WorldWideScience

Sample records for length polymorphism rflp

  1. Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP).

    Science.gov (United States)

    Prakash, Om; Pandey, Prashant K; Kulkarni, Girish J; Mahale, Kiran N; Shouche, Yogesh S

    2014-09-01

    Terminal restriction fragment length polymorphism (T-RFLP) is a rapid, robust, inexpensive and simple tool for microbial community profiling. Methods used for DNA extraction, PCR amplification and digestion of amplified products have a considerable impact on the results of T-RFLP. Pitfalls of the method skew the similarity analysis and compromise its high throughput ability. Despite a high throughput method of data generation, data analysis is still in its infancy and needs more attention. Current article highlights the limitations of the methods used for data generation and analysis. It also provides an overview of the recent methodological developments in T-RFLP which will assist the readers in obtaining real and authentic profiles of the microbial communities under consideration while eluding the inherent biases and technical difficulties.

  2. Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP).

    Science.gov (United States)

    Watanarojanaporn, N; Longtonglang, A; Boonkerd, N; Tittabutr, P; Lee, J; Teaumroong, N

    2014-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol-chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.

  3. Characterization of Campylobacter jejuni and Campylobacter coli genotypes in poultry flocks by restriction fragment length polymorphism (RFLP) analysis.

    Science.gov (United States)

    Carreira, Ana Cláudia; Cunha, Mónica V

    2015-01-01

    We describe a simple, rapid, and discriminatory methodology that allows the routine molecular characterization of Campylobacter jejuni and Campylobacter coli isolates. The proposed approach is built on one of the earliest and simplest molecular typing methods ever, consisting on the analysis of the fragments of different lengths generated by digestion of homologous DNA sequences with specific restriction endonucleases, a process known as restriction fragment length polymorphism (RFLP) analysis. The strategy underneath the workflow reported here is meant to explore the polymorphisms of Campylobacter spp. flaA gene (flaA-RFLP) that allows the local investigation of the genetic diversity and distribution of C. coli and C. jejuni isolates from different sources, namely, chickens' caeca. Although not appropriate for global and long-term epidemiological studies as a single approach, flaA-RFLP analysis can be very useful in surveys limited in space and time and, for specific epidemiological settings, an alternative to more modern and resource-demanding techniques.

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

    Science.gov (United States)

    Rohit, Anusha; Maiti, Biswajit; Shenoy, Shalini; Karunasagar, Indrani

    2016-01-01

    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. Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture) and PCR-RFLP. 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. 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.

  5. 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...

  6. Comparison between terminal-restriction fragment length polymorphism (T-RFLP) and quantitative culture for analysis of infants' gut microbiota.

    Science.gov (United States)

    Sjöberg, Fei; Nowrouzian, Forough; Rangel, Ignacio; Hannoun, Charles; Moore, Edward; Adlerberth, Ingegerd; Wold, Agnes E

    2013-07-01

    The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism (T-RFLP). A database was constructed for direct identification of T-RFLP peaks by analysis of pure-culture bacteria and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at >10⁶ CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts. In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be identified using the database. T-RFLP readily identified bacteria to the genus level and also provided some sub-genus discrimination. Both T-RFLP and culture identified Bifidobacterium, Clostridium and Bacteroides spp. among the most common colonizers of the infantile microbiota throughout the first year of life. T-RFLP analysis showed that microbiota complexity was high in the first weeks of life, declined to a minimum at 1-2 months of age, and thereafter increased again. Principal component analysis revealed that early samples (1 week-6 months) chiefly differed between individual infants, while 12-month samples were similar between children, but different from the early samples. Our results indicate that T-RFLP has high sensitivity and adequate taxonomic discrimination capacity for analysis of

  7. A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker for the identification of Amaranthus cruentus species.

    Science.gov (United States)

    Park, Young-Jun; Nishikawa, Tomotaro; Matsushima, Kenichi; Minami, Mineo; Nemoto, Kazuhiro

    2014-12-01

    A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5'-T/TAA-3' in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. 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...

  11. An improved PCR-restriction fragment length polymorphism (RFLP) method for the identification of cry1-type genes.

    Science.gov (United States)

    Shu, Changlong; Liu, Dongming; Zhou, Zishan; Cai, Jilin; Peng, Qi; Gao, Jiguo; Song, Fuping; Zhang, Jie

    2013-11-01

    The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.

  12. Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

    OpenAIRE

    Wood, S.; Starr, T V; Shukin, R J

    1986-01-01

    We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes dete...

  13. Rapid Identification Method of Omphalotus japonicus by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).

    Science.gov (United States)

    Sugano, Yohei; Sakata, Kozue; Nakamura, Kosuke; Noguchi, Akio; Fukuda, Nozomi; Suzuki, Tomohiro; Kondo, Kazunari

    2017-01-01

    Omphalotus japonicus is a poisonous mushroom that grows in Japan. It can be mistaken for edible mushrooms (Shiitake, Hiratake and Mukitake), and if ingested, it causes food poisoning within 30 min to 1 hr. We established a rapid detection method using PCR-RFLP to identify O. japonicus by restriction digestion of the amplified ITS region. By using Sau96I, Bpu10I, SfcI or DrdI/HincII as a restriction enzyme, it was possible to rapidly identify and discriminate O. japonicus based on the fragment length. This study also provided a short PCR-RFLP system comprising amplification and digestion of a short 200-bp DNA fragment within the ITS region. The system could identify and discriminate O. japonicus after in vitro gastric digestion of native and heated mushroom samples as a model of food poisoning. In addition, a confirmatory assay using real-time PCR was developed to achieve more sensitive detection of O. japonicus.

  14. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea.

    Science.gov (United States)

    Kong, Hyun-Hee; Shin, Ji-Yeol; Yu, Hak-Sun; Kim, Jin; Hahn, Tae-Won; Hahn, Young-Ho; Chung, Dong-Il

    2002-04-01

    We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43 Acanthamoeba environmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A. castellanii complex. The most predominant (48.8%) type was A. castellanii KA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those of A. castellanii Castellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those of A. castellanii Ma, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those of A. lugdunensis L3a, KA/E2, and KA/E12. The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.

  15. Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli.

    Science.gov (United States)

    Sugimoto, Norihiko; Shima, Kensuke; Hinenoya, Atsushi; Asakura, Masahiro; Matsuhisa, Akio; Watanabe, Haruo; Yamasaki, Shinji

    2011-07-01

    In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.

  16. Genetic Typing of Bovine Viral Diarrhoea Virus (BVDV by Restriction Fragment Length Polymorphism (RFLP and Identification of a New Subtype in Poland

    Directory of Open Access Journals (Sweden)

    Kuta Aleksandra

    2015-04-01

    Full Text Available Restriction fragment length polymorphism (RFLP analysis was developed for genetic typing of Polish strains of bovine viral diarrhoea virus (BVDV. The method was applied using 60 BVDV isolates, which included BVDV genotype 1, subtypes a, b, d, e, f, and g, and genotype 2a. RT-PCR products of the 5’untranslated region (5’UTR were digested using three enzymes. Restriction patterns classified the strains into seven groups, each with a specific and different pattern from other subtypes. These findings were confirmed by nucleotide sequencing and phylogenetic analysis. The results suggest that RFLP analysis is a simple, reliable, and fast genotyping method for BVDV strains in comparison with sequencing. This method can distinguish six subtypes of BVDV-1 including a new subtype 1e, identified exclusively by this method, and it allows differentiation of BVDV-1 from BVDV-2 genotype.

  17. Towards the molecular characterisation of parasitic nematode assemblages: an evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis.

    Science.gov (United States)

    Lott, M J; Hose, G C; Power, M L

    2014-09-01

    Identifying factors which regulate temporal and regional structuring within parasite assemblages requires the development of non-invasive techniques which facilitate both the rapid discrimination of individual parasites and the capacity to monitor entire parasite communities across time and space. To this end, we have developed and evaluated a rapid fluorescence-based method, terminal restriction fragment length polymorphism (T-RFLP) analysis, for the characterisation of parasitic nematode assemblages in macropodid marsupials. The accuracy with which T-RFLP was capable of distinguishing between the constituent taxa of a parasite community was assessed by comparing sequence data from two loci (the ITS+ region of nuclear ribosomal DNA and the mitochondrial CO1) across ∼20 species of nematodes (suborder Strongylida). Our results demonstrate that with fluorescent labelling of the forward and reverse terminal restriction fragments (T-RFs) of the ITS+ region, the restriction enzyme Hinf1 was capable of generating species specific T-RFLP profiles. A notable exception was within the genus Cloacina, in which closely related species often shared identical T-RFs. This may be a consequence of the group's comparatively recent evolutionary radiation. While the CO1 displayed higher sequence diversity than the ITS+, the subsequent T-RFLP profiles were taxonomically inconsistent and could not be used to further differentiate species within Cloacina. Additionally, several of the ITS+ derived T-RFLP profiles exhibited unexpected secondary peaks, possibly as a consequence of the restriction enzymes inability to cleave partially single stranded amplicons. These data suggest that the question of T-RFLPs utility in monitoring parasite communities cannot be addressed without considering the ecology and unique evolutionary history of the constituent taxa.

  18. Amplification of a single-locus variable-number direct repeats with restriction fragment length polymorphism (DR-PCR/RFLP) for genetic typing of Acinetobacter baumannii strains.

    Science.gov (United States)

    Nowak-Zaleska, Alicja; Krawczyk, Beata; Kotłowski, Roman; Mikucka, Agnieszka; Gospodarek, Eugenia

    2008-01-01

    In search of an effective DNA typing technique for Acinetobacter baumannii strains for hospital epidemiology use, the performance and convenience of a new target sequence was evaluated. Using known genomic sequences of Acinetobacter baumannii strains AR 319754 and ATCC 17978, we developed single-locus variable-number direct-repeat analysis using polymerase chain reaction-restriction fragment length polymorphism (DR-PCR/RFLP) method. A total of 90 Acinetobacter baumannii strains isolated from patients of the Clinical Hospital in Bydgoszcz, Poland, were examined. Initially, all strains were typed using macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE). Digestion of the chromosomal DNA with the ApaI endonuclease and separation of the fragments by PFGE revealed 21 unique types. Application of DR-PCR/RFLP resulted in recognition of 12 clusters. The results showed that the DR-PCR/RFLP method is less discriminatory than REA-PFGE, however, the novel genotyping method can be used as an alternative technique for generating DNA profiles in epidemiological studies of intra-species genetic relatedness of Acinetobacter baumannii strains.

  19. Terminal restriction fragment length polymorphism (T-RFLP) profiling of bacterial 16S rRNA genes.

    Science.gov (United States)

    Osborne, Catherine A

    2014-01-01

    T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacterial communities.

  20. Keragaman Genotip dan Jarak Genetik Sapi Madura Berdasarkan Restriction Fragment Length Polymorphism-DNA (RFLP-DNA

    Directory of Open Access Journals (Sweden)

    Marleny Leasa

    2013-02-01

    Full Text Available Penelitian ini bertujuan untuk mengetahui variasi genotip dan jarak genetik sapi Madura di Kabupaten Sampang dan Balai Besar Inseminasi Buatan (BBIB Singosari berdasarkan teknik RFLP. Digesti DNA genom dengan enzim EcoRI dan PstI menghasilkan fragmen DNA dengan ukuran yang bervariasi baik pada induk, pedet, dan pejantan unggul dengan kisaran antara 10000 bp sampai 980 bp dan 10000 bp sampai 1250 bp. Analisis MVSP1 dengan metode UPGMA untuk jarak genetik ditemukan bahwa sampel sapi Madura berada dalam 2 cluster dan 1 outgroup. Persentase jarak genetik berada pada rentangan 0 sampai 25%.   (Kata kunci: Variasi genotip, Jarak genetik, Sapi Madura, RFLP

  1. Pros and cons of ion-torrent next generation sequencing versus terminal restriction fragment length polymorphism T-RFLP for studying the rumen bacterial community.

    Science.gov (United States)

    de la Fuente, Gabriel; Belanche, Alejandro; Girwood, Susan E; Pinloche, Eric; Wilkinson, Toby; Newbold, C Jamie

    2014-01-01

    The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T-RFLP). Although absolute number differed, there was a high correlation between NGS and T-RFLP in terms of richness and diversity with R values of 0.836 and 0.781 for richness and Shannon-Wiener index, respectively. Dendrograms for both datasets were also highly correlated (Mantel test = 0.742). Eighteen OTUs and ten genera were significantly impacted by the addition of rumen protozoa, with an increase in the relative abundance of Prevotella, Bacteroides and Ruminobacter, related to an increase in free ammonia levels in the rumen. Our findings suggest that classic fingerprinting methods are still valuable tools to study microbial diversity and structure in complex environments but that NGS techniques now provide cost effect alternatives that provide a far greater level of information on the individual members of the microbial population.

  2. Comparison of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay to pulsed-field gel electrophoresis to determine the effect of repeated subculture and prolonged storage on RFLP patterns of Shiga toxin-producing Escherichia coli O157:H7.

    Science.gov (United States)

    Shima, Kensuke; Wu, Yuluo; Sugimoto, Norihiko; Asakura, Masahiro; Nishimura, Kazuhiko; Yamasaki, Shinji

    2006-11-01

    In this study, we compared a recently developed PCR-restriction fragment length polymorphism (PCR-RFLP) assay with pulsed-field gel electrophoresis (PFGE) using three different Shiga toxin-producing Escherichia coli (STEC) strains to understand whether repeated subculture in vitro and prolonged storage at room temperature affect the RFLP patterns of STEC. The PFGE profiles of the STEC strains changed by 1 to 8 fragments after repeated subculture and prolonged storage; one strain was no longer clonal after repeated subculture compared to the original isolate according to the Tenover criteria. In contrast, RFLP patterns obtained by PCR-RFLP were identical after repeated subculture and prolonged storage. These data clearly indicate that the PCR-RFLP assay which is based on the diversity of region V, a regulatory region of Stx-phage, was not affected by repeated subculture and prolonged storage and is a more practical and reliable method for molecular typing of STEC strains.

  3. Screening of cytoplasmic DNA diversity between and within Lupinus mutabilis Sweet and Lupinus albus sensu lato by restriction fragment length polymorphism (RFLP).

    Science.gov (United States)

    Olczak, T; Rurek, M; Jańska, H; Augustyniak, H; Sawicka-Sienkiewicz, E J

    2001-01-01

    Seven populations and five mutant lines of the Andean lupin and four species from the section Albus were screened for their mitochondrial and chloroplast polymorphisms. For this purpose the RFLP method with EcoRI as a restriction enzyme was used. Lupinus luteus, Lupinus albus and Phaseolus vulgaris organellar clones as well as amplified fragments were used as probes. We found that mitochondrial probes were more suitable than chloroplast probes for identification of inter- and intra-specific variations within the examined material. Most mitochondrial probes differentiate the two species investigated. A high level of mitochondrial polymorphism was observed among the populations of L. mutabilis in contrast to monomorphism among the species in the section Albus. A limited polymorphism was detected between the mutant lines of L. mutabilis. We conclude from this study that the mitochondrial RFLP analysis is a valuable tool for identification of variability among Andean lupin populations.

  4. Phylogenetic congruence of Sarcocystis neurona Dubey et al., 1991 (Apicomplexa: Sarcocystidae) in the United States based on sequence analysis and restriction fragment length polymorphism (RFLP).

    Science.gov (United States)

    Elsheikha, Hany M; Murphy, Alice J; Mansfield, Linda S

    2005-07-01

    The objectives of the present study were to assess the genetic diversity, phylogeny and phylogeographical relationships of available Sarcocystis neurona isolates from different localities in the United States. All 13 Sarcocystis isolates from different hosts were subjected to polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses using two published DNA markers (25/396 and 33/54). The 334 bp sequence of the 25/396 marker of these isolates and Besnoitia darlingi, B. bennetti, Toxoplasma gondii and Neospora caninum were sequenced and compared. Phylogenetic analysis was performed using neighbour-joining (NJ), maximum parsimony (MP) and minimum evolution (ME) methods based on the sequences of the 25/396 marker of the 13 Sarcocystis isolates obtained in this study and sequences of 10 related isolates from GenBank. Phylogenetic trees revealed a close relatedness among S. neurona isolates in the US (nucleotide sequence diversity neurona into two separate groups: a northern US group and a Southern US group. These findings suggest a correlation between grouping of the isolates and geographical segregation and were consistent with a genetic bottleneck hypothesis during opossum colonisation of North America. These data do not support either the view of S. neurona as a single super-species or its division into multiple subspecies.

  5. Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis.

    Science.gov (United States)

    Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

    2014-10-01

    This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

  6. Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis

    Science.gov (United States)

    Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

    2014-01-01

    This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment. PMID:25178301

  7. A combined sequence-based and fragment-based characterization of microbial eukaryote assemblages provides taxonomic context for the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method.

    Science.gov (United States)

    Kim, Diane Y; Countway, Peter D; Yamashita, Warren; Caron, David A

    2012-12-01

    Microbial eukaryotes in seawater samples collected from two depths (5 m and 500 m) at the USC Microbial Observatory off the coast of Southern California, USA, were characterized by cloning and sequencing of 18S rRNA genes, as well as DNA fragment analysis of these genes. The sequenced genes were assigned to operational taxonomic units (OTUs), and taxonomic information for the sequence-based OTUs was obtained by comparison to public sequence databases. The sequences were then subjected to in silico digestion to predict fragment sizes, and that information was compared to the results of the T-RFLP method applied to the same samples in order to provide taxonomic context for the environmental T-RFLP fragments. A total of 663 and 678 sequences were analyzed for the 5m and 500 m samples, respectively, which clustered into 157 OTUs and 183 OTUs. The sequences yielded substantially fewer taxonomic units as in silico fragment lengths (i.e., following in silico digestion), and the environmental T-RFLP resulted in the fewest unique OTUs (unique fragments). Bray-Curtis similarity analysis of protistan assemblages was greater using the T-RFLP dataset compared to the sequence-based OTU dataset, presumably due to the inability of the fragment method to differentiate some taxa and an inability to detect many rare taxa relative to the sequence-based approach. Nonetheless, fragments in our analysis generally represented the dominant sequence-based OTUs and putative identifications could be assigned to a majority of the fragments in the environmental T-RFLP results. Our empirical examination of the T-RFLP method identified limitations relative to sequence-based community analysis, but the relative ease and low cost of fragment analysis make this method a useful approach for characterizing the dominant taxa within complex assemblages of microbial eukaryotes in large datasets.

  8. Terminal-restriction fragment length polymorphism (T-RFLP) analysis for changes in the gut microbiota profiles of indomethacin- and rebamipide-treated mice.

    Science.gov (United States)

    Imaeda, Hirotsugu; Fujimoto, Takehide; Takahashi, Kenichiro; Kasumi, Eiji; Fujiyama, Yoshihide; Andoh, Akira

    2012-01-01

    We investigated the effects of indomethacin and rebamipide on the gut microbiota profiles using terminal restriction fragment polymorphism (T-RFLP) analysis. Female C57BL/6J mice were given indomethacin (10 mg/kg, s.c.) once a day and 2.5 mg rebamipide orally 3 times a day. After 7 days, they were sacrificed, and luminal contents were obtained from the ileum and cecum. The gut microbiota communities were analyzed by T-RFLP analysis with BslI digestion. T-RFLP analyses showed that rebamipide and indomethacin had no significant effects on the gut microbiota profiles in the ileum and cecum. In contrast, the combination of rebamipide + indomethacin induced a significant change in the gut microbiota. The changes in the microbiota composition induced by the combination of rebamipide + indomethacin were characterized by the increase in the orders Bifidobacteriales and Lactobacillales, the genera Bacteroides and Prevotella and the family Clostridiaceae. The diversity of the gut microbiota community generated by the combination of rebamipide + indomethacin was significantly higher than those induced by either rebamipide or indomethacin alone. The combination of rebamipide + indomethacin induces remarkable changes in the gut microbiota composition and diversity. The clinical activity of rebamipide on nonsteroidal anti-inflammatory drug-induced intestinal injury may be exerted through a modulation of the gut microbiota. Copyright © 2012 S. Karger AG, Basel.

  9. 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...

  10. 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...

  11. PCR-based restriction fragment length polymorphism (RFLP) analysis of Campylobacter jejuni isolates from humans, chickens and dogs in northern Taiwan.

    Science.gov (United States)

    Tsai, Hsiang-Jung; Huang, Huang-Chi; Tsai, Huei-Lin; Chang, Cha-Chin

    2006-08-01

    Two hundred and twenty strains of Campylobacter jejuni (70 human, 51 canine and 99 chicken strains) were isolated from September 2003 to September 2004 in northern Taiwan. These strains were subtyped by PCR-RFLP analysis of the flagellin (FlaA) gene. On the basis of restrictive digest, six types were identified with AfaI, seven types with MboI and five types with HaeIII. With the combination of these three enzymes, 47 distinct PCR-RFLP patterns were observed-25 each from human and chicken isolates, and 9 from canine isolates. In human strains, the most frequently occurring types were Cj-28 (14.3%), Cj-17 (10%), Cj-16 (8.6%), Cj-37 (7.1%) and Cj-46 (7.1%). In canine strains, the most prevalent types were Cj-1 (33.3%), Cj-26 (19.6%), Cj-3 (15.7%), Cj-2 (9.8%) and Cj-10 (9.8%). In chicken strains, the most frequently occurring types were Cj-46 (40.4%), Cj-29 (9.1%), Cj-45 (7.1%) and Cj-41 (5.1%). The results suggest that poultry is a source, but not the sole source, of C. jejuni infection in humans. Two RFLP types, Cj-17 and Cj-37, frequently occurring in human isolates in this study have also been found to be prevalent in human isolates in Japan, China and the Czech Republic, indicating a possible international clonal spread.

  12. A novel Tth111I restriction fragment length polymorphism (RFLP) allows tracing of X-chromosome inactivation in the (Xid) hetrozygote

    Energy Technology Data Exchange (ETDEWEB)

    Shanmugam, V.; Sell, W.; Saha, B.K. [Emory Univ. of School of Medicine, Atlanta, GA (United States)] [and others

    1996-02-01

    The X-linked immunodeficiency (Xid) in CBA/N mice serves as a model for the X-linked agammaglobulinemia (XLA) syndrome in man. X-chromosome inactivation in F{sub 1} heterozygotes derived from CBA/N (X{sup xid}/X{sup xid}) and B6.Pgk-1a (X{sup +}/Y) was investigated by monitoring the methylation status of the individual Pgk-1 alleles, Pgk-1b and Pgk-1a, respectively, using a novel Tth111I RFLP. Results indicate that in circulating B lymphocytes of female heterozygotes, only the X chromosomes carrying the normal alleles (X{sup +}) are active (nonrandom inactivation of the X chromosome), whereas in non-B cells both the X chromosomes (X{sup +} and X{sup xid}) are active (random inactivation of the X chromosome). These results were further confirmed by direct evaluation of transcription of the Btk gene, the gene mutated both in Xid and in XLA. 36 refs., 2 figs., 2 tabs.

  13. Genome Polymorphisms Between Indica and Japonica Revealed by RFLP

    Institute of Scientific and Technical Information of China (English)

    WANG Song-wen; LIU Xia; XU Cai-guo; SHI Li-li; ZHANG Xin; DING De-liang; WANG Yong

    2007-01-01

    Revealing the genome polymorphisms between indica and japonica subspecies; RFLP markers, which are located across 12 chromosomes of rice, were used to analyze indica-japonica differentiation in different rice varieties. At the same time, genome sequence variations of screened loci were analyzed by bioinformatics method. Twenty-eight RFLP probes, which can classify indica-japonica rice, were confirmed. Subspecies genome polymorphisms of screened loci were found by analyzing the publication of the genome sequences data of rice. The study indicated that these screened markers can be used for classifying indica-japonica subspecies. With the publication of the genome sequences of rice, marker polymorphisms between indica and japonica subspecies can be revealed by genome differentiation.

  14. An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

    Science.gov (United States)

    Gedil, M A; Wye, C; Berry, S; Segers, B; Peleman, J; Jones, R; Leon, A; Slabaugh, M B; Knapp, S J

    2001-04-01

    Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.

  15. 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.

    Science.gov (United States)

    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.

  16. Use of restriction fragment length polymorphism as a genetic marker for typing Mycobacterium avium strains.

    Science.gov (United States)

    Roiz, M P; Palenque, E; Guerrero, C; Garcia, M J

    1995-05-01

    Restriction fragment length polymorphism (RFLP) was used to study 75 clinical isolates identified as Mycobacterium avium. Two repetitive insertion sequences, IS1311 and IS900, were used as DNA probes. Although less than 25% of isolates showed RFLP patterns with IS900, all strains gave banding patterns with IS1311. M. avium strains isolated from patients with AIDS exhibited marked polymorphism with both probes.

  17. Use of restriction fragment length polymorphism as a genetic marker for typing Mycobacterium avium strains.

    Science.gov (United States)

    Roiz, M P; Palenque, E; Guerrero, C; Garcia, M J

    1995-01-01

    Restriction fragment length polymorphism (RFLP) was used to study 75 clinical isolates identified as Mycobacterium avium. Two repetitive insertion sequences, IS1311 and IS900, were used as DNA probes. Although less than 25% of isolates showed RFLP patterns with IS900, all strains gave banding patterns with IS1311. M. avium strains isolated from patients with AIDS exhibited marked polymorphism with both probes. PMID:7615764

  18. Frequencies of VNTR and RFLP polymorphisms associated with factor VIII gene in Singapore

    Energy Technology Data Exchange (ETDEWEB)

    Fong, I.; Lai, P.S.; Ouah, T.C. [National Univ. of Singapore (Malaysia)] [and others

    1994-09-01

    The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1 = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.

  19. Cultivar identification in T. aestivum using highly polymorphic RFLP probes.

    Science.gov (United States)

    Vaccino, P; Accerbi, M; Corbellini, M

    1993-08-01

    Two probes, specific for HMW-glutenins and γ-gliadins have been used to identify 50 common wheat Italian cultivars, most of which are closely related, and four common wheat cultivars originating outside Italy. The probes revealed complex polymorphic patterns; three probe/enzyme combinations had the necessary sensitivity for the identification of all 54 cultivars. As already shown for potato and barley, the use of four-cutter restriction enzymes and polyacrylamide gels proved particularly useful for detecting polymorphism.

  20. Association of apolipoprotein E (RFLP polymorphism with myopia

    Directory of Open Access Journals (Sweden)

    Himabindu P

    2006-01-01

    Full Text Available BACKGROUND: Myopia or nearsightedness is a spherical error of refraction, whereby the images are focused in front of retina. Eye, being an organ rich in activated oxygen species, requires a high level of antioxidants to protect the unsaturated fatty acids. Apolipoprotein E (APOE is one of the proteins that is produced by Muller cells within the retina and is also endowed with antioxidant properties. Genetic polymorphism of APO E is controlled by three common alleles e3, e2 and e4 and rare e1, e4v at the APOE structural gene locus. Different isoforms of APO E differ in their antioxidant properties, and the e4 allele has lesser ability to combat oxidative stress. AIMS: Myopia being a disease influenced by oxidative stress, the present study was undertaken to find association of myopia with APO E polymorphism. MATERIALS AND METHODS: A total of 187 myopic cases and 192 controls were genotyped for apolipoprotein E polymorphism. RESULTS: In both controls and myopic cases, E3/3 genotype was found to be the most frequent one. There was an increase in E3/4 genotype frequency among male probands, high myopia cases and probands with early age at onset, suggesting that the E3/4 genotype might confer risk for myopia development. CONCLUSION: This association with E3/4 genotype might predispose susceptible individuals to develop high myopia and early onset myopia.

  1. 棘阿米巴土壤分离株CB/S1的线粒体DNA限制性片段长度多态性%MITOCHONDRIAL DNA RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) OF ACANTHAMOEBA SP. CB/S1 ISOLAT ED FROM SOIL

    Institute of Scientific and Technical Information of China (English)

    崔春权; 玄英花; 郑善子; 金松竹; 杨香

    2012-01-01

    [目的]观察棘阿米巴土壤分离株CB/S1的线粒体DNA限制性片段长度多态性(RFLP).[方法]从棘阿米巴CB/S1提取线粒体DNA用Eco RI酶切,与土壤分离株CJY/S4比较观察限制性片段长度多态性.用地衣红-卡红染色观察棘阿米巴内共生细菌.[结果]棘阿米巴土壤分离株CB/S1线粒体DNA RFLP与CJY/S4相比除了额外的片段(extra bands),两个分离株具有相同的片段模式.经地衣红染色观察CB/S1内共生细菌呈黑色、棒状、不规则的分布在胞质内.[结论]额外的片段表明棘阿米巴CB/S1内含有内共生细菌的环状DNA,并且除了内共生细菌的存在棘阿米巴CB/S1与土壤分离株CJY/S4具有密切的亲缘关系.线粒体DNA限制性片段长度多态性是棘阿米巴分类及发现内共生细菌的简便而有效的方法.%[Objective] To survey characterization of Mitochondrial DNA Restriction Fragment Length Polymorphism (MtDNA RFLP) of Aeanthamoeba sp. CB/SI isolated from soil of china. [Methods] The extracted MlDNA was digested with the Eco RI restriction enzyme, electrophoresed in 0.7% agarose gel. The endosymbiont of Acanthamoeba sp. was characterized with orcein-camiime staining [Results] Acanthamoeba sp. CB/SI showED extra bands as weli as common bands with the other Aeanthamoeba sp. CJY/S4 strain isolated from soil, rode-shaped endosymbionts were randomly distributed within the cytoplasm of trophozoite of Aeanthamoeba sp. CB/SI. [Conclusion] These results indicated that the.Acanlhamoeba sp. CB/SI may possi-bly harbor intracellular endosymbiotic bacteria containing circular DNAs. With the exception of the presence of endosymbionts, Aeanthamoeba sp. CB/S1 with Aeanthamoeba CJY/S4 were found to be closely phylogenetically related.

  2. 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, Jesper; 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...

