This study used PCR-RFLP method, whichamplification of the κ-Casein gene resulted an amplicon with length of 157 bp, located in exon 4. Theamplified fragment were digested with EcoRV restriction enzyme, which cut the κ-Casein gene in exon4 at nucleotides of GAT|ATC, revealed the presence of one polymorphism at the base position of 23 bpthat occurs with a substitution of Ile (ATC of the T genetic variant into Thr (ACC of the C geneticvariant. Genotyping κ-Casein gene in riverine buffalo produced two types of allele, namely C allele (157bp and T allele (136 and 21 bp. These two alleles resulted in three types of genotypes, namely CC, CT,and TT. Frequency of the C allele was dominant to T allele. κ-Casein gene in swamp buffalo wasmonomorphic with one allele, namely C allele. Heterozygosity value of riverine and swamp buffaloeswere low. PIC value in riverine and swamp buffalo ranged 0.000-0.288. Fixation index of κ-Casein genein riverine buffalo was low (Siborong-borong SBBC = -0,0036; Deli Serdang = -0.025, but in swampbuffalo was in fixation. This study showed that κ-Casein|EcoRV were polymorphic in riverine buffaloand monomorphic in swamp buffalo.

  18. Polymorphism of the IL13 gene may be associated with Uterine leiomyomas in Slovenian women

    Directory of Open Access Journals (Sweden)

    Krsteski J

    2016-12-01

    Full Text Available Uterine leiomyomas (ULM are a common cause of solid pelvic tumors in women. Their etiopathogenesis remains unclear. Interleukins (ILs and their receptors can influence tumor biology of ULM. The aim of this study was to evaluate single nucleotide polymorphisms (SNPs exhibited in the genes IL4 (rs2070874, IL4R (rs1801275, IL12RB1 (rs11575934, IL12B (rs6887695, IL13 (rs20541 and IL23R (rs7517847 as risk factors for ULM in Slovenian women and to identify associations between corresponding clinical parameters and the analyzed SNPs. In addition, solitary and multiple ULM were compared to identify clinical and/or genetic parameters influencing their occurrence. We conducted a case-control study that included 181 women with leiomyomas and 133 control subjects. Genotyping of selected SNPs was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and high resolution melting (HRM techniques. The TT genotype of rs20541 (IL13 was significantly associated with decreased risk of ULM compared to both the CC and CT genotypes [p = 0.018; odds ratio (OR = 0.184; 95% confidence interval (95% CI = 0.048-0.7121. Using genetic and clinical data to develop a predictive model with logistic regression, we found that adenomyosis, higher age at diagnosis, family history of ULM occurrence, earlier menarche, lower number of pregnancies and lower age at first sexual intercourse, the G allele and genotypes AG and GG of rs1801275 (IL4R were associated with an increased risk of multiple ULM occurrence. We also found an association between rs20541 (IL13 and 17ß-estradiol serum levels in patients with multiple ULM (p 0.003. Our study showed, for the first time, that rs20541 (IL13 may contribute to susceptibility of ULM development and that rs1801275 (IL4R can predispose patients to develop multiple ULM.

  19. Polymorphism of growth hormone receptor (GHR gene in Holstein Friesian dairy cattle

    Directory of Open Access Journals (Sweden)

    Restu Misrianti

    2011-12-01

    Full Text Available Growth hormone gene have a critical role in the regulation of lactation, mammary gland development and growth process through its interaction with a specific receptor. Growth hormone (GH is an anabolic hormone which is synthesized and secreted by somatotrop cell in pituitary anterior lobe, and interacts with a specific receptor on the surface of the target cells. Growth hormone receptor (GHR has been suggested as candidate gene for traits related to milk production in Bovidae. The purpose of this study was to identify genetic polymorphism of the Growth Hormone Receptor (GHR genes in Holstein Friesian (HF cattle. Total of 353 blood samples were collected from five populations belonging to Cikole Dairy Cattle Breeding Station (BPPT-SP Cikole (88 samples, Pasir Kemis (95 samples, Cilumber (98 samples, Cipelang Livestock Embryo Center (BET Cipelang (40 samples, Singosari National Artificial Insemination Centre (BBIB Singosari (32 samples and 17 frozen semen samples from Lembang Artificial Insemination Center (BIB Lembang. Genomic DNAs were extracted by a standard phenol-chloroform protocol and amplified by a polymerase chain reaction (PCR techniques then PCR products were genotyped by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP methods. There were two allele dan three genotypes were found namely: allele A and G, Genotype AA, AG and GG repectively. Allele A frequency (0.70-0.82 relatively higher than allele G frequency (0.18-0.30. Chi square test show that on group of BET Cipelang, BIB Lembang and BBIB Singosari population were not significantly different (0.00-0.93, while on group of BET Cipelang, BIB Lembang dan BBIB Singosari population were significantly different (6.02-11.13. Degree of observed heterozygosity (Ho ranged from 0.13-0.42 and expected heterozygosity (He ranged from 0.29-0.42.

  20. Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils.

    Science.gov (United States)

    Edel-Hermann, Véronique; Dreumont, Christiane; Pérez-Piqueres, Ana; Steinberg, Christian

    2004-03-01

    Monitoring the structure and dynamics of fungal communities in soils under agricultural and environmental disturbances is currently a challenge. In this study, a terminal restriction fragment length polymorphism (T-RFLP) fingerprinting method was developed for the rapid comparison of fungal community structures. The terminal restriction fragment polymorphism of different regions of the small-subunit (SSU) ribosomal RNA (rRNA) gene was simulated by sequence comparison using 10 restriction enzymes, and analyzed among three different soils using fungal-specific primers. Polymerase chain reaction amplification of the 3' end of the SSU rRNA gene with the primer nu-SSU-0817-5' and with the fluorescently labelled primer nu-SSU-1536-3', and digestion of the amplicons with AluI and MboI were found to be optimal and were used in a standardized T-RFLP procedure. Both the number and the intensity of terminal restriction fragments detected by capillary gel electrophoresis were integrated in correspondence analyses. Three soils with contrasting physicochemical properties were differentiated according to the structure of their fungal communities. Assessment of the impact on the fungal community structure of the amendment of two soils with compost or manure confirmed the reproducibility and the sensitivity of the method. Shifts in the community structure were detected between non-amended and amended soil samples. In both soils, the shift differed with the organic amendment applied. In addition, the fungal community structures of the two soils were affected in a different way by the same organic amendment. The fingerprinting method provides a rapid tool to investigate the effect of various perturbations on the fungal communities in soils.

  1. Evaluating the effect of 3 glucocorticoid receptor gene polymorphisms on risk of relapse in 100 Iranian children with acute lymphoblastic leukemia: a case-control study.

    Science.gov (United States)

    Namazi, Soha; Zareifar, Soheila; Monabati, Ahmad; Ansari, Shahla; Karimzadeh, Iman

    2011-03-01

    Glucocorticoids are an important component of treatment for childhood acute lymphoblastic leukemia (ALL). To induce antileukemic effects, glucocorticoids have to bind their intracellular receptors. Little is known about probable mechanisms of glucocorticoid resistance in ALL. The aim of this study was to evaluate the possible association between 3 prominent glucocorticoid receptor gene polymorphisms-BclI, N363S, and ER22/23EK-and the risk of relapse in children with ALL. We conducted a case-control study on 100 children with ALL, aged 0 to 15 years, including 50 nonrelapsed (control) and 50 relapsed (case) subjects. Required patient information such as demographic characteristics; relevant clinical and paraclinical findings at diagnosis; chemotherapy protocols used at diagnosis; and relapse properties, including time interval from date of initial diagnosis to relapse and number, type, and site of relapse, were gathered from patients' medical files. Genotyping of BclI, N363S, and ER22/23EK polymorphisms was carried out by polymerase chain reaction restriction fragment-length polymorphism (PCR-RFLP). Statistical analysis was performed. The distribution of BclI, N363S, and ER22/23EK polymorphism genotypes in our population and in populations examined in similar studies was compared using the χ(2) test or Fischer exact test. One hundred children with ALL, consisting of 65 males and 35 females, were recruited into this study. Their mean (SD) age was 5.39 (3.02) years. No relapsed or nonrelapsed individuals were homozygous for N363S and ER22/23EK polymorphisms. The allelic frequencies of mutant alleles of BclI, N363S, and ER22/23EK polymorphisms in all patients were 0.195, 0.02, and 0.005, respectively. No statistically significant association between BclI, N363S, and ER22/23EK polymorphisms and risk of relapse in children with ALL was observed (P = 0.104 [BclI], not calculated for the last 2 polymorphisms [N363S and ER22/23EK]). The incidence of BclI polymorphism in

  2. Genotyping of human and porcine Yersinia enterocolitica, Yersinia intertmedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Kuehni-Boghenbor, Kathrin; On, Stephen L.W.; Kokotovic, Branko

    2006-01-01

    In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping ba...

  3. Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing

    NARCIS (Netherlands)

    Huyen, Mai N. T.; Kremer, Kristin; Lan, Nguyen T. N.; Buu, Tran N.; Cobelens, Frank G. J.; Tiemersma, Edine W.; de Haas, Petra; van Soolingen, Dick

    2013-01-01

    In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of

  4. Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing.

    NARCIS (Netherlands)

    Huyen, M.N.; Kremer, K.; Lan, N.T.; Buu, T.N.; Cobelens, F.G.; Tiemersma, E.W.; Haas, P. de; Soolingen, D. van

    2013-01-01

    BACKGROUND: In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard

  5. Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis

    DEFF Research Database (Denmark)

    Nielsen, H. S.; Storgaard, Torben; Oleksiewicz, M.B.

    2000-01-01

    A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment length polymorphism a...

  6. Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

    DEFF Research Database (Denmark)

    Høgdall, Estrid; Vuust, Jens; Lind, Peter

    2000-01-01

    of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T...

  7. THE HUMAN FUMARYLACETOACETATE GENE : CHARACTERIZATION OF RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISMS AND IDENTIFICATION OF HAPLOTYPES IN TYROSINEMIA TYPE-1 AND PSEUDODEFICIENCY

    NARCIS (Netherlands)

    ROOTWELT, H; KVITTINGEN, EA; HOIE, K; AGSTERIBBE, E; HARTOG, M; BERGER, R

    Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia

  8. On the power to detect differences between male and female mutation rates for Duchenne muscular dystrophy, using classical segregation analysis and restriction fragment length polymorphisms

    NARCIS (Netherlands)

    Karel, E.R.; te Meerman, G J; Ten Kate, L P

    The power to detect departures from the theoretical proportion of new mutants in X-linked lethal disorders has been analyzed for several types of segregation analysis, including methods based on completely linked restriction fragment length polymorphisms. It is shown that all methods require large

  9. Association between AA-NAT gene polymorphism and reproductive performance in sheep

    OpenAIRE

    Ding-ping,Bai; Cheng-jiang,Yu; Yu-lin,Chen

    2012-01-01

    Arylalkylamine N-acetyltransferase (AA-NAT) is critical enzyme in Melatonin (MLT) biosynthesis for MLT regulating the animal seasonal breeding. In this study, DNA sequencing methods were applied to detect the polymorphisms of the AA-NAT gene in 179 Chinese sheep belonging to two non-seasonal reproduction breeds and two seasonal reproduction breeds. One mutation at exon 3 (NM_001009461:c.486A > G) was firstly described at the sheep AA-NAT locus. Hence, we described the SmaI PCR-RFLP m...

  10. Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai).

    Science.gov (United States)

    Nie, H-T; Li, Q; Kong, L-F

    2014-06-01

    Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker-centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72-h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second-division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y-values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y-values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species. © 2014 Stichting International Foundation for Animal Genetics.

  11. Selection criteria for scoring amplified fragment length polymorphisms (AFLPs) positively affect the reliability of population genetic parameter estimates.

    Science.gov (United States)

    Herrmann, Doris; Poncet, Bénédicte N; Manel, Stéphanie; Rioux, Delphine; Gielly, Ludovic; Taberlet, Pierre; Gugerli, Felix

    2010-04-01

    A reliable data set is a fundamental prerequisite for consistent results and conclusions in population genetic studies. However, marker scoring of genetic fingerprints such as amplified fragment length polymorphisms (AFLPs) is a highly subjective procedure, inducing inconsistencies owing to personal or laboratory-specific criteria. We applied two alternative marker selection algorithms, the newly developed script scanAFLP and the recently published AFLPScore, to a large AFLP genome scan to test how population genetic parameters and error rates were affected. These results were confronted with replicated random selections of marker subsets. We show that the newly developed marker selection criteria reduced the mismatch error rate and had a notable influence on estimates of genetic diversity and differentiation. Both effects are likely to influence biological inference. For example, genetic diversity (HS) was 29% lower while genetic differentiation (FST) was 8% higher when applying scanAFLP compared with AFLPScore. Likewise, random selections of markers resulted in substantial deviations of population genetic parameters compared with the data sets including specific selection criteria. These randomly selected marker sets showed surprisingly low variance among replicates. We conclude that stringent marker selection and phenotype calling reduces noise in the data set while retaining patterns of population genetic structure.

  12. IS1245 restriction fragment length polymorphism typing of Mycobacterium avium from patients admitted to a reference hospital in Campinas, Brazil

    Directory of Open Access Journals (Sweden)

    A.C. Panunto

    2003-10-01

    Full Text Available Mycobacterium avium is an important pathogen among immunodeficient patients, especially patients with AIDS. The natural history of this disease is unclear. Several environmental sources have been implicated as the origin of this infection. Polyclonal infection with this species is observed, challenging the understanding of its pathogenesis and treatment. In the present study 45 M. avium strains were recovered from 39 patients admitted to a reference hospital between 1996 and 1998. Species identification was performed using a species-specific nucleic acid hybridization test (AccuProbe® from Gen-Probe®. Strains were genotyped using IS1245 restriction fragment length polymorphism typing. Blood was the main source of the organism. In one patient with disseminated disease, M. avium could be recovered more than once from potentially sterile sites. Strains isolated from this patient had different genotypes, indicating that the infection was polyclonal. Four patient clones were characterized in this population, the largest clone being detected in eight patients. This finding points to a common-source transmission of the organism.

  13. Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots.

    Science.gov (United States)

    Sakai, Masao; Matsuka, Akira; Komura, Taichi; Kanazawa, Shinjiro

    2004-10-01

    Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods. In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids. To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment length polymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples. Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis. In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots. Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.

  14. Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism.

    Science.gov (United States)

    Bounamous, Azzedine; Lehrter, Véronique; Hadj-Henni, Leila; Delecolle, Jean-Claude; Depaquit, Jérôme

    2014-07-01

    A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

  15. Amplified fragment length polymorphism used to investigate genetic variability of the stable fly (Diptera: Muscidae) across North America.

    Science.gov (United States)

    Kneeland, K M; Skoda, S R; Foster, J E

    2013-09-01

    The stable fly, Stomoxys calcitrans (L.), is a cosmopolitan pest of livestock and humans. The pestiferous nature and painful bite cause stress to cattle and other animals. The stress and resulting avoidance behaviors manifest as reductions in weight gain or milk production in cattle; estimated annual economic loss in the United States exceeds US$2 billion. Understanding the population genetics of stable flies could provide information on their population dynamics, origins of outbreaks, and geographical patterns of insecticide resistance, resulting in a tactical advantage for developing management strategies. Previous studies, mostly on a local scale, reported a high level of gene flow between locations. Here, we report results wherein amplified fragment length polymorphism was used to determine genetic diversity of stable fly samples consisting of 11-40 individuals from 12 locations representing the United States, Canada, and Panama. The Analysis of Molecular Variance showed that the majority of genetic diversity was within groups; very little was among groups. The F(ST) and G(ST) values were low ( 1.0). The tests of neutrality suggested population expansion, and no genetic differentiation was found between locations. These results show that stable flies have a high level of gene flow on a continental scale, with limited isolation owing to distance or geographical barriers.

  16. Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti

    Energy Technology Data Exchange (ETDEWEB)

    Severson, D.W.; Thathy, V.; Mori, A. [Univ. of Wisconsin, Madison, WI (United States)] [and others

    1995-04-01

    Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F{sub 2} populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs [2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filaria nematode Brugia malayi and the yellow fever virus. 35 refs., 2 figs., 3 tabs.

  17. Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    Azzedine Bounamous

    2014-07-01

    Full Text Available A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b, t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

  18. Prevalence of Trichomonas spp. in domestic pigeons in Shandong Province, China, and genotyping by restriction fragment length polymorphism.

    Science.gov (United States)

    Jiang, Xiyue; Sun, Jingjing; Wang, Fangkun; Li, Hongmei; Zhao, Xiaomin

    2016-05-01

    Oropharyngeal swabs (n = 609) were collected randomly from 80,000 domestic pigeons (Columba livia domestica) on five pigeon farms and at one pigeon slaughterhouse in Shandong Province, China, from September 2012 to July 2013. Trichomonas spp. were detected in 206/609 (33.8%) samples. The prevalence was 14.9-31.1%, depending on different levels of sanitation and management, and was 4.8% in nestling pigeons, 13.6% in breeding pigeons and 35.2% in adolescent pigeons. Trichomonas gallinae genotypes A and B, and Trichomonas tenax-like isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing of the 5.8S rDNA-internal transcribed spacer (ITS) regions. RFLP analysis with the restriction enzyme BsiEI generated different RFLP band patterns between T. gallinae and T.tenax-like isolates. When BsiEI RFLP analysis was combined with HaeIII RFLP analysis, all infection types of T. gallinae and T.tenax-like isolates could be identified. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

    Directory of Open Access Journals (Sweden)

    Diaz R

    2001-01-01

    Full Text Available The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55% had unique IS6110 restriction fragment length polymorphism (RFLP patterns, while isolates from 23 others (45% had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

  20. Application of Fluorescent Amplified Fragment Length Polymorphism for Comparison of Human and Animal Isolates of Yersinia enterocolitica

    Science.gov (United States)

    Fearnley, Catherine; On, Stephen L. W.; Kokotovic, Branko; Manning, Georgina; Cheasty, Tom; Newell, Diane G.

    2005-01-01

    An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype. PMID:16151073

  1. Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

    Science.gov (United States)

    Demezas, David H.; Reardon, Terry B.; Watson, John M.; Gibson, Alan H.