  3. Typing of Human Mycobacterium avium Isolates in Italy by IS1245-Based Restriction Fragment Length Polymorphism Analysis

    Science.gov (United States)

    Lari, Nicoletta; Cavallini, Michela; Rindi, Laura; Iona, Elisabetta; Fattorini, Lanfranco; Garzelli, Carlo

    1998-01-01

    All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern. PMID:9817900

  4. CHARACTERIZATION OF APHYLLOPHORALES CAUSING BROWN AND WHITE ROT IN WOODS: ANALYSIS OF ITS RESTRICTION POLYMORPHISM (PCR/RFLP

    Directory of Open Access Journals (Sweden)

    Alba ZAREMSKI

    2015-03-01

    Full Text Available Intraspecific polymorphism exists for many fungus species, making their macroscopic identification difficult. Easily usable molecular markers providing access to information about variability have boosted the development of fungus diversity analyses. For this study, on wood-decaying Aphyllophorales, the main problem to be solved consisted in detecting any existence of intraspecific polymorphism in these fungi, in order to complete and clarify their identification. In order to characterize 98 strains in the CIRAD collection, we developed a rapid discriminant analysis method based on analysing the diversity of partial ribosomal DNA sequences independently of the macro- and microscopic morphological traits of the fruiting bodies required for the identification of genera and species. Adaptation of the Qiagen kit for higher plants made it possible to extract fungal DNA from a few milligrams of mycelium; with most of the strains studied, these DNA extracts enabled us to obtain an ITS amplificate of a size varying from 550 to 895 base pairs. We also found a large margin of error, of around 12%, for the DNA molecular weight readings on gel, leading us to prefer direct sequencing for precise determination of amplificate lengths. PCR-RFLP showed a good ability to characterize a given strain by providing different profiles obtained using several restriction enzymes. We thus demonstrated that studying ITS diversity using PCR-RFLP is a discriminant method making it possible to distinguish between strains. However, this method calls for the compilation of a database whose use remains partly subjected to interpretation, which cannot be used to work on a material unknown to the database. This simple, rapid technique is efficient and perfectly suitable for monitoring known strains in controlled trials.

  5. Genetic diversity among elite Sorghum lines revealed by restriction fragment length polymorphisms and random amplified polymorphic DNAs.

    Science.gov (United States)

    Vierling, R A; Xiang, Z; Joshi, C P; Gilbert, M L; Nguyen, H T

    1994-02-01

    The genetic diversity of sorghum, as compared to corn, is less well characterized at the genetic and molecular levels despite its worldwide economic importance. The objectives of this study were to: (1) investigate genetic diversity for restriction fragment length polymorphism (RFLPs) and random amplified polymorphic DNAs (RAPDs) in elite sorghum lines, (2) compare similarities based on molecular markers with pedigree relationships, and (3) examine the potential of RFLPs and RAPDs for assigning sorghum lines to the A/B (sterile) and R (restorer) groups. Using four restriction enzymes, polymorphism was detected with 61% of the RFLP probes used, compared to 77% of the random primers. One hundred and sixteen (64%) probe-enzyme combinations yielded multiple-band profiles compared to 98% of the random primers. RFLP profiles generated 290 polymorphic bands compared to 177 polymorphic RAPDs. Pair-wise comparisons of polymorphic RFLPs and RAPDs were used to calculate Nei and Jaccard coefficients. These were employed to generate phenograms using UPGMA and neighborjoining clustering methods. Analysis of RFLP data with Jaccard's coefficient and neighbor-joining clustering produced the phenogram with the closest topology to the known pedigree.

  6. Detection of the rs10250202 polymorphism in protection of telomeres 1 gene through introducing a new restriction enzyme site for PCR-RFLP assay.

    Science.gov (United States)

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

    2016-01-01

    Human protection of telomeres 1 (POT1) gene is a single stranded telomere binding proteins with a critical role in ensuring chromosome stability. There have been variants of POT1 gene, and the polymorphisms of POT1 gene were associated with some diseases. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a traditional method to detect the single nucleotide polymorphism (SNP), and it can be used to detect the polymorphism of rs10250202. But the restriction enzymes required for the detection of the polymorphism of rs10250202 are expensive. So we designed a novel PCR-RFLP assay for genotyping the POT1 rs10250202 SNP. In the study, a new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and the restriction enzyme BclI for CRS-PCR was cheaper than other enzymes. After detecting Han Chinese workers, Allele frequencies were found to be 51.54 % for allele A and 48.46 % for allele C respectively. The PCR results were confirmed by DNA sequencing. CRS-PCR provides a simple, low-cost, practical, and reproducible method.

  7. 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.

  8. 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

    Sulpha agents, which act by inhibiting the enzyme dihydropteroate synthase (DHPS), are used widely for the treatment and prophylaxis of Pneumocystis carinii pneumonia (PCP). Recently, we have shown that mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis carinii f.sp hominis...... 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...... 55 and 57 of the P. carinii DHPS gene. The RFLP-assay was compared with direct DNA sequencing on 27 PCP isolates from HIV-1 positive patients with a mixture of wildtype and mutant DHPS types. In all samples the RFLP-assay correctly identified wildtype or DHPS mutation at codon 55 or 57. Combined...

  9. Typing and clustering of Yersinia pseudotuberculosis isolates by restriction fragment length polymorphism analysis using insertion sequences.

    Science.gov (United States)

    Voskresenskaya, E; Savin, C; Leclercq, A; Tseneva, G; Carniel, E

    2014-06-01

    Yersinia pseudotuberculosis is an enteropathogen that has an animal reservoir and causes human infections, mostly in temperate and cold countries. Most of the methods previously used to subdivide Y. pseudotuberculosis were performed on small numbers of isolates from a specific geographical area. One aim of this study was to evaluate the typing efficiency of restriction fragment length polymorphism of insertion sequence hybridization patterns (IS-RFLP) compared to other typing methods, such as serotyping, ribotyping, and multilocus sequence typing (MLST), on the same set of 80 strains of Y. pseudotuberculosis of global origin. We found that IS100 was not adequate for IS-RFLP but that both IS285 and IS1541 efficiently subtyped Y. pseudotuberculosis. The discriminatory index (DI) of IS1541-RFLP (0.980) was superior to those of IS285-RFLP (0.939), ribotyping (0.944), MLST (0.861), and serotyping (0.857). The combination of the two IS (2IS-RFLP) further increased the DI to 0.998. Thus, IS-RFLP is a powerful tool for the molecular typing of Y. pseudotuberculosis and has the advantage of exhibiting well-resolved banding patterns that allow for a reliable comparison of strains of worldwide origin. The other aim of this study was to assess the clustering power of IS-RFLP. We found that 2IS-RFLP had a remarkable capacity to group strains with similar genotypic and phenotypic markers, thus identifying robust populations within Y. pseudotuberculosis. Our study thus demonstrates that 2IS- and even IS1541-RFLP alone might be valuable tools for the molecular typing of global isolates of Y. pseudotuberculosis and for the analysis of the population structure of this species.

  10. Typing and Clustering of Yersinia pseudotuberculosis Isolates by Restriction Fragment Length Polymorphism Analysis Using Insertion Sequences

    Science.gov (United States)

    Voskresenskaya, E.; Savin, C.; Leclercq, A.; Tseneva, G.

    2014-01-01

    Yersinia pseudotuberculosis is an enteropathogen that has an animal reservoir and causes human infections, mostly in temperate and cold countries. Most of the methods previously used to subdivide Y. pseudotuberculosis were performed on small numbers of isolates from a specific geographical area. One aim of this study was to evaluate the typing efficiency of restriction fragment length polymorphism of insertion sequence hybridization patterns (IS-RFLP) compared to other typing methods, such as serotyping, ribotyping, and multilocus sequence typing (MLST), on the same set of 80 strains of Y. pseudotuberculosis of global origin. We found that IS100 was not adequate for IS-RFLP but that both IS285 and IS1541 efficiently subtyped Y. pseudotuberculosis. The discriminatory index (DI) of IS1541-RFLP (0.980) was superior to those of IS285-RFLP (0.939), ribotyping (0.944), MLST (0.861), and serotyping (0.857). The combination of the two IS (2IS-RFLP) further increased the DI to 0.998. Thus, IS-RFLP is a powerful tool for the molecular typing of Y. pseudotuberculosis and has the advantage of exhibiting well-resolved banding patterns that allow for a reliable comparison of strains of worldwide origin. The other aim of this study was to assess the clustering power of IS-RFLP. We found that 2IS-RFLP had a remarkable capacity to group strains with similar genotypic and phenotypic markers, thus identifying robust populations within Y. pseudotuberculosis. Our study thus demonstrates that 2IS- and even IS1541-RFLP alone might be valuable tools for the molecular typing of global isolates of Y. pseudotuberculosis and for the analysis of the population structure of this species. PMID:24671793

  11. Restriction fragment length polymorphism in the adhesin gene hpaA of Helicobacter pylori.

    Science.gov (United States)

    Evans, D G; Evans, D J; Lampert, H C; Graham, D Y

    1995-08-01

    To assess the degree of restriction fragment length polymorphism (RFLP) in the Helicobacter pylori adhesin gene hpaA and to determine the molecular basis of RFLP in this gene. A 375-bp, polymerase chain reaction-amplified internal sequence of hpaA, obtained from 50 different H. pylori isolates, was restricted with Sau3A and HinfI, individually. Polymerase chain reaction products representing different RFLP types were sequenced. Seven different polymorphic types were found in hpaA. Base substitutions at only four positions, two in Sau3A and two in HinfI sites, account for all of the RFLP types, including the size of the restriction fragments determined by gel electrophoresis. Most, 90%, of the base substitutions are very conservative, i.e., either do not change the encoded amino acid or substitute a homologous amino acid, and cause no detectable antigenic or functional effect on hpaA. The region of hpaA encoding the receptor-binding motif was particularly well conserved. RFLP typing of hpaA using Sau3A and HinfI provides an additional tool for comparing the genetic relatedness of H. pylori isolates collected during epidemiological and/or treatment studies.

  12. Identification of processed Japanese green tea based on polymorphisms generated by STS-RFLP analysis.

    Science.gov (United States)

    Kaundun, Shiv S; Matsumoto, Satoru

    2003-03-26

    In Japan, tea is generally sold blended, though 90% of the total production is clonal. Due to the increasingly strict consumer need and taste, however, more and more Japanese green teas are being sold under their particular cultivar name. Moreover, tea made from Yabukita, a much appreciated cultivar originally developed in Japan, has recently been produced and imported from a neighboring country. This paper describes a simple and inexpensive methodology capable of identifying fresh and processed Japanese green teas to discourage its fraudulent commercialization. The study was based on 46 main tea cultivars, and polymorphism detected through STS-RFLP analysis of the coding and noncoding DNA regions of three genes, namely phenylalanine ammonia-lyase, chalcone synthase, and dihydroflavonol 4-reductase, for which nucleotide information was available. All 46 tea cultivars analyzed could be easily distinguished using a combination of codominant DNA markers. Yabukita displayed a unique profile when PAL intron was digested with DdeI, thus allowing its rapid authentication at low cost.

  13. Genetic diversity among Puntius sophore complex using restriction fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    Smitha Balaraj

    2012-08-01

    Full Text Available Restriction fragment length polymorphism (RFLP analysis of genomic DNA was employed to characterize species of Puntius sophore complex, a benthopelagic freshwater fish species. Samples of five species of Puntius viz, Puntius sophore, Puntius amphibius, Puntius chola, Puntius bimaculatus and Puntius dorsalis from the East-flowing Tamiraparani and Kuzhithurai and West-flowing Tamiraparani river basins (Western Ghats were analyzed. The restriction enzyme employed (Hind III generated species-specific DNA patterns for each of the five species. DNA exhibited considerable polymorphism among species. All samples showed relatively high values of diversity. The relation between species is discussed.

  14. Characterization of Pseudomonas aeruginosa in Burn Patients Using PCR- Restriction Fragment Length Polymorphism and Random Amplified Polymorphic DNA Analysis

    Directory of Open Access Journals (Sweden)

    Abdolaziz Rastegar Lari

    2010-09-01

    Full Text Available One of the major opportunistic pathogens in patients with burninjuries is Pseudomonas aeruginosa, which causes severe infectionsin burned patients. The objective of the study was to examinethe molecular epidemiology of P. aeruginosa colonization inthe burn unit of Shahid Motahari Hospital in Tehran, Iran. Restrictionfragment length polymorphism (RFLP and random amplifiedpolymorphic DNA (RAPD analysis were employed tostudy 127 clinical and two environmental P. aeruginosa isolatescollected from January to June 2008. In RFLP, the PCR productsof 16S rRNA gene were digested with restriction enzyme Alu I,Hae III, and Rsa I, and the fragments generated were analyzed byagarose electrophoresis. Molecular typing by RFLP did show nodiscriminatory power for P. aeruginosa isolates, but RAPD-PCRrevealed eight different genotypes; RAPD1to RAPD8 in clinicaland environmental isolates. RAPD1 was the major genotype inclinical (n=64, 50.4% and environmental isolates (n=1, 50%.The findings suggest that RAPD might have a superior typeabilityand discriminatory power over RFLP to study P. aeruginusa.Moreover, they highlight the need for further attention to the controlof infection sources in Burn Units to prevent the transmissionof the bacterium.

  15. Restriction fragment length polymorphism of the HLA-DP subregion and correlations to HLA-DP phenotypes

    DEFF Research Database (Denmark)

    Hyldig-Nielsen, J J; Morling, Niels; Ødum, Niels;

    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 alpha and DP beta 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 beta (14 different DNA markers defined by individual fragments or clusters thereof) and the DP alpha (8 markers) probes, especially when enzymes recognizing only four base pairs were used. A few...

  16. 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.

  17. Evaluation of Semiautomated IS6110-Based Restriction Fragment Length Polymorphism Typing for Mycobacterium tuberculosis in a High-Burden Setting.

    Science.gov (United States)

    Said, Halima M; Krishnamani, Keshav; Omar, Shaheed V; Dreyer, Andries W; Sansom, Bianca; Fallows, Dorothy; Ismail, Nazir A

    2016-10-01

    The manual IS6110-based restriction fragment length polymorphism (RFLP) typing method is highly discriminatory; however, it is laborious and technically demanding, and data exchange remains a challenge. In an effort to improve IS6110-based RFLP to make it a faster format, DuPont Molecular Diagnostics recently introduced the IS6110-PvuII kit for semiautomated typing of Mycobacterium tuberculosis using the RiboPrinter microbial characterization system. This study aimed to evaluate the semiautomated RFLP typing against the standard manual method. A total of 112 isolates collected between 2013 and 2014 were included. All isolates were genotyped using manual and semiautomated RFLP typing methods. Clustering rates and discriminatory indexes were compared between methods. The overall performance of semiautomated RFLP compared to manual typing was excellent, with high discriminatory index (0.990 versus 0.995, respectively) and similar numbers of unique profiles (72 versus 74, respectively), numbers of clustered isolates (33 versus 31, respectively), cluster sizes (2 to 6 and 2 to 5 isolates, respectively), and clustering rates (21.9% and 17.1%, respectively). The semiautomated RFLP system is technically simple and significantly faster than the manual RFLP method (8 h versus 5 days). The analysis is fully automated and generates easily manageable databases of standardized fingerprints that can be easily exchanged between laboratories. Based on its high-throughput processing with minimal human effort, the semiautomated RFLP can be a very useful tool as a first-line method for routine typing of M. tuberculosis isolates, especially where Beijing strains are highly prevalent, followed by manual RFLP typing if resolution is not achieved, thereby saving time and labor.

  18. 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

    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.

  19. Genetic analysis of the fungus, Bremia lactucae, using restriction fragment length polymorphisms.

    Science.gov (United States)

    Hulbert, S H; Ilott, T W; Legg, E J; Lincoln, S E; Lander, E S; Michelmore, R W

    1988-12-01

    Restriction fragment length polymorphisms (RFLPs) were developed as genetic markers for Bremia lactucae, the biotrophic Oomycete fungus which causes lettuce downy mildew. By using 55 genomic and cDNA probes, a total of 61 RFLP loci were identified among three heterothallic isolates of B. lactucae. Of these 61 RFLP loci, 53 were heterozygous in at least one of the three strains and thus were informative for linkage analysis in at least one of two F1 crosses that were performed. Analysis of the cosegregation of these 53 RFLPs, eight avirulence loci and the mating type locus allowed the construction of a preliminary genetic linkage map consisting of 13 small linkage groups. Based on the extent of linkage detected among probes, the genome of B. lactucae can be estimated to be approximately 2000 cM. Linkage was detected between a RFLP locus and an avirulence gene, providing a potential starting point for chromosome walking to clone an avirulence gene. The high frequency of DNA polymorphism in naturally occurring isolates and the proper Mendelian segregation of loci detected by low copy number probes indicates that it will be possible to construct a detailed genetic map of B. lactucae using RFLPs as markers. The method of analysis employed here should be applicable to many other outbreeding, heterozygous species for which defined inbred lines are not available.

  20. Evaluation of rs62527607 [GT] single nucleotide polymorphism located in BAALC gene in children with acute leukemia using mismatch PCR-RFLP.

    Science.gov (United States)

    Nadimi, Motahareh; Rahgozar, Soheila; Moafi, Alireza; Tavassoli, Manoochehr; Mesrian Tanha, Hamzeh

    2016-01-01

    Acute leukemia is the most common cancer in children and involves several factors that contribute to the development of multidrug resistance and treatment failure. According to our recent studies, the BAALC gene is identified to have high mRNA expression levels in childhood acute lymphoblastic leukemia (ALL) and those with multidrug resistance. Several polymorphisms are associated with the expression of this gene. To date, there has been no study on the rs62527607 [GT] single nucleotide polymorphism (SNP) of BAALC gene and its link with childhood acute lymphoblastic and myeloid leukemia (AML). The purpose of this study is to evaluate the prevalence of this polymorphism in pediatric acute leukemia, as well as its relationship with prognosis. DNA samples were extracted from bone marrow slides of 129 children with ALL and 16 children with AML. The rs62527607 [GT] SNP was evaluated using mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based analysis. The association between the SNP alleles and patient disease-free survival was then assessed. The prevalence of the T-allele of rs62527607 [GT] SNP in childhood T-ALL and pre-B-ALL was 28.3% and 11.2%, respectively. In the pre-B-ALL patients, 3 year disease free survival was associated with the GG genotype. Results showed a robust association between the rs62527607 SNP and the risk of relapse in ALL, but not AML, patients. T-ALL patients with the GT genotype had an 8.75 fold higher risk of relapse. The current study demonstrates a significant association between the genotype GT and the polymorphic allele G424T, and introduces this SNP as a negative prognostic factor in children with ALL.

  1. Genetic variability of Chlamydophila abortus strains assessed by PCR-RFLP analysis of polymorphic membrane protein-encoding genes.

    Science.gov (United States)

    Sait, Michelle; Clark, Ewan M; Wheelhouse, Nicholas; Spalding, Lucy; Livingstone, Morag; Sachse, Konrad; Markey, Bryan K; Magnino, Simone; Siarkou, Victoria I; Vretou, Evangelia; Caro, María R; Yaga, Raja; Lainson, F Alex; Smith, David G E; Wright, Frank; Longbottom, David

    2011-08-05

    This study used PCR-RFLP to investigate the genetic variability of pmp-encoding genes from fifty-two Chlamydophila abortus (C. abortus) strains originating from abortion cases from various geographical regions and host species. Six primer pairs were used to PCR-amplify DNA fragments encoding eighteen pmps. PCR products were digested using four restriction endonucleases and Bayesian methodologies were used to compare RFLP profiles and assign strains to a RFLP genotype. Strains could be assigned to 2 genotypes in the region encoding pmp18D, 3 genotypes in the regions encoding pmp1A-pmp2B, pmp3E-pmp6H and pmp11G-pmp15G, 4 genotypes in the region encoding pmp7G-pmp10G and 5 genotypes in the region encoding pmp16G-pmp17G. In all regions, the majority of strains (88.4-96.1%) had the same genotype as the reference strain S26/3. No correlation could be made between genotype, host species or geographical origin except for the two variant Greek strains, LLG and POS, which formed a discrete genotype in all pmp-encoding regions except pmp18D. Relative rates of evolution calculated for each pmp-encoding gene locus suggest that differing selective pressures and functional constraints may exist on C. abortus polymorphic membrane proteins. These findings suggest that although intraspecies heterogeneity of pmp-encoding genes in C. abortus is low, the sequence heterogeneity should be an important consideration when using pmps as the basis for novel diagnostics or vaccine development.

  2. 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 i

  3. Characterization of microbial communities found in the human vagina by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

    NARCIS (Netherlands)

    Coolen, MJL; Post, E; Davis, CC; Forney, LJ

    2005-01-01

    To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the h

  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

    Directory of Open Access Journals (Sweden)

    Huyen Mai NT

    2013-02-01

    Full Text Available Abstract 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 in typing of Mycobacterium tuberculosis. However, some reports indicated that VNTR typing may be less suitable for Beijing genotype isolates. We therefore compared the performance of internationally standardized RFLP and 24 loci VNTR typing to discriminate among 100 Beijing genotype isolates from the Southern Vietnam. Methods Hundred Beijing genotype strains defined by spoligotyping were randomly selected and typed by RFLP and VNTR typing. The discriminatory power of VNTR and RFLP typing was compared using the Bionumerics software. Results Among 95 Beijing strains available for analysis, 14 clusters were identified comprising 34 strains and 61 unique profiles in 24 loci VNTR typing ((Hunter Gaston Discrimination Index (HGDI = 0.994. 13 clusters containing 31 strains and 64 unique patterns in RFLP typing (HGDI = 0.994 were found. Nine RFLP clusters were subdivided by VNTR typing and 12 VNTR clusters were split by RFLP. Five isolates (5% revealing double alleles or no signal in two or more loci in VNTR typing could not be analyzed. Conclusions Overall, 24 loci VNTR typing and RFLP typing had similar high-level of discrimination among 95 Beijing strains from Southern Vietnam. However, loci VNTR 154, VNTR 2461 and VNTR 3171 had hardly added any value to the level of discrimination.

  5. An Authentic RFLP Lab for High School or College Biology Students.

    Science.gov (United States)

    Guilfoile, Patrick G.; Plum, Stephen

    1998-01-01

    Explains how students can perform an alternative authentic DNA fingerprinting analysis. Presents restriction fragment length polymorphism (RFLP) analysis and can serve as a simulated molecular epidemiology laboratory or as a simulated forensic laboratory exercise. (DDR)

  6. 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

    -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......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......) sequences of the VHSV glycoprotein (G) gene, were used to predict restriction fragment length polymorphism (RFLP) patterns that were subsequently grouped and compared with a phylogenetic analysis of the G-gene sequences of the same set of isolates. Digestion of PCR amplicons from the full-length G...

  7. Differentiation of Helicobacter pylori isolates by polymerase chain reaction-restriction fragment length polymorphism

    Institute of Scientific and Technical Information of China (English)

    SHI Li; SUN Yong; ZHANG Ya-li; ZHANG Zhen-shu; ZHOU Dian-yuan

    2002-01-01

    Objective: To investigate the association between the diversity of urease gene and urease activity of clinical isolates of Helicobacter pylori (H. pylori). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of urease gene and rapid urease activity test were used to study the urease activity of different clinical isolates of H. pylori. Results: H. pylori clinical isolates were divided into 4types according to their PCR-RFLP results of urease gene and urease activity. Type I , possessing strong urease activity (0. 11) and presented 1 fragment of 1.7 kb by PCR-RFLP, had close relations with gastric ulcer; type Ⅱ , with the weakest urease activity (0. 07) and 2 fragments (1.3 and 0. 4 kb respectively), was associated with duodenal bulb ulcer; type Ⅱ , with the strongest urease activity (0. 12) and 2 fragments (0. 4and 0. 17 kb) with or without 1 fragment (0. 23 or 0. 37 kb) , was responsible for gastritis; type Ⅳ, with weak urease activity (0. 09) and 2 fragments (1.5 and 0. 2 kb), was shown to be related to both gastric and duodenal bulb ulcers. Conclusion: The diversity of urease gene decides different urease activities of different clinical isolates of H. pylori, hence the different possibilities of pathogenesis due to this bacteria.

  8. Generation of a Restriction Fragment Length Polymorphism Linkage Map for Toxoplasma Gondii

    Science.gov (United States)

    Sibley, L. D.; LeBlanc, A. J.; Pfefferkorn, E. R.; Boothroyd, J. C.

    1992-01-01

    We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen. PMID:1360931

  9. 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.

  10. 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.

  11. 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…

  12. 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…

  13. Molecular Fingerprinting of Mycobacterium tuberculosis Isolates Obtained in Havana, Cuba, by IS6110 Restriction Fragment Length Polymorphism Analysis and by the Double-Repetitive-Element PCR Method

    Science.gov (United States)

    Montoro, Ernesto; Valdivia, José; Leão, Sylvia Cardoso

    1998-01-01

    Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis. PMID:9738082

  14. 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.

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

    Science.gov (United States)

    Santos, M S; Souza, E S; S Junior, R M; Talhari, S; Souza, J V B

    2010-08-01

    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.

  16. The Polymorphism of Exon 2 of GOLA-DRB3 Gene in Nadushan Goat Using PCR-RFLP

    Directory of Open Access Journals (Sweden)

    E Abbaszadeh

    2012-02-01

    Full Text Available This study was conducted to determine polymorphism of GOLA-DRB3 in Nadushan goat using PCR-RFLP. Blood samples were randomly taken from 100 goats. DNA was extracted from the blood samples using DIAtom DNA prep Kit. Spectrophotometer and 1% agarose gel were used for determining its quantities and qualities. Exon 2 of DRB 3.2 gene encompassing 285 bp was amplified with heminested-PCR method in two rounds and PCR products were digested with TaqI. Digested PCR products were electrophoresed in 10% Acryl Amide gel or 2% Agarose gel and gels stained with ethidium bromide. Digested PCR products with TaqI included 2 fragments at 122bp and 163bp (T restriction pattern or undigested fragment at 285bp (t restriction pattern. Population was in Hardy-Weinberg equilibrium (p>0.05. Shanon index, Nei index, observed and expected heterozygosity were 0.68, 0.49, 0.55 and 0.49, respectively. With regard to results of this study, GOLA-DRB3 marker has good efficiency for genetic diversity determining. Using results of this study and former studies and records and quantitative information of locus in this population, we can perform determining and identifying QTL

  17. 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

    2017-07-08

    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 DNA polymerase ζ and SNPs in this gene are associated with altered susceptibility to cancer. This newly designed experiment is composed of three parts, including genomic DNA extraction, gene amplification by PCR, and genotyping by RFLP. By combining these activities, the students are not only able to learn a series of biotechniques in molecular biology, but also acquire the ability to link the learned knowledge with practical applications. This comprehensive experiment will help the medical students improve the conceptual understanding of SNP and the technical understanding of SNP detection. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):299-304, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

  18. New chicken Rfp-Y haplotypes on the basis of MHC class II RFLP and MLC analyses

    DEFF Research Database (Denmark)

    Juul-Madsen, H R; Zoorob, R; Auffray, C;

    1997-01-01

    New chicken Rfp-Y haplotypes were determined by the use of restriction fragment length polymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B15, B19, B21, B201. The RFLP polymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps...... near a polymorphic lectin gene located in the Rfp-Y system and DNA from families with known segregation of the implicated RFLP polymorphism. For the first time it is shown that major histocompatibility complex class II genes in the Rfp-Y system have functional implications. Sequence information...

  19. 16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

    Directory of Open Access Journals (Sweden)

    Silvio D Brugger

    Full Text Available A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7 and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

  20. Identification of weakly beta-hemolytic porcine spirochetes by biochemical reactions, PCR-based restriction fragment length polymorphism analysis and species-specific PCR.

    Science.gov (United States)

    Ohya, Tatsuo; Araki, Hiroshi; Sueyoshi, Masuo

    2008-08-01

    We examined the usefulness of PCR-based restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR combined with a newly devised rapid biochemical test using microplates for identifying weakly beta-hemolytic intestinal spirochetes (WBHIS) isolated from pigs. WBHIS strains showing atypical biochemical characteristics were decisively identified at the species level by PCR-RFLP and species-specific PCR. Identification of WBHIS at the species level in routine diagnostic work will certainly contribute to clarifying the pathogenicity of WBHIS.

  1. 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.

  2. Genotypic change of porcine circovirus type 2 on Japanese pig farms as revealed by restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Takahagi, Yoichi; Nishiyama, Yasutaka; Toki, Shinji; Yonekita, Taro; Morimatsu, Fumiki; Murakami, Hiroshi

    2008-06-01

    Porcine circovirus type 2 (PCV2) has been recognized as the causal agent of postweaning multisystemic wasting syndrome and can be divided into two major genotypic groups. We developed a method of restriction fragment length polymorphism (RFLP) analysis of PCV2 open reading frame 2 for easy discrimination between the two major groups. Genotyping of PCV2 isolates from 10 Japanese commercial pig farms was performed, and the analysis revealed that both PCV2 groups and at least five RFLP types of PCV2 are prevalent in Japan. On two farms, the genotypes of the PCV2 isolates in the spring of 2007 were different from those in the autumn of 2006. One genotype may have become dominant within only six months on these farms.

  3. 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.

  4. UTILIZATION OF T-RFLP (TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM) TO CHARACTERIZE MIXED ECTOMYCORRHIZAL FUNGAL COMMUNITIES

    Science.gov (United States)

    Studies of ectomycorrhizal community structure have used a variety of analytical regimens including sole or partial reliance on gross morphological characterization of colonized root tips. Depending on the rigor of the classification protocol, this technique can incorrectly assig...

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

    Science.gov (United States)

    Fatima, Akeela; Bashir, Gulnaz; Wani, Tehmeena; Jan, Abiroo; Kohli, Amrish; Khan, Mosin S

    2017-01-01

    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. 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. Hospital-based cross-sectional study. 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. Kappa test for agreement. 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. 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.

  6. 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.

  7. Effect of ANXA2 gene single nucleotide polymorphism (SNP) on the development of osteonecrosis in Indian sickle cell patient: a PCR-RFLP approach.

    Science.gov (United States)

    Pandey, Sanjay; Ranjan, Ravi; Pandey, Sweta; Mishra, Rahasya Mani; Seth, Tulika; Saxena, Renu

    2012-07-01

    Osteonecrosis is a serious complication in sickle cell patients. The common sites of the necrosis are femoral head, head of the humerus and acetabulam. Annexin A2 (ANXA2) protein mainly functions in bone formation and bone resorption. Alteration of ANXA2 gene may affect the manifestations of osteonecrosis in the patients. PCR-RFLP is a common applicable technique for the detection of known mutation/polymorphisms. Here we are presenting application of the PCR-RFLP technique for determination of the ANXA2 gene single nucleotide polymorphism frequency and their clinical association among Indian sickle cell patients. Five known SNPs of ANXA2 gene (rs7170178, rs73435133, rs73418020, rs72746635 and rs73418025) were determined using the HpyCH4V, DdeI, HpyCH4III and Sau 961 restriction enzyme respectively. Restriction enzyme DdeI was common for rs73435133 and rs72746635 SNP. Only the rs7170178 SNP was detected among patient and control and the other four SNPs were absent in the studied groups. The frequency of ANXA2 gene rs7170178 SNP (A/G, G/G) was comparatively higher in sickle cell patients than controls and it was clinically associated with sickle cell osteonecrosis. The P value of heterozygotes (A/G) and homozygotes (G/G) genotypes were <0.001 and 0.001 respectively, which were highly significant. This study established the application of PCR-RFLP in detection of ANXA2 SNPs in sickle cell patients.