    1991-01-01

    Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships. PMID:16348600

  2. Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes

    International Nuclear Information System (INIS)

    Hyldig-Nielsen, J.J.; Morling, N.; Oedum, N.; Ryder, L.P.; Platz, P.; Jakobsen, B.; Svejgaard, A.

    1987-01-01

    The restriction fragment length polymorphism (RFLP) of the class II HLA-DP subregion of the major histocompatibility complex (MHC) of humans has been unraveled by Southern blotting using DP/sub α/ and DP/sub β/ probes in a study of 46 unrelated individuals with known HLA-DP types. Contrary to earlier preliminary findings with a limited number of enzymes, the RFLP appears to be quite extensive both with the DP/sub β/ (14 different DNA markers defined by individual fragments or clusters thereof) and the DP/sub α/ (8 markers) probes, especially when enzyme recognizing only four base pairs were used. A few markers were absolutely or strongly associated with individual DP antigens, whereas most were associated with two or more DP antigens as defined by primed lymphocyte typing. Thus, Southern blotting seems feasible for typing for most DP determinants by specific fragments or subtraction between the various more broadly reactive DNA markers, and the RFLP provides further information on the DP subregion in addition to that provided by primed lymphocyte typing. In two recombinant families, the DP/sub β/ and DP/sub α/ DNA markers segregated with DP antigens, whereas the DR/sub β/, DQ/sub β/, DQ/sub α/, and DX/sub α/ markers followed the DR and DQ antigens

  3. Identification of Echinococcus granulosus strains using polymerase chain reaction-restriction fragment length polymorphism amongst livestock in Moroto district, Uganda.

    Science.gov (United States)

    Chamai, Martin; Omadang, Leonard; Erume, Joseph; Ocaido, Michael; Oba, Peter; Othieno, Emmanuel; Bonaventure, Straton; Kitibwa, Annah

    2016-07-29

    A descriptive study was conducted to identify the different strains of Echinococcus granulosus occurring in livestock in Moroto district, Uganda. Echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment length polymorphism and Sanger DNA sequencing. Echinococcus granulosus genotypes or strains were identified through use of Bioinformatics tools: BioEdit, BLAST and MEGA6. The major finding of this study was the existence of a limited number of E. granulosus genotypes from cattle, goats, sheep and camels. The most predominant genotype was G1 (96.05%), corresponding to the common sheep strain. To a limited extent (3.95%), the study revealed the existence of Echinococcus canadensis G6/7 in three (n = 3) of the E. granulosus-positive samples. No other strains of E. granulosus were identified. It was concluded that the common sheep strain of Echinococcus sensu stricto and G6/7 of E. canadensis were responsible for echinococcal disease in Moroto district, Uganda.

  4. Characterization of Bois noir isolates by restriction fragment length polymorphism of a Stolbur-specific putative membrane protein gene.

    Science.gov (United States)

    Pacifico, D; Alma, A; Bagnoli, B; Foissac, X; Pasquini, G; Tessitori, M; Marzachì, C

    2009-06-01

    Bois noir phytoplasma (BNp), widespread in wine-producing areas of Europe and endemic in France and Italy, is classified in the 16SrXII-A subgroup, whose members are referred to as Stolbur phytoplasmas. The 16S rDNA gene of Stolbur phytoplasma shows low variability, and few non-ribosomal genes are available as markers to assess variation among isolates. We used the Stolbur-specific stol-1H10 gene, encoding a putative membrane-exposed protein, to investigate genetic diversity of French and Italian BNp isolates from plants and insects. Amplification of stol-1H10 from infected grapevines, weeds, and Hyalesthes obsoletus produced fragments of three sizes, and restriction fragment length polymorphism analysis divided these amplicons further into 12 profiles (V1 to V12). French BNp isolates were more variable than Italian ones, and different profiles were present in infected grapevines from France and Italy. Isolate V3, most abundant among Italian affected grapes but present among French ones, was found in one Urtica dioica sample and in all H. obsoletus collected on this species. Four Italian-specific profiles were represented among infected Convolvulus arvensis, the most frequent of which (V12) was also detected in H. obsoletus collected on this species. Most of the variability in the stol-1H10 sequence was associated with type II on the tuf gene.

  5. Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    Caldeira Roberta L

    2003-01-01

    Full Text Available Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2 and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

  6. Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQwl and DQw3 alleles of the HLA-D region

    Energy Technology Data Exchange (ETDEWEB)

    Szafer, F.; Brautbar, C.; Tzfoni, E.; Frankel, G.; Sherman, L.; Cohen, I.; Hacham-Zadeh, S.; Aberer, W.; Tappeiner, G.; Holubar, K.; Steinman, L.

    1987-09-01

    Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ..beta.., DQ..cap alpha.., and DR..beta.. cDNA probes. Hybridization with the DQ..beta.. probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ..cap alpha.. and DR..beta.. probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.

  7. APOE and CETP TaqIB polymorphisms influence metabolic responses to Hibiscus sabdariffa L. and Gynostemma pentaphyllum Makino tea consumption in hypercholesterolemic subjects.

    Science.gov (United States)

    Jeenduang, Nutjaree; Sangkaew, Boonnisa; Chantaracha, Pacharee; Chanchareonsri, Sirada; Plyduang, Thunyaluk; Thitdee, Wanida; Samae, Cathaleeya; Pitumanon, Wacharaporn

    2017-03-01

    Hibiscus sabdariffa L. (HS) and Gynostemma pentaphyllum Makino (GP) have been used as traditional medicines to treat diabetes and hypercholesterolemia. Nevertheless, there is interindividual variation in the metabolic responses to HS and GP consumption. This may be due to genetic factors. The aim of this study was to investigate the effects of HS and GP tea consumption on anthropometric data, fasting blood glucose (FBG), and lipid concentrations in hypercholesterolemia subjects with different genotypes of the APOE and CETP TaqIB polymorphisms. Forty-eight subjects with hypercholesterolemia were given either HS or GP tea for 30 days. Anthropometric and biochemical variables were determined, and APOE and CETP TaqIB polymorphisms were analyzed using the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). E4 (p=0.008) and homozygous B1B1 (p=0.010) carriers had significantly decreased HDL-C concentrations after HS consumption; in addition, B2 carriers who consumed HS showed significantly decreased triglyceride (TG) concentrations (p=0.039). Regarding GP consumption, non-E4 carriers had significantly decreased HDL-C (p=0.009) and FBG (p=0.042) concentrations. Furthermore, B2 carriers had significantly decreased total cholesterol (TC) (p=0.045), HDL-C (p=0.004), and FBG (p=0.026) concentrations. HS consumption may have beneficial effects with respect to TG concentrations in the B2 carriers, but it may adversely affect HDL-C concentrations in homozygous B1B1 and E4 carriers. In contrast, GP consumption may have favorable effects on TC and FBG concentrations but not on HDL-C concentrations for B2 and/or non-E4 carriers.

  8. Association of polymorphic variants in IL1B gene with secretion of IL-1β protein and inflammatory markers in north Indian rheumatoid arthritis patients.

    Science.gov (United States)

    Jahid, Mohd; Rehan-Ul-Haq; Chawla, Diwesh; Avasthi, Rajnish; Ahmed, Rafat Sultana

    2018-01-30

    The proinflammatory cytokine interleukin-1beta (IL-1β) is a key mediator of inflammation which affects cell proliferation and differentiation. IL-1β is considered to contribute to the pathophysiology of rheumatoid arthritis (RA). Polymorphisms in cytokine genes are highly influenced by ethnicity. Hence, in this study polymorphism of the IL1B-511(C/T) within promoter region was analyzed by using polymerase chain reaction-restriction fragment length Polymorphism (PCR-RFLP) in 187 RA patients and 214 controls. The prevalence of different genotypes and allelic frequency distribution was compared in RA patients and controls. Levels of inflammatory markers and serum levels of IL-1β were estimated by ELISA The serum inflammatory markers levels were significantly higher in RA patients as compared to controls (RF=127.3±21.3U/mL, Anti-CCP=17.8±8.3U/mL, CRP=17.86±7.1mg/L and IL-1β=21.25±4.19pg/mL in RA patients pCCP (r=0.207) and CRP (r=0.166) among RA patients were found. The levels of anti-CCP were significantly higher in homozygous mutant variants (TT) as well as the heterozygous mutant variants (C/T) in comparison to the wild variants (CC) (pCCP and IL-1β levels as observed in RA patients and hence disease susceptibility. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. The potential effect of metallothionein 2A -5A/G single nucleotide polymorphism on blood cadmium, lead, zinc and copper levels.