  8. Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

    OpenAIRE

    Rachman, Cinta; Kabadjova, Petia; Valcheva, Rosica; Prévost, Hervé; Dousset, Xavier

    2004-01-01

    The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in ord...

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism.

    Science.gov (United States)

    Şahin, Ergin

    2015-12-01

    Antiviral therapies with nucleotide analogues (NA) is crucial in the treatment of chronic hepatitis B as it substantially protects patients from the complications of the disease . However in most of the available NA therapies, resistance emerges in the patients' HBV populations. Therefore, detection of antiviral resistance as early as possible by means of genotypically monitoring the patients' HBV pool during NA therapy is critical to manage treatment regime. In this research study we have investigated the sensitivity and specificity of the terminal restriction fragment length polymorphism (T-RFLP) method in detecting HBV subpopulations carrying antiviral resistance mutations. For this aim, differentiation of mutant strains from wild type strains was demonstrated by PCR-RFLP method. With using recombinant plasmids containing mutant and wild type HBV genomes, we constructed artificial HBV genome populations in order to determine the sensitivity of PCR-T-RFLP method in detecting antiviral resistant minor HBV populations. Finally by comparing with the DNA sequencing method, we demonstrated the specificity of T-RFLP method in genotyping HBV populations. As a result we showed that T-RFLP is able to detect HBV subpopulations representing as low as 1 % of the whole viral population. Additionally T-RFLP showed 100 % concordance with the DNA sequencing method in genotyping HBV populations. As a conclusion, considering the other genotyping methods used in evaluating HBV populations, T-RFLP showed high sensitivity and specificity profiles in detecting antiviral resistant HBV subpopulations. Therefore T-RFLP method can be easily employed in genotypic evaluation of patients' HBV populations during the course of antiviral treatment.

  12. Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR.

    Science.gov (United States)

    Küchler, Erika Calvano; Tannure, Patricia Nivoloni; Falagan-Lotsch, Priscila; Lopes, Taliria Silva; Granjeiro, Jose Mauro; Amorim, Lidia Maria Fonte

    2012-01-01

    The aim of this study was to evaluate, by PCR-RFLP and real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by dna extraction. The concentration, purity and integrity of the dna were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 a/g (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using real-time PCR. There was no significant difference of dna yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and real time pcr reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by real-time pcr presented c allele for irf6 gene polymorphism (homozygous: cc; heterozygous: Ct) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and pcr amplified after storage in mouthwash solution at room temperature.

  13. Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Erika Calvano Küchler

    2012-08-01

    Full Text Available OBJECTIVE: The aim of this study was to evaluate, by PCR-RFLP and real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. MATERIAL AND METHODS: A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by dna extraction. The concentration, purity and integrity of the dna were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 a/g (rs 4444903 polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215 using real-time PCR. RESULTS: There was no significant difference of dna yield (p=0.75 and purity (p=0.86 among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and real time pcr reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by real-time pcr presented c allele for irf6 gene polymorphism (homozygous: cc; heterozygous: Ct and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. CONCLUSION: We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and pcr amplified after storage in mouthwash solution at room temperature.

  14. 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.

  15. The human tyrosine aminotransferase gene: characterization of restriction fragment length polymorphisms and haplotype analysis in a family with tyrosinemia type II.

    Science.gov (United States)

    Westphal, E M; Natt, E; Grimm, T; Odievre, M; Scherer, G

    1988-07-01

    Deficiency in hepatic tyrosine aminotransferase (TAT) causes tyrosinemia type II, an autosomal recessively inherited disorder. Using a TAT cosmid clone, we have identified an MspI restriction fragment length polymorphism (RFLP) 5' to the TAT gene, with allele frequencies of 0.63 and 0.37. Analysis of the cloned maternal and paternal TAT alleles from a patient with tyrosinemia type II led to the identification of a HaeIII RFLP at the 3' end of the TAT gene, with allele frequencies of 0.94 and 0.06. The two RFLPs are 27 kb apart and in no allelic association. From haplotype frequencies, a polymorphism information content (PIC) value of 0.44 was obtained. The two RFLPs have allowed the unambiguous identification of the mutant TAT alleles in the patient's pedigree by haplotype analysis.

  16. A specific polymerase chain reaction based on the gyrB gene sequence and subsequent restriction fragment length polymorphism analysis of Pasteurella pneumotropica isolates from laboratory mice.

    Science.gov (United States)

    Hayashimoto, Nobuhito; Ueno, Masami; Takakura, Akira; Itoh, Toshio

    2007-03-01

    For a molecular identification and typing tool, we developed a specific polymerase chain reaction (PCR) based on the gyrB gene sequence and a subsequent restriction fragment length polymorphism (RFLP) analysis using the products amplified from the specific PCR to facilitate discrimination of biotypes of Pasteurella pneumotropica from laboratory mice. Appropriate PCR products, a 1039-basepair fragment for biotype Jawetz and a 1033-basepair fragment for biotype Heyl, were amplified by use of the primers CZO-1 and NJO-2 from all 105 P. pneumotropica isolates tested and the 2 reference strains but not from other bacterial species tested. MspI digestion of PCR-generated products showed 3 RFLP patterns among the 105 isolates, and these patterns correlated with the biotype of the isolate (RFLP pattern 1, biotype Jawetz; RFLP pattern 2, biotype Heyl; and RFLP pattern 3, biotype Jawetz with Beta-hemolytic activity). Our procedure identifies and biotypes isolates of P. pneumotropica from laboratory mice, using simple PCR and enzymatic restriction techniques. Therefore, this procedure is useful as a rapid identification and biotyping tool for isolates of P. pneumotropica from laboratory mice.

  17. 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

    2015-01-01

    Background: 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. Objectives: The aim of this study was to investigate the polymorphism of thermophilic Campylobacter isolated from broiler fecal samples in Shiraz, southern Iran. Materials and Methods: 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. Results: 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. Conclusions: 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. PMID:26060566

  18. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS ASSOCIATED WITH FACTOR Ⅷ CARRIER DETECTION USING DNA RFLPs IN CHINESE

    Institute of Scientific and Technical Information of China (English)

    汪运山; 王德芬; 曾畿生

    1992-01-01

    RFLPs for XbaI, BelI and BglI sites of human FⅧwere informative for 48%, 41% and 15% of females studied, respectively. BglI RFLP is different from that reported by Chan et al, a fact suggests Yangtze River region population of China would be at variance with the Southern Chinese population in certain RFLP distribution. TaqI allelic system Ⅰin the DXS52 region also shows the same variance among them, but heterozygous rate 0f 71% for system Ⅰ(alleles 1 to 8) and 49% for system Ⅱ(αand βalleles) were very similar. Using the Bell/XbaI RFLPs, accurate information could be obtained from this study for 56% of women who were at risk for hemophilia A (HA) carriership. The carrier of the remaining 44% could be determined by utilizing the TaqI RFLP. In addition, we report a new intergenie polymorphism (9%) at DXS115 as a marker for detection of heterozygotes in families at risk for HA. The advantage of using the XbaI/KpnI RFLP is that both the intragemie RFLP and the new intergenie RFLP can be evaluated on the same blot at the same time.

  19. [Species identification of grouper and snapper in Taiwan Strait using polymerase chain reaction-restriction fragment length polymorphism analysis and lab-on-a-chip system].

    Science.gov (United States)

    Chen, Shuangya; Zhang, Jin; Chen, Weiling; Xu, Dunming; Zhou, Yu

    2011-07-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-a-chip system were used to identify grouper and snapper species in Taiwan Strait. A fragment of 464 bp length of mitochondrial cytochrome b gene was amplified by PCR and the products were digested with restriction enzymes Dde I , Hae III and NLa III, individually. The fragments generated after digestion were further resolved on the DNA Chip. Eight grouper species and five snapper species were successfully identified. The results demonstrated that PCR-RFLP analysis and lab-on-a-chip system provide a fast, easy, automated, and reliable analysis approach. This approach is potential for the purpose of fish adulteration control.

  20. Association between FokI, ApaI and TaqI RFLP polymorphisms in VDR gene and Hashimoto's thyroiditis: preliminary data from female patients in Serbia.

    Science.gov (United States)

    Djurovic, J; Stojkovic, O; Ozdemir, O; Silan, F; Akurut, C; Todorovic, J; Savic, K; Stamenkovic, G

    2015-06-01

    Hashimoto's thyroiditis (HT) is the most prevalent autoimmune thyroid disorder caused by an interaction between genes and environmental triggers. Intrathyroid lymphocytic infiltration may lead to progressive destruction of thyroid tissue and consequently to hypothyroidism. Many studies in different populations have shown association between vitamin D receptor (VDR) gene polymorphisms and various autoimmune diseases, including HT. The study included 44 female patients (mean age ± standard deviation 38 ± 5.4) with Hashimoto's thyroiditis and 32 healthy age-matched, sex-matched and geographically matched controls without personal history of autoimmune and endocrine diseases. Genomic DNA was isolated from peripheral blood-EDTA, and the target VDR gene was genotyped by PCR-RFLP technique after VDR-FokI (rs2228570), VDR-ApaI (rs7975232) and VDR-TaqI (rs731236) restriction enzymes digestion. We used spss 20.0 integrated software for data analysis and found a significant difference in the genotype distribution of VDR-FokI polymorphism between patients with HT and controls (P = 0.009). For ApaI and TaqI, we observed a higher frequency of variant allele in patients with HT, which was not significantly different compared to control women (P > 0.05). The current first and preliminary results identified the association between VDR-FokI gene polymorphism and Hashimoto's thyroiditis in Serbian population. Results need to be supported by further investigations that define haplotype patterns for VDR gene polymorphisms in a larger group of HT patients of both sexes.

  1. Tools for T-RFLP data analysis using Excel.

    Science.gov (United States)

    Fredriksson, Nils Johan; Hermansson, Malte; Wilén, Britt-Marie

    2014-11-08

    Terminal restriction fragment length polymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed. The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data. The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.

  2. 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...

  3. ANALYSIS OF POLYMORPHISM OF BETA CASEIN OF SLOVAK PINZGAU CATTLE BY PCR-RFLP FOR ALLELS A1 AND A2

    Directory of Open Access Journals (Sweden)

    MARTINA MILUCHOVÁ

    2013-07-01

    Full Text Available The work was oriented to identification of -casein gene polymorphism and analysis of genotype structure in population of Slovak Pinzgau cattle. The material involved 89 cattle. Bovine genomic DNA was isolated by fenol-chlorophorm deprotenization and ethanol precipitation and used in order to estimate  - casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study there were homozygote genotype A1A1 (27 animals, heterozygote genotype A1A2 (46 animals and homozygote genotype A2A2 (16 animals. In the total population of cattle heterozygotes A1A2 – 0.5168 were the most frequent, while homozygotes A2A2 – 0.1798 were the least frequent ones. This suggests a slight superiority of allele A1– 0.5618.

  4. 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.

  5. 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.

  6. CUTANEOUS MYCOBACTERIUM MARINUM INFECTION DIAGNOSED BY PCR-RFLP ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    LI Xiao-jie; WANG Hong-sheng; TAO Shi-qin; WU Qin-xue; LIU Wei-da

    2009-01-01

    Objective To identify Mycobacterium marinum (M. marinum) inducing misdiagnosis and treatment failure.Methods The lesional specimen of patient with cutaneous M. marinum were cultivated on Lwenstein-Jensen medium. The isolate was identified by biochemical tests and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene.Results Smooth and non-pigmented colonies were noted after incubation at 32 ℃ for 2 weeks. The isolate was acid-fast bacilli and confirmed as M. marinum by biochemical tests and PCR-RFLP.Conclusion For a correct diagnosis of cutaneous M. marinum infection, it is crucial for clinicians to have a high index of suspicion, obtain the history of exposure and trauma and understand growth characteristics of the organism. Compared with conventional biochemical techniques, PCR-RFLP analysis is a more rapid, accurate and reliable method for mycobacterial identification to species level.

  7. Utility of MtCOI polymerase chain reaction-restriction fragment length polymorphism in differentiating between Q and B whitefly Bemisia tabaci biotypes

    Institute of Scientific and Technical Information of China (English)

    Wei-Hua Ma; Xian-Chun Li; Timothy J. Dennehy; Chao-Liang Lei; Mo Wang; Benjamin A. Degain; Robert L. Nichols

    2009-01-01

    The invasive, insecticide-resistant, Q whitefly biotype, has gradually spread to other countries including the US via human-mediated movement of plant materials. We assessed the utility of the VspI-based mtCOl (mitochondrion cytochrome oxidase I) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) tech-nique as a rapid, cost-effective, and reliable alternative for differentiating the Q from the dominant B biotype in Arizona. Using the standard mtCOI gene sequencing and mtCOI PCR-RFLP techniques, we biotyped eight whitefly strains of five individuals each collected from poinsettia and cotton at different locations in Arizona. Complete concordance was observed between the two methods, with three strains being identified as the Q biotype and five samples as the B biotype. We also scanned the mtCOI gene sequences for VspI polymorphisms in the B and Q biotype whiteflies currently available in the GenBank database. This global screening revealed the existence of three and four VspI polymorphic types for the Q and B biotypes, respectively. Nevertheless, all three VspI polymorphic Q biotype whiteflies shared a common and unique VspI site that can be used to differentiate Q biotype from the four VspI polymorphic B biotype whiteflies identified. These results demonstrate that the VspI-based mtCOI gene PCR-RFLP provides a reliable diagnostic tool for differentiating the Q and B biotype whiteflies in the US and elsewhere.

  8. Amplified Fragment Length Polymorphism Diversity in Cephalosporium maydis from Egypt.

    Science.gov (United States)

    Saleh, Amgad A; Zeller, Kurt A; Ismael, Abou-Serie M; Fahmy, Zeinab M; El-Assiuty, Elhamy M; Leslie, John F

    2003-07-01

    ABSTRACT Cephalosporium maydis, the causal agent of late wilt of maize, was first described in Egypt in the 1960s, where it can cause yield losses of up to 40% in susceptible plantings. We characterized 866 isolates of C. maydis collected from 14 governates in Egypt, 7 in the Nile River Delta and 7 in southern (Middle and Upper) Egypt, with amplified fragment length polymorphism (AFLP) markers. The four AFLP primer-pair combinations generated 68 bands, 25 of which were polymorphic, resulting in 52 clonal haplotypes that clustered the 866 isolates into four phylogenetic lineages. Three lineages were found in both the Nile River Delta and southern Egypt. Lineage IV, the most diverse group (20 haplotypes), was recovered only from governates in the Nile River Delta. In some locations, one lineage dominated (up to 98% of the isolates recovered) and, from some fields, only a single haplotype was recovered. Under field conditions in Egypt, there is no evidence that C. maydis reproduces sexually. The nonuniform geographic distribution of the pathogen lineages within the country could be due to differences in climate or in the farming system, because host material differs in susceptibility and C. maydis lineages differ in pathogenicity.

  9. 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......-based cluster analysis. The tree resulting from the subgroup analysis clearly separated all subgroups with high bootstrap values with the species S. bongori being the most distantly related of the subgroups. The tree resulting from the analysis of the SARB collection showed that some serotypes are very clonal...... whereas others are highly divergent. Conclusions: AFLP clearly clustered strains representing the subgroups of Salmonella together with high bootstrap values and the serotypes of subspecies enterica were divided into polyphyletic or monophyletic types corresponding well with multilocus enzyme...

  10. Screening for JH1 genetic defect carriers in Jersey cattle by a polymerase chain reaction and restriction fragment length polymorphism assay.

    Science.gov (United States)

    Zhang, Yi; Guo, Gang; Huang, Hetian; Lu, Lu; Wang, Lijie; Fang, Lingzhao; Liu, Lin; Wang, Yachun; Zhang, Shengli

    2015-09-01

    An autosomal recessive genetic defect termed JH1 has been associated with early embryonic loss in the Jersey cattle breed. The genetic basis has been identified as a cytosine to thymine mutation in the CWC15 gene that changes an amino acid from arginine to a stop code. To screen for JH1 carriers in an imported Jersey population in China, a method based on a polymerase chain reaction amplification followed by a restriction fragment length polymorphism assay (PCR-RFLP) was developed for the accurate diagnosis of the JH1 allele. A total of 449 randomly chosen cows were examined with the PCR-RFLP assay, and 31 were identified as JH1 carriers, corresponding to a carrier frequency of 6.9%. The PCR-RFLP method was validated by DNA sequencing of 8 positive and 13 negative samples, with all 21 samples giving the expected DNA sequence. In addition, 3 negative and 3 positive samples were confirmed by a commercial microarray-based single nucleotide polymorphism assay. Finally, samples from 9 bulls in the United States of known status were correctly identified as carriers (5 bulls) or noncarriers (4 bulls). As the JH1 defect has most likely spread worldwide, implementing routine screening is necessary to avoid the risk of carrier-to-carrier matings and to gradually eradicate the deleterious gene.

  11. Characterization of field strains of infectious laryngotracheitis virus in China by restriction fragment length polymorphism and sequence analysis.

    Science.gov (United States)

    Yan, Zhuanqiang; Li, Shengpeng; Xie, Qingmei; Chen, Feng; Bi, Yingzuo

    2016-01-01

    Nineteen strains of infectious laryngotracheitis virus (ILTV; Gallid herpesvirus 1) were isolated from dead or diseased birds in chicken flocks from different areas of China between 2010 and 2014 and used to investigate ILTV epidemiology. These strains were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns and sequence analysis of the thymidine kinase (TK) gene. PCR-RFLP analysis showed that the TK gene generated 2 patterns when digested with restriction endonuclease enzymes. Pattern A corresponded to 2 virulent field strains, while pattern B was characteristic of 2 virulent field strains, 15 low pathogenicity field strains, and all vaccine strains. Sequence analysis of the TK gene indicated that the messenger RNA polyadenylation signals could be identified in some isolates where amino acid 252 was threonine, and in those with methionine at that position. The present study has demonstrated that most of the outbreaks of ILT in China were caused either by low virulence strains or by vaccine-related strains, and also emphasizes the importance of reinforcing ILTV surveillance in both vaccinated and nonvaccinated flocks.

  12. 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.

  13. A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons

    Institute of Scientific and Technical Information of China (English)

    Guo-Bing Zeng; Shu-Juan Wen; Zhan-Hui Wang; Li Yan; Jian Sun; Jin-Lin Hou

    2004-01-01

    AIM: Traditional hepatitis B virus (HBV) genotyping methods using restriction fragment length polymorphism (RFLP) can reliably identify genotypes A to F. As HBV genotypes G and H have been recently identified, this study was to establish an accurate and simple genotyping method for all eight HBV genotypes (A to H).METHODS: Two hundred and forty HBV small S sequences obtained from GeneBank were analysed for restriction enzyme sites that would be genotype-specific. Restriction patterns following digestion with restriction enzymes BsrⅠ,StyⅠ, DpnⅠ, HpaⅡ, and EaeⅠ, were determined to identify all eight HBV genotypes. Mixed genotype infections were confirmed by cloning and further RFLP analysis.RESULTS: The new genotyping method could identify HBV genotypes A to H. Genotypes B and C could be determined by a single step digestion with BsrⅠ and StyⅠ in parallel. This was particularly useful in the Far East where genotypes B and C are predominant. Serum samples from 187 Chinese HBV carriers were analysed with this genotyping system, and the genotype distribution was 1.1% (2), 51.9% (97), 40.6% (76) and 4.8%(9) for genotypes A, B, C, and D, respectively. Mixed genotypes were found in only 3 patients (1.6%). Sequence data analysis confirmed the validity of this new method.CONCLUSION: This HBV genotyping system can identify all eight HBV genotypes. It is accurate and simple, and can be widely used for studies on HBV genotyping.

  14. Taenia saginata: differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay.

    Science.gov (United States)

    Nunes, Cáris Maroni; Dias, Ana Karina Kerche; Dias, Francisca Elda Ferreira; Aoki, Sérgio Moraes; de Paula, Henrique Borges; Lima, Luis Gustavo Ferraz; Garcia, José Fernando

    2005-08-01

    Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different DraI-RFLP pattern: a two-band pattern (421 and 100 bp) for T. saginata and a three-band pattern (234, 188, and 99 bp) for T. solium was observed allowing the two species to be separated. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T. saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). Of these, three showed a T. solium pattern and five a T. saginata pattern.

  15. Molecular characterization of Mycobacterium tuberculosis isolates from Tehran, Iran by restriction fragment length polymorphism analysis and spoligotyping.

    Science.gov (United States)

    Feyisa, Seifu Gizaw; Haeili, Mehri; Zahednamazi, Fatemeh; Mosavari, Nader; Taheri, Mohammad Mohammad; Hamzehloo, Gholamreza; Zamani, Samin; Feizabadi, Mohammad Mehdi

    2016-04-01

    INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.

  16. Terminal Restriction Fragment Length Polymorphism for the Identification of Spirorchiid Ova in Tissues from the Green Sea Turtle, Chelonia mydas.

    Science.gov (United States)

    Chapman, Phoebe A; Traub, Rebecca J; Kyaw-Tanner, Myat T; Owen, Helen; Flint, Mark; Cribb, Thomas H; Mills, Paul C

    2016-01-01

    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 epidemiological and

  17. Terminal Restriction Fragment Length Polymorphism for the Identification of Spirorchiid Ova in Tissues from the Green Sea Turtle, Chelonia mydas

    Science.gov (United States)

    Chapman, Phoebe A.; Traub, Rebecca J.; Kyaw-Tanner, Myat T.; Owen, Helen; Flint, Mark; Cribb, Thomas H.; Mills, Paul C.

    2016-01-01

    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 epidemiological and

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Expression of tumor related gene NAG6 in gastric cancer and restriction fragment length polymorphism analysis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Mei Zhang; Shou-Rong Sheng; Xiao-Yan Wang; Liang-Hua Bin; Jie-Ru Wang; Gui-Yuan Li

    2004-01-01

    AIM: NAG6 gene is a novel tumor related gene identified recently. This study was designed to examine the expression of this gene in gastric cancer and corresponding normal tissues, and to investigate its role in the occurrence and development of gastric cancer, also to study if the genetic structure of NAG6 was altered in gastric cancer.METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis and dot hybridization were used to compare the expression level of NAG6 gene in 42cases of gastric cancer tissues with their corresponding normal tissues of the same patients respectively. In addition,restriction fragment length polymorphism (RFLP) analysis was adopted to study if the genetic structure of NAG6 was altered in gastric carcinomas.RESULTS: The expression of NAG6 in 57.1% gastric cancer tissues (25/42) was absent by RT-PCR analysis. The downregulation rate of NAG6 in gastric cancer tissues was significantly higher than that in corresponding normal tissues (P<0.01). However no correlation between the downregulation of NAG6 and lymph-node and/or distance metastasis was found in this study (P>0.05). Dot hybridization confirmed the results of RT-PCR. Furthermore,the results of EcoRI RFLP analysis of NAG6 gene demonstrated that 3 of 7 cases of gastric cancer showed loss of 5 kb fragment in comparison with their corresponding normal tissues.CONCLUSION: NAG6 gene is significantly down regulated in gastric cancer. The loss of genetic materials may be the cause of down-regulation of NAG6 expression. This seems to suggest that NAG6 may represent a candidate of putative tumor suppressor gene at 7q31-32 loci associated with gastric carcinoma. The down-regulation of this gene may play a role in occurrence and development of this disease, however it may not be associated with lymph node and/or distance metastasis.

  20. A Rapid PCR-RFLP Method for Monitoring Genetic Variation among Commercial Mushroom Species

    Science.gov (United States)

    Martin, Presley; Muruke, Masoud; Hosea, Kenneth; Kivaisi, Amelia; Zerwas, Nick; Bauerle, Cynthia

    2004-01-01

    We report the development of a simplified procedure for restriction fragment length polymorphism (RFLP) analysis of mushrooms. We have adapted standard molecular techniques to be amenable to an undergraduate laboratory setting in order to allow students to explore basic questions about fungal diversity and relatedness among mushroom species. The…

  1. 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.

  2. PCR-RFLP analysis of mitochondrial DNA in tench Tinca tinca.

    Science.gov (United States)

    Lo Presti, R; Gasco, L; Lisa, C; Zoccarato, I; Di Stasio, L

    2010-02-01

    Polymorphism was detected at ND1, ND6, D-loop and cyt b segments of mtDNA in 105 tench (Tinca tinca L.), using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and five composite haplotypes were identified. The diversity indices and the results of the population comparisons revealed that the identified markers provide a powerful tool for further studies on this species.

  3. Amplification refractory mutation system polymerase chain reaction versus optimized polymerase chain reaction restriction-fragment length polymorphism for apolipoprotein E genotyping of majorly depressed patients.

    Science.gov (United States)

    You, Hongmin; Chen, Jin; Zhou, Jingjing; Huang, Hua; Pan, Junxi; Wang, Ziye; Lv, Lin; Zhang, Lujun; Li, Juan; Qin, Bin; Yang, Yongtao; Xie, Peng

    2015-11-01

    Major depressive disorder (MDD) is a prevalent, debilitating mood disorder that has been associated with several genetic polymorphisms. One such polymorphism, namely that of apolipoprotein E (APOE), has three allelic forms (ε2, ε3 and ε4) that encode for six unique isoforms of the APOE protein. A growing number of techniques have been developed for APOE genotyping; however, not all polymerase chain reaction (PCR)‑based genotyping techniques are equally accurate or cost‑effective. In order to find a more accurate and cost‑effective APOE genotyping method for MDD screening in large populations, the present study comparatively evaluated two genotyping methods, amplification refractory mutation system PCR (ARMS‑PCR) and optimized PCR restriction‑fragment length polymorphism (PCR‑RFLP), in blood samples taken from a population of 708 MDD patients. Although either of the two methods were able to detect all six unique APOE genotypes, comparisons of the two methods with Sanger sequencing demonstrated that ARMS‑PCR (94%) was significantly more accurate than optimized PCR‑RFLP (82%). ARMS‑PCR should prove useful in quickly verifying ambiguous results obtained by other APOE genotyping methods and can be cost-effectively performed in the setting of a small laboratory or a population-based screening program.

  4. Study on the Polymorphism of Porcine Hormone-sensitive Lipase Gene Exon I by PCR-RFLP in Different Breeds%猪品种间HSL基因外显子I PCR-RFLP的研究

    Institute of Scientific and Technical Information of China (English)

    雷明刚; 吴珍芳; 邓昌彦; 熊远著

    2001-01-01

    Hormone-sensitive lipase (HSL) is the key enzyme responsible for the mobilization of free acids from adipose tissue, and it is also the most important enzyme that affects fat deposition. In this paper, the polymorphism of HSL gene in exon I was detected by PCR-RFLP. It revealed a BsaHI-site polymorphism in different pig breeds. There exist two genotypes (AA,AG) in the fat-type Tongcheng pig and Qingping pig with allele frequencies of 0.917A,0.083G and 0.893,0.107,respectively. Duroc has two genotypes (AG,GG) with allele frequencies 0.15A and 0.85G. However, There exists only GG genotype in lean-type Large White and Landrace pig.%激素敏感脂肪酶(Hormone-sensitive lipase,HSL)是负责分解脂肪组织中甘油三酯释放游离脂肪酸的关键酶。本研究利用PCR*.RFLP(polymerase chain reaction-restriction fragment length polymorphism)方法,分析了猪HSL基因外显子I区多态性,发现不同品种猪间存在多态性。瘦肉型大白猪和长白猪全部表现为GG基因型;杜洛克猪表现为AG和GG两种基因型,其等位基因A和G的频率是15%和85%;脂肪型通城猪和清平猪表现为AA和AG两种基因型,其等位基因A、G的频率分别是91.67%、8.33%和89.83%、10.17%。

  5. 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.

  6. Differentiation of non-pylori Helicobacter species based on PCR-restriction fragment length polymorphism of the 23S rRNA gene.

    Science.gov (United States)

    Yadegar, Abbas; Alebouyeh, Masoud; Lawson, Andy J; Mirzaei, Tabassom; Nazemalhosseini Mojarad, Ehsan; Zali, Mohammad Reza

    2014-06-01

    Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A-C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR-RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.

  7. Restriction fragment length polymorphism analysis of rotavirus VP7-encoding gene from humans and animals of Northeast India: a relative study of Indian and global isolates.

    Science.gov (United States)

    Chakraborty, P; Barman, N N; Sharma, I

    2015-09-01

    A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic relationship between 67 (29 Indian, 38 global) rotavirus isolates of human, bovine and porcine neonates. The assay involved direct digestion of RT-PCR amplified VP7 cDNAs with three restriction enzymes (VspI, HaeIII, NlaIV) independently. Forty-eight RFLP patterns were identified for all 67 strains, and of these 20 patterns were associated with Indian isolates. A correlation between the restriction patterns and G type was apparent through deduction of enzyme restriction sites from known sequences. Major G serotypes (G1, G2, G6, G8) with a few mixed types could be differentiated where there was a positive assortment of intrinsic serotypes from multiple host origin, and certain single or combined enzyme profiles were highly dominant in the population. Significant genetic variations were established between global and Indian isolates and none of the RFLP patterns were shared between them. These data suggest that the Indian wild-type rotavirus population is distinguishable based on the VP7 gene, and co-circulation of distinct strains in different hosts is foremost, indicating the possible likelihood of inter-species transmission.

  8. Intestinal microbiota is different in women with preterm birth: results from terminal restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Shiozaki, Arihiro; Yoneda, Satoshi; Yoneda, Noriko; Yonezawa, Rika; Matsubayashi, Takamichi; Seo, Genichiro; Saito, Shigeru

    2014-01-01

    Preterm birth is a leading cause of perinatal morbidity and mortality. Studies using a cultivation method or molecular identification have shown that bacterial vaginosis is one of the risk factors for preterm birth. However, an association between preterm birth and intestinal microbiota has not been reported using molecular techniques, although the vaginal microbiota changes during pregnancy. Our aim here was to clarify the difference in intestinal and vaginal microbiota between women with preterm birth and women without preterm labor. 16S ribosomal ribonucleic acid genes were amplified from fecal and vaginal DNA by polymerase chain reaction. Using terminal restriction fragment length polymorphism (T-RFLP), we compared the levels of operational taxonomic units of both intestinal and vaginal flora among three groups: pregnant women who delivered term babies without preterm labor (non-PTL group) (n = 20), those who had preterm labor but delivered term babies (PTL group) (n = 11), and those who had preterm birth (PTB group) (n = 10). Significantly low levels of Clostridium subcluster XVIII, Clostridium cluster IV, Clostridium subcluster XIVa, and Bacteroides, and a significantly high level of Lactobacillales were observed in the intestinal microbiota in the PTB group compared with those in the non-PTL group. The levels of Clostridium subcluster XVIII and Clostridium subcluster XIVa in the PTB group were significantly lower than those in the PTL group, and these levels in the PTL group were significantly lower than those in non-PTL group. However, there were no significant differences in vaginal microbiota among the three groups. Intestinal microbiota in the PTB group was found to differ from that in the non-PTL group using the T-RFLP method.

  9. Rapid detection of clarithromycin resistant Helicobacter pylori strains in Spanish patients by polymerase chain reaction-restriction fragment length polymorphism

    Science.gov (United States)

    Agudo, Sonia; Pérez-Pérez, Guillermo; Alarcón, Teresa; López-Brea, Manuel

    2014-01-01

    Introduction The aim of this study was to characterize the mutations types present in the 23S rRNA gene related to H. pylori clarithromycin-resistance strains in Spain and evaluate a novel PCR-RFLP method for detection of the most frequent point mutation in our population. Methods Gastric biopsies were obtained by endoscopy from patients with gastric symptoms. H. pylori was cultured according to standard microbiological procedures and clarithromycin resistance was determined by E-test. DNA extraction was performed by NucliSens platform with the NucliSens magnetic extraction reagents (bioMérieux) according to the manufacturer instructions. Analyses for point mutations in 23S rRNA gene strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using BsaI enzyme to detect restriction sites that correspond to the mutation (A2143G). Result We found 42 out of 118 (35.6%) strains resistant to clarithromycin by E-test. E-test results were confirmed for the presence of point mutation in 34 (88.1%) of these strains. Mutation A2143G was found in 85.3% of the strains. Analyses with the restriction enzyme BsaI was able to confirm the presence of A2143G mutation. There were 8 H. pylori strains resistant to clarithromycin by E-test but without any point mutation in the 23 rRNA gene. Conclusion We conclude that PCR-RFLP is a reliable method to detect clarithromycin-resistance H. pylori strains in countries with a high prevalence of clarithromycin-resistance as Spain It may be useful before choosing regimens of H. pylori eradication. PMID:21412667

  10. Intestinal microbiota is different in women with preterm birth: results from terminal restriction fragment length polymorphism analysis.

    Directory of Open Access Journals (Sweden)

    Arihiro Shiozaki

    Full Text Available Preterm birth is a leading cause of perinatal morbidity and mortality. Studies using a cultivation method or molecular identification have shown that bacterial vaginosis is one of the risk factors for preterm birth. However, an association between preterm birth and intestinal microbiota has not been reported using molecular techniques, although the vaginal microbiota changes during pregnancy. Our aim here was to clarify the difference in intestinal and vaginal microbiota between women with preterm birth and women without preterm labor. 16S ribosomal ribonucleic acid genes were amplified from fecal and vaginal DNA by polymerase chain reaction. Using terminal restriction fragment length polymorphism (T-RFLP, we compared the levels of operational taxonomic units of both intestinal and vaginal flora among three groups: pregnant women who delivered term babies without preterm labor (non-PTL group (n = 20, those who had preterm labor but delivered term babies (PTL group (n = 11, and those who had preterm birth (PTB group (n = 10. Significantly low levels of Clostridium subcluster XVIII, Clostridium cluster IV, Clostridium subcluster XIVa, and Bacteroides, and a significantly high level of Lactobacillales were observed in the intestinal microbiota in the PTB group compared with those in the non-PTL group. The levels of Clostridium subcluster XVIII and Clostridium subcluster XIVa in the PTB group were significantly lower than those in the PTL group, and these levels in the PTL group were significantly lower than those in non-PTL group. However, there were no significant differences in vaginal microbiota among the three groups. Intestinal microbiota in the PTB group was found to differ from that in the non-PTL group using the T-RFLP method.

  11. Community terminal restriction fragment length polymorphisms reveal insights into the diversity and dynamics of leaf endophytic bacteria

    Directory of Open Access Journals (Sweden)

    Ding Tao

    2013-01-01

    Full Text Available Abstract Background Plant endophytic bacteria play an important role benefiting plant growth or being pathogenic to plants or organisms that consume those plants. Multiple species of bacteria have been found co-inhabiting plants, both cultivated and wild, with viruses and fungi. For these reasons, a general understanding of plant endophytic microbial communities and their diversity is necessary. A key issue is how the distributions of these bacteria vary with location, with plant species, with individual plants and with plant growing season. Results Five common plant species were collected monthly for four months in the summer of 2010, with replicates from four different sampling sites in the Tallgrass Prairie Preserve in Osage County, Oklahoma, USA. Metagenomic DNA was extracted from ground, washed plant leaf samples, and fragments of the bacterial 16S rDNA genes were amplified for analysis of terminal restriction fragment length polymorphism (T-RFLP. We performed mono-digestion T-RFLP with restriction endonuclease DdeI, to reveal the structures of leaf endophytic bacterial communities, to identify the differences between plant-associated bacterial communities in different plant species or environments, and to explore factors affecting the bacterial distribution. We tested the impacts of three major factors on the leaf endophytic bacterial communities, including host plant species, sampling dates and sampling locations. Conclusions Results indicated that all of the three factors were significantly related (α = 0.05 to the distribution of leaf endophytic bacteria, with host species being the most important, followed by sampling dates and sampling locations.

  12. Evaluation of Microbial Diversity in Wetland through Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP)

    Science.gov (United States)

    2006-06-01

    the United States Marine Corps, United States Air Force, Department of Defense, or the United States Government. AFIT/GES/ENV/06J-02 EVALUATION OF...would have never come to completion. I am also appreciative to the United States Marine Corps for providing this opportunity for professional...variety of organisms including bacteria (e.g. Bacillus subtilis, Bacillus anthracis), fungi (e.g. yeasts, molds), algae and Actinomycetes (e.g

  13. 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), syste...

  14. Veronaea botryosa: molecular identification with amplified fragment length polymorphism (AFLP) and in vitro antifungal susceptibility

    NARCIS (Netherlands)

    Badali, H.; Yazdanparast, S.A.; Bonifaz, A.; Mousavi, B.; de Hoog, G.S.; Klaassen, C.H.W.; Meis, J.F.G.M.

    2013-01-01

    Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated in

  15. Veronaea botryosa: molecular identification with amplified fragment length polymorphism (AFLP) and in vitro antifungal susceptibility

    NARCIS (Netherlands)

    Badali, H.; Yazdanparast, S.A.; Bonifaz, A.; Mousavi, B.; de Hoog, G.S.; Klaassen, C.H.W.; Meis, J.F.G.M.

    2013-01-01

    Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated in

  16. 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...

  17. AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF MYCOBACTERIUM AVIUM COMPLEX ISOLATES RECOVERED FROM SOUTHERN CALIFORNIA

    Science.gov (United States)

    Fine-scale genotyping methods are necessary in order to identify possible sources of human exposure to opportunistic pathogens belonging to the Mycobacterium avium complex (MAC). In this study, amplified fragment length polymorphism (AFLP) analysis was evaluated for fingerprintin...

  18. Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry.

    Science.gov (United States)

    Miyaguchi, Hajime

    2016-09-20

    An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 ± 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

  19. Polymerase chain reaction-restriction fragment length polymorphism method for differentiation of uropathogenic specific protein gene types.

    Science.gov (United States)

    Lai, Yun Mei; Zaw, Myo Thura; Shamsudin, Shamsul Bahari; Lin, Zaw

    2016-08-01

    The putative pathogenicity island (PAI) containing the uropathogenic specific protein (usp) gene and three small open reading frames (orfU1, orfU2, and orfU3) encoding 98, 97, and 96 amino acid proteins is widely distributed among uropathogenic Escherichia coli (UPEC) strains. This PAI was designated as PAIusp. Sequencing analysis of PAIusp has revealed that the usp gene can be divided into two types - uspI and uspII - based on sequence variation at the 3' terminal region and the number and position of orfUs differ from strain to strain. Based on usp gene types and orfU sequential patterns, PAIusp can be divided into four subtypes. Subtyping of PAIusp is a useful method to characterize UPEC strains. In this study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to differentiate usp gene types. This method could correctly identify the usp gene type in usp-positive UPEC strains in our laboratory.

  20. Lactic acid bacterial population dynamics during fermentation and storage of Thai fermented sausage according to restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Wanangkarn, Amornrat; Liu, Deng-Cheng; Swetwiwathana, Adisorn; Jindaprasert, Aphacha; Phraephaisarn, Chirapiphat; Chumnqoen, Wanwisa; Tan, Fa-Jui

    2014-09-01

    This study applied restriction fragment length polymorphism (RFLP) analysis to identify the lactic acid bacteria (LAB) isolated from "mum" Thai fermented sausages during fermentation and storage. A total of 630 lactic acid bacteria were isolated from the sausages prepared using 2 methods. In Method 1, after stuffing, the sausages were stored at 30 °C for 14 days. In Method 2, after stuffing and storage at 30 °C for 3 days, the sausages were vacuum-packed and stored at 4 °C until Day 28. The sausages were sampled on Days 0, 3, 14, and 28 for analyses. The 16S rDNA was amplified and digested using restriction enzymes. Of the restriction enzymes evaluated, Dde I displayed the highest discrimination capacity. The LAB were classified and 7 species were identified For Methods 1 and 2, during fermentation, the Lactobacillus sakei and Lactobacillus plantarum species were dominant. For Method 2, the proportion of Leuconostoc mesenteroides markedly increased during storage, until L. sakei and Ln. mesenteroides represented the dominant species. The identification of LAB in the sausage samples could facilitate the selection of appropriate microorganisms for candidate starter cultures for future controlled mum production.

  1. SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping

    Directory of Open Access Journals (Sweden)

    Chang Hsueh-Wei

    2010-04-01

    Full Text Available Abstract Background PCR-restriction fragment length polymorphism (RFLP assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2. Results The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels, gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system. Conclusions The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.

  2. Mutagenic primer design for mismatch PCR-RFLP SNP genotyping using a genetic algorithm.

    Science.gov (United States)

    Yang, Cheng-Hong; Cheng, Yu-Huei; Yang, Cheng-Huei; Chuang, Li-Yeh

    2012-01-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is useful in small-scale basic research studies of complex genetic diseases that are associated with single nucleotide polymorphism (SNP). Designing a feasible primer pair is an important work before performing PCR-RFLP for SNP genotyping. However, in many cases, restriction enzymes to discriminate the target SNP resulting in the primer design is not applicable. A mutagenic primer is introduced to solve this problem. GA-based Mismatch PCR-RFLP Primers Design (GAMPD) provides a method that uses a genetic algorithm to search for optimal mutagenic primers and available restriction enzymes from REBASE. In order to improve the efficiency of the proposed method, a mutagenic matrix is employed to judge whether a hypothetical mutagenic primer can discriminate the target SNP by digestion with available restriction enzymes. The available restriction enzymes for the target SNP are mined by the updated core of SNP-RFLPing. GAMPD has been used to simulate the SNPs in the human SLC6A4 gene under different parameter settings and compared with SNP Cutter for mismatch PCR-RFLP primer design. The in silico simulation of the proposed GAMPD program showed that it designs mismatch PCR-RFLP primers. The GAMPD program is implemented in JAVA and is freely available at http://bio.kuas.edu.tw/gampd/.

  3. Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.

    Directory of Open Access Journals (Sweden)

    Huidan Zhang

    Full Text Available Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA. In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%, significantly higher than that obtained by direct sequencing (13, 13.3%. Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient's age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical

  4. Restriction fragment length polymorphism of adhesin gene hpaA from different Helicobacter pylori strains of Chongqing, China

    Institute of Scientific and Technical Information of China (English)

    Yu Hong; Xu-Hu Mao; Wei-Kun Zeng; Li-Ming Ma; Shen-Rong Jing; Quan-Ming Zou

    2005-01-01

    AIM: To assess the variability of adhesin gene hpaA between different Helicobacter pylori ( H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). METHODS: Twelve different H pylori strains were chosento amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809,CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains(abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains,were digested by HhaⅠ and HaeⅢ individually and analyzed by agarose gel electrophoresis. RESULTS: In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18T vector respectively, then the recombinant plasmids were digested simultaneously with NcoⅠ and XhoⅠ to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Hae Ⅲ could be seen as 4 types of bands and 5 types with Hha Ⅰ. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group Ⅰ, NCTC11637 and SS1; group Ⅱ, CCS9809, which RFLP type digested with HaeⅢ wasthe same as strains of group Ⅰ, but HhaⅠ RFLP showeddifference compared with the other groups; group Ⅲ,CCS9810; group Ⅳ, CCS9803; group Ⅴ: CCS9801,CCS9802, CCS9806, CCS9813, MG1, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence

  5. Restriction fragment length polymorphism of adhesin gene hpaA from different Helicobacter pylori strains of Chongqing, China.

    Science.gov (United States)

    Hong, Yu; Mao, Xu-Hu; Zeng, Wei-Kun; Ma, Li-Ming; Jing, Shen-Rong; Zou, Quan-Ming

    2005-05-07

    To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by HhaI and HaeIII individually and analyzed by agarose gel electrophoresis. In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with NcoI and XhoI to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Hae III could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIII was the same as strains of group I, but HhaI RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS

  6. [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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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

  9. Application of restriction fragment length polymorphism analysis to simple and rapid genotyping of bovine viral diarrhea virus strains isolated in Japan.

    Science.gov (United States)

    Seki, Yoshihisa; Seimiya, Yukio M; Motokawa, Masato; Yaegashi, Gakuji; Nagai, Makoto; Hayashi, Michiko

    2008-04-01

    The E2 regions of 177 bovine viral diarrhea virus (BVDV) strains isolated in Japan between 1957 and 2006 were analyzed for genotyping. The strains were classified into 8 genotypes (1a, 1b, 1c, 1d, 1e, 1f, So and 2a) based on the phylogenetic analysis. The restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products using 6 selected enzymes (Apo I, Mly I, BstAP I, Pvu II, Ear I, EcoR V) disclosed the cutting patterns classified into 11 groups (I-XI), each of that consisted of strains belonging to a single genotype. Namely, groups-I and -II were composed by genotype-1a strains, groups-III and -IV by 1b strains, and groups-V and -VI by 1c strains. Other groups-VII, -VIII, -IX, -X and -XI comprised genotypes-1d, -1e, -1f, -So and -2a strains, respectively. The results suggest that the RFLP analysis can simply and rapidly differentiate the 8 genotypes of BVDV strains.

  10. Genotypic characterization of Indian isolates of infectious bursal disease virus strains by reverse transcription-polymerase chain reaction combined with restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Priyadharsini, C V; Senthilkumar, T M A; Raja, P; Kumanan, K

    2016-03-01

    The reverse transcription PCR (RT-PCR) combined with restriction fragment length polymorphism (RFLP) is used for the differentiation of classical virulent (cv), virulent (v) and very virulent (vv) strains of infectious bursal disease virus (IBDV) isolates from chicken bursal tissues in southern states of India. In the present study, six different isolates (MB11, HY12, PY12, BGE14, VCN14 and NKL14) of IBDV strains were subjected for genotyping along with vaccine virus (Georgia, intermediate strain) using RT-PCR for amplification of a 743 bp sequence in the hypervariable region of VP2 gene followed by restriction enzyme digestion with 5 different restriction enzymes (BspMI, SacI, HhaI, StuI and SspI). The RT-PCR products obtained from vvIBDV strains were digested by SspI enzyme except PY12, BGE14 and MB11 isolates. The SacI digested the isolate MB11, PY12 and the vaccine strain, but it did not cleave the very virulent isolates of IBDV. HhaI cleaved all the isolates with different restriction profile patterns. StuI digested all the vvIBDV isolates and BspMI was not able to differentiate field isolates from vaccine strain. Though RT-PCR combined with RFLP is a genotypic method, further confirmation of serotypes to distinguish the vvIBDV from cvIBDV has to be carried out using pathogenicity studies.

  11. Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae.

    Science.gov (United States)

    Awasthi, Sharda Prasad; Asakura, Masahiro; Neogi, Sucharit Basu; Hinenoya, Atsushi; Ramamurthy, T; Yamasaki, Shinji

    2014-05-01

    Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

  12. Comparison of human gut microbiota in control subjects and patients with colorectal carcinoma in adenoma: Terminal restriction fragment length polymorphism and next-generation sequencing analyses.

    Science.gov (United States)

    Kasai, Chika; Sugimoto, Kazushi; Moritani, Isao; Tanaka, Junichiro; Oya, Yumi; Inoue, Hidekazu; Tameda, Masahiko; Shiraki, Katsuya; Ito, Masaaki; Takei, Yoshiyuki; Takase, Kojiro

    2016-01-01

    Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in Japan. The etiology of CRC has been linked to numerous factors including genetic mutation, diet, life style, inflammation, and recently, the gut microbiota. However, CRC-associated gut microbiota is still largely unexamined. This study used terminal restriction fragment length polymorphism (T-RFLP) and next-generation sequencing (NGS) to analyze and compare gut microbiota of Japanese control subjects and Japanese patients with carcinoma in adenoma. Stool samples were collected from 49 control subjects, 50 patients with colon adenoma, and 9 patients with colorectal cancer (3/9 with invasive cancer and 6/9 with carcinoma in adenoma) immediately before colonoscopy; DNA was extracted from each stool sample. Based on T-RFLP analysis, 12 subjects (six control and six carcinoma in adenoma subjects) were selected; their samples were used for NGS and species-level analysis. T-RFLP analysis showed no significant differences in bacterial population between control, adenoma and cancer groups. However, NGS revealed that i), control and carcinoma in adenoma subjects had different gut microbiota compositions, ii), one bacterial genus (Slackia) was significantly associated with the control group and four bacterial genera (Actinomyces, Atopobium, Fusobacterium, and Haemophilus) were significantly associated with the carcinoma-in-adenoma group, and iii), several bacterial species were significantly associated with each type (control: Eubacterium coprostanoligens; carcinoma in adenoma: Actinomyces odontolyticus, Bacteroides fragiles, Clostridium nexile, Fusobacterium varium, Haemophilus parainfluenzae, Prevotella stercorea, Streptococcus gordonii, and Veillonella dispar). Gut microbial properties differ between control subjects and carcinoma-in-adenoma patients in this Japanese population, suggesting that gut microbiota is related to CRC prevention and development.

  13. Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism.

    Science.gov (United States)

    Fortuna, Ann-Marie; Marsh, Terence L; Honeycutt, C Wayne; Halteman, William A

    2011-08-01

    Terminal restriction fragment length polymorphism (T-RFLP) can be used to assess how land use management changes the dominant members of bacterial communities. We compared T-RFLP profiles obtained via amplification with forward primers (27, 63F) each coupled with the fluorescently labeled reverse primer (1392R) and multiple restriction enzymes to determine the best combination for interrogating soil bacterial populations in an agricultural soil used for potato production. Both primer pairs provide nearly universal recognition of a 1,400-bp sequence of the bacterial domain in the V(1)-V(3) region of the 16S ribosomal RNA (rRNA) gene relative to known sequences. Labeling the reverse primer allowed for direct comparison of each forward primer and the terminal restriction fragments' relative migration units obtained with each primer pair and restriction enzyme. Redundancy analysis (RDA) and nested multivariate analysis of variance (MANOVA) were used to assess the effects of primer pair and choice of restriction enzyme on the measured relative migration units. Our research indicates that the 63F-1392R amplimer pair provides a more complete description with respect to the bacterial communities present in this potato (Solanum tuberosum L.)-barley (Hordeum vulgare L.) rotation over seeded to crimson clover (Trifolium praense L.). Domain-specific 16S rRNA gene primers are rigorously tested to determine their ability to amplify across a target region of the gene. Yet, variability within or between T-RFLP profiles can result from factors independent of the primer pair. Therefore, researchers should use RDA and MANOVA analyses to evaluate the effects that additional laboratory and environmental variables have on bacterial diversity.

  14. Polymorphism Analysis of NCOA1 Gene in Pigs by PCR-RFLP%猪NCOA1基因PCR-RFLP多态性分析

    Institute of Scientific and Technical Information of China (English)

    侯永刚; 闫守庆; 何晓波; 柏蒙蒙; 孙泰雷; 吴明明; 孙金海

    2010-01-01

    [Objective] The research aimed to study the polymorphism of NCOA1 gene in pigs by PCR-RFLP.[Method] Using 81 Landrace pigs from Liuhe pig breeding farm as research objects,the genome DNA was extracted from ear tissue of pigs by conventional Phenol-chloroform extraction method.The genomic DNA was used as template to make PCR amplification and a 440 bp target fragment was obtained.PCR amplification products were digested by restriction nuclease Rsa I.The agarose gel electrophoresis detection was made.And the gene frequency and the genotype frequency were calculated.[Result] The agarose gel electrophoresis detection showed that the enzyme digestion products had three genotypes of AA (440 bp),AB (440 bp,282 bp,158 bp) and BB (282 bp,158 bp): The genotype frequencies of AA,AB and BB were 0.54,0.43 and 0.03,respectively: The frequencies of allele A and B were 0.76 and 0.24,respectively.[Conclusion] In the studied pig herd,A is the dominant allele and the genotype frequency of AA is higher.%[目的]研究猪NCOA1基因的PCR-RFLP多态性,为猪的分子育种和标记辅助选择提供理论依据.[方法]以81头山东寿光六和原种猪场的长白猪为研究对象,采取常规酚-氯仿抽提法从猪耳组织提取基因组DNA,以基因组DNA为模板进行PCR扩增获得440 bp的NCOA1基因目的片段,利用限制性内切酶Rsa Ⅰ对PCR产物进行酶切和琼脂凝胶电泳检测,并计算各等位基因频率和基因型频率.[结果]琼脂糖电泳检测酶切产物出现3种基因型:AA型(1条带:440 bp)、AB型(3条带:440、282、158 bp)、BB型(2条带:282、158 bp);基因型频率分别为0.54、0.43和0.03;A和B 2个等位基因的基因频率分别为0.76和0.24.[结论]该研究猪群中,A为优势等位基因,AA基因型频率较高.

  15. Containment of extended length polymorphisms in silk proteins.

    Science.gov (United States)

    Chinali, Alberto; Vater, Wolfram; Rudakoff, Baerbel; Sponner, Alexander; Unger, Eberhard; Grosse, Frank; Guehrs, Karl-Heinz; Weisshart, Klaus

    2010-04-01

    The spider silk gene family to the current date has been developed by gene duplication and homogenization events as well as conservation of crucial sequence parts. These evolutionary processes have created an amazing diversity of silk types each associated with specific properties and functions. In addition, they have led to allelic and gene variants within a species as exemplified by the major ampullate spidroin 1 gene of Nephila clavipes. Due to limited numbers of individuals screened to date little is known about the extent of these heterogeneities and how they are finally manifested in the proteins. Using expanded sample sizes, we show that sequence variations expressed as deletions or insertions of tri-nucleotides lead to different sized and structured repetitive units throughout a silk protein. Moreover, major ampullate spidroins 1 can quite dramatically differ in their overall lengths; however, extreme variants do not spread widely in a spider population. This suggests that a certain size range stabilized by purifying selection is important for spidroin 1 gene integrity and protein function. More than one locus for spidroin 1 genes possibly exist within one individual genome, which are homogenized in size, are differentially expressed and give a spider a certain degree of adaptation on silk's composition and properties. Such mechanisms are shared to a lesser extent by the second major ampullate spidroin gene.

  16. Comparative Analysis of Human and Canine Campylobacter upsaliensis Isolates by Amplified Fragment Length Polymorphism

    DEFF Research Database (Denmark)

    Damborg, Peter; Guardabassi, Luca; Pedersen, Karl

    2008-01-01

    Human (n = 33) and canine (n = 53) Campylobacter upsaliensis isolates from seven countries were genotyped by a new amplified fragment length polymorphism method. We observed 100% typeability and high overall diversity. The majority of human strains (23/33) clustered separately from canine strains...

  17. 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...

  18. Identification of Common Species of Dermatophytes by PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    HE Ganlin; LI Jiawen; DING Juan; TAN Zhijan

    2005-01-01

    To establish a simple, sensitive and effective technique for the identification of six common dermatophytes, polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase Ⅱ gene were used. The DNA of 6 common dermatophytes was amplified by primer dPsD1 and then primers dPsD2. The products generated by dPsD2were digested with restriction enzyme Hinc Ⅱ and Hinf Ⅰ separately. A DNA fragment of about 3390 bp was amplified by using primer dPsD1 from the genomic DNA of each dermatophyte species. The product of dPsD2 was 2380 bp and the restriction profiles of Hinc Ⅱ and Hinf Ⅰ were between 58- 1670 bp. By using PCR-RFLP, all of the 6 dermatophytoses were diagnosed to species level and no obvious difference identification between Hinc Ⅱ and Hinf Ⅰ. It is concluded that the PCR-RFLP identification of dermatophytes by Hinc Ⅱ or Hinf Ⅰ is efficient and rapid in clinical practice.

  19. Vegetative compatibility and RFLP analysis of Colletotrichum destructivum isolates from alfalfa and red clover

    Directory of Open Access Journals (Sweden)

    Vasić Tanja

    2016-01-01

    Full Text Available A total of 17 isolates of Colletotrichum from alfalfa (Medicago sativa L. and red clover (Trifolium pratense L. plants with anthracnose symptoms were collected from 11 districts in Serbia during 2005-2010 and tested for variability in vegetative compatibility groups (VCGs and restriction fragment length polymorphisms (RFLP. Nitrate nonutilising (nit mutants were isolated from each of investigated C. destructivum isolates by selecting chlorate-resistant sectors on medium with chlorate. The isolates were grouped in five VCGs while one isolate was self-incompatible. No relationship was found between VCGs and geographical origin of the isolates. Restriction Fragment Length Polymorphism (RFLP analysis of a 900 bp intron of the glutamine synthetase (GS gene revealed a unique polymorphic profile of C. destructivum isolates, distinct from the profiles of other Colletotrichum species. An identical profile was produced for all C. destructivum isolates, regardless of their host and geographical origin. PCR-RFLP failed to detect some the Serbian C. destructivum isolates. [Projekat Ministarstva nauke Republike Srbije, br. TR31057

  20. A genetic map of chromosome 20q12-q13. 1: Multiple highly polymorphic microsatellite and RFLP markers linked to the maturity-onset diabetes of the Young (MODY) locus

    Energy Technology Data Exchange (ETDEWEB)

    Rothschild, C.B.; Akots, G.; Hayworth, R.; Pettenati, M.J.; Rao, P.N.; Wood, P. (Wake Forest Univ., Winston-Salem, NC (United States)); Stolz, F.M.; Hansmann, I. (Universitaet Goettingen (Germany)); Serino, K.; Keith, T.P. (Collaborative Research Inc., Waltham, MA (United States)); Fajans, S.S. (Univ. of Michigan Medical Center, Ann Arbor (United States))

    1993-01-01

    Multiple highly polymorphic markers have been used to construct a genetic map of the q12-q13.1 region of chromosome 20 and to map the location of the maturity-onset diabetes of the young (MODY) locus. The genetic map encompasses 23 cM and includes 11 loci with PIC values >.50, seven of which have PICs >.70. New dinucleotide repeat polymorphisms associated with the D20S17, PPGB, and ADA loci have been identified and mapped. The dinucleotide repeat polymorphisms have increased the PIC of the ADA locus to .89 and, with an additional RFLP at the D20S17 locus, the PIC of the D20S17 locus to .88. The order of the D20S17 and ADA loci determined genetically (cen-ADA-D20S17-qter) was confirmed by multicolor fluorescence in situ hybridization. The previously unmapped PPGB marker is closely linked to D20S17, with a two-point lod score of 50.53 at [cflx [theta

  1. 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......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...... 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...

  2. 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.

  3. Restriction fragment length polymorphism of ovine casein genes: close linkage between the alpha s1-, alpha s2-, beta- and kappa-casein loci.

    Science.gov (United States)

    Leveziel, H; Metenier, L; Guerin, G; Cullen, P; Provot, C; Bertaud, M; Mercier, J C

    1991-01-01

    Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.

  4. Sequence length polymorphisms within primate amelogenin and amelogenin-like genes: usefulness in sex determination.

    Science.gov (United States)

    Morrill, Benson H; Rickords, Lee F; Schafstall, Heather J

    2008-10-01

    Sequence length polymorphisms between the amelogenin (AMELX) and the amelogenin-like (AMELY) genes both within and between several mammalian species have been identified and utilized for sex determination, species identification, and to elucidate evolutionary relationships. Sex determination via polymerase chain reaction (PCR) assays of the AMELX and AMELY genes has been successful in greater apes, prosimians, and two species of old world monkeys. To date, no sex determination PCR assay using AMELX and AMELY has been developed for new world monkeys. In this study, we present partial AMELX and AMELY sequences for five old world monkey species (Mandrillus sphinx, Macaca nemestrina, Macaca fuscata, Macaca mulatta, and Macaca fascicularis) along with primer sets that can be used for sex determination of these five species. In addition, we compare the sequences we generated with other primate AMELX and AMELY sequences available on GenBank and discuss sequence length polymorphisms and their usefulness in sex determination within primates. The mandrill and four species of macaque all share two similar deletion regions with each other, the human, and the chimpanzee in the region sequenced. These two deletion regions are 176-181 and 8 nucleotides in length. In analyzing existing primate sequences on GenBank, we also discovered that a separate six-nucleotide polymorphism located approximately 300 nucleotides upstream of the 177 nucleotide polymorphism in sequences of humans and chimps was also present in two species of new world monkeys (Saimiri boliviensis and Saimiri sciureus). We designed primers that incorporate this polymorphism, creating the first AMELX and AMELY PCR primer set that has been used successfully to generate two bands in a new world monkey species.

  5. Evaluation of genetic diversity in jackfruit (Artocarpus heterophyllus Lam.) based on amplified fragment length polymorphism markers.

    Science.gov (United States)

    Shyamalamma, S; Chandra, S B C; Hegde, M; Naryanswamy, P

    2008-07-22

    Artocarpus heterophyllus Lam., commonly called jackfruit, is a medium-sized evergreen tree that bears high yields of the largest known edible fruit. Yet, it has been little explored commercially due to wide variation in fruit quality. The genetic diversity and genetic relatedness of 50 jackfruit accessions were studied using amplified fragment length polymorphism markers. Of 16 primer pairs evaluated, eight were selected for screening of genotypes based on the number and quality of polymorphic fragments produced. These primer combinations produced 5976 bands, 1267 (22%) of which were polymorphic. Among the jackfruit accessions, the similarity coefficient ranged from 0.137 to 0.978; the accessions also shared a large number of monomorphic fragments (78%). Cluster analysis and principal component analysis grouped all jackfruit genotypes into three major clusters. Cluster I included the genotypes grown in a jackfruit region of Karnataka, called Tamaka, with very dry conditions; cluster II contained the genotypes collected from locations having medium to heavy rainfall in Karnataka; cluster III grouped the genotypes in distant locations with different environmental conditions. Strong coincidence of these amplified fragment length polymorphism-based groupings with geographical localities as well as morphological characters was observed. We found moderate genetic diversity in these jackfruit accessions. This information should be useful for tree breeding programs, as part of our effort to popularize jackfruit as a commercial crop.