    Science.gov (United States)

    Kayaaltı, Zeliha; Aliyev, Vugar; Söylemezoğlu, Tülin

    2011-10-01

    Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins. Because of their rich thiol groups, MTs bind to the biologically essential metals and perform these metals' homeostatic regulations; absorb the heavy metals and assist with their transportation and extraction. The aim of this study was to investigate the association between the metallothionein 2A (MT2A) core promoter region -5 A/G single nucleotide polymorphism (SNP) and Cd, Pb, Zn and Cu levels in the blood samples. MT2A polymorphism was determined by the standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique using the 616 blood samples and the genotype frequencies were found as 86.6% homozygote typical (AA), 12.8% heterozygote (AG) and 0.6% homozygote atypical (GG). Metal levels were analyzed by dual atomic absorption spectrophotometer system and the average levels of Cd, Pb, Zn and Cu in the blood samples were 1.69±1.57 ppb, 30.62±14.13 ppb, 0.98±0.49 ppm and 1.04±0.45 ppm, respectively. As a result; highly statistically significant associations were detected between the -5 A/G core promoter region SNP in the MT2A gene and Cd, Pb and Zn levels (p=0.004, p=0.012 and p=0.002, respectively), but no association was found with Cu level (p=0.595). Individuals with the GG genotype had statistically lower Zn level and higher Cd and Pb levels in the blood samples than individuals with AA and AG genotypes. This study suggests that having the GG genotype individuals may be more sensitive for the metal toxicity and they should be more careful about protecting their health against the toxic effects of the heavy metals. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Relationship between Occupational Stress, 5-HT2A Receptor Polymorphisms and Mental Health in Petroleum Workers in the Xinjiang Arid Desert: A Cross-Sectional Study.

    Science.gov (United States)

    Jiang, Ting; Ge, Hua; Sun, Jian; Li, Rong; Han, Rui; Liu, Jiwen

    2017-04-10

    At present, there is growing interest in research examining the relationship between occupational stress and mental health. Owing to the socioeconomic impact of occupational stress and the unique environment of petroleum workers in Xinjiang, a cross-sectional study was carried out between April and December 2015 to investigate the relationship between occupational stress, 5-hydroxytryptamine receptor (5-HTR2A) genotype, and mental health. A total of 1485 workers were selected. The Symptom Checklist 90 was used to assess nine classes of psychological symptoms. Work-related stressors were evaluated using the Occupational Stress Inventory-Revised Edition. Levels of 5-HTR2A (the Tl02C and A-1438G single nucleotide polymorphism in the 5-HTR2A gene) were measured by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The findings of the present study revealed a high prevalence rate of mental health problems (40.29%) in petroleum workers stationed in the arid desert, and suggested a strong correlation between occupational stress and mental health. The TC and CC genotype of Tl02C were found to be protective factors against mental health problems (odds ratio (OR) = 0.455, 95% confidence interval (CI): = 0.269-0.771, odds ratio (OR) = 0.340, 95% confidence interval (CI): 0.162-0.716). AG and GG genotype of A-1438G [odds ratio (OR) 1 = 2.729, 95% confidence interval (CI): 1.433-5.195; odds ratio (OR) 2 = 2.480, 95% confidence interval (CI): 1.221-5.037] were revealed as risk factors. These data provide evidence that occupational stress and 5-HTR2A gene polymorphism contributes to the incidence of mental health problems.

  11. Mediterranean dietary pattern and VEGF +405 G/C gene polymorphisms in patients with metabolic syndrome: An aspect of gene-nutrient interaction.

    Science.gov (United States)

    Hajiluian, Ghazaleh; Abbasalizad Farhangi, Mahdieh; Jahangiry, Leila

    2017-01-01

    To evaluate the relationship between Mediterranean dietary pattern, anthropometric and metabolic biomarkers and vascular endothelial growth factor (VEGF) +405 G/C gene polymorphism in patient with metabolic syndrome (Mets). In this study 150 patients with Mets and 50 healthy subjects were enrolled. Dietary intakes were evaluated with a semi-quantitative food-frequency questionnaire (FFQ) and Mediterranean dietary quality index (Med-DQI) was assessed. Anthropometric assessments and blood pressure measurement were performed. Biochemical assays including fasting serum glucose (FSG), matrix metalloproteinase-3 (MMP-3), liver enzymes and lipid profiles were also assessed. Polymorphism of +405 G/C VEGF gene was determined utilizing polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP) method. Serum high density lipoprotein-cholesterol (HDL-C) was significantly lower and low density lipoprotein cholesterol (LDL-C), triglyceride (TG), total cholesterol (TC) concentrations and FSG were significantly higher in metabolic syndrome patients compared with control group (P consumption of "cholesterol" had significantly upper serum TG; also high consumption of "fish" and "vegetables-fruits" was associated with a significantly lower serum LDL concentrations. In metabolic syndrome patients with CC genotype, mean score of "saturated fatty acid" subgroup was significantly higher compared with other genotypes; whereas, in healthy individuals, mean score of "fruit-vegetable" subgroup in individuals of CC and GG genotype was significantly higher (P<0.05). Our findings indicated a significant relationship between Mediterranean dietary quality index and both anthropometric and metabolic risk factors. We also indicated a higher "saturated fatty acid" intake in CC genotype among metabolic syndrome patients.

  12. Relationship between Occupational Stress, 5-HT2A Receptor Polymorphisms and Mental Health in Petroleum Workers in the Xinjiang Arid Desert: A Cross-Sectional Study

    Directory of Open Access Journals (Sweden)

    Ting Jiang

    2017-04-01

    Full Text Available At present, there is growing interest in research examining the relationship between occupational stress and mental health. Owing to the socioeconomic impact of occupational stress and the unique environment of petroleum workers in Xinjiang, a cross-sectional study was carried out between April and December 2015 to investigate the relationship between occupational stress, 5-hydroxytryptamine receptor (5-HTR2A genotype, and mental health. A total of 1485 workers were selected. The Symptom Checklist 90 was used to assess nine classes of psychological symptoms. Work-related stressors were evaluated using the Occupational Stress Inventory-Revised Edition. Levels of 5-HTR2A (the Tl02C and A-1438G single nucleotide polymorphism in the 5-HTR2A gene were measured by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP. The findings of the present study revealed a high prevalence rate of mental health problems (40.29% in petroleum workers stationed in the arid desert, and suggested a strong correlation between occupational stress and mental health. The TC and CC genotype of Tl02C were found to be protective factors against mental health problems (odds ratio (OR = 0.455, 95% confidence interval (CI: = 0.269–0.771, odds ratio (OR = 0.340, 95% confidence interval (CI: 0.162–0.716. AG and GG genotype of A-1438G [odds ratio (OR 1 = 2.729, 95% confidence interval (CI: 1.433–5.195; odds ratio (OR 2 = 2.480, 95% confidence interval (CI: 1.221–5.037] were revealed as risk factors. These data provide evidence that occupational stress and 5-HTR2A gene polymorphism contributes to the incidence of mental health problems.

  13. Genetic relationship and diversity in a sesame (Sesamum indicum L. germplasm collection using amplified fragment length polymorphism (AFLP

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2006-02-01

    Full Text Available Abstract Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia, and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP. Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA. This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres

  14. Genetic relationship and diversity in a sesame (Sesamum indicum L.) germplasm collection using amplified fragment length polymorphism (AFLP)

    Science.gov (United States)

    Laurentin, Hernán E; Karlovsky, Petr

    2006-01-01

    Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia), and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP). Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA). This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres. Future germplasm

  15. Association of a common allelic polymorphism (C677T) in the methylene tetrahydrofolate reductase gene with a reduced risk of osteoporotic fractures. A case control study in Danish postmenopausal women

    DEFF Research Database (Denmark)

    Jørgensen, H L; Madsen, J S; Madsen, B

    2002-01-01

    Twin studies indicate a substantial genetic component in the development of osteoporosis. One of the latest studied candidate genes is the one coding for methylene tetrahydrofolate reductase (MTHFR) (C677T) in which a point mutation gives rise to a thermolabile variant of MTHFR. The aim of this s...... associated with BMD at the lower forearm or with ultrasound parameters measured at the calcaneus. However, a significant increase in the odds ratio of fracture was found for the wild-type C-allele....... of this study was to investigate the influence of this mutation on peripheral measures of bone density and on the odds ratios (OR) for hip and lower forearm fracture in a case control study of Danish postmenopausal women. A total of 74 women with lower forearm fracture, 41 women with hip fracture, and 207 age...... fragment length polymorphism (PCR-RFLP). Only 2 of 21 individuals with the TT genotype had sustained a fracture as opposed to 46 of 142 with the CT genotype and 67 of 159 with the CC genotype (P = 0.007). Using logistic regression, the following odds ratios were found when comparing the individuals...

  16. Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

    Science.gov (United States)

    Erhardt, Georg; Shuiep, El Tahir Salih; Lisson, Maria; Weimann, Christina; Wang, Zhaoxin; El Zubeir, Ibtisam El Yas Mohamed; Pauciullo, Alfredo

    2016-06-01

    Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants.

  17. Analysis of the Association Between Apolipoprotein E Polymorphism and Cardiovascular Risk Factors in an Elderly Population with Longevity

    Directory of Open Access Journals (Sweden)

    Schwanke Carla Helena Augustin

    2002-01-01

    Full Text Available OBJECTIVE: To establish the allelic and genotypic frequencies related to apolipoprotein E (ApoE polymorphism and association of the genotypes with risk factors and cardiovascular morbidity in an elderly population with longevity. METHODS: We analyzed 70 elderly patients aged 80 years or more who were part of the Projeto Veranópolis. We used the gene amplification technique through the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and cleavage with the restriction enzyme Hha I to identify the ApoE genotypes. The most frequent genotypes were compared considering biological variables and cardiovascular risks and morbidity. RESULTS: The frequencies of the E2, E3, and E4 alleles were 0.05, 0.84, and 0.11, respectively, and of the genotypes were as follows: E3E3 (0.70, E3E4 (0.22, E2E3 (0.06, and E2E2 (0.02. Individuals with the E3E4 had a mean age greater than those with the E3E3. No association was observed between the genotypes and the variables analyzed, except for obesity, which was associated with the E3E3 genotype. Individuals with the E3E4 genotype had high levels of LDL-cholesterol and fibrinogen as compared with those with the E3E3 genotype. CONCLUSION: The results suggest that the E4E4 genotype may be associated with early mortality. A balance between the protective or neutral factors and the cardiovascular risk factors may occur among the individuals with different genotypes, attenuating the negative effects of the E4 allele.