  6. 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.

  7. 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....

  8. A genetic map of chromosome 20q12-q13.1: multiple highly polymorphic microsatellite and RFLP markers linked to the maturity-onset diabetes of the young (MODY) locus.

    Science.gov (United States)

    Rothschild, C B; Akots, G; Hayworth, R; Pettenati, M J; Rao, P N; Wood, P; Stolz, F M; Hansmann, I; Serino, K; Keith, T P

    1993-01-01

    Multiple highly polymorphic markers have been used to construct a genetic map of the q12-q13.1 region of chromosome 20 and to map the location of the maturity-onset diabetes of the young (MODY) locus. The genetic map encompasses 23 cM and includes 11 loci with PIC values > .50, seven of which have PICs > .70. New dinucleotide repeat polymorphisms associated with the D20S17, PPGB, and ADA loci have been identified and mapped. The dinucleotide repeat polymorphisms have increased the PIC of the ADA locus to .89 and, with an additional RFLP at the D20S17 locus, the PIC of the D20S17 locus to .88. The order of the D20S17 and ADA loci determined genetically (cen-ADA-D20S17-qter) was confirmed by multicolor fluorescence in situ hybridization. The previously unmapped PPGB marker is closely linked to D20S17, with a two-point lod score of 50.53 at theta = .005. These markers and dinucleotide repeat markers associated with the D20S43, D20S46, D20S55, D20S75, and PLC1 loci and RFLPs at the D20S16, D20S17, D20S22, and D20S33 have been used to map the MODY locus on chromosome 20 to a 13-cM (sex averaged) interval encompassing ADA, D20S17, PPGB, D20S16, and D20S75 on the long arm of chromosome 20 and to create a genetic framework for additional genetic and physical mapping studies of the region. With these multiple highly polymorphic loci, any MODY family of appropriate size can be tested for the chromosome 20 linkage.

  9. Animal Species Identification by PCR – RFLP of Cytochrome b

    Directory of Open Access Journals (Sweden)

    Tomáš Minarovič

    2010-05-01

    Full Text Available An alternative DNA detection system is based on the polymerase chain reaction (PCR amplification of a segment of the mitochondrial cytochrome b gene. Subsequent cleavage by a restriction enzymes gives rise to a specie-specific pattern on an agarose gel. We used five animal species (Mustela vison, Mustela putorius furo, Sus scrofa domesticus, Oryctolagus cuninculus, Anser anser. Length of PCR product was 359 bp and we used universal primers. Restriction fragment length polymorphism was analyzed by using the restriction endonuclease AluI. Results of cleavage were visualized by using electrophoresis and UV transiluminator. Every animal specie has a unique combination of restriction fragments i.e. Mustela vison 81 bp, 109 bp and 169 bp, Mustela putorius furo 169 bp and 190 bp, Sus scrofa domesticus 115 bp and 244 bp, Oryctolagus cunninculus is not cleaved by AluI so it has whole 359 bp fragment on agarose gel, Anser anser 130 bp and 229 bp. The results suggest that the method of PCR - RFLP is rapid and simple method for identification of species. PCR – RFLP can reliably identify chosen species. Application of genetic methods is very useful for breeding of livestock and protection of biodiversity.

  10. Suitability of IS6110-RFLP and MIRU-VNTR for Differentiating Spoligotyped Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from Sichuan in China

    Directory of Open Access Journals (Sweden)

    Chao Zheng

    2014-01-01

    Full Text Available Genotypes of Mycobacterium tuberculosis complex (MTBC vary with the geographic origin of the patients and can affect tuberculosis (TB transmission. This study was aimed to further differentiate spoligotype-defined clusters of drug-resistant MTBC clinical isolates split in Beijing (n=190 versus non-Beijing isolates (n=84 from Sichuan region, the second high-burden province in China, by IS6110-restriction fragment length polymorphism (RFLP and 24-locus MIRU-VNTRs. Among 274 spoligotyped isolates, the clustering ratio of Beijing family was 5.3% by 24-locus MIRU-VNTRs versus 2.1% by IS6110-RFLP, while none of the non-Beijing isolates were clustered by 24-locus MIRU-VNTRs versus 9.5% by IS6110-RFLP. Hence, neither the 24-locus MIRU-VNTR was sufficient enough to fully discriminate the Beijing family, nor the IS6110-RFLP for the non-Beijing isolates. A region adjusted scheme combining 12 highly discriminatory VNTR loci with IS6110-RFLP was a better alternative for typing Beijing strains in Sichuan than 24-locus MIRU-VNTRs alone. IS6110-RFLP was for the first time introduced to systematically genotype MTBC in Sichuan and we conclude that the region-adjusted scheme of 12 highly discriminative VNTRs might be a suitable alternative to 24-locus MIRU-VNTR scheme for non-Beijing strains, while the clusters of the Beijing isolates should be further subtyped using IS6110-RFLP for optimal discrimination.

  11. Identification of Salmonella serotypes isolated from cantaloupe and chile pepper production systems in Mexico by PCR-restriction fragment length polymorphism.

    Science.gov (United States)

    Gallegos-Robles, Miguel A; Morales-Loredo, Alberto; Alvarez-Ojeda, Genoveva; Vega-P, Adrián; Chew-M, Yazmín; Velarde, Sixto; Fratamico, Pina

    2008-11-01

    A study was conducted in 2006 to determine the prevalence of Salmonella on three cantaloupe farms in Matamoros, Coahuila, Mexico, and on one farm that cultivates chile peppers var. Bell in Culiacán, Sinaloa, Mexico. Samples from cantaloupe farms consisted of cantaloupe rinses, irrigation water, water from furrows in the field, and workers' hands. Samples from the chile pepper farm consisted of rinses of chile peppers obtained at the field, pepper rinses obtained at the packing house, and irrigation water from the field. A total of 55 samples were obtained from both production systems. Twelve and 10 samples from the cantaloupe and chile pepper production systems, respectively, tested positive for Salmonella according to a traditional culture method. The difference between the proportion of Salmonella-positive samples from the cantaloupe production system (12 of 28 = 0.43) and the chile pepper production system (10 of 27 = 0.37) was not statistically significant (P > 0.05). A PCR-restriction fragment length polymorphism (RFLP) method based on the fliC gene was used to determine the serotype of the isolates. Salmonella Typhimurium was the only serotype found associated with the cantaloupe production system, whereas both Salmonella Typhimurium and Enteritidis serotypes were found associated with the chile pepper production system. Results showed that 91% (20 of 22) and 9% (2 of 22) of the isolates from both agricultural systems matched with the Salmonella Typhimurium and Salmonella Enteritidis reference strain restriction profiles, respectively. This study demonstrates the utility of the PCR-RFLP technique for determining the serotypes of Salmonella isolates obtained from cantaloupe and chile pepper production systems.

  12. Genetic diversity and relationship in American and African oil palm as revealed by RFLP and AFLP molecular markers

    Directory of Open Access Journals (Sweden)

    Barcelos Edson

    2002-01-01

    Full Text Available The objective of this work was to evaluate the genetic diversity, its organization and the genetic relationships within oil palm (Elaeis oleifera (Kunth Cortés, from America, and E. guineensis (Jacq., from Africa germplasm using Restriction Fragment Length Polymorphism (RFLP and Amplified Fragment Length Polymorphism (AFLP. In complement to a previous RFLP study on 241 E. oleifera accessions, 38 E. guineensis accessions were analyzed using the same 37 cDNA probes. These accessions covered a large part of the geographical distribution areas of these species in America and Africa. In addition, AFLP analysis was performed on a sub-set of 40 accessions of E. oleifera and 22 of E. guineensis using three pairs of enzyme/primer combinations. Data were subjected to Factorial Analysis of Correspondence (FAC and cluster analysis, with parameters of genetic diversity being also studied. Results appeared congruent between RFLP and AFLP. In the E. oleifera, AFLP confirmed the strong structure of genetic diversity revealed by RFLP, according to geographical origin of the studied material, with the identification of the same four distinct genetic groups: Brazil, French Guyana/Surinam, Peru, north of Colombia/Central America. Both markers revealed that genetic divergence between the two species is of the same magnitude as that among provenances of E. oleifera. This finding is in discrepancy with the supposed early tertiary separation of the two species.

  13. A NEW APPROACH TO GENE DIAGNOSIS OF DUCHENNE/BECKER MUSCULAR DYSTROPHY AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS

    Institute of Scientific and Technical Information of China (English)

    许顺斌; 黄尚志; 罗会元

    1994-01-01

    Four (CA), repeats, located in introns,44,45,49 and 50 of the dystrophin gene,were evaluated in Chinese.These loci are highly polymorphic,with polymorphism information contents of 0.872,0.772,0.870 and 0.718,respectively.All four loci can be easily amplified and labelled using two duplex PCR reactions with α-32P-dCTP and can be detected by denaturing polyacrylamide gel electrophoresis.Using these four loci and the two polymorphic(CA)n repeats located at the 5′ and 3′ ends of the dystrophin gene,we have developed a new PCR-based procedure-Amp-FLP( amplified fragment length polymorphism)linkage analysis for the gene diagnosis of DMD/BMD.This method can detect intragenic recombination rapidly and efficiently and greatly improves the success rate of carrier deterction and prenatal diagnosis in non-deletion DMD/BMD families.All of the loci used in this procedure are intragenic.In addition ,the loci in introns 44,45,49 and 50 are located in the deletion-prone region of the dystrophin gene,making them valuable and usefui in the identification of deletion mutations.Here we report one case of deletion detection using these four loci.

  14. Random amplified polymorphic DNA and amplified fragment length polymorphism assessment of genetic variation in Nicaraguan populations of Pinus oocarpa.

    Science.gov (United States)

    Díaz, V; Muñiz, L M; Ferrer, E

    2001-11-01

    Pinus oocarpa is the most widely distributed pine species of Mexico and Central America. The natural populations of Nicaragua have been affected by extensive human activities. As a consequence, their size has been reduced, and there is a serious threat to the development of mature woodland. Knowledge of population structures and the genetic diversity of the species is required for the design of sustainable use and conservation strategies. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic variation among 10 populations from three geographical regions of Nicaragua. Both markers revealed high levels of diversity in these populations. G(ST) values and analyses of molecular variance (AMOVA) found that most variation was within populations but there is still a significant differentiation between populations indicating that the populations sampled cannot be considered a single panmictic unit. The partitions created by AMOVA also showed that there was little differentiation between populations of different regions, although cluster analyses based on RAPDs and AFLPs indicated a closer relationship among most of the populations from a same geographical region. Management of P. oocarpa in Nicaragua should be aimed to maintain the high degree of genetic variation within individual populations that is still observed even in some of these highly degraded populations.

  15. 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...

  16. 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...

  17. 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.

  18. A specific PCR-assay for resistance to Biotypes 1 and 2 of the rosy leaf curling aphid in apple based on an RFLP marker closely linked to the Sd1 gene

    NARCIS (Netherlands)

    Roche, P.; Arkel, van G.; Heusden, van A.W.

    1997-01-01

    This report describes the conversion of a restriction fragment length polymorphism (RFLP) marker (the 2B12a locus), linked to the Sd1 aphid resistance gene, to a polymerase chain reaction (PCR) based marker. A section of the 2B12 probe was sequenced and two primers were designed to amplify this

  19. A specific PCR-assay for resistance to Biotypes 1 and 2 of the rosy leaf curling aphid in apple based on an RFLP marker closely linked to the Sd1 gene

    NARCIS (Netherlands)

    Roche, P.; Arkel, van G.; Heusden, van A.W.

    1997-01-01

    This report describes the conversion of a restriction fragment length polymorphism (RFLP) marker (the 2B12a locus), linked to the Sd1 aphid resistance gene, to a polymerase chain reaction (PCR) based marker. A section of the 2B12 probe was sequenced and two primers were designed to amplify this sequ

  20. Association between telomere length and CYP19 TTTA repetition polymorphism in healthy and breast cancer-diagnosed women

    Science.gov (United States)

    Murillo-Ortiz, Blanca; Martínez-Garza, Sandra; Suárez García, David; Castillo Valenzuela, Rosa del Carmen; García Regalado, Juan Francisco; Cano Velázquez, Gerardo

    2017-01-01

    Introduction Several studies have reported an increase in breast cancer (BC) risk when patients are carriers of the CYP19 TTA polymorphism with ≥10 repeats; moreover, it has been reported that telomere length is associated with a higher susceptibility of developing cancer. Objective The objective of this study was to understand the relationship between CYP19 TTTA repetition polymorphism and telomere length and its effects on serum estradiol, estrone and follicle-stimulating hormone (FSH). Materials and methods A total of 180 postmenopausal healthy and 70 BC-diagnosed women were included. Telomere length was determined through real-time quantitative polymerase chain reaction, and aromatase polymorphism was analyzed through DNA; both samples were obtained from circulating leukocytes. Serum estrone, estradiol and FSH were determined by enzyme-linked immunosorbent assay. Results Patients with a BC diagnosis showed >10 repetitions more frequently, compared with that of healthy women (50% vs 23%, χ2 = 11.44, p = 0.0007). A significant difference in telomere length between healthy and BC women was observed (5,042.7 vs 2,256.7 pb, Z = 4.88, p 10 repeats. Moreover, telomere length showed an inverse relationship with the number of repeats of the aromatase polymorphism in healthy women (R2 = 0.04, r = −0.24); in contrast, BC patients did not display this relationship. In addition, telomere length presented an inverse relationship with serum levels of estradiol and estrone in BC patients (p = 0.02). Conclusion Telomere length is shorter in BC patients than in healthy patients. The CYP19 TTTA repeat polymorphism is associated with serum levels of estradiol and estrone in both healthy women and BC patients, being higher in those with polymorphism carriers >10 repeats. Telomere length has an inverse correlation with the number of repeats of the aromatase polymorphism in healthy women but not in BC women. Estradiol and estrone levels in BC women have an inverse relationship

  1. T-REX: software for the processing and analysis of T-RFLP data

    Directory of Open Access Journals (Sweden)

    Culman Steven W

    2009-06-01

    Full Text Available Abstract Background Despite increasing popularity and improvements in terminal restriction fragment length polymorphism (T-RFLP and other microbial community fingerprinting techniques, there are still numerous obstacles that hamper the analysis of these datasets. Many steps are required to process raw data into a format ready for analysis and interpretation. These steps can be time-intensive, error-prone, and can introduce unwanted variability into the analysis. Accordingly, we developed T-REX, free, online software for the processing and analysis of T-RFLP data. Results Analysis of T-RFLP data generated from a multiple-factorial study was performed with T-REX. With this software, we were able to i label raw data with attributes related to the experimental design of the samples, ii determine a baseline threshold for identification of true peaks over noise, iii align terminal restriction fragments (T-RFs in all samples (i.e., bin T-RFs, iv construct a two-way data matrix from labeled data and process the matrix in a variety of ways, v produce several measures of data matrix complexity, including the distribution of variance between main and interaction effects and sample heterogeneity, and vi analyze a data matrix with the additive main effects and multiplicative interaction (AMMI model. Conclusion T-REX provides a free, platform-independent tool to the research community that allows for an integrated, rapid, and more robust analysis of T-RFLP data.

  2. Genotyping of Giardia duodenalis isolates from human subjects in Zabul, using PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Maryam Abedi

    2016-09-01

    Full Text Available Objective: To uncover the molecular prevalence of Giardia duodenalis by PCR-restriction fragment length polymorphism (RFLP in Zabul city, Iran. Methods: Twenty-four stool samples were collected from 215 patients with suspected giardiasis by microscopic examination. To increase the sensitivity of the PCR, the total genomic DNA from isolates was extracted by applying glass beads and the QIAamp Kit. A one-step PCRRFLP method, targeting the glutamate dehydrogenase gene, was utilized to differentiate the assemblages A and B among isolates. Results: The PCR fragment was determined from 30 isolates, RFLP assay of 24 isolates showed 24 (100 isolates as Genotype B group BIII. Conclusions: The results with the glutamate dehydrogenase gene assay demonstrated that the predominant subtype of Giardia duodenalis in the area is BIII, which showed animals are the main reservoir of the isolates in this area.

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

    Science.gov (United States)

    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.

  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. Restriction Fragment Length Polymorphism Analysis of Large Subunit rDNA of Symbiotic Dinoflagellates from Scleractinian Corals in the Zhubi Coral Reef of the Nansha Islands

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Zooxanthellae are very important for the coral reef ecosystem. The diversity of coral hosts is high in the South China Sea, but the diversity of zooxanthellae has not yet been investigated. We chose the Zhubi Coral Reef of the Nansha Islands as the region to be surveyed in the present study because it represents a typical tropical coral reef of the South China Sea and we investigated zooxanthellae diversity in 10 host scleractinian coral species using polymerase chain reaction (PCR) of the large subunit rRNA and restriction fragment length polymorphism (RFLP) patterns. Pocillopora verrucosa, Acropora pelifera, Acropora millepora, Fungia fungites, Galaxea fascicularis, and Acropora pruinosa harbor Clade C, Goniastrea aspera harbors Clade D, and Acropora formosa harbors Clades D and C. Therefore, the Clade C is the dominant type in the Zhubi Coral Reef of the NanshaIslands. Furthermore, the results of the present also disprove what has been widely accepted, namely that one coral host harbors only one algal symbiont. The coral-algal symbiosis is flexible, which may be an important mechanism for surviving coral bleaching. Meanwhile, on the basis of the results of the present study, we think that Symbiodinium Clade D may be more tolerant to stress than Symbiodinium Clade C.

  6. Comparison Between AFLP and RFLP Markers in Detecting the Diversity of Rice (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    CHEN Liang; LIANG Chun-yang; SUN Chuan-qing; JIN De-min; JIANG Ting-bo; WANG Bin; WANG Xiang-kun

    2002-01-01

    AFLP and RFLP were used to study the diversity of 20 rice cultivars. 15 primer combinations were used in the AFLP analysis and 47 - 118 bands were amplified in each lane. A total of 107 polymorphic bands were detected in the RFLP analysis using 49 RFLP probes. The cluster analysis based on RFLP data showed that 20 rice cultivars could be divided into an indica group and a japonica group, as did the AFLP data; however AFLP is more suitable in detecting the difference of pedigree and ecotype among the 20 cultivars.The genetic distance based on AFLP and RFLP data showed the same tendency, but AFLP markers increased the measure of genetic distance among intra-subspecific cultivars and decreased the measure of genetic distance among inter-subspecific cultivars relative to RFLP markers. That indicated that AFLP is more suitable than RFLP in the diversity study and RFLP is more suitable to study the indica-japonica differentiation of cultivars.

  7. PCR-RFLP analysis of chitinase genes enables efficient genotyping of Metarhizium anisopliae var. anisopliae.

    Science.gov (United States)

    Enkerli, Jürg; Ghormade, Vandana; Oulevey, Catherine; Widmer, Franco

    2009-10-01

    A new genotyping tool has been developed and evaluated for Metarhizium anisopliae var. anisopliae. The tool is based on Restriction Fragment Length Polymorphism (RFLP) analysis of three chitinase genes that are functionally linked to insect-pathogenicity of this fungus. It allowed for discrimination of 14 genotypes among 22 M. anisopliae var. anisopliae strains of a world wide collection. Analyses revealed that the approach may also be applicable to other Metarhizium varieties. The new tool will be useful for genetic characterization of M. anisopliae var. anisopliae strains, and it is applicable for laboratories with limited access to molecular diagnostic equipment.

  8. Long telomere length and a TERT-CLPTM1 locus polymorphism association with melanoma risk.

    Science.gov (United States)

    Llorca-Cardeñosa, Marta J; Peña-Chilet, Maria; Mayor, Matias; Gomez-Fernandez, Cristina; Casado, Beatriz; Martin-Gonzalez, Manuel; Carretero, Gregorio; Lluch, Ana; Martinez-Cadenas, Conrado; Ibarrola-Villava, Maider; Ribas, Gloria

    2014-12-01

    Telomere length has been associated with the development of cancer. Studies have shown that shorter telomere length may be related to a decreased risk of cutaneous melanoma. Furthermore, deregulation of the telomere-maintaining gene complexes, has been related to this oncogenic process. Some variants in these genes seem to be correlated with a change in telomerase expression. We examined the effect of 10 single nucleotide polymorphisms (SNPs) in the TERT gene (encoding telomerase), one SNP in the related TERT-CLPTM1L locus and one SNP in the TRF1 gene with telomere length, and its influence on melanoma risk in 970 Spanish cases and 733 Spanish controls. Genotypes were determined using KASP technology, and telomere length was measured by quantitative polymerase chain reaction (PCR) on DNA extracted from peripheral blood leucocytes. Our results demonstrate that shorter telomere length is associated with a decreased risk of melanoma in our population (global p-value, 2.69×10(-11)), which may be caused by a diminution of proliferative potential of nevi (melanoma precursor cells). We also obtained significant results when we tested the association between rs401681 variant (TERT-CLPTM1L locus) with melanoma risk (Odds ratio, OR; 95% confidence interval, CI=1.24 (1.08-1.43); p-value, 3×10(-3)). This is the largest telomere-related study undertaken in a Spanish population to date. Furthermore, this study represents a comprehensive analysis of some of the most relevant telomere pathway genes in relation to cutaneous melanoma susceptibility.

  9. Application of PCR-RFLP technique on the classification and identification of parasite%PCR-RFLP技术在寄生虫分类鉴定中的应用

    Institute of Scientific and Technical Information of China (English)

    刘爱波; 杨毅梅

    2010-01-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique is a simple and rapid method to determine the classification of parasites. At present, PCR-RFLP technique has been widely applied in the field of parasitology. The article reviewed the application of PCR-RFLP technique on the classification and identification of medical protozoa and helminths.%PCR-限制性片段长度多态性(PCR-RFLP)技术是一种简便、快速的鉴定寄生虫种类的方法.目前,PCR-RFLP技术广泛应用于寄生虫学领域.该文综述了PCR-RFLP技术在医学原虫和蠕虫分类鉴定中的应用现状.

  10. Genetic diversity of the edible mushroom Pleurotus sp. by amplified fragment length polymorphism.

    Science.gov (United States)

    Pawlik, Anna; Janusz, Grzegorz; Koszerny, Joanna; Małek, Wanda; Rogalski, Jerzy

    2012-10-01

    Pleurotus strains are the most important fungi used in the agricultural industry. The exact characterization and identification of Pleurotus species is fundamental for correct identification of the individuals and exploiting their full potential in food industry. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of 21 Pleurotus isolates of Asian and European origin. Using one PstI restriction endonuclease and four selective primers in an AFLP assay, 371 DNA fragments were generated, including 308 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished 21 Pleurotus sp. fungi. The coefficient of Jaccard's genome profile similarity between the analyzed strains ranged from 0.0 (Pleurotus sp. I vs. P. sajor-caju 237 and P. eryngii 238) to 0.750 (P. ostreatus 246 vs. P. ostreatus 248), and the average was 0.378. The AFLP-based dendrogram generated by the UPGMA method grouped all the Pleurotus fungi studied into two major clusters and one independent lineage located on the outskirt of the tree occupied by naturally growing Pleurotus species strain I. The results of the present study suggest the possible applicability of the AFLP-PstI method in effective identification and molecular characterization of Pleurotus sp. strains.

  11. Differentiation of Salmonella enteritidis isolates by fluorescent amplified fragment length polymorphism.

    Science.gov (United States)

    Kober, Márcia Vargas; Abreu, Marina Bystronski; Bogo, Maurício Reis; Ferreira, Carlos Alexandre Sanchez; Oliveira, Sílvia Dias

    2011-01-01

    Salmonella Enteritidis is responsible for human gastroenteritis outbreaks worldwide, and the molecular characterization of isolates is an important tool for epidemiological studies. Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 31 Salmonella Enteritidis strains from South Brazil isolated from human, foods, swine, broiler carcasses, and other poultry-related samples to subtype isolates in comparison to pulsed-field gel electrophoresis (PFGE) analysis. Five strains of Salmonella Enteritidis from different geographical regions, Salmonella Enteritidis ATCC 13076, and four isolates of different Salmonella serovars were also tested. Among the 41 isolates tested, 96 polymorphic AFs and 40 distinct profiles were obtained, displaying a Simpson's index of diversity of 0.99; whereas the PFGE analysis presented 13 patterns and the resulting Simpson's index was 0.55. Nine FAFLP and seven PFGE clusters could be inferred based in Dice similarity coefficient. FAFLP clustering readily identified different serotypes of Salmonella but did not distinguish isolates epidemiologically nonrelated or distinct phage types. Therefore, these results indicate that FAFLP is a rapid method for epidemiological investigations of Salmonella outbreaks, presenting a high discriminatory power for subtyping of Salmonella Enteritidis.

  12. Typing Candida Species Using Microsatellite Length Polymorphism and Multilocus Sequence Typing.

    Science.gov (United States)

    Garcia-Hermoso, Dea; Desnos-Ollivier, Marie; Bretagne, Stéphane

    2016-01-01

    To gain more insight into the epidemiological relationships between isolates of Candida spp. obtained from various origins, several molecular typing techniques have been developed. Two methods have emerged in the 2000s as soon as enough knowledge of the Candida spp. genomes was available to choose adequate loci and primers, namely microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST). To contrast with previous PCR-based methods, specific amplifications with stringent conditions easily reproducible are the basis of MLP and MLST. MLST relies on Sanger sequencing to detect single-nucleotide polymorphisms within housekeeping genes. MLP needs a first in silico step to select tandemly repeated stretches of two to five nucleotides. One of the two primers used to amplify a microsatellite locus is labeled and fragment sizing is automatically performed using high-resolution electrophoresis platforms. MLST provides results easily comparable between laboratories and active MLST schemes are publicly available for the main Candida species. For comparative studies, MLP needs standards to compensate for the electrophoretic variations depending on the platforms used. Both methods can help us gain insight into the genetic relatedness of fungal isolates, both with advantages and drawbacks, and the choice of one method rather than the other depends on the task in question.

  13. Genetic diversity of Actinobacillus pleuropneumoniae assessed by amplified fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Kokotovic, Branko; Angen, Øystein

    2007-01-01

    Amplified fragment length polymorphism (AFLP) was evaluated as a method for genotypic characterization and subtyping within the bacterial species Actinobacillus pleuropneumoniae. A total of 155 isolates of A. pleuropneumoniae, representing the serotypic variation described to occur within...... this species, were analyzed. In order to elucidate the species boundaries, six strains of the phylogenetically closely related species Actinobacillus lignieresii were also included. Furthermore, the ability of AFLP to subtype was studied using 42 isolates of serovar 2 and the performance compared...... to that obtained by pulsed-field gel electrophoresis (PFGE). AFLP analysis provided a clear separation of A. lignieresii and A. pleuropneumoniae and divided the isolates of A. pleuropneumoniae into 20 clusters. Most of the serovars of A. pleuropneumoniae were represented by single and quite homogeneous clusters...

  14. Selection of enzymes for terminal restriction fragment length polymorphism analysis of fungal internally transcribed spacer sequences.

    Science.gov (United States)

    Alvarado, Pablo; Manjón, Jose L

    2009-07-01

    Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.

  15. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA.

    Science.gov (United States)

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-04-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

  16. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

    Directory of Open Access Journals (Sweden)

    Leonardo Silva Barbedo

    2016-04-01

    Full Text Available Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

  17. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

    Science.gov (United States)

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-01-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories. PMID:27074256

  18. Characterization of gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism: gut microbiota could be a diagnostic marker of coronary artery disease.

    Science.gov (United States)

    Emoto, Takuo; Yamashita, Tomoya; Kobayashi, Toshio; Sasaki, Naoto; Hirota, Yushi; Hayashi, Tomohiro; So, Anna; Kasahara, Kazuyuki; Yodoi, Keiko; Matsumoto, Takuya; Mizoguchi, Taiji; Ogawa, Wataru; Hirata, Ken-Ichi

    2017-01-01

    The association between atherosclerosis and gut microbiota has been attracting increased attention. We previously demonstrated a possible link between gut microbiota and coronary artery disease. Our aim of this study was to clarify the gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism (T-RFLP). This study included 39 coronary artery disease (CAD) patients and 30 age- and sex- matched no-CAD controls (Ctrls) with coronary risk factors. Bacterial DNA was extracted from their fecal samples and analyzed by T-RFLP and data mining analysis using the classification and regression algorithm. Five additional CAD patients were newly recruited to confirm the reliability of this analysis. Data mining analysis could divide the composition of gut microbiota into 2 characteristic nodes. The CAD group was classified into 4 CAD pattern nodes (35/39 = 90 %), while the Ctrl group was classified into 3 Ctrl pattern nodes (28/30 = 93 %). Five additional CAD samples were applied to the same dividing model, which could validate the accuracy to predict the risk of CAD by data mining analysis. We could demonstrate that operational taxonomic unit 853 (OTU853), OTU657, and OTU990 were determined important both by the data mining method and by the usual statistical comparison. We classified the gut microbiota profiles in coronary artery disease patients using data mining analysis of T-RFLP data and demonstrated the possibility that gut microbiota is a diagnostic marker of suffering from CAD.

  19. Molecular typing of sand fly species (Diptera, Psychodidae, Phlebotominae) from areas endemic for Leishmaniasis in Ecuador by PCR-RFLP of 18S ribosomal RNA gene.

    Science.gov (United States)

    Terayama, Yoshimi; Kato, Hirotomo; Gomez, Eduardo A; Uezato, Hiroshi; Calvopiña, Manuel; Iwata, Hiroyuki; Hashiguchi, Yoshihisa

    2008-09-01

    Surveillance of the distribution of sand fly species is important for prediction of the risk and expansion of Leishmania infection in endemic and surrounding areas. In the present study, a simple and reliable method of typing New World Lutzomyia species circulating in endemic areas in Ecuador was established by using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique. PCR-RFLP of 18S ribosomal RNA (rRNA) genes with the restriction enzyme AfaI and subsequently HinfI successfully identified seven sand fly species in nine endemic areas in Ecuador. Although intraspecific genetic-diversity affecting the RFLP-patterns was detected in a species, the patterns were species specific. The method promises to be a powerful tool for the classification of New World Lutzomyia species.