  18. Close correlation between restriction fragment length polymorphism of the L-MYC gene and metastasis of human lung cancer to the lymph nodes and other organs

    International Nuclear Information System (INIS)

    Kawashima, Kazuko; Shikama, Hiroshi; Imoto, Kazuhiko; Izawa, Mitsuo; Nishimura, Susumu; Naruke, Tsuguo; Okabayashi, Kenzo

    1988-01-01

    Restriction length fragment polymorphism of the L-MYC gene was examined in DNAs from lung cancer tissues and normal tissues of 51 Japanese patients with lung cancer. In individual patients, no difference was seen between the restriction length fragments of the two alleles of L-MYC [6-kilobase (kb)] and 10-kb fragments in EcoRI digests in lung cancer tissues and normal tissues. But a striking correlation was found between the restriction length fragment polymorphism pattern of L-MYC and the extent of metastasis, particularly to the lymph nodes at the time of surgery: Patients with only the L band (10 kb) had few lymph node metastatic lesions, whereas patients with either the S band (6 kb) or the S and L bands almost always had lymph node metastatic lesion. A similar correlation was found between the presence of the S band and metastases to other organs. This correlation was particularly marked in cases of adenocarcinoma. These results indicate a clear genetic influence on metastases and a consequent poor prognosis for certain patients of lung cancer; L-MYC restriction length fragment polymorphism is thus shown to be a useful marker for predicting the metastatic potential of human lung cancer

  19. Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae.

    Science.gov (United States)

    Castro-Carrera, T; Toral, P G; Frutos, P; McEwan, N R; Hervás, G; Abecia, L; Pinloche, E; Girdwood, S E; Belenguer, A

    2014-03-01

    Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition

  20. Terminal Restriction Fragment Length Polymorphism for the Identification of Spirorchiid Ova in Tissues from the Green Sea Turtle, Chelonia mydas.

    Directory of Open Access Journals (Sweden)

    Phoebe A Chapman

    Full Text Available Blood flukes are among the most common disease causing pathogens infecting vertebrates, including humans and some of the world's most globally endangered fauna. Spirorchiid blood flukes are parasites of marine turtles, and are associated with pathology, strandings and mortalities worldwide. Their ova embolize in tissues and incite significant inflammatory responses, however attempts to draw correlations between species and lesions are frustrated by difficulties in identifying ova beyond the genus level. In this study, a newly developed terminal restriction fragment length polymorphism (T-RFLP method was validated as a tool for differentiating between mixed spirorchiid ova in turtle tissue. Initially, a multiplex PCR was used to differentiate between the five genera of spirorchiid flukes. Following this, PCR was performed using genus/genera-specific fluorescently tagged primer pairs and PCR products digested analysis using restriction endonucleases. Using capillary electrophoresis, this T-RFLP method could differentiate between twelve species and genotypes of spirorchiid flukes in turtles. It was applied to 151 tissue samples and successfully identified the spirorchiid species present. It was found to be more sensitive than visual diagnosis, detecting infections in 28 of 32 tissues that were negative on histology. Spirorchiids were present in 96.7% of tissues tested, with Neospirorchis genotype 2 being the most prevalent, present in 93% of samples. Mixed infections were common, being present in 60.7% of samples tested. The method described here is, to our knowledge, the first use of the T-RFLP technique on host tissues or in an animal ecology context, and describes a significant advancement in the clinical capacity to diagnose a common cause of illness in our environment. It is proven as a sensitive, specific and cost-efficient means of identifying spirorchiid flukes and ova in turtles, with the potential to contribute valuable information to

  1. Association of TNF-Alpha gene polymorphisms and susceptibility to hepatitis B virus infection in Egyptians.

    Science.gov (United States)

    Talaat, Roba M; Abdelkhalek, Mohamed S; El-Maadawy, Eman A; Abdel-Mageed, Wael S; El-Shenawy, Soha Z; Osman, Mohamed A

    2017-11-01

    Tumor necrosis factor alpha (TNF-α) is one of the important cytokine in generating an immune response against hepatitis B virus (HBV). Genetic polymorphisms might influence gene transcription, leading to disturbance in cytokine production. We hypothesized that single nucleotide polymorphism (SNPs) in TNF-α gene could affect the pathogenesis of HBV. To test this hypothesis, we investigated the role of TNF-α polymorphism [-863C/A (rs1800630), -308G/A (rs1800629), -376G/A (rs1800750), -857C/T (rs1799724) and +489G/A (rs1800610)] in the susceptibility to chronic hepatitis B (CHB) infection. Polymorphisms of the TNF-α (-863C/A (rs1800630), -308G/A) were analyzed by Polymerase chain reaction sequence specific primer (PCR-SSP) while TNF-α (-376G/A, -857C/T and +489G/A) by PCR-restriction fragment length polymorphism (PCR-RFLP) in 104 patients with CHB and 104 healthy controls. The plasma level of TNF-α was measured using Enzyme-linked immunosorbent assay (ELISA). The study showed a significant increase in the frequency of -863CC, -376GA, -857CC, -857TT and +489GA genotypes and -863C, -376A, -857C, and +489A alleles in CHB patients compared to controls. In addition, CAGCG haplotype had a highest frequency in CHB patients. A strong Linkage Disequilibrium (LD) between TNF-α -863C/A (rs1800630) and -376G/A (D' = 0.7888, r 2  = 0.0200); -308G/A and -857C/T (D' = 0.9213, r 2  = 0.1770); -308G/A and +489G/A (D' = 0.9088, r 2  = 0.1576) was demonstrated. CHB patients had significantly lower levels of TNF-α compared to controls. In conclusion, our preliminary results suggest that -863C/A (rs1800630), -308G/A, -376G/A, and +489G/A of the TNF-α gene may play a role in HBV susceptibility in Egyptians. The significant reduction in TNF-α in CHB patient was independent of any particular genotype/haplotype in TNF-α. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  2. Association of HLA class II alleles and CTLA-4 polymorphism with type 1 diabetes

    Directory of Open Access Journals (Sweden)

    Rana J EI Wafai

    2011-01-01

    Full Text Available Type-1 diabetes mellitus (T1DM is a progressive complex autoimmune disease in which combinations of environmental as well as genetic factors contribute to T-cell mediated destruction of insulin-secreting β-cells of the pancreas. HLA class II alleles on chromosome 6p21 [insulin dependent diabetes mellitus 1 (IDDM1], especially DR and DQ, show strong association with T1DM. In addition, several studies have suggested that polymorphisms in the CTLA-4 gene (IDDM12 on chromosome 2q33 form part of the genetic susceptibility for type 1 diabetes. The aim of this study was to analyze HLA alleles of the DQB1 and DRB1 genes using polymerase chain reaction using sequence specific primers (PCR-SSP technique and to investigate the asso-ciation of the A49G CTLA-4 polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis in Lebanese T1DM patients. The study was conduc-ted on 39 Lebanese T1DM patients. Results of HLA typing showed an increased frequency of the HLA-DQB1FNx010201, HLA-DQB1FNx010302, HLA-DRB1FNx010301 and HLA-DRB1FNx010401 alleles, sugges-ting risk association and thus can be considered as susceptibility alleles. On the other hand, strong protection against the disease was conferred by the HLA-DRB1FNx01110101, HLA-DQB1FNx010301 and HLADQB1FNx010601 alleles. RFLP analysis of the A49G polymorphism showed a significant increase in the G allele and GG genotype frequencies in patients, suggesting that CTLA-4 may be considered as a susceptibility gene for the development of T1DM in the Lebanese population. Analysis of the two polymorphisms showed no detectable association between the two genes. However, a significant negative association of the G allele with the DQB1FNx010201 allele was ob-served. This might indicate that the two genetic risk factors, namely HLA and CTLA-4, act independently of each other with no additive effect.

  3. Telomere Length Polymorphisms: A Potential Factor Underlying Increased Risk of Prostate Cancer in African American Men and Familial Prostate Cancer. Addendum

    Science.gov (United States)

    2009-12-01

    assessed in biopsies, polymorphisms in genes involved in inflammation and response to infection , and presence of antibodies against infectious...A.M., Lillemoe, K.D., Schulick, R., Hruban, R.H., Maitra, A., Argani, P. Telomere length variation in biliary tract metaplasia, dysplasia, and...Meeker AK. Dual-label centromere and telomere FISH identifies human, rat, and mouse cell contribution to Multispecies recombinant urogenital sinus

  4. Application of Amplified Fragment Length Polymorphism Fingerprinting for Taxonomy and Identification of the Soft Rot Bacteria Erwinia carotovora and Erwinia chrysanthemi

    OpenAIRE

    Avrova, Anna O.; Hyman, Lizbeth J.; Toth, Rachel L.; Toth, Ian K.

    2002-01-01

    The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (c...