  20. Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods

    Directory of Open Access Journals (Sweden)

    Cláudia de Moura

    2013-04-01

    Full Text Available Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT. The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

  1. 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.

  2. 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...

  3. Identification of Avian Leukosis Virus Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism(PCR/RFLP)%PCR/RFLP鉴别禽白血病病毒

    Institute of Scientific and Technical Information of China (English)

    刘公平; 刘福安; 赵振芬

    2001-01-01

    用PCR方法从禽白血病病毒(avian leukosis virus,ALV)亚群毒株RAV-1、RAV-2感染或未感染的SPF鸡胚成纤维细胞(CEF)分别扩增出1.2 kb基因片段。RAV-1囊膜基因可被BglⅡ分为2条相近的600 bp片段,RAV-2囊膜基因被BamHⅠ分为2条约600 bp片段,对照组基因片段(内源性病毒)不被BglⅡ和BamHⅠ切割。结果显示,ALV囊膜基因的PCR/RFLP可用于诊断禽白血病和鉴别不同的ALV毒株。

  4. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    Science.gov (United States)

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  5. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies

    NARCIS (Netherlands)

    Bergen, van M.A.P.; Simons, G.; Graaf-van Bloois, van der L.; Putten, van J.P.; Rombout, J.; Wesley, I.; Wagenaar, J.A.

    2005-01-01

    Differentiation of Campylobacter fetus into C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv) is important for both clinical and economic reasons. In the past, several molecular typing methods have been used for differentiation, including amplified fragment length polymorphism (AFLP).

  6. Differentiation of Sheeppox and Goatpox Viruses by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

    Institute of Scientific and Technical Information of China (English)

    Gnanavel Venkatesan; Vinayagamurthy Balamurugan; Revaniah Yogisharadhya; Amit Kumar; Veerakyathappa Bhanuprakash

    2012-01-01

    In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains.A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene.The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels.Phylogenetic analysis showed three distinct clusters of SPPV,GTPV and Lumpy skin disease virus (LSDV) isolates.Further,multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I,which is present in SPPV isolates studied.This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains.The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV.The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.

  7. 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.

  8. Differentiation of polyvalent bacteriophages specific to uropathogenic Proteus mirabilis strains based on the host range pattern and RFLP.

    Science.gov (United States)

    Maszewska, Agnieszka; Wójcik, Ewelina; Ciurzyńska, Aneta; Wojtasik, Arkadiusz; Piątkowska, Iwona; Dastych, Jarosław; Różalski, Antoni

    2016-01-01

    Urinary tract infections (UTIs) caused by P. mirabilis are difficult to cure because of the increasing antimicrobial resistance of these bacteria. Phage therapy is proposed as an alternative infection treatment. The aim of this study was to isolate and differentiate uropathogenic P. mirabilis strain specific polyvalent bacteriophages producing polysaccharide depolymerases (PDs). 51 specific phages were obtained. The plaques of 29 bacteriophages were surrounded by halos, which indicated that they produced PDs. The host range analysis showed that, except phages 58B and 58C, the phage host range profiles differed from each other. Phages 35 and 45 infected all P. mirabilis strains tested. Another 10 phages lysed more than 90% of isolates. Among these phages, 65A, 70, 66 and 66A caused a complete lysis of the bacterial lawn formed by 62% to 78% of strains. Additionally, phages 39A and 70 probably produced PDs. The phages' DNA restriction fragment length polymorphism (RFLP) analysis demonstrated that genomes of 51 isolated phages represented 34 different restriction profiles. DNA of phage 58A seemed to be resistant to selected EcoRV endonuclease. The 33 RFLP-EcoRV profiles showed a Dice similarity index of 38.8%. 22 RFLP patterns were obtained from single phage isolates. The remaining 12 restriction profiles consisted of 2 to 4 viruses. The results obtained from phage characterization based on the pattern of phage host range in combination with the RFLP method enabled effective differentiation of the studied phages and selection of PD producing polyvalent phages for further study.

  9. Veronaea botryosa: molecular identification with amplified fragment length polymorphism (AFLP) and in vitro antifungal susceptibility.

    Science.gov (United States)

    Badali, Hamid; Yazdanparast, Seyed Amir; Bonifaz, Alexandro; Mousavi, Bita; de Hoog, G Sybren; Klaassen, Corné H W; Meis, Jacques F

    2013-06-01

    Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC90s across all strains in increasing order posaconazole (0.25 μg/ml), itraconazole (1 μg/ml), voriconazole (4 μg/ml), terbinafine (4 μg/ml), caspofungin (8 μg/ml), anidulafungin (8 μg/ml), isavuconazole (16 μg/ml), amphotericin B (16 μg/ml), and fluconazole (32 μg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05).

  10. Genomic diversity amongst Vibrio isolates from different sources determined by fluorescent amplified fragment length polymorphism.

    Science.gov (United States)

    Thompson, F L; Hoste, B; Vandemeulebroecke, K; Swings, J

    2001-12-01

    The genomic diversity among 506 strains of the family Vibrionaceae was analysed using Fluorescent Amplified Fragments Length Polymorphisms (FAFLP). Isolates were from different sources (e.g. fish, mollusc, shrimp, rotifers, artemia, and their culture water) in different countries, mainly from the aquacultural environment. Clustering of the FAFLP band patterns resulted in 69 clusters. A majority of the actually known species of the family Vibrionaceae formed separate clusters. Certain species e.g. V. alginolyticus, V. cholerae, V. cincinnatiensis, V. diabolicus, V. diazotrophicus, V. harveyi, V. logei, V. natriegens, V. nereis, V. splendidus and V. tubiashii were found to be ubiquitous, whereas V. halioticoli, V. ichthyoenteri, V. pectenicida and V. wodanis appear to be exclusively associated with a particular host or geographical region. Three main categories of isolates could be distinguished: (1) isolates with genomes related (i.e. with > or =45% FAFLP pattern similarity) to one of the known type strains; (2) isolates clustering (> or =45% pattern similarity) with more than one type strain; (3) isolates with genomes unrelated (<45% pattern similarity) to any of the type strains. The latter group consisted of 236 isolates distributed in 31 clusters indicating that many culturable taxa of the Vibrionaceae remain as yet to be described.

  11. 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.

  12. 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.

  13. Polymerase chain reaction-restriction fragment length polymorphism method to distinguish three mealybug groups within the Planococcus citri-P. minor species complex (Hemiptera: Coccoidea: Pseudococcidae).

    Science.gov (United States)

    Rung, A; Miller, D R; Scheffer, S J

    2009-02-01

    The mealybug species Planococcus citri (Risso) and Planococcus minor (Maskell) (Hemiptera: Coccoidea: Pseudococcidae) have special significance to U.S. quarantine and U.S. agriculture. Commonly intercepted at U.S. ports-of-entry, they are difficult to identify based on morphological characters. This study presents a molecular method for distinguishing P. citri, P. minor, and a genetically distinct group that is morphologically identical to P. citri, from Hawaii. This method uses polymerase chain reaction (PCR) followed by restriction fragment polymorphism analysis (RFLP) using the restriction enzymes BspH1, BsmH1, and HpH1. The resulting band patterns can be visualized in a 2% agarose gel and are sufficient to differentiate between the three entities mentioned above. PCR-RFLP diagnostics can be used for all life stages and is cheaper and faster than DNA sequencing.

  14. Detection of deletion and mutation on pig Mx1 gene (gene resistance to influenza virus) with PCR-RFLP Nar I restriction

    OpenAIRE

    Cece Sumantri; T Morzumi; N Hamashima

    2001-01-01

    The study was done to detect the incident of deletion and mutation in exon 14th of Mx1 gene in pig. Six hundred base pairs at the position (1937 to 2537) of the 14th exon of the pig Mx1 gene was amplified by polymerase chain reaction (PCR) from 15 breed of pig DNA sample. The amplified PCR products were digested by Nar I enzyme that called restriction fragment length polymorphism (PCR-RFLP) technique. The results show genetic polymorphism at the 14th exon of pig Mx1 gene. The Nar I digested r...

  15. Comparison of four-colour IS6110-fAFLP with the classic IS6110-RFLP on the ability to detect recent transmission in the city of Barcelona, Spain.

    Science.gov (United States)

    Borrell, Sònia; Thorne, Nicola; Español, Montserrat; Mortimer, Chloe; Orcau, Angels; Coll, Pere; Gharbia, Saheer; González-Martín, Julian; Arnold, Catherine

    2009-05-01

    The purpose of the study was to compare the IS6110-RFLP (RFLP) results obtained in a previous epidemiological study in the city of Barcelona, Spain [Borrell S, Espanol M, Orcau A, Tudo G, March F, Cayla JA, et al. Factors associated with differences between conventional contact tracing and molecular epidemiology in study of tuberculosis transmission and analysis in the city of Barcelona, Spain. J Clin Microbiol 2009 Jan;47(1):198-204.] with the results obtained with IS6110-fAFLP, [Thorne N, Evans JT, Smith EG, Hawkey PM, Gharbia S, Arnold C. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110-restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting. Clin Microbiol Infect 2007 Oct;13(10):964-70.] on the ability to detect recent transmission. fAFLP was applied to DNA samples of RFLP clustered strains, with and without known epidemiological links, with the additional inclusion of four nucleotide-specific fluorophores to further increase the discrimination of the fragments obtained. Four-colour fAFLP was performed on 123 RFLP clustered strains with no epidemiological link (NELC) and on 28 epidemiologically linked RFLP clustered (ELC) strains grouped into 48 and 13 clusters respectively. Clustering results obtained by the two methods were highly congruent in ELC strains with fAFLP allocating 92.3% of the ELCs. For the NELCs, RFLP results were confirmed in 39/48 (81.2%) of fAFLP-clusters with 0-1 different fragments and 9/48 (18.8%) differed in 2-4 fragments, which are considered genetically related but not recently transmitted. In conclusion, overestimation of recent tuberculosis transmission can occur because of the inaccurate analysis of RFLP results. Four-colour fAFLP allows us to differentiate between recent transmission strains and epidemiologically unrelated but genetically related strains.

  16. Genotyping of Mycobacterium avium subsp. avium isolates from naturally infected lofts of domestic pigeons in Ahvaz by IS901 RFLP

    Directory of Open Access Journals (Sweden)

    Kaveh Parvandar Asadollahi

    2015-12-01

    Full Text Available Background and Objectives: Avian tuberculosis is one of the most important infections affecting most species of birds.Mycobacterium avium can not only infect all species of birds, but also infect some domesticated mammals.The most crucial aspect of control and eradication scheme is identification of infection sources and transmission routs. Mo- lecular techniques such as restriction fragment length polymorphism and pulse field gel electrophoresis have been shown to be much more discriminatory and suitable for use in the epidemiological study.Materials and Methods: Eighty suspected pigeons to avian tuberculosis based on their clinical signs, were subjected to the study. Forty Mycobacterium avium subsp. avium isolates out of a total of 51 identified isolates were subjected to the test.Results: IS901-RFLP using Pvu II was successfully conducted and produced 7 patterns. The majority of isolates (60% were RFLP type PI.1. This type was the most similar type to standard strain. However, all the patterns obtained in this study were different from the standard strain.Conclusion: The result of this study indicate that these isolates probably are limited to Khuzestan region. We recommend DNA fingerprinting differentiation of non tuberculous Mycobacteria particularly Mycobacterium avium complex isolated from infected birds and human to possibly find source of infections. Keywords: Mycobacterium avium, RFLP, polymorphism, avian tuberculosis

  17. 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.

  18. Genomic Fingerprinting of the Vaccine Strain of Clostridium Tetani by Restriction Fragment Length Polymorphism Technique

    Directory of Open Access Journals (Sweden)

    Naser Harzandi

    2013-05-01

    Full Text Available Background: Clostridium tetani or Nicolaier’s bacillus is an obligatory anaerobic, Gram-positive, movable with terminal or sub terminal spore. The chromosome of C. tetani contains 2,799,250 bp with a G+C content of 28.6%. The aim of this study was identification and genomic fingerprinting of the vaccine strain of C. tetani.Materials and Methods: The vaccine strain of C. tetani was provided by Razi Vaccine and Serum Research Institute. The seeds were inoculated into Columbia blood agar and grown for 72 h and transferred to the thioglycolate broth medium for further 36 h culturing. The cultures were incubated at 35ºC in anaerobic conditions. DNA extraction with phenol/ chloroform method was performed. After extraction, the consistency of DNA was assayed. Next, the vaccine strain was digested using pvuII enzyme and incubated at 37ºC for overnight. The digested DNA was gel-electrophoresed by 1% agarose for a short time. Then, the gel was studied with Gel Doc system and transferred to Hybond N+membrane using standard DNA blotting techniques.Results: The vaccine strain of C. tetani genome was fingerprinted by RFLP technique. Our preliminary results showed no divergence exists in the vaccine strain used for the production tetanus toxoid during the periods of 1990-2011.Conclusion: Observation suggests that there is lack of significant changes in RFLP genomic fingerprinting profile of the vaccine strain. Therefore, this strain did not lose its efficiency in tetanus vaccine production. RFLP analysis is worthwhile in investigating the nature of the vaccine strain C. tetani.

  19. Restriction Fragment Length Polymorphism Separates Species of the Xiphinema americanum Group.

    Science.gov (United States)

    Vrain, T C

    1993-09-01

    The Xiphinema americanum group of species is responsible for vectoring several important virus diseases to perennial crops. Variability of transmission of viruses by different species, and difficulties in separating species by morphometric measurements alone, make it essential to reassess the taxonomic position of several species in the group. The measurement of DNA sequence variability is a sensitive assay that can re-evaluate the separation of species and populations from each other. This study describes how an RFLP approach, in which the restriction sites in transcribed spacers of ribosomal repeats were detected, confirmed the separation of 16 populations of these species into X. americanum, X. rivesi, X. pacificum, and X. bricolensis.

  20. Genetic Association Between Androgen Receptor Gene CAG Repeat Length Polymorphism and Male Infertility: A Meta-Analysis.

    Science.gov (United States)

    Pan, Bihui; Li, Rui; Chen, Yao; Tang, Qiuqin; Wu, Wei; Chen, Liping; Lu, Chuncheng; Pan, Feng; Ding, Hongjuan; Xia, Yankai; Hu, Lingqing; Chen, Daozhen; Sha, Jiahao; Wang, Xinru

    2016-03-01

    The association between polymorphism of androgen receptor gene CAG (AR-CAG) and male infertility in several studies was controversial. Based on studies on association between AR-CAG repeat length and male infertility in recent years, an updated meta-analysis is needed. We aimed to evaluate the association between AR-CAG repeat length and male infertility in advantage of the data in all published reports.We searched for reports published before August 2015 using PubMed, CNKI, VIP, and WanFang. Data on sample size, mean, and standard deviation (SD) of AR-CAG repeat length were extracted independently by 3 investigators.Forty-four reports were selected based on criteria. The overall infertile patients and azoospermic patients were found to have longer AR-CAG repeat length (standard mean difference (SMD) = 0.19, 95% confidence interval (CI): 0.10-0.28, P CAG repeat length was longer in infertile men in Asian, Caucasian, and mixed races (SMD = 0.25, 95% CI: 0.08-0.43, P CAG repeat length was associated with male infertility. The subgroup study on races shows that increased AR-CAG repeat length was associated with male infertility in Asian, Caucasian, and mixed races. Increased AR-CAG repeat length was also associated with azoospermia.This meta-analysis supports that increased androgen receptor CAG length is capable of causing male infertility susceptibility.

  1. DNA polymorphism of HLA class II genes in pauciarticular juvenile rheumatoid arthritis

    DEFF Research Database (Denmark)

    Morling, N; Friis, J; Fugger, L;

    1991-01-01

    We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) class II genes: HLA-DRB, -DQA, -DQB, DPA, and -DPB in 54 patients with pauciarticular juvenile rheumatoid arthritis (PJRA) and in healthy Danes. The frequencies of DNA fragments a...

  2. DNA polymorphism of HLA class II genes in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Cowland, J B; Andersen, V; Halberg, P

    1994-01-01

    We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) genes: HLA-DRB, -DQA, -DQB, -DPB in 24 Danish patients with systemic lupus erythematosus (SLE) and in 102 healthy Danes. A highly significant increase of the frequency of the DR3...

  3. Polymerase Chain Reaction- Restriction Fragment Length Polymorphism in the Detection of Leu262Val Polymorphism of PARL Gene%PCR-RFLP方法检测PARL基因Leu262Val多态性的实验研究

    Institute of Scientific and Technical Information of China (English)

    代莉; 李会芳; 王玉明; 宋滇平

    2011-01-01

    Objective To determine the optimum experimental conditions of the Leu262Val polymorphism of PARL gene detected by polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP).Method In the PCR, PCR program was optimized by touchdown PCR (TD-PCR) ; four kinds of primers'concentration (0.32 umol/l, 0.4μmol/L, 0.6μmol/L, 0.8 μmol/L) were used, PCR products were observed in 2% agarose. In the RFLP, three kinds of PCR product quantity (5 μL, 10μL, 15 μL) and five kinds of incubation time (12 h, 8 h, 4 h, 2 h,1 h) were set when the incision enzyme quantity was unchanging, PCR products were observed in 2% agarose. Results (1) TD-PCR showed no non-specific band and simplified the process for seeking the optimum annealing temperature in ordinary amplification. ( 2) When the primers concentration was 0.4 μ mol/L. PCR production level was higher than others and had less dimer of primers. (3) It was 10 μl PCR production amount that was digested could creat a clear digested electropherogram. (4) It was 4 hours that was demanded in incubated reaction at 37 ℃ . Conclusions The design of primers is the key of PCR, the optimum primer's concentration is 0.4 μmol/L. TD-PCR can optimize connditions of ordinary PCR, it is more simple and effective than the ordinary PCR; The effective RFLP protocol: the system is 20 μL including 10μL PCR reaction mixture and 10 U enzyme are incubated at 37 ℃ for 4 hours.%目的 探讨聚合酶链反应-限制性片段长度多态性方法(PCR-RFLP)检测PARL基因Leu262Val多态性的最适实验条件.方法 在PCR实验中,运用降落PCR(touchdown PCR,TD-PCR)方法优化PCR条件;对引物终浓度设定0.8 μmol/L、0.6 μmol/L、0.4 μmol/L和0.32 μmol/L四个浓度梯度进行扩增;通过2%琼脂糖凝胶电泳观察结果.在RFLP实验中,在内切酶量10 U不变的情况下,PCR产物量分别设定为5 μL、10 μL、15 μL;酶切时间分别设定为12 h、8 h、4 h、2 h、1 h

  4. Genetic Association Between Androgen Receptor Gene CAG Repeat Length Polymorphism and Male Infertility: A Meta-Analysis

    OpenAIRE

    Pan, Bihui; Li, Rui; CHEN, YAO; Tang, Qiuqin; Wu, Wei; Chen, Liping; Lu, Chuncheng; Pan, Feng; Hongjuan DING; Xia,Yankai; Hu, Lingqing; Chen, Daozhen; Sha, Jiahao; Wang, Xinru

    2016-01-01

    Abstract The association between polymorphism of androgen receptor gene CAG (AR-CAG) and male infertility in several studies was controversial. Based on studies on association between AR-CAG repeat length and male infertility in recent years, an updated meta-analysis is needed. We aimed to evaluate the association between AR-CAG repeat length and male infertility in advantage of the data in all published reports. We searched for reports published before August 2015 using PubMed, CNKI, VIP, an...

  5. Analysis of Single Nucleotide Polymorphism in Human Paraoxonase 1 Gene(Q192R) with Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Introduction Single nucleotide polymorphisms (SNPs) are the most abundant DNA markers in the human genome occurring at a frequency of one in every 500-1000 nucleotides[1]. A variety of methods have been used for the analysis of single nucleotide polymorphisms, including restriction fragment length polymorphism (RFLP) [2], direct sequencing by using laser-induced fluorescence detection[3], fluorescence energy transfer[4], MALDI-TOF MS combined with primer extension or invasive cleavage[5,6] , and fluorescence polarization[7].

  6. Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping

    Directory of Open Access Journals (Sweden)

    Roussel Sophie

    2013-01-01

    Full Text Available Abstract Background Listeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES’s Laboratory for Food safety has been the European Union Reference Laboratory (EURL for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecular subtyping methods to PFGE, and integrate their use within the National Reference Laboratories (NRL network. Since 2008, the United Kingdom (UK-NRL for L. monocytogenes at the Health Protection Agency (HPA, London, has used fluorescent Amplified Fragment Length Polymorphism (fAFLP for the routine surveillance of L. monocytogenes isolated from human clinical cases, food and food processing environments in the UK. This study compares fAFLP with PFGE for subtyping L. monocytogenes. Results A panel of 109 L. monocytogenes isolates from either human cases of listeriosis, foods, food processing environments and animals were used for the comparative evaluation. Among these, 2 strains were tested from duplicate culture by both methods. The panel also included field isolates, isolates associated with outbreaks or sporadic cases and reference strains. The two strains tested in duplicate displayed the same fAFLP and PFGE types. Strains known to be epidemiologically associated with one another were found to have unique PFGE and fAFLP types. FAFLP and PFGE divided the strains into 76 and 82 distinct profiles, or types, respectively. The discriminatory index calculated was 0.993 and 0.996 for fAFLP and PFGE, respectively. Conclusions The discriminatory ability of fAFLP was similar to that of PFGE for the subtyping of L. monocytogenes isolates. As a less labour intensive technique fAFLP may be a better method to use than PFGE in investigating outbreaks of human

  7. Rapid Identification of Pathogenic Bacteria by means of Two Conservative Gene Loci' Specific PCR-CE-RFLP

    Institute of Scientific and Technical Information of China (English)

    高鹏; 张卓然; 徐维家; 安万新; 张晓慧; 戴兵; 范艳萍; 王运锋; 李萍; 温杰; 于卫健; 高向仪; 谢凡迪; 王永海

    2003-01-01

    To establish a rapid identification method for common pathogenic bacteria on the basis of molecular biology and to construct a preliminary Polymerase Chain Reaction-Capillary Electrophoresis-Restriction Fragment Length Polymorphism (PCR-CE-RFLP) database of bacteria isolated from clinical specimens frequently, 183 strains collected from clinical samples belonging to 12 genera and 19 species whose biochemical characterizations corresponded to the typical ones were examined.The genomic DNAs were amplified by two pairs of fluorescence labeled primers aiming at 16S rRNA gene and 16S-23S rRNA spacer region gene respectively at the same time. PCR products were then digested by restriction endonuclease Hae Ⅲ in-completely before taking capillary electrophoresis. The results with the PCR-CE-RFLP patterns of 16S rRNA genes were just alike within some genera, but when it comes to 16S-23S rRNA spacer region genes, each bacterium showed a unique pattern, which can be distinguished from each other easily. It seems that PCR-CE-RFLP patterns of 16S rRNA gene could only be used to classify the bacteria into family level, whereas the data of 16S-23S rRNA spacer region gene could be utilized to identify the whole microorganisms as precisely as the species level. In spite of the data of the spacer region gene alone can be sufficiently to verify the whole bacteria, we insist that the 16S rRNA gene could be of some assistant in case that there should be lots of families of bacteria, in which some similar ones, with the same RFLP data of 16S-23S rRNA spacer region gene, may coexist. This study proves that the utility of PCR-CE-RFLP is a convenient, rapid method to identify pathogenic bacteria, and is also a quick diagnosis measure for application to clinical use.

  8. A PCR-RFLP assay to detect and type cytolethal distending toxin (cdt) genes in Campylobacter hyointestinalis

    Science.gov (United States)

    HATANAKA, Noritoshi; KAMEI, Kazumasa; SOMROOP, Srinuan; AWASTHI, Sharda Prasad; ASAKURA, Masahiro; MISAWA, Naoaki; HINENOYA, Atsushi; YAMASAKI, Shinji

    2016-01-01

    Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We have recently identified two types of cytolethal distending toxin (cdt) gene in C. hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay that can differentiate Chcdt-I from Chcdt-II. When the PCR-RFLP assay was applied to 17 other Campylobacter strains and 25 non-Campylobacter strains, PCR products were not obtained irrespective of their cdt gene-possession, indicating that the specificity of the PCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C. hyointestinalis strains including 23 analyzed in the previous study and 12 newly isolated from pigs and bovines, all of them showed the presence of cdt genes. Furthermore, a restriction digest by EcoT14-I revealed that 29 strains contained both Chcdt-I and Chcdt-II and 6 strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly, however, PCR products obtained from 7 C. hyointestinalis strains were not completely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigested PCR product was homologous to cdtB but not to Chcdt-IB or Chcdt-IIB, indicating the presence of another cdt gene-variant. Then, we further digested the PCR products with DdeI in addition to EcoT14-I, showing that all three cdt genes, including a possible new Chcdt variant, could be clearly differentiated. Thus, the PCR-RFLP assay developed in this study is a valuable tool for evaluating the Chcdt gene-profile of bacteria. PMID:27916784

  9. Sequence Length Polymorphisms within Cervid AmelogeninX and AmelogeninY Genes: Use in Sex Determination

    Directory of Open Access Journals (Sweden)

    Benson H. Morrill

    2009-12-01

    Full Text Available Sequence length polymorphisms between the amelogeninX and amelogeninY genes have been utilized for the design of rapid PCR sex determination assays for various mammalian species, including four cervid species (Capreolus capreolus, Cervus elaphus, Cervus nippon, and Dama dama. Several of these assays have been shown to be able to determine sex from non-invasively collected samples such as hair roots, feces, or skeletal remains. Such assays can be helpful for both zoo and wildlife biologists for sex determination, and could possibly be further developed to aid in species identification and SNP analyses if sequence information is also available. In order to further study the amelogeninX and amelogeninY gene polymorphisms that exist within cervids, partial sequences from these genes were generated and analyzed in multiple amelogeninX and amelogeninY samples of mule deer (Odocoileus hemionus and Rocky Mountain elk (Cervus elaphus nelsoni; and single amelogeninX and amelogeninY samples of sika deer (Cervus nippon, fallow deer (Dama dama and axis deer (Axis axis. Within these five species, three sequence length polymorphism regions (two or more nucleotides inserted or deleted between samples were found. The lengths of these polymorphisms were 45, 9, and 9 nucleotides. In addition, a SNP was found in the amelogeninY sequences of the mule deer samples that were analyzed. Lastly, the primers used to amplify the regions of the amelogeninX and amelogeninY sequences were able to determine sex in all five species that were studied.

  10. 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/.

  11. ANALISIS KEANEKARAGAMAN KULTIVAR PISANG MENGGUNAKAN PENANDA PCR-RFLP PADA INTERNAL TRANSCRIBED SPACER (ITS DNA RIBOSOM

    Directory of Open Access Journals (Sweden)

    T.W.D. Ekasari

    2012-09-01

    Full Text Available Pisang merupakan bahan makanan pokok keempat terpenting di negara berkembang yang memiliki keanekaragaman sangat tinggi. Penanda DNA mikrosatelit dapat membedakan kultivar pisang yang memiliki genom A dengan kultivar pisang bergenom B. Namun penanda mikrosatelit memiliki beberapa keterbatasan, yaitu membutuhkan primer spesifik dan membutuhkan preparasi yang lebih rumit, sehingga membutuhkan waktu dan biaya yang cukup mahal. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP terhadap DNA internal transcribed spacer (ITS ribosom mampu mengklasifikasikan kultivar-kultivar pisang berdasarkan pita restriksi daerah ITS yang dipotong dengan enzim RsaI. Koleksi DNA dari 15 kultivar pisang di Laboratorium Genetika dan Molekular Jurusan Biologi UNNES sudah diklasifikasikan genomnya berdasarkan mikrosatelit. DNA kultivar pisang diamplifikasi menggunakan primer ITS L dan ITS 4 menghasilkan fragmen ITS sebesar 700 pb. Pemotongan fragmen ITS DNA ribosom dengan enzim RsaI menghasilkan fragmen  530 pb yang spesifik untuk genom A, fragmen 350 pb dan 180 pb spesifik untuk genom B. Hasil perbandingan klasifikasi genomik berdasarkan mikrosatelit dan PCR-RFLP dari daerah ITS DNA ribosom menunjukkan bahwa klasifikasi genomnya serupa. Banana is the fourth most important staple foods in developing countries which has very high diversity. Microsatellite markers can be able to differentiate bananas cultivars which have A and B genomes, but this marker has restrictions. It requires a specific primer which is takes time and the costs expensive enough. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP of the ribosomal DNA internal transcribed spacer (ITS was able to classify banana cultivars based on the restriction band ITS regions cut by RsaI enzyme. The DNA collection from 15 banana cultivars from the Laboratory of Genetics and Molecular Biology Department of Biological Science UNNES have been classified its

  12. The PML gene is linked to a megabase-scale insertion/deletion restriction fragment length polymorphism

    Energy Technology Data Exchange (ETDEWEB)

    Goy, A.; Xiao, Y.H.; Passalaris, T. [Memorial Sloan-Kettering Cancer Center, New York, NY (United States)] [and others

    1995-03-20

    The PML gene located on chromosome band 15q22 is involved with the RAR{alpha} locus (17q21) in a balanced reciprocal translocation uniquely observed in acute promyelocytic leukemia. Physical mapping studies by pulsed-field gel electrophoresis revealed that the PML gene is flanked by two CpG islands that are separated by a variable distance in normal individuals. Several lines of evidence demonstrate that this is the consequence of a large insertion/deletion polymorphism linked to the PML locus: (1) overlapping fragments obtained with a variety of rare-cutting restriction enzymes demonstrated the same variability in distance between the flanking CpG islands; (2) mapping with restriction enzymes insensitive to CpG methylation confirmed that the findings were not a consequence of variable methylation of CpG dinucleotides; (3) the polymorphism followed a Mendelian inheritance pattern. This polymorphism is localized 3{prime} to the PML locus. There are five common alleles, described on the basis of BssHII fragments, ranging from 220 to 350 kb with increments of approximately 30 kb between alleles. Both heterozygous (61%) and homozygous (391%) patterns were observed in normal individuals. Mega-base-scale insertion/deletion restriction fragment length polymorphisms are very rare and have been described initially in the context of multigene families. Such structures have been also reported as likely regions of genetic instability. High-resolution restriction mapping of this particular structure linked to the PML locus is underway. 47 refs., 4 figs., 1 tab.

  13. Polymorphic CAG and GGC repeat lengths in the androgen receptor gene and prostate cancer risk: analysis of a Brazilian population.