  5. Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Part 1: Fungal field study

    Science.gov (United States)

    Grant T. Kirker; M. Lynn Prewitt; Tor P. Schultz; Susan V. Dieh

    2012-01-01

    The effects of chlorothalonil (CTN), butylated hydroxytoluene (BHT), and ammoniacal copper quat (ACQ-C) on the fungal community on southern yellow pine (SYP) were assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis over 15 months. Field stakes, treated with 0.25 and 0.37 % ACQ-C, 0.1 and 0.25 % CTN, 2 % BHT alone, 0.1 and 0.25 % CTN...

  6. Detection and characterization of a dehalogenating microorganism by terminal restriction fragment length polymorphism fingerprinting of 16S rRNA in a sulfidogenic, 2-bromophenol-utilizing enrichment.

    Science.gov (United States)

    Fennell, Donna E; Rhee, Sung-Keun; Ahn, Young-Beom; Häggblom, Max M; Kerkhof, Lee J

    2004-02-01

    Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.

  7. Association of MDR1 gene polymorphisms with the risk of hepatocellular carcinoma in the Chinese Han population

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jian [Tianjin Medical University, Tianjin (China)

    2013-03-15

    The multidrug resistance 1 gene (MDR1) is an important candidate gene for influencing susceptibility to hepatocellular carcinoma (HCC). The objective of the present study was to evaluate the association of MDR1 polymorphisms with the risk of HCC in the Chinese Han population. A total of 353 HCC patients and 335 healthy subjects were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR) and DNA sequencing methods were used to identify MDR1 gene polymorphisms. Two allelic variants (c.335T>C and c.3073A>C) were detected. The CC genotype of the c.335T>C polymorphism was associated with an increased risk of developing HCC compared to the TT genotype (OR = 2.161, 95%CI = 1.350-3.459, χ{sup 2} = 10.55, P = 0.0011). The risk of HCC was significantly higher for the CC genotype in the c.3073A>C polymorphism compared to the AA genotype in the studied populations (CC vs AA: OR = 2.575, 95%CI = 1.646-4.028, χ{sup 2} = 17.64, P < 0.0001). The C allele of the c.335T>C and c.3073A>C variants may contribute to the risk of HCC (C vs T of c.335T>C: OR = 1.512, 95%CI = 1.208-1.893, χ{sup 2} = 13.07, P = 0.0003, and C vs A of c.3073A>C: OR = 1.646, 95%CI = 1.322-2.049, χ{sup 2} = 20.03, P < 0.0001). The c.335T>C and c.3073A>C polymorphisms of the MDR1 gene were associated with the risk of occurrence of HCC in the Chinese Han population. Further investigations are needed to confirm these results in larger different populations.

  8. Alternative genotyping method for the single nucleotide polymorphism A2959G (AF159246 of the bovine CAST gene Método alternativo de genotipagem do polimorfismo de nucleotídeo único A2959G (AF159246 do gene CAST bovino

    Directory of Open Access Journals (Sweden)

    Rogério Abdallah Curi

    2008-05-01

    Full Text Available The objective of this work was to genotype the single nucleotide polymorphism (SNP A2959G (AF159246 of bovine CAST gene by PCR-RFLP technique, and to report its use for the first time. For this, 147 Bos indicus and Bos taurus x Bos indicus animals were genotyped. The accuracy of the method was confirmed through the direct sequencing of PCR products of nine individuals. The lowest frequency of the meat tenderness favorable allele (A in Bos indicus was confirmed. The use of PCR-RFLP for the genotyping of the bovine CAST gene SNP was shown to be robust and inexpensive, which will greatly facilitate its analysis by laboratories with basic structure.O objetivo deste trabalho foi genotipar o polimorfismo de nucleotídeo único ("single nucleotide polymorphism" - SNP A2959G (AF159246 do gene CAST bovino, pela técnica de PCR-RFLP, e reportar a sua utilização pela primeira vez. Para tanto, 147 animais Bos indicus e Bos taurus x Bos indicus foram genotipados. A acurácia do método foi confirmada por meio do seqüenciamento direto de produtos de PCR de nove indivíduos. A menor freqüência do alelo A, favorável à maciez da carne, foi confirmada nos animais Bos indicus. O uso da PCR-RFLP, para a genotipagem do SNP do gene CAST bovino, mostrou-se consistente e de baixo custo, o que permite a sua análise por laboratórios dotados de estrutura básica.

  9. ABO Gene Polymorphism and Thrombomodulin -33G>A Polymorphism Were Not Risk Factors for Myocardial Infarction in Javanese Men.

    Science.gov (United States)

    Lukitasari, Mifetika; Sadewa, Ahmad Hamim; Rohman, Mohammad Saifur

    2017-01-01

    Genetic factors contribute to about a half of coronary artery diseases. During the last several decades, some studies suggested that non-O blood group and thrombomodulin polymorphism -33G>A are the risk factors of coronary artery disease especially in Asia. There was no prior study in Indonesia regarding this issue. Hence, this study was designed to investigate the correlation of ABO polymorphism and thrombomodulin polymorphism -33G>A with the incidence of acute myocardial infarction (AMI). A total of 192 subjects were enrolled in this case control study. AMI patients were diagnosed based on World Health Organization criteria. Healthy patients were subjects with AMI risk factor without any sign and symptoms of AMI. Patients with diabetes mellitus, cancer, and arrhythmia were excluded from this study. Genotyping for both polymorphisms was performed by PCR RFLP methods. The result of this study suggested that ABO polymorphism and thrombomodulin polymorphism -33G>A were not risk factors of AMI, p = 0.727 and p = 0.699, respectively. Furthermore, the analysis to identify the synergy of these polymorphisms failed to prove their correlation with AMI ( p = 0.118). Conclusively, this study showed that ABO polymorphism and thrombomodulin polymorphism -33G>A were not risk factors of AMI.

  10. Differences in genotype and allele frequency distributions of polymorphic drug metabolizing enzymes CYP2C19 and CYP2D6 in mainland Chinese Mongolian, Hui and Han populations.

    Science.gov (United States)

    Yin, S-J; Ni, Y-B; Wang, S-M; Wang, X; Lou, Y-Q; Zhang, G-L

    2012-06-01

    Cytochrome P450 2C19 (CYP2C19) and CYP2D6 are important xenobiotic metabolic enzymes and both show considerable genetic variability between Orientals and Caucasians. There are known marked heterogeneity in susceptibility to various cancers and hypertension among Chinese Mongolian, Hui and Han ethnic groups, but the molecular mechanisms are unknown. Our objective was to investigate the patterns of distribution of CYP2C19 and CYP2D6 polymorphisms among healthy Chinese subjects to determine whether any observed inter-ethnic variability might be worth further investigation as possible contributors to the known differences in disease prevalence. Blood samples were collected from 454 unrelated Chinese healthy subjects (214 Han, 111 Hui, 129 Mongolian) for genotyping analysis. The single nucleotide polymorphisms (SNPs) CYP2C19*2 (681G>A in exon 5), CYP2C19*3 (636G>A in exon 4) and CYP2D6*10 (188C>T in exon 1) were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Significantly higher frequencies of the CYP2C19 poor metabolic genotypes were observed in Chinese Han (18·7%), Chinese Hui (25·0%) and Chinese Mongolian (10·9%) subjects than has been reported for Caucasians (1·7-3·0%, P Hui (32·4%) and Han (29·7%) than in Chinese Mongolian (18·2%, P Hui ethnic groups than have been reported for Caucasians (11·1-16·3% and 0-0·2%, P Hui (40·6% and 30·7%) and Mongolian subjects (31·3% and 9·6%, both P Hui and Han populations. These differences may be important in explaining reported inter-ethnic differences in disease prevalence and response to drugs. © 2011 Blackwell Publishing Ltd.

  11. Examination of Growth Hormone (GH) Gene Polymorphism and its Association with Body Weight and Selected Body Dimensions in Ducks.

    Science.gov (United States)

    Mazurowski, Artur; Frieske, Anna; Kokoszynski, Dariusz; Mroczkowski, Sławomir; Bernacki, Zenon; Wilkanowska, Anna

    2015-01-01

    The main objective of the study was to assess the polymorphism in intron 2 of the GH gene and its association with some morphological traits (body weight--BW, length of trunk with neck--LTN, length of trunk--LT, chest girth--CG, length of breast bone--LBB, length of shank--LS). Polymorphism in intron 2 of the GH gene was evaluated for four duck populations (Pekin ducks AF51, Muscovy ducks from a CK and CRAMMLCFF mother and Mulard ducks). Genetic polymorphism was determined with the PCR-RFLP method using the BsmFI restriction enzyme. In the studied duck sample two alleles (GH(C) and GH(T)) and three genotypes (GH/TT, GH/CT, GH/CC) were found at locus GH/BsmFI. In both groups of Muscovies and in Mulards the dominant allele was GH(T). On the contrary in Pekin ducks AF51, the frequency of both alleles was found to be similar. The most frequent genotype in the examined ducks was GH/TT. In Pekin ducks AF51 three genotypes were observed, while in Mulard ducks and in male Muscovy ducks from a mother marked as CK, two genotypes (GH/TT and GH/CT) were identified. Muscovy duck females from a CK mother and all males and females of Muscovy duck from a CRAMMLCFF mother were monomorphic with only the GH/TTgenotype detected. The results showed that males of Pekin duck AF51 with the GH/TT genotype were characterized by higher (P GH/CC and GH/CTgenotype. In females of Pekin ducks AF51, this same trend was observed; individuals with GH/TT genotype were superior (P GH/TT genotype were distinguished by higher values of all evaluated traits compared to ducks with GH/CT and GH/CC genotypes, however most of the recorded differences were not significant. The only trait markedly impacted (P GH gene intron 2 was the LS value in males.