    Science.gov (United States)

    Silva Neto, Brasil; Koff, Walter J; Biolchi, Vanderlei; Brenner, Cleber; Biolo, Karlo D; Spritzer, Poli Mara; Brum, Ilma S

    2008-02-01

    Variations in transcriptional activity of the androgen receptor (AR) are related to polymorphic CAG and GGC repeats in exon 1 of the AR gene. We investigated the association between CAG and GGC repeat length and the risk of prostate cancer in a case-control study from a Brazilian population. We evaluated 49 patients and 51 healthy controls. DNA was extracted from peripheral leukocytes and the AR gene was analyzed by fragment analysis (GeneMapper software, Applied Biosystems, Foster City, California, USA). CAG and GGC mean lengths were not different between cases and controls. The risk for prostate cancer was higher for CAG repeats repeat lengths (CAG + GGC) repeats ( 17) were not associated with risk for prostate cancer (OR = 1.13 [95% CI 0.47-2.75]). In conclusion, fewer number of CAG repeats and total repeats (CAG + GGC) in the AR gene may be associated with increased risk for prostate cancer.

  14. Telomere length is associated with ACE I/D polymorphism in hypertensive patients with left ventricular hypertrophy

    DEFF Research Database (Denmark)

    Fyhrquist, Frej; Eriksson, Anders; Saijonmaa, Outi

    2013-01-01

    and association of telomere length with cardiovascular risk is affected by ACE (I/D) genotype. METHODS: We measured leucocyte telomere length (LTL) by Southern blot and analysed ACE I/D genotypes in 1249 subjects with hypertension and left ventricular hypertrophy (LVH). We examined interactions of ACE I......INTRODUCTION: Short telomeres are often associated with cardiovascular risk factors and age-related diseases, while the angiotensin converting enzyme (ACE) gene insertion/deletion polymorphism (DD, ID, II) has shown such associations less consistently. We hypothesized that telomere length....../D genotype with LTL and cardiovascular risk. RESULTS: Mean LTL in DD or ID genotype was shorter (8.15 and 8.14 kb, respectively), than in II genotype (8.27 kb, p=0.0005). This difference was significant in the younger subjects (55-64 years, p=0.02) but not in the older group (65-80 years, p=0.56 ). In DD...

  15. Relationships among androgen receptor CAG repeat polymorphism, sex hormones and penile length in Han adult men from China: a cross-sectional study

    Directory of Open Access Journals (Sweden)

    Yan-Min Ma

    2014-06-01

    Full Text Available This study aimed to investigate the correlations among androgen receptor (AR CAG repeat polymorphism, sex hormones and penile length in healthy Chinese young adult men. Two hundred and fifty-three healthy men (aged 22.8 ± 3.1 years were enrolled. The individuals were grouped as CAG short (CAG S if they harbored repeat length of ≤20 or as CAG long (CAG L if their CAG repeat length was >20. Body height/weight, penile length and other parameters were examined and recorded by the specified physicians; CAG repeat polymorphism was determined by the polymerase chain reaction (PCR method; and the serum levels of the sex hormones were detected by radioimmunoassay. Student's t-test or linear regression analysis was used to assess the associations among AR CAG repeat polymorphism, sex hormones and penile length. This investigation showed that the serum total testosterone (T level was positively associated with the AR CAG repeat length (P = 0.01; whereas, no significant correlation of T or AR CAG repeat polymorphism with the penile length was found (P = 0.593. Interestingly, an inverse association was observed between serum prolactin (PRL levels and penile length by linear regression analyses (β= −0.024, P = 0.039, 95% confidence interval (CI: −0.047, 0. Collectively, this study provides the first evidence that serum PRL, but not T or AR CAG repeat polymorphism, is correlated with penile length in the Han adult population from northwestern China.

  16. Relationships among androgen receptor CAG repeat polymorphism, sex hormones and penile length in Han adult men from China: a cross-sectional study.

    Science.gov (United States)

    Ma, Yan-Min; Wu, Kai-Jie; Ning, Liang; Zeng, Jin; Kou, Bo; Xie, Hong-Jun; Ma, Zhen-Kun; Wang, Xin-Yang; Gong, Yong-Guang; He, Da-Lin

    2014-01-01

    This study aimed to investigate the correlations among androgen receptor (AR) CAG repeat polymorphism, sex hormones and penile length in healthy Chinese young adult men. Two hundred and fifty-three healthy men (aged 22.8 ± 3.1 years) were enrolled. The individuals were grouped as CAG short (CAG S ) if they harbored repeat length of ≤ 20 or as CAG long (CAG L ) if their CAG repeat length was >20. Body height/weight, penile length and other parameters were examined and recorded by the specified physicians; CAG repeat polymorphism was determined by the polymerase chain reaction (PCR) method; and the serum levels of the sex hormones were detected by radioimmunoassay. Student's t-test or linear regression analysis was used to assess the associations among AR CAG repeat polymorphism, sex hormones and penile length. This investigation showed that the serum total testosterone (T) level was positively associated with the AR CAG repeat length (P = 0.01); whereas, no significant correlation of T or AR CAG repeat polymorphism with the penile length was found (P = 0.593). Interestingly, an inverse association was observed between serum prolactin (PRL) levels and penile length by linear regression analyses (β= -0.024, P = 0.039, 95% confidence interval (CI): -0.047, 0). Collectively, this study provides the first evidence that serum PRL, but not T or AR CAG repeat polymorphism, is correlated with penile length in the Han adult population from northwestern China.

  17. Relationships among androgen receptor CAG repeat polymorphism, sex hormones and penile length in Han adult men from China:a cross-sectional study

    Institute of Scientific and Technical Information of China (English)

    YanMin Ma; DaLin He; KaiJie Wu; Liang Ning; Jin Zeng; Bo Kou; HongJun Xie; ZhenKun Ma; XinYang Wang; YongGuang Gong

    2014-01-01

    This study aimed to investigate the correlations among androgen receptor (AR) CAG repeat polymorphism, sex hormones and penile length in healthy Chinese young adult men. Two hundred and iffty-three healthy men (aged 22.8 ± 3.1 years) were enrolled. The individuals were grouped as CAG short (CAGS) if they harbored repeat length of≤20 or as CAG long (CAGL) if their CAG repeat length was>20. Body height/weight, penile length and other parameters were examined and recorded by the speciifed physicians;CAG repeat polymorphism was determined by the polymerase chain reaction (PCR) method;and the serum levels of the sex hormones were detected by radioimmunoassay. Student’s t-test or linear regression analysis was used to assess the associations among AR CAG repeat polymorphism, sex hormones and penile length. This investigation showed that the serum total testosterone (T) level was positively associated with the AR CAG repeat length (P= 0.01); whereas, no signiifcant correlation of T or AR CAG repeat polymorphism with the penile length was found (P= 0.593). Interestingly, an inverse association was observed between serum prolactin (PRL) levels and penile length by linear regression analyses (b=-0.024, P= 0.039, 95%conifdence interval (CI):-0.047, 0). Collectively, this study provides the ifrst evidence that serum PRL, but not T or AR CAG repeat polymorphism, is correlated with penile length in the Han adult population from northwestern China.

  18. Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis.

    Science.gov (United States)

    Olivares-Fuster, Oscar; Shoemaker, Craig A; Klesius, Phillip H; Arias, Covadonga R

    2007-04-01

    Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.

  19. Bridging the Gap Between Large-scale Data Sets and Analyses: Semi-automated Methods to Facilitate Length Polymorphism Scoring and Data Analyses.

    Science.gov (United States)

    Amplified fragment length polymorphism (AFLP) markers can be developed more quickly and at a lower cost than microsatellite and single nucleotide polymorphism markers, which makes them ideal markers for large-scale studies of understudied taxa — such as species at risk. However,...

  20. Amplified fragment length polymorphism: an adept technique for genome mapping, genetic differentiation, and intraspecific variation in protozoan parasites.

    Science.gov (United States)

    Kumar, Awanish; Misra, Pragya; Dube, Anuradha

    2013-02-01

    With the advent of polymerase chain reaction (PCR), genetic markers are now accessible for all organisms, including parasites. Amplified fragment length polymorphism (AFLP) is a PCR-based marker for the rapid screening of genetic diversity and intraspecific variation. It is a potent fingerprinting technique for genomic DNAs of any origin or complexity and rapidly generates a number of highly replicable markers that allow high-resolution genotyping. AFLPs are convenient and reliable in comparison to other markers like random amplified polymorphic DNA, restriction fragment length polymorphism, and simple sequence repeat in terms of time and cost efficiency, reproducibility, and resolution as it does not require template DNA sequencing. In addition, AFLP essentially probes the entire genome at random, without prior sequence knowledge. So, AFLP markers have emerged as an advance type of genetic marker with broad application in genomic mapping, population genetics, and DNA fingerprinting and are ideally suited as screening tool for molecular markers linked with biological and clinical traits. This review describes the AFLP procedure and its applications and overview in the fingerprinting of a genome, which has been currently used in parasite genome research. We outline the AFLP procedure adapted for Leishmania genome study and discuss the benefits of AFLPs for assessing genetic variation and genome mapping over other existing molecular techniques. We highlight the possible use of AFLPs as genetic markers with its broad application in parasitological research because it allows random screening of the entire genome for linkage with genetic and clinical properties of the parasite. In this review, we have taken a pragmatic approach on the study of AFLP for genome mapping and polymorphism in protozoan parasites and conclude that AFLP is a very useful tool.

  1. 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.

  2. Carrier detection and early diagnosis of Wilson's disease by restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Figus, A; Lampis, R; Devoto, M; Ristaldi, M S; Ideo, A; de Virgilis, S; Nurchi, A M; Corrias, A; Corda, R; Lai, M E

    1989-01-01

    Wilson's disease, a rare autosomal recessive disorder, has been recently mapped to the long arm of chromosome 13 (q14.1). In this study, we carried out linkage analysis between three chromosome 13 DNA markers, D13S1, D13S10, D13S2, the locus for the red cell enzyme esterase D (ESD), and the Wilson's disease locus (WND) in 17 Wilson's disease families of Italian descent, mostly from Sardinia. We confirmed a tight linkage [theta = 0.00, Z (theta) = 4.07] between the WND and ESD loci, and provided suggestive evidence for linkage [theta = 0.00, Z(theta) = 1.85] of the WND locus with D13S10. Multipoint linkage analysis indicated the following order: centromere-D13S1-D13S10-WND-ESD-D13S2. RFLP analysis at these two loci in our families allowed us either to define the carrier status (50%) or to exclude the homozygous state (25%) in the great majority of unaffected sibs. PMID:2563776

  3. Authentication of Panax ginseng from its adulterants by PCR-RFLP and ARMS.

    Science.gov (United States)

    Diao, Ying; Lin, Xian-Ming; Liao, Chao-Lin; Tang, Chun-Zi; Chen, Zhong-Jian; Hu, Zhong-Li

    2009-04-01

    As a widely used and expensive herbal medicine, Panax ginseng has many adulterants in the commercial market. PCR-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) based on 5S rDNA sequence analysis were applied to identify two common adulterants of P. ginseng. The sizes of 5S rRNA gene non-transcribed spacers (NTS) sequences in P. ginseng and its adulterants were determined, ranging from 143 to 424 bp. The PCR product of P. ginseng only could be digested among the tested specimens because of its specific SpeI restriction site found in the 5S rDNA sequence. In addition, P. ginseng was successfully identified from compound medicinal preparations and from the Single-Taste medicines. These results suggest that the methods are able to authenticate P. ginseng.

  4. Identification of genotypes of Giardia duodenalis human isolates in Isfahan, Iran, using polymerase chain reaction - Restriction Fragment Length polymorphism

    Directory of Open Access Journals (Sweden)

    Nader Pestehchian

    2012-01-01

    Conclusions: PCR - RFLP assay targeting gdh locus is a sensitive tool and discriminates genotypes, sub genotypes and mixed type of G.duodenalis. Results of our study suggest both anthroponotic and zoonotic origins for the infections respectively.

  5. Relative diversity and community structure analysis of rumen protozoa according to T-RFLP and microscopic methods.

    Science.gov (United States)

    Tymensen, Lisa; Barkley, Cindy; McAllister, Tim A

    2012-01-01

    Protozoa are common inhabitants of the rumen where they play roles in host nutrition and methanogenesis. Knowledge of how changes in the composition of protozoa communities affect these processes is limited in part due to a lack of efficient methods for protozoa community analysis. In this study, a terminal-restriction fragment length polymorphism (T-RFLP) assay targeting the 18S rRNA gene was developed for comparative analysis of rumen protozoa communities. Comparison of diversity and structure of protozoa communities from hay-fed versus silage/grain-fed cattle via T-RFLP analysis yielded similar overall results to microscopy analysis. According to both methods, Entodinium spp. were more abundant in the silage/grain-fed cattle and protozoa diversity (as calculated using the Shannon index) was higher for the hay-fed cattle due to greater species evenness. Type B protozoa were more prevalent in the hay-fed cattle, whereas Type A protozoa were more prevalent in the silage/grain-fed cattle. Analysis of similarity (ANOSIM) indicated that the protozoa communities from hay-fed and silage/grain-fed cattle were different, and multivariate analysis indicated that pen mates (i.e., cattle fed the same diet and housed together) tended to have similar protozoa communities types. In summary, we present a T-RFLP method for analyzing rumen protozoa communities which complements traditional microscopy approaches but has the advantage of being amenable to high-throughput. Copyright © 2011. Published by Elsevier B.V.

  6. Evaluasi Keragaman Genetik Gen Hormon Pertumbuhan (GH pada Sapi Pesisir Sumatera Barat Menggunakan Penciri PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Jakaria

    2007-04-01

    Full Text Available A total of 134 Pesisir cattle were genotyped for growth hormone (GH gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The genotype and allele frequencies of the GH MspI and GH AluI Pesisir cattle were determined. The GH MspI gene frequencies for the C and T allele were 0.209 and 0.791 respectively, while GH AluI gene frequencies for the L and V allele were 0.992 and 0.008 respectively. The chi-square analysis indicated that this population is not in Hardy-Weinberg Equilibrium status. Expected heterozygosis value (He for GH MspI and GH AluI were 0.3306±0.0266 and 0.0149±0.0073 respectively. The PCR-RFLP GH MspI marker has higher genetic variability compare to PCR-RFLP AluI marker. This finding showed that GH MspI T allele was favorable as a GH marker for Bos indicus breeds.

  7. Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

    Science.gov (United States)

    Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

    2016-09-01

    We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05).

  8. Use of restriction fragment length polymorphism to identify Candida species, related to onychomycosis

    Science.gov (United States)

    Mohammadi, Rasoul; Badiee, Parisa; Badali, Hamid; Abastabar, Mahdi; Safa, Ahmad Hosseini; Hadipour, Mahboubeh; Yazdani, Hajar; Heshmat, Farnaz

    2015-01-01

    Background: Onychomycosis is one of the most common clinical forms of fungal infections due to both filamentous fungi and yeasts. The genus of Candida is one of the most prominent causes of onychomycosis in all around the world. Although Candida albicans is still the most frequent cause of nail infections, use of broad-spectrum antifungal agents has led to a shift in the etiology of C. albicans to non-albicans species. The aim of the present study is rapid and precise identification of candida species isolated from nail infection by using of PCR-RFLP technique. Materials and Methods: A total of 360 clinical yeast strains were collected from nail infections in Iran. Genomic DNA was extracted using FTA; cards. ITS1-5.8SrDNA-ITS2 region was amplified using universal primers and subsequently products were digested with the restriction enzyme MspI. For identification of newly described species (C. parapsilosis complex), the SADH gene was amplified, followed by digestion with Nla III restriction enzyme. Results: Candida albicans was the most commonly isolated species (41.1%), followed by C. parapsilosis (21.4%), C. tropicalis (12.8%), C. kefyr (9.4%), C. krusei (5.5%), C. orthopsilosis (4.1%), C. glabrata (2.8%), C. guilliermondii (1.4%), C. rugosa (0.8%), and C. lusitaniae (0.5%). Patients in the age groups of 51-60 and 81-90 years had the highest and lowest distribution of positive specimens, respectively. Conclusion: Rapid and precise identification of Candida species from clinical specimens lead to appropriate therapeutic plans. PMID:26015921

  9. Multiplex genotype determination at a DNA sequence polymorphism cluster in the human immunoglobulin heavy-chain region

    Energy Technology Data Exchange (ETDEWEB)

    Li, H.; Hood, L. [California Institute of Technology, Pasadena, CA (United States)

    1995-03-20

    We have developed a method for multilocus genotype determination. The method involves using restriction fragment length polymorphisms (RFLPs) for allele discrimination. If a polymorphism is not an RFLP, it is converted into an RFLP during the polymerase chain reaction (PCR). After amplification and restriction enzyme digestion, samples are analyzed by sequential gel loading during electrophoresis. The efficiency of this method was demonstrated by determining the genotypes of 108 semen samples at seven DNA sequence polymorphic sites identified in the human immunoglobulin heavy-chain variable region. It was shown that more than 1000 PCR products could be easily analyzed per day per investigator. To show the reliability of this method, some of the typing results were confirmed by DNA sequence analysis. By computer simulation, most (98%) polymorphisms were shown to be natural or convertible (by changing 1 bp close to or next to each polymorphic site) RFLPs for the commercially available 4-base cutters. 47 refs., 4 figs., 3 tabs.

  10. Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays

    Directory of Open Access Journals (Sweden)

    Marize Quinhones Pires

    2014-04-01

    Full Text Available Introduction During a diagnostic evaluation of canine visceral leishmaniasis (VL, two of seventeen dogs were found to be co-infected by Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi. Methods Specific polymerase chain reaction (PCR and restriction fragment length polymorphism-PCR (RFLP-PCR assays were performed. Results PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi in those fragments. Conclusions This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

  11. Identification and characterization of novel single nucleotide polymorphism markers for fat deposition in muscle tissue of pigs using amplified fragment length polymorphism.

    Science.gov (United States)

    Supakankul, Pantaporn; Kumchoo, Tanavadee; Mekchay, Supamit

    2017-03-01

    This study was conducted to identify and evaluate the effective single nucleotide polymorphism (SNP) markers for fat deposition in the longissimus dorsi muscles of pigs using the amplified fragment length polymorphism (AFLP) approach. Sixty-four selective primer combinations were used to identify the AFLP markers in the 20 highest- and 20 lowest-intramuscular fat (IMF) content phenotypes. Five AFLP fragments were converted into simple codominant SNP markers. These SNP markers were tested in terms of their association with IMF content and fatty acid (FA) composition traits in 620 commercially crossbred pigs. The SSC7 g.4937240C>G marker showed an association with IMF content (pIMF content and arachidonic levels (pA marker revealed an association with palmitoleic and ω9 FA levels (pT marker showed a significant association with IMF content and FA levels of palmitoleic, eicosenoic, arachidonic, monounsaturated fatty acids, and ω9 FA levels. However, no significant association of SSC8 g.47338181G>A was observed with any IMF and FA levels in this study. Four SNP markers (SSC7 g.4937240C>G, SSC9 g.5496647_5496662insdel, SSC10 g.71225134G>A, and SSC17 g.61976696G>T) were found to be associated with IMF and/or FA content traits in commercially crossbred pigs. These findings provide evidence of the novel SNP markers as being potentially useful for selecting pigs with the desirable IMF content and FA composition.

  12. Size polymorphism of chicken major histocompatibility complex-encoded B-G molecules is due to length variation in the cytoplasmic heptad repeat region

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K;

    1990-01-01

    that the extracellular regions of these molecules are very similar and that the length polymorphism is due to variations in the cytoplasmic regions. Inspection of the cDNA-derived protein sequence in this region shows many heptad repeats, which may allow variation in length by step deletion and alternative splicing...

  13. Clinical Identification of Common Species of Dermatophytes by PCR and PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    丁娟; 李家文; 刘志香; 谭志建

    2004-01-01

    To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase Ⅱ gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc Ⅱ. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans.From the restriction profiles of Hinc Ⅱ , 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCRRFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase Ⅱ gene is rapid and efficient.

  14. Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins.

    Science.gov (United States)

    Ziebell, Kim A; Read, Susan C; Johnson, Roger P; Gyles, Carlton L

    2002-06-01

    This study evaluated two generic polymerase chain reaction (PCR) protocols, and nine subtyping protocols and three PCR-restriction fragment length polymorphism (RFLP) protocols for detection of stx genes. The PCR protocols were evaluated by testing 12 reference isolates and 496 field strains of Shiga toxin-producing Escherichia coli (STEC). Both generic methods detected all stx genes. In tests with the reference isolates, all methods detected stx1 and stx2, seven subtyping methods detected stx2v(EH250), seven detected stx2e and only two detected stx2f. Four of the subtyping protocols identified stx genes in all of the field isolates. The PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested. The observed limitations of the protocols were shown to be due to nucleotide sequence variation in the region of the PCR primers. One subtyping protocol that detected the virulence-related genes, eae and ehxA, and all stx except for the stx2f gene, was modified by newly designed primers so that it identified all stx genes. This modified protocol provides comprehensive characterization of STEC in a single multiplex reaction.

  15. Spatial and seasonal variations in bacterial communities of the Yellow Sea by T-RFLP analysis

    Institute of Scientific and Technical Information of China (English)

    Hongyuan WANG; Xiaolu JIANG; Ya HE; Huashi GUAN

    2009-01-01

    Four typical coastal sites (rocky shore, sandy shore, mud flat shore, and artificial harbor) at the Yellow Sea were chosen to investigate the spatial and seasonal variations in bacterial communities. This was accomplished by using terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR amplified 16S rDNA fragments. Two kinds of tetrameric restriction enzymes, HhaI and MspI, were used in the experiment to depict the bacterial community diversity in different marine environments. It was found that the community compositions digested by the two enzymes separately were different. However, the results of bacterial community diversity derived from them were similar. The MDA analysis results of T-RFLP profiles coming from HhaI and MspI both exhibited a significant seasonal community shift for bacteria and a relatively low spatial variation among the four locations. With HhaI as the sample, the pair wise T-tests also revealed that variations were minor between each pair of marine environments, with R ranging from 0.198 to 0.349. However, the bacterial community structure in the mud flat site depicted a larger difference than each of the other three sites (R ranging from 0.282 to 0.349).

  16. [PCR-RFLP/Hsp70 for identification and tipification of leishmania from the tropical region].

    Science.gov (United States)

    Margarita Montalvo, Ana; Fraga, Jorge; Aylema Romero, Jaqueline; Monzote, Lianet; Ivon Montano, Ing; Dujardin, Jean Claude

    2006-01-01

    The optimization of the PCR conditions for amplification of the gene coding for the 70 kDa (HSp70) heat shock protein as well as the analysis of the restriction fragment length polymorphism (RFLP) were carried out. DNA from a reference strain of Leishmania mexicana was used as template. Analytical sensitivity and specificity, and reproducibility of PCR using DNA from L. mexicana, Lamazonensis, L. guyanensis and L. lainsoni were determined. A 1.3 kp band was obtained, which confirmed gene amplification. The band patterns derived from Haelll enzyme digestion allowed differentiating several species. L. guyanensis and L. lainsoni were different from each other, while L. mexicana and L. amazonensis, which shared a common pattern, were different from the other two species. Analytical sensitivity and specificity were adequate. The enzymatic restriction of the PCR product made it possible to differentiate Leishmania spp. from T. cruzi. The feasibility of identifying and typifying species from the American continent through PCR-RFLP/Hsp70 and of using enzymatic restriction of amplified product to distinguish Leishmania spp. from Trypanosornma cruzi was shown. This was the first step in implementing these molecular methods in the reference laboratory of the Institute.

  17. Common 5' beta-globin RFLP haplotypes harbour a surprising level of ancestral sequence mosaicism.

    Science.gov (United States)

    Webster, Matthew T; Clegg, John B; Harding, Rosalind M

    2003-07-01

    Blocks of linkage disequilibrium (LD) in the human genome represent segments of ancestral chromosomes. To investigate the relationship between LD and genealogy, we analysed diversity associated with restriction fragment length polymorphism (RFLP) haplotypes of the 5' beta-globin gene complex. Genealogical analyses were based on sequence alleles that spanned a 12.2-kb interval, covering 3.1 kb around the psibeta gene and 6.2 kb of the delta-globin gene and its 5' flanking sequence known as the R/T region. Diversity was sampled from a Kenyan Luo population where recent malarial selection has contributed to substantial LD. A single common sequence allele spanning the 12.2-kb interval exclusively identified the ancestral chromosome bearing the "Bantu" beta(s) (sickle-cell) RFLP haplotype. Other common 5' RFLP haplotypes comprised interspersed segments from multiple ancestral chromosomes. Nucleotide diversity was similar between psibeta and R/T-delta-globin but was non-uniformly distributed within the R/T-delta-globin region. High diversity associated with the 5' R/T identified two ancestral lineages that probably date back more than 2 million years. Within this genealogy, variation has been introduced into the 3' R/T by gene conversion from other ancestral chromosomes. Diversity in delta-globin was found to lead through parts of the main genealogy but to coalesce in a more recent ancestor. The well-known recombination hotspot is clearly restricted to the region 3' of delta-globin. Our analyses show that, whereas one common haplotype in a block of high LD represents a long segment from a single ancestral chromosome, others are mosaics of short segments from multiple ancestors related in genealogies of unsuspected complexity.

  18. A genetic map of chromosome 20q12-q13.1: Multiple highly polymorphic microsatellite and RFLP markers linked to the maturity-onset diabetes of the young (MODY) locus

    OpenAIRE

    1993-01-01

    Multiple highly polymorphic markers have been used to construct a genetic map of the q12-q13.1 region of chromosome 20 and to map the location of the maturity-onset diabetes of the young (MODY) locus. The genetic map encompasses 23 cM and includes 11 loci with PIC values >.50, seven of which have PICs >.70. New dinucleotide repeat polymorphisms associated with the D20S17, PPGB, and ADA loci have been identified and mapped. The dinucleotide repeat polymorphisms have increased the PIC of the AD...

  19. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of simple PCR-RFLP for preliminary differentiation.

    Science.gov (United States)

    Mirhendi, H; Zarei, F; Motamedi, M; Nouripour-Sisakht, S

    2016-03-01

    This work aimed to identify the species distribution of common clinical and environmental isolates of black Aspergilli based on simple restriction fragment length polymorphism (RFLP) analysis of the β-tubulin gene. A total of 149 clinical and environmental strains of black Aspergilli were collected and subjected to preliminary morphological examination. Total genomic DNAs were extracted, and PCR was performed to amplify part of the β-tubulin gene. At first, 52 randomly selected samples were species-delineated by sequence analysis. In order to distinguish the most common species, PCR amplicons of 117 black Aspergillus strains were identified by simple PCR-RFLP analysis using the enzyme TasI. Among 52 sequenced isolates, 28 were Aspergillus tubingensis, 21 Aspergillus niger, and the three remaining isolates included Aspergillus uvarum, Aspergillus awamori, and Aspergillus acidus. All 100 environmental and 17 BAL samples subjected to TasI-RFLP analysis of the β-tubulin gene, fell into two groups, consisting of about 59% (n=69) A. tubingensis and 41% (n=48) A. niger. Therefore, the method successfully and rapidly distinguished A. tubingensis and A. niger as the most common species among the clinical and environmental isolates. Although tardy, the Ehrlich test was also able to differentiate A. tubingensis and A. niger according to the yellow color reaction specific to A. niger. A. tubingensis and A. niger are the most common black Aspergillus in both clinical and environmental isolates in Iran. PCR-RFLP using TasI digestion of β-tubulin DNA enables rapid screening for these common species.

  20. 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.

  1. Validation of Simple Sequence Length Polymorphism Regions of Commonly Used Mouse Strains for Marker Assisted Speed Congenics Screening

    Directory of Open Access Journals (Sweden)

    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.

  2. 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

  3. Delineation of Campylobacter concisus genomospecies by amplified fragment length polymorphism analysis and correlation of results with clinical data

    DEFF Research Database (Denmark)

    Aabenhus, R.; On, Stephen L.W.; Siemer, Berit L.;

    2005-01-01

    phenotypically indistinguishable but genetically distinct taxa (i.e., genornospecies) that may vary in pathogenicity. We examined 62 C concisus strains by amplified fragment length polymorphism (AFLP) profiling and correlated the results with clinical data. All C. concisus strains gave unique AFLP profiles......, and numerical analysis of these data distributed the strains among four clusters. The clustering was of taxonomic significance: two clusters contained, respectively, the type strain (of oral origin) and a reference strain (from diarrhea) of each of the known genomospecies. Genomospecies 2 strains were more....... All genomospecies 4 strains were of the same protein profile group and failed to react with a C concisus species-specific PCR assay based on 23S rRNA gene sequences: the taxonomic position of this group requires closer investigation. Campylobacter concisus is genetically and taxonomically diverse...

  4. Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Fry, N K; Bangsborg, Jette Marie; Bernander, S;

    2000-01-01

    The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation...... by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each...... using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1...

  5. 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.

  6. Discrimination of Paederus fuscipes and Paederus littoralis by mtDNA-COI PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Sahar Bazrafkan

    2016-10-01

    Full Text Available Background: Linear dermatitis is endemic in Iran where most cases occur in the Caspian Sea coast and Fars prov­ince. The disease is caused by beetles of the genus Paederus which are active from early spring to beginning of au­tumn although its incidence rises from May to August. The classic taxonomy of Paederus spp. is based on the male genitalia that is very complex and needs expertise. In this study, we report a DNA-based method to discriminate Paederus fuscipes and Paederus littoralis (=syn: P. lenkoranus, P. ilsae.Methods: Type specimens were collected from north and south of Iran. Molecular typing of the species was per­formed using restriction fragment length polymorphism (RFLP analysis of polymerase chain reaction (PCR-am­plified fragments of mtDNA-COI.Results: Sequence analyses of the data obtained in this study showed significant DNA polymorphisms. There were 89 substitutions between COI sequences of the two species. The mtDNA-COI fragment comprises several useful species-specific restriction sites comprising HaeIII that could result in distinctively different species-specific PCR–RFLP profiles. The HaeIII enzyme cuts the 872 bp PCR amplicon of P. littoralis into 737 and 100 bp and two small nonvisible bands whereas it does not cut P. fuscipes amplicon into fragments.Conclusion: This study demonstrates that molecular typing is useful method and allows one to differentiate between two species and is recommended for discrimination of other Paederus species, which morphologically are indistin­guishable or very difficult to be distinguished.