  12. Association between H-RAS T81C genetic polymorphism and gastrointestinal cancer risk: A population based case-control study in China

    International Nuclear Information System (INIS)

    Zhang, Yongjing; Jin, Mingjuan; Liu, Bing; Ma, Xinyuan; Yao, Kaiyan; Li, Qilong; Chen, Kun

    2008-01-01

    Gastrointestinal cancer, such as gastric, colon and rectal cancer, is a major medical and economic burden worldwide. However, the exact mechanism of gastrointestinal cancer development still remains unclear. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours and the single nucleotide polymorphism at H-RAS cDNA position 81 was demonstrated to contribute to the risks of bladder, oral and thyroid carcinoma. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to gastrointestinal cancer as well, and we conducted this study to test the hypothesis in Chinese population. A population based case-control study, including 296 cases with gastrointestinal cancer and 448 healthy controls selected from a Chinese population was conducted. H-RAS T81C polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay. In the healthy controls, the TT, TC and CC genotypes frequencies of H-RAS T81C polymorphism, were 79.24%, 19.87% and 0.89%, respectively, and the C allele frequency was 10.83%. Compared with TT genotype, the TC genotype was significantly associated with an increased risk of gastric cancer (adjusted OR = 3.67, 95%CI = 2.21–6.08), while the CC genotype showed an increased risk as well (adjusted OR = 3.29, 95%CI = 0.54–19.86), but it was not statistically significant. In contrast, the frequency of TC genotype was not significantly increased in colon cancer and rectal cancer patients. Further analysis was performed by combining TC and CC genotypes compared against TT genotype. As a result, a statistically significant risk with adjusted OR of 3.65 (95%CI, 2.22–6.00) was found in gastric cancer, while no significant association of H-RAS T81C polymorphism with colon cancer and rectal cancer was observed. These findings indicate, for the first time, that there is an H-RAS T81C polymorphism existing in Chinese population

  13. Association between H-RAS T81C genetic polymorphism and gastrointestinal cancer risk: A population based case-control study in China

    Directory of Open Access Journals (Sweden)

    Li Qilong

    2008-09-01

    Full Text Available Abstract Background Gastrointestinal cancer, such as gastric, colon and rectal cancer, is a major medical and economic burden worldwide. However, the exact mechanism of gastrointestinal cancer development still remains unclear. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours and the single nucleotide polymorphism at H-RAS cDNA position 81 was demonstrated to contribute to the risks of bladder, oral and thyroid carcinoma. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to gastrointestinal cancer as well, and we conducted this study to test the hypothesis in Chinese population. Methods A population based case-control study, including 296 cases with gastrointestinal cancer and 448 healthy controls selected from a Chinese population was conducted. H-RAS T81C polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP assay. Results In the healthy controls, the TT, TC and CC genotypes frequencies of H-RAS T81C polymorphism, were 79.24%, 19.87% and 0.89%, respectively, and the C allele frequency was 10.83%. Compared with TT genotype, the TC genotype was significantly associated with an increased risk of gastric cancer (adjusted OR = 3.67, 95%CI = 2.21–6.08, while the CC genotype showed an increased risk as well (adjusted OR = 3.29, 95%CI = 0.54–19.86, but it was not statistically significant. In contrast, the frequency of TC genotype was not significantly increased in colon cancer and rectal cancer patients. Further analysis was performed by combining TC and CC genotypes compared against TT genotype. As a result, a statistically significant risk with adjusted OR of 3.65 (95%CI, 2.22–6.00 was found in gastric cancer, while no significant association of H-RAS T81C polymorphism with colon cancer and rectal cancer was observed. Conclusion These findings indicate, for the first time, that there

  14. Ethnic differences in the prevalence of polymorphisms in CYP7A1, CYP7B1 AND CYP27A1 enzymes involved in cholesterol metabolism

    Directory of Open Access Journals (Sweden)

    Vera Dias

    2011-01-01

    Full Text Available It is well known that drug disposition and response are greatly determined by the activities of drug metabolizing enzymes, which are polymorphic. Some of these polymorphisms are clinically relevant and presented an ethnic-dependent pattern of distribution. The characterization of the genetic distribution of different populations allows the selection of therapeutic options in accordance with the genetic background, with the objective to avoid adverse reactions and inefficacy of the treatment. In this work, we studied selected genetic polymorphisms in drug metabolizing enzymes in three different ethnic groups - Portugal, Mozambique and Colombia. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP genotyping methods were developed for drug metabolizing enzymes, namely, cholesterol 7α-hydroxylase (CYP7A1 (−203A>C, −346C>T, −496C>T, N233S, G347S, sterol 27-hydroxylase (CYP27A1 (R164W, A169V, D273N, V400A and oxysterol 7α-hydroxylase (CYP7B1 (−116C>G, R324H, 1774C>T to characterize the allelic distribution of these polymorphisms among three different ethnic/geographic origins. A total of 12 CYP7A1, CYP27A1 and CYP7B1 genetic variants were genotyped in a sample of 92 Portuguese, 151 Mozambican and 91 Colombian subjects. The variants N233S in CYP7A1 and 1774C>T in CYP7B1 were not detected in any population studied. The promoter polymorphisms in CYP7A1 (−203A>C, −346C>T, −496C>T had high frequency in the three ethnic groups. G347S (CYP7A1, R164W, A169V and V400A (CYP27A1 were present in a low frequency but with a similar distribution in the three ethnic groups. Significant differences were observed for D273N (CYP27A1, −346C>T (CYP7A1, −116C>G and R324H (CYP7B1Our results demonstrate a high variability of drug metabolizing enzymes between the different populations analyzed, indicating that at least some of these polymorphisms are ethnic specific.

  15. Ethnic differences in the prevalence of polymorphisms in CYP7A1, CYP7B1 AND CYP27A1 enzymes involved in cholesterol metabolism.

    Science.gov (United States)

    Dias, Vera; Ribeiro, V

    2011-07-01

    It is well known that drug disposition and response are greatly determined by the activities of drug metabolizing enzymes, which are polymorphic. Some of these polymorphisms are clinically relevant and presented an ethnic-dependent pattern of distribution. The characterization of the genetic distribution of different populations allows the selection of therapeutic options in accordance with the genetic background, with the objective to avoid adverse reactions and inefficacy of the treatment. In this work, we studied selected genetic polymorphisms in drug metabolizing enzymes in three different ethnic groups - Portugal, Mozambique and Colombia. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping methods were developed for drug metabolizing enzymes, namely, cholesterol 7α-hydroxylase (CYP7A1) (-203A>C, -346C>T, -496C>T, N233S, G347S), sterol 27-hydroxylase (CYP27A1) (R164W, A169V, D273N, V400A) and oxysterol 7α-hydroxylase (CYP7B1) (-116C>G, R324H, 1774C>T) to characterize the allelic distribution of these polymorphisms among three different ethnic/geographic origins. A total of 12 CYP7A1, CYP27A1 and CYP7B1 genetic variants were genotyped in a sample of 92 Portuguese, 151 Mozambican and 91 Colombian subjects. The variants N233S in CYP7A1 and 1774C>T in CYP7B1 were not detected in any population studied. The promoter polymorphisms in CYP7A1 (-203A>C, -346C>T, -496C>T) had high frequency in the three ethnic groups. G347S (CYP7A1), R164W, A169V and V400A (CYP27A1) were present in a low frequency but with a similar distribution in the three ethnic groups. Significant differences were observed for D273N (CYP27A1), -346C>T (CYP7A1), -116C>G and R324H (CYP7B1)Our results demonstrate a high variability of drug metabolizing enzymes between the different populations analyzed, indicating that at least some of these polymorphisms are ethnic specific.

  16. [Association between ameloblastin gene polymorphisms and the susceptibility to dental fluorosis].

    Science.gov (United States)

    Jiao, Yong-zhuo; Mu, Li-hong; Wang, Ying-xiong; An, Wei; Jiang, Miao

    2013-01-01

    To study the distribution of ameloblastin (AMBN) gene polymorphism in coal-fire caused fluorosis (CFCF) in Chongqing municipality and the relationship between AMBN gene polymorphism and the susceptibility to dental fluorosis. Under a case-control study, 100 children aged 8 - 12 and 30 adults with dental fluorosis were enrolled in Wushan and Fengjie counties of Chongqing from December 2010 to February 2011. Another 100 children aged 8 - 12 and 30 adults with non-dental fluorosis were chosen as internal control groups together with 50 children and 30 adults without dental fluorosis were selected as external control groups in the non-epidemic area of Yubei district. DNA was extracted from peripheral blood sample of these people. Genotype of AMBN gene 7 extron 538_540delGGA, 10 extron 657A > G and 13 extron 986C > T loci were detected using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The rates of 7 extron 538_540delGGA loci among case, internal and external control groups were as follows: GGA/GGA-/- 61.2% (74/121), 78.5% (102/130), 74.3% (52/70) ; GGA/-: 24.0% (29/121), 15.4% (20/130), 22.9% (16/70) ; -/-: 14.8% (18/121), 6.1% (8/130), 2.8% (2/70), the difference was statistically significant (χ(2) = 14.353 P 0.05). CC appeared as 81.0% (98/121), 90.0% (117/130), 87.1% (61/70) while CT as 19.0% (23/121), 10.0% (13/130), 12.9% (9/70), with difference not statistically significant (χ(2) = 4.319, P > 0.05). In comparing with the two control groups, the frequency of GGA/GGA was decreasing (χ(2) values were 8.957, 3.405, respectively, P fluorosis (OR values were 2.7, 5.9, respectively, P fluorosis (OR = 2.1, P T loci polymorphism in AMBN gene might serve as the susceptibility factors causing the coal-fired fluorosis.