  7. Androgen receptor CAG repeats length polymorphism and the risk of polycystic ovarian syndrome (PCOS.

    Directory of Open Access Journals (Sweden)

    Singh Rajender

    Full Text Available OBJECTIVE: Polycystic ovarian syndrome (PCOS refers to an inheritable androgen excess disorder characterized by multiple small follicles located at the ovarian periphery. Hyperandrogenism in PCOS, and inverse correlation between androgen receptor (AR CAG numbers and AR function, led us to hypothesize that CAG length variations may affect PCOS risk. METHODS: CAG repeat region of 169 patients recruited following strictly defined Rotterdam (2003 inclusion criteria and that of 175 ethnically similar control samples, were analyzed. We also conducted a meta-analysis on the data taken from published studies, to generate a pooled estimate on 2194 cases and 2242 controls. RESULTS: CAG bi-allelic mean length was between 8.5 and 24.5 (mean = 17.43, SD = 2.43 repeats in the controls and between 11 and 24 (mean = 17.39, SD = 2.29 repeats in the cases, without any significant difference between the two groups. Further, comparison of bi-allelic mean and its frequency distribution in three categories (short, moderate and long alleles did not show any significant difference between controls and various case subgroups. Frequency distribution of bi-allelic mean in two categories (extreme and moderate alleles showed over-representation of extreme sized alleles in the cases with marginally significant value (50.3% vs. 61.5%, χ(2 = 4.41; P = 0.036, which turned insignificant upon applying Bonferroni correction for multiple comparisons. X-chromosome inactivation analysis showed no significant difference in the inactivation pattern of CAG alleles or in the comparison of weighed bi-allelic mean between cases and controls. Meta-analysis also showed no significant correlation between CAG length and PCOS risk, except a minor over-representation of short CAG alleles in the cases. CONCLUSION: CAG bi-allelic mean length did not differ between controls and cases/case sub-groups nor did the allele distribution. Over-representation of short

  8. Semi-automated genetic analyses of soil microbial communities: comparison of T-RFLP and RISA based on descriptive and discriminative statistical approaches.

    Science.gov (United States)

    Hartmann, Martin; Frey, Beat; Kölliker, Roland; Widmer, Franco

    2005-06-01

    Cultivation independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. This approach is based on direct extraction of total soil DNA followed by PCR amplification of selected marker genes and subsequent genetic fingerprint analyses. Semi-automated genetic fingerprinting techniques such as terminal restriction fragment length polymorphism (T-RFLP) and ribosomal intergenic spacer analysis (RISA) yield high-resolution patterns of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring, when rapid high throughput screening for differences or changes is more important than phylogenetic identification of organisms affected. Our objective was to perform profound statistical analysis to evaluate the cultivation independent approach and the consistency of results from T-RFLP and RISA. As a model system, we used two different heavy metal treated soils from an open top chamber experiment. Bacterial T-RFLP and RISA profiles of 16S rDNA were converted into numeric data matrices in order to allow for detailed statistical analyses with cluster analysis, Mantel test statistics, Monte Carlo permutation tests and ANOVA. Analyses revealed that soil DNA-contents were significantly correlated with soil microbial biomass in our system. T-RFLP and RISA yielded highly consistent and correlating results and both allowed to distinguish the four treatments with equal significance. While RISA represents a fast and general fingerprinting method of moderate cost and labor intensity, T-RFLP is technically more demanding but offers the advantage of phylogenetic identification of detected soil microorganisms. Therefore, selection of either of these methods should be based on the specific research question under investigation.

  9. Risk of multiple myeloma is associated with polymorphisms within telomerase genes and telomere length

    DEFF Research Database (Denmark)

    Campa, Daniele; Martino, Alessandro; Varkonyi, Judit;

    2015-01-01

    that a TERT variant, rs2242652, is associated with reduced MM susceptibility (OR = 0.81; 95% CI: 0.72-0.92; p = 0.001). In addition we measured the leukocyte telomere length (LTL) in a subgroup of 140 cases who were chemotherapy-free at the time of blood donation and 468 controls, and found that MM patients...... had longer telomeres compared to controls (OR = 1.19; 95% CI: 0.63-2.24; ptrend  = 0.01 comparing the quartile with the longest LTL versus the shortest LTL). Our data suggest the hypothesis of decreased disease risk by genetic variants that reduce the efficiency of the telomerase complex. This reduced...... efficiency leads to shorter telomere ends, which in turn may also be a marker of decreased MM risk....

  10. 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

  11. Variability among inbred lines and RFLP mapping of sunflower isozymes

    Directory of Open Access Journals (Sweden)

    Carrera Alicia D.

    2002-01-01

    Full Text Available Eight isozyme systems were used in this study: acid phosphatase (ACP, alcohol dehydrogenase (ADH, esterase (EST, glutamate dehydrogenase (GDH, malate dehydrogenase (MDH, phosphoglucoisomerase (PGI, 6-phosphogluconate dehydrogenase (PGD, and phosphoglucomutase (PGM. The polymorphism of these enzyme systems was studied in 25 elite inbred lines. A total of 19 loci were identified, but only eight of them were polymorphic in the germplasm tested. The polymorphic index for the eight informative markers ranged from 0.08 to 0.57, with a mean value of 0.36. Five isozyme loci were mapped in F2:3 populations with existing RFLP data. Est-1, Gdh-2 and Pgi-2 were mapped to linkage groups 3, 14 and 9, respectively. As in previous reports, an ACP locus and a PGD locus were found to be linked, both located in linkage group 2 of the public sunflower map.

  12. Relationships among androgen receptor CAG repeat polymorphism, sex hormones and penile length in Han adult men from China: a cross-sectional study

    OpenAIRE

    Yan-Min Ma; Kai-Jie Wu; Liang Ning; Jin Zeng; Bo Kou; Hong-Jun Xie; Zhen-Kun Ma; Xin-Yang Wang; Yong-Guang Gong; Da-Lin He

    2014-01-01

    This study aimed to investigate the correlations among androgen receptor (AR) CAG repeat polymorphism, sex hormones and penile length in healthy Chinese young adult men. Two hundred and fifty-three healthy men (aged 22.8 ± 3.1 years) were enrolled. The individuals were grouped as CAG short (CAG S ) if they harbored repeat length of ≤20 or as CAG long (CAG L ) if their CAG repeat length was >20. Body height/weight, penile length and other parameters were examined and recorded by the specified ...

  13. Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

    OpenAIRE

    Kabadjova, Petia; Dousset, Xavier; Le Cam, Virginie; Prevost, Hervé

    2002-01-01

    A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763T a...

  14. IDENTIFIKASI DAGING BABI MENGGUNAKAN METODE PCR-RFLP GEN Cytochrome b DAN PCR PRIMER SPESIFIK GEN AMELOGENIN (Pork Identification Using PCR-RFLP of Cytochrome b Gene and Species Specific PCR of Amelogenin Gene

    Directory of Open Access Journals (Sweden)

    Yuny Erwanto

    2013-03-01

    Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP and species specific PCR methods had been applied for identifying pork in mixture of meat. Pork sample in various levels (1, 3, 5 and 10% was prepared in mixture with beef, chicken and mutton. The primary CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b b (cytochrome b gene and PCR successfully amplified fragments of 359 bp. To distinguish pig species existence, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed that pig mitochondrial DNA was cut into 131 and 228 bp fragments. A polymerase chain reaction (PCR method based on the nucleotide sequence variation in the amelogenin gene has been chosen for the specific identification of pork DNAs in mixture meat. The primers designed generated specific fragments of 353 and 312 bp length for pork. The specificity of the primary designed was tested on 4 animal species including pig, cattle, chicken and goat species. Analysis of experimental mixture meat demonstrated that 1% of raw pork tissues could be detected using PCR-RFLP with BseDI restriction enzyme but detection using species-specific PCR showed the cross reactivity to beef, chicken and mutton. The cytochrome b PCR-RFLP species identification assay yielded excellent results for identification of pig species. PCR-RFLP is a potentially reliable technique for detection of the existence of pork in animal food product for Halal authentication. Keywords: Pork identification, cytochrome b, amelogenin, polymerase chain reaction   ABSTRAK   Penelitian ini dilakukan untuk mengaplikasikan metode deteksi daging babi dalam campuan daging dengan sapi, kambing dan ayam melalui PCR-RFLP dan PCR dengan primer spesifik untuk babi. Level kontaminasi daging babi dibuat sebesar 1, 3, 5 dan 10% dari total daging dalam campuran. Metode PCR-RFLP menggunakan sepasang primer yaitu gen cytochrome b dari mitokondria yang

  15. A Study on Campylobacter jejuni and Campylobacter coli through Commercial Broiler Production Chains in Thailand: Antimicrobial Resistance, the Characterization of DNA Gyrase Subunit A Mutation, and Genetic Diversity by Flagellin A Gene Restriction Fragment Length Polymorphism.

    Science.gov (United States)

    Thomrongsuwannakij, Thotsapol; Blackall, Patrick J; Chansiripornchai, Niwat

    2017-06-01

    chain reaction-restriction fragment length polymorphism of the flagellin A gene (flaA-RFLP) to determine their genetic relationships. Ten distinct clusters were recognized by flaA-RFLP typing. The results showed that horizontal transmission was the major route of Campylobacter transmission in this study. In conclusion, the emergence of MDR and high resistance rates to several antimicrobials are major concerns identified in this study. The prudent use of these agents and active surveillance of resistance at the farm level are essential steps to reduce the public health risks identified in this work.

  16. Morphological diversity and polymorphism of self-assembling collagen peptides controlled by length of hydrophobic domains.

    Science.gov (United States)

    McGuinness, Kenneth; Khan, I John; Nanda, Vikas

    2014-12-23

    Synthetic collagen mimetic peptides are used to probe the role of hydrophobic forces in mediating protein self-assembly. Higher order association is an integral property of natural collagens, which assemble into fibers and meshes that comprise the extracellular matrix of connective tissues. The unique triple-helix fold fully exposes two-thirds of positions in the protein to solvent, providing ample opportunities for engineering interaction sites. Inclusion of just a few hydrophobic groups in a minimal peptide promotes a rich variety of self-assembly behaviors, resulting in hundred-nanometer to micron size nanodiscs and nanofibers. Morphology depends primarily on the length of hydrophobic domains. Peptide discs contain lipophilic domains capable of sequestering small hydrophobic dyes. Combining multiple peptide types result in composite structures of discs and fibers ranging from stars to plates-on-a-string. These systems provide valuable tools to shed insight into the fundamental principles underlying hydrophobicity-driven higher order protein association that will facilitate the design of self-assembling systems in biomaterials and nanomedical applications.

  17. Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods Identificação de novas flagelinas codificadas por fliC em Escherichia coli isoladas de animais domésticos utilizando RFLP-PCR e sequenciamento

    Directory of Open Access Journals (Sweden)

    Cláudia de Moura

    2013-04-01

    Full Text Available Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT. The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.A identificação da Escherichia coli requer conhecimento sobre os sorotipos e fatores de virulência prevalentes permitindo a classificação em patogênico/não patogênico. No entanto, algumas destas bactérias não expressam o antígeno flagelar in vitro. Neste caso, o PCR-restriction fragment length polymorphism (RFLP-PCR e o sequenciamento do gene fliC podem ser adequados para a identificação desses antígenos, substituindo a sorologia tradicional. Nesta pesquisa foram estudadas 17 amostras de E. coli isoladas de animais e que apresentavam antígeno H não tipável (HNT. Os antígenos H foram caracterizados por PCR-RFLP e sequenciamento do gene fliC. Três novos genes da flagelina foram identificados, para os quais anti-soros específicos foram obtidos. A técnica PCR-RFLP mostrou-se mais rápida que a sorotipagem do antígeno H em E. coli, fornecendo informações sobre algumas características desses antígenos e indicou a presença de novos genes fliC.

  18. 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...

  19. Characterization of Mycoplasma hyosynoviae strains by amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis and 16S ribosomal DNA sequencing

    DEFF Research Database (Denmark)

    Kokotovic, Branko; Friis, N.F.; Ahrens, Peter

    2002-01-01

    , were investigated by analysis of amplified fragment length polymorphisms of the Bgl II and Mfe I restriction sites and by pulsed-field gel electrophoresis of a Bss HII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory...

  20. Molecular epidemiology of contagious bovine pleuropneumonia in Tanzania based on amplified fragment length polymorphism and pulsed-field gel electrophoresis analysis

    DEFF Research Database (Denmark)

    Kusiluka, L.J.M.; Ojeniyi, B.; Friis, N.F.

    2001-01-01

    . The strains were analysed fur variations in the EcoRI and Csp6I restriction sites in the genomic DNA using the amplified fragment length polymorphism (AFLP) technique, and variations in the BamHI restriction sites using pulscd-field gel electrophoresis (PFGE). Six AFLP I) pes were detected among the analysed...

  1. Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: Strain diversity, host range, and recombination

    NARCIS (Netherlands)

    Schouls, L.M.; Reulen, S.; Duim, B.; Wagenaar, J.A.; Willems, R.J.L.; Dingle, K.E.; Colles, F.M.; Embden, van J.D.A.

    2003-01-01

    Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic

  2. Biodiversity and ITS-RFLP characterisation of Aspergillus section Nigri isolates in grapes from four traditional grape-producing areas in Greece.

    Directory of Open Access Journals (Sweden)

    Dimosthenis Kizis

    Full Text Available A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS. Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP, four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC and Enzyme Linked Immuno Sorbent Assay (ELISA, resulting in significant interspecies differences in OTA production.

  3. 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.

  4. Clinical applicability of PCR-RFLP in detection of mycobacterium infections of skin%PCR-RFLP法直接检测分枝杆菌皮肤感染的临床应用

    Institute of Scientific and Technical Information of China (English)

    张彩萍; 丁克云; 王洪生; 冯雨苗; 林麟; 崔盘根; 陈敏; 吴勤学

    2013-01-01

    目的:评价分枝杆菌热休克蛋白65(Hsp65)基因检测分枝杆菌皮肤感染标本的敏感性和特异性.方法:多聚酶链反应-限制性片段长度多态性(PCR-RFLP)的10%非变性聚丙烯酰胺凝胶法直接检测Hsp65基因,PCR-RFLP检测阳性的标本再经Hsp65基因和16S rRNA DNA基因测序验证.结果:与培养法相比,PCR-RFLP法检测临床分枝杆菌皮肤感染的敏感性为30% (3/10),特异性为100%(10/10).结论:分枝杆菌PCR-RFLP的10%非变性聚丙烯酰胺凝胶法可用于分枝杆菌皮肤感染的临床检测.%Objective: To assess the sensitivity and specificity of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)based on hsp65 by 10% non-denaturizing polyacrylamide gel in the detection of mycobacterium infections of skin. Methods: Twenty clinical skin specimens suspected of mycobacterium infections were detected and identified by culture and PCR - RFLP directly and confirmed by sequencing of hsp65 and 16S rRNA DNA genes. Results: Compared with culture method, the sensitivity and the specificity of PCR - RFLP were 30% (3/10) and 100% (10/10). Conclusion: PCR- RFLP based on hsp65 with 10% polyacrylamide gel can be clinically applied in the detection of mycobacterium infections of skin.

  5. Comparison of microsatellite length polymorphism and multilocus sequence typing for DNA-Based typing of Candida albicans.

    Science.gov (United States)

    Garcia-Hermoso, Dea; Cabaret, Odile; Lecellier, Gael; Desnos-Ollivier, Marie; Hoinard, Damien; Raoux, Dorothée; Costa, Jean-Marc; Dromer, Françoise; Bretagne, Stéphane

    2007-12-01

    For genotyping Candida albicans isolates, two PCR-based methods have recently emerged: multilocus sequence typing (MLST), based on the sequence of selected genes, and microsatellite length polymorphism (MLP), based on the length of PCR products containing variable numbers of short DNA repeats. To compare the two methods in their abilities to differentiate and group C. albicans isolates, we selected 50 independent isolates collected at the National Reference Center for Mycoses and Antifungals. MLST typing was performed using sequencing of seven loci as described at (http://test1.mlst.net). The MLP method consisted of a single multiplex PCR testing three different loci. Dendrograms were constructed by the unweighted pair group cluster method with Euclidean metric for both methods. The correlation between the distance matrices was performed with a Mantel test tested with 1,000 random permutations. The sensitivity and specificity of the MLP typing system were determined after allocating MLST groups for the greater number of isolates of each distinct MLP group. The discriminatory power index was >0.99, and the distances between the isolates were highly correlated with both systems. The Mantel coefficient and the Pearson product-moment correlation coefficient were 35,699 and 0.32, respectively (P < or = 1.2 x 10(-6)). Using MLP, the average specificity and sensitivity of clustering compared to MLST were 83% and 73%, respectively, when the singletons were excluded. The two methods are similarly discriminatory and can be interchangeable depending on the objectives. MLP is less expensive and faster than MLST. However, MLST is currently more accurate and additional standardization is needed for MLP.

  6. 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.

  7. Amplified fragment length polymorphism of Streptococcus suis strains correlates with their profile of virulence-associated genes and clinical background.

    Science.gov (United States)

    Rehm, Thomas; Baums, Christoph G; Strommenger, Birgit; Beyerbach, Martin; Valentin-Weigand, Peter; Goethe, Ralph

    2007-01-01

    Amplified fragment length polymorphism (AFLP) typing was applied to 116 Streptococcus suis isolates with different clinical backgrounds (invasive/pneumonia/carrier/human) and with known profiles of virulence-associated genes (cps1, -2, -7 and -9, as well as mrp, epf and sly). A dendrogram was generated that allowed identification of two clusters (A and C) with different subclusters (A1, A2, C1 and C2) and two heterogeneous groups of strains (B and D). For comparison, three strains from each AFLP subcluster and group were subjected to multilocus sequence typing (MLST) analysis. The closest relationship and lowest diversity were found for patterns clustering within AFLP subcluster A1, which corresponded with sequence type (ST) complex 1. Strains within subcluster A1 were mainly invasive cps1 and mrp+ epf+ (or epf*) sly+ cps2+ strains of porcine or human origin. A new finding of this study was the clustering of invasive mrp* cps9 isolates within subcluster A2. MLST analysis suggested that A2 correlates with a single ST complex (ST87). In contrast to A1 and A2, subclusters C1 and C2 contained mainly pneumonia isolates of genotype cps7 or cps2 and epf- sly-. In conclusion, this study demonstrates that AFLP allows identification of clusters of S. suis strains with clinical relevance.

  8. Usefulness of restriction fragment length polymorphism and spoligotyping for epidemiological studies of Mycobacterium bovis in Madagascar: description of new genotypes.

    Science.gov (United States)

    Rasolofo Razanamparany, Voahangy; Quirin, René; Rapaoliarijaona, Andriampenomaka; Rakotoaritahina, Hugues; Vololonirina, Elie Jeanne; Rasolonavalona, Tiana; Ferdinand, Séverine; Sola, Christophe; Rastogi, Nalin; Ramarokoto, Herimanana; Chanteau, Suzanne

    2006-04-16

    Tuberculosis is highly prevalent in cattle in Madagascar. An epidemiological study based on genotyping of Mycobacterium bovis and its transmission to humans was carried out. The restriction fragment length polymorphism (IS6110 and DR markers) and spoligotyping were used to assess the genetic diversity of strains from different regions of Madagascar. One of these strains was isolated from goat, the other strains were isolated from zebu cattle. Nine IS6110 profiles, 20 DR profiles and 12 spoligotypes were obtained. About 90% of all isolates gave a single IS6110 band at about 1.8 kb. Most strains had the same spoligotype. M. bovis strains commonly lack spacers 39-43, and all Malagasy strains also lacked spacers 3-5, 8-10 and 16. This pattern has not been reported elsewhere. DR was the most discriminatory of the three markers. The patterns obtained with the three markers were combined to identify 34 different genotypes, one of which was found in 35% of the strains. No region-specific M. bovis genotype was identified, but the genotyping of 18 M. bovis strains isolated from patients showed that the human and bovine strains were identical, suggesting possible human contamination from zebu cattle.

  9. 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.

  10. Leishmania spp. identification by polymerase chain reaction-restriction fragment length polymorphism analysis and its applications in French Guiana.

    Science.gov (United States)

    Simon, Stéphane; Veron, Vincent; Carme, Bernard

    2010-02-01

    Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism-polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains (Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.

  11. 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.

  12. Culturable actinobacteria from the marine sponge Hymeniacidon perleve: isolation and phylogenetic diversity by 16S rRNA gene-RFLP analysis.

    Science.gov (United States)

    Zhang, Haitao; Lee, Yoo Kyung; Zhang, Wei; Lee, Hong Kum

    2006-08-01

    A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification-restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.

  13. PCR-RFLP Analysis of 28 SrDNA for Specification of Fasciola gigantica (Cobbold, 1855) in the Infected Lymnaea auricularia (Linnaeus, 1785) Snails from Northwestern Iran.

    Science.gov (United States)

    Yakhchali, Mohammad; Malekzadeh-Viayeh, Reza; Imani-Baran, Abass

    2014-09-01

    Fasciolosis in livestock is a crucial concern in the globe, mainly due to its impact on human health. The aim of this study was to determine the rate of infection with Fasciola gigantica (Cobbold, 1855) larvae in the field-collected snails of Lymnaea auricularia (Linnaeus, 1785) from northwestern Iran using a molecular approach. A total of 6,759 pond snails were collected from 28 freshwater bodies in West Azarbaijan. PCR was performed to amplify a 618-bp fragment of the nuclear 28 SrRNA gene of Fasciola. The PCR products were digested by AvaII restriction enzyme to create restriction fragment length polymorphism (RFLP) patterns specific for the detection of F. gigantica. Of the total collected snails 496 (7.34 %) were L. auricularia, among which 4.64% (23 out of 496) were infected with a Fasciola species according to the PCR analysis. Only 2.22% (11 out of 496) of the infected snails were from the mountainous areas. The highest Fasciola infection rate recorded in the snails of a single site was 1.81% (9 out of 496 snails). Based on the RFLP pattern, F. gigantica accounted for 2.42% of the infection rates in the study sites. Application of PCR-RFLP was proven to be a useful approach for valid and robust detection of the infection with F. gigantica in its intermediate host snails. These findings may therefore be applicable for establishment of the control programs against dissemination of the infection in different regions.

  14. Application of RFLP-PCR-Based Identification for Sand Fly Surveillance in an Area Endemic for Kala-Azar in Mymensingh, Bangladesh

    Directory of Open Access Journals (Sweden)

    Mohammad Shafiul Alam

    2012-01-01

    Full Text Available Mymensingh is the most endemic district for kala-azar in Bangladesh. Phlebotomus argentipes remains the only known vector although a number of sand fly species are prevalent in this area. Genotyping of sand flies distributed in a VL endemic area was developed by a PCR and restriction-fragment-length polymorphism (RFLP of 18S rRNA gene of sand fly species. Using the RFLP-PCR analysis with AfaI and HinfI restriction enzymes, P. argentipes, P. papatasi, and Sergentomyia species could be identified. Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found. Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection. The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh.

  15. Efficiency of IRAP and ITS-RFLP marker systems in accessing genetic variation of Pyrenophora graminea

    Directory of Open Access Journals (Sweden)

    Imad Zein

    2010-01-01

    Full Text Available The usefulness of IRAP (inter-retrotransposon amplified polymorphism and ITS-RFLP (restriction of PCR-amplified internal transcribed spacers of the rDNA markers in the analysis of 39 Pyrenophora graminea isolates was determined. Each marker system could discriminate between all of the isolates in detecting polymorphism, albeit with variable efficiency. IRAP and ITS-RFLP produced 85% and 77% polymorphic bands, respectively, with a corresponding mean polymorphic information content (PIC of 0.38 and 0.36. The IRAP marker index ratio (2.41 was higher than ITS-RFLP (1.50. On one hand, the quality nature of data (QND was higher for ITS-RFLP (0.169 than IRAP (0.093. However, correlation between both marker similarity matrices was significant (r = 0.34, p < 0.05. These findings suggest their combined use in phylogenetic analysis. To our knowledge, this is the first report of a comparison involving these two advanced DNA marker systems.

  16. PCR-RFLP Polymorphism of Promoter Region of MSTN Gene in Southwest Pony%西南矮马肌肉生长抑制素基因启动子区PCR-RFLP多态性检测

    Institute of Scientific and Technical Information of China (English)

    徐龙鑫; 杨胜林; 杨海兵; 李爱萍; 王忠霞

    2012-01-01

    In this present study, promoter region of myostation(MSTN) gene of Guizhou pony, Debao pony and Ningqiang pony were analyzed by PCR-RFLP. The results indicated that there was one Ssp I digestion site in the 204 bp amplified fragment. An T→C mutation at the site 156 bp of this gene by DNA sequencing which produced two sorts of genotypes, AA and AB, however, BB genotype was not founded in this study. The genotype frequency of AB in Guizhou pony and Ningqiang pony were higher than AA, while AA genotype was preponderant in Debao pony. The allele frequency of A were higher than B in all these three pony breeds. Guizhou pony and Debao pony were at Hardy-Weinberg equilibrium (P>0. 05) , but Ningqiang pony was significantly different (P<0. 05).%利用PCR-RFLP技术对贵州矮马、德保矮马和宁强矮马3个品种的45个个体的肌肉生长抑制素(myostatin,MSTN)基因的启动子区进行多态性分析.结果表明,大小为204 bp的扩增片段经限制性内切酶Ssp I酶切后,不同基因型经过测序发现156T→C突变,产生AA和AB两种基因型,没有发现BB基因型.贵州矮马和宁强矮马中AB基因型频率大于AA基因型,德保矮马中AA基因型占优势,在3种矮马品种A等位基因频率均大于B等位基因.卡方检测结果发现,贵州和德保矮马在该酶切位点处于平衡状态(P>0.05),而宁强矮马在该位点分布差异显著(P<0.05).

  17. 慢性乙肝患者HBV核心基因一组单核苷酸多态性与血清HBV DNA水平的相关性研究%Study on the correlation between a set of single nucleotide polymorphisms of hepatitis B virus core gene and serum HBV DNA levels in patients with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    李一荣; 王雪平; 陈凤花; 袁琳; 马荣红; 温晓波; 周志明

    2011-01-01

    Objective To investigate the relation between a set of single nucleotide polymorphisms (SNP) in core gene of HBV in chronic hepatitis B patients and HBV DNA levels. Methods PCR restriction fragment length polymorphism(PCR-RFLP) assay and restriction enzyme Tsp509I were adopted to determine HBV SNP in HBV core gene. Nucleotide sequences of core gene were determined using the dideoxy chain termination method. HBV DNA levels were quantitated with real-time PCR. Results Five typical RFLP patterns, RFLP-C, RFLP-D, RFLP-E, RFLP-G and RFLP-C/G mixture were found and the distribution of HBV RFLP patterns was as follows: C, 61. 5% ; D, 2. 6% ; E, 9.6%; G, 16.7%; C/G mixture, 9.6%. Five SNPs, A165T, A336C, A336T, T337C and T385C, were found to be associated with RFLP patterns change and only SNP A336C or A336T caused the substitution of Glu-83 with Asp in HBcAg. The serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P =0. 02) and RFLP-C/G group(P = 0. 006) , respectively, furthermore, the positive rate of serum ALT in RFLP-C/G group was lower than that in RFLP-C, RFLP-E and RFLP-G group, respectively. Under the condition of HBeAg-positive, the serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P = 0. 015) and RFLP-C/G group(P =0.008) , respectively. Conclusion PCR-RFLP used in this study can be adopted to determine HBV SNPs, not genotypes in Chinese patients with chronic hepatitis B. The serum HBV DNA level in RFLP-C group higher than that in RFLP-G or RFLP-C/G group maybe associated with amino acid mutation, Glu83 Asp.%目的 研究慢性乙肝患者HBV核心基因的单核苷酸多态性(SNP)与血清HBV DNA水平的相关关系.方法 采用PCR-RFLP和限制性内切酶Tsp509I榆测HBV核心基因的SNP,采用双脱氧终止法对核心基因进行序列测定,实时荧光PCR技术用于HBVDNA水平的定量检测.结果 慢性乙肝人群中发现5种典型的PCR-RFLP图谱,分别是RFLP-C、RFLP-D、RFLP-E、RFLP-G和RFLP-C/G,各

  18. Species identification of grouper and snapper in Taiwan Strait using polymerase chain reaction-restriction fragment length polymorphism analysis and lab-on-a-chip system%聚合酶链式反应-限制性片段长度多态性和芯片生物分析技术用于台湾海峡常见石斑鱼和鲷鱼的品种鉴定

    Institute of Scientific and Technical Information of China (English)

    陈双雅; 张津; 陈伟玲; 徐敦明; 周昱

    2011-01-01

    Polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP )analysis and lab-on-a-chip system were used to identify grouper and snapper species in Taiwan Strait. A fragment of 464 bp length of mitochondrial cytochrome b gene was amplified by PCR and the products were digested with restriction enzymes Dde Ⅰ, Hae Ⅲ and Nla Ⅲ , individually. The fragments generated after digestion were further resolved on the DNA Chip. Eight grouper species and five snapper species were successfully identified. The results demonstrated that PCR-RFLP analysis and lab-on-a-chip system provide a fast , easy, automated, and reliable analysis approach. This approach is potential for the purpose of fish adulteration control.%应用聚合酶链式反应-限制性片段长度多态性分析(PCR-RFLP)和芯片生物分析系统建立了台湾海峡常见石斑鱼和鲷鱼的分子生物学品种鉴定新方法.首先提取鱼的基因组脱氧核糖核酸(DNA)进行细胞色素b基因特定片段的PCR扩增,然后用Dde Ⅰ、Hae Ⅲ和Nla Ⅲ 3种限制性内切酶进行酶切,在Agilent DNA 1000芯片上对酶切片段进行分离.该方法成功鉴定了台湾海峡常见的8种石斑鱼品种和5种鲷鱼品种,是一种快速、简便、有效的鱼类品种鉴定分析手段.

  19. PCR-RFLP Provides Discrimination for Total flaA Sequence Analysis in Clinical Campylobacter jejuni Isolates.

    Science.gov (United States)

    Ghorbanalizadgan, Mahdi; Bakhshi, Bita; Najar-Peerayeh, Shahin

    2016-09-21

    The aims of this study were to determine the genetic relatedness among 20 clinical Campylobacter jejuni samples isolated from children with diarrhea in Iran and to introduce the best method of discrimination based on flagellin gene (flaA) sequence divergence. A total of 400 stool specimens were obtained from children under 5 years of age from July 2012 to June 2013. Primers were designed based on conserved sequences flanking the flaA gene that encompassed and amplified the entire flaA gene and followed by sequencing and data analysis with MEGA version 6.0.6 software. Ninety amino acids and 560 nucleotide polymorphic sequences were detected within 1,681 bp of the flaA sequence of which 43 (2.5%) and 12 (0.7%) were singletons, respectively. New repeat boxes within the flaA sequences were found in this study. Unweighted Pair Group Method with Arithmetic Mean dendrogram based on nucleotides of the full length flaA gene, the flaA short variable region gene and the in silico flaA phylogenic tree of DdeI restriction fragment length polymorphism (RFLP) profiles produced very similar