  17. Polymorphisms in MTHFR, MS and CBS genes and homocysteine levels in a Pakistani population.

    Directory of Open Access Journals (Sweden)

    Mohsin Yakub

    Full Text Available BACKGROUND: Hyperhomocysteinemia (>15 µmol/L is highly prevalent in South Asian populations including Pakistan. In order to investigate the genetic determinants of this condition, we studied 6 polymorphisms in genes of 3 enzymes--methylenetetrahydrofolate reductase (MTHFR; C677T; A1298C, methionine synthase (MS; A2756G, cystathionine-β-synthase (CBS; T833C/844ins68, G919A involved in homocysteine metabolism and investigated their interactions with nutritional and environmental factors in a Pakistani population. METHODOLOGY/PRINCIPAL FINDINGS: In a cross-sectional survey, 872 healthy adults (355 males and 517 females; age 18-60 years were recruited from a low-income urban population in Karachi. Fasting venous blood was obtained and assessed for plasma/serum homocysteine; folate, vitamin B12, pyridoxal phosphate and blood lead. DNA was isolated and genotyping was performed by PCR-RFLP (restriction-fragment-length-polymorphism based assays. The average changes in homocysteine levels for MTHFR 677CT and TT genotypes were positive [β(SE β, 2.01(0.63 and 16.19(1.8 µmol/L, respectively]. Contrary to MTHFR C677T polymorphism, the average changes in plasma homocysteine levels for MS 2756AG and GG variants were negative [β(SE β, -0.56(0.58 and -0.83(0.99 µmol/L, respectively]. The average change occurring for CBS 844ins68 heterozygous genotype (ancestral/insertion was -1.88(0.81 µmol/L. The combined effect of MTHFR C677T, MS A2756G and CBS 844ins68 genotypes for plasma homocysteine levels was additive (p value <0.001. Odds of having hyperhomocysteinemia with MTHFR 677TT genotype was 10-fold compared to MTHFR 677CC genotype [OR (95%CI; 10.17(3.6-28.67]. Protective effect towards hyperhomocysteinemia was observed with heterozygous (ancestral/insertion genotype of CBS 844ins68 compared to homozygous ancestral type [OR (95% CI; 0.58 (0.34-0.99]. Individuals with MTHFR 677CT or TT genotypes were at a greater risk of hyperhomocysteinemia in folate and

  18. ABCB1 3435C>T and 2677G>T/A polymorphisms in Polish and Bosnian patients with Crohn’s disease – A preliminary report

    Directory of Open Access Journals (Sweden)

    Krzysztof Borecki

    2017-11-01

    Full Text Available The role of ABCB1 single nucleotide polymorphisms (SNPs in the development of Crohn’s disease (CD remains unclear. Due to inconsistent results of several European population-based studies and limited information on populations from Poland and Bosnia and Herzegovina (B&H, we conducted a preliminary association study of two main ABCB1 SNPs and CD. ABCB1 3435C>T and 2677G>T/A SNPs were analyzed in Polish and Bosnian patients with CD (n = 85 and n = 30, respectively and controls (n = 82 and n = 30, respectively using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP for 3435C>T and allele-specific PCR for 2677G>A/T SNP. A deviation from Hardy-Weinberg equilibrium was found for both SNPs in Polish patients with CD, and for 2677G>A/T in Polish control group. The allele and genotype frequencies of the two ABCB1 SNPs were not significantly different between the CD patients and controls in both populations (p > 0.05. Similarly, the genotype distribution of 3435C>T and 2677G>T/A SNPs was not significantly different between Polish and Bosnian patients with CD (p > 0.05. At least one mutated ABCB1 allele was carried by 97.7% of Polish and 90.0% of Bosnian patients with CD. No association was found between the ABCB1 SNPs and CD in the two populations. In conclusion, the two ABCB1 SNPs may not contribute to CD susceptibility in the populations of Poland and B&H. Further studies with larger samples in both populations are warranted.

  19. Comparative Study of IS6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands, Based on a 5-Year Nationwide Survey

    NARCIS (Netherlands)

    Beer, J.L. de; Ingen, J. van; Vries, G. de; Erkens, C.; Sebek, M.; Mulder, A.; Sloot, R.; Brandt, A.M. van den; Enaimi, M.; Kremer, K.; Supply, P.; Soolingen, D. van

    2013-01-01

    In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a

  20. Comparative study of IS6110 restriction fragment length polymorphism and variable-number tandem-repeat typing of Mycobacterium tuberculosis isolates in the Netherlands, based on a 5-year nationwide survey

    NARCIS (Netherlands)

    de Beer, Jessica L.; van Ingen, Jakko; de Vries, Gerard; Erkens, Connie; Sebek, Maruschka; Mulder, Arnout; Sloot, Rosa; van den Brandt, Anne-Marie; Enaimi, Mimount; Kremer, Kristin; Supply, Philip; van Soolingen, Dick

    2013-01-01

    In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a

  1. Polymorphisms of 5,10-methylenetetralydrofolate reductase (MTHFR), fruit and vegetable intake, and the risk of stomach cancer.

    Science.gov (United States)

    Mu, L N; Cao, W; Zhang, Z F; Yu, S Z; Jiang, Q W; You, N C; Lu, Q Y; Zhou, X F; Ding, B G; Chang, J; Chen, C W; Wei, G R; Cai, L

    2007-01-01

    Stomach cancer is a serious public health problem in China. 5,10-Methylenetetralydrofolate reductase (MTHFR) may be involved in both DNA methylation and DNA synthesis. Folate deficiency is associated with cancer risk that may be modulated by a genetic variation in the MTHFR gene in folate metabolism. The main goal of this study was to evaluate the association between polymorphisms of the MTHFR gene and the risk of stomach cancer. This study also explored the modification effects of fruit and vegetable intake (one of the main constituents is folate) on the risk of this disease. A population-based case-control study was conducted in Taixing, China, consisting of 206 newly diagnosed cases with primary stomach cancer and 415 healthy population controls. Polymorphisms of MTHFR C677T and A1298C were assayed by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) techniques. The data were analysed using the logistic regression model. No obvious association between the MTHFR A1298C polymorphism and the risk of stomach cancer was observed in this study. The frequencies of 677 C/C, C/T, and T/T were 34.5, 50.9, and 14.6%, respectively, in controls. The frequency of the MTHFR 677 wild homozygotic genotype was 25.8% in cases, which was lower than that in controls (34.5%). The adjusted odds ratio (OR) for the MTHFR 677 any T genotype was 2.05 (95% confidence interval (CI), 1.26-3.34) when compared with the C/C genotype. In the low fruit and vegetable intake group an increasing trend was observed with the T allele exposure, p = 0.0056. The adjusted ORs were 1.68 (95% CI = 0.86-3.29) for the C/T genotype and 3.58 (95% CI = 1.46-8.75) for the T/T genotype, respectively. The MTHFR 677 any T genotype was associated with an increased risk of primary stomach cancer among the Chinese population. Folate deficiency might modify the MTHFR gene polymorphism and influence the risk of stomach cancer.

  2. Development of intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase for discriminating Curcuma species.

    Science.gov (United States)

    Kita, Tomoko; Komatsu, Katsuko; Zhu, Shu; Iida, Osamu; Sugimura, Koji; Kawahara, Nobuo; Taguchi, Hiromu; Masamura, Noriya; Cai, Shao-Qing

    2016-03-01

    Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Development of rapid PCR-RFLP technique for identification of ...

    African Journals Online (AJOL)

    Species differentiation was realized by digestion of the amplified ~195 bp fragments with Sse9I restriction enzyme. The results indicate that 7/7 of Kebab loghmeh, 9/10 of minced meat, 4/8 of beef burger and 2/5 samples of canned stew samples, were contaminated with one of prohibited ruminant species residual.

  4. Difficulties with conventional phytoplasma diagnostic using PCR/RFLP analyses

    Czech Academy of Sciences Publication Activity Database

    Fránová, Jana

    2011-01-01

    Roč. 64, Suppl. (2011), s. 287-288 ISSN 1721-8861 R&D Projects: GA MŠk OC09021; GA AV ČR(CZ) 1QS500510558; GA MZe QH71145 Institutional research plan: CEZ:AV0Z50510513 Keywords : DNA extraction * PCR * primers * RFLP Subject RIV: EE - Microbiology, Virology Impact factor: 0.592, year: 2011

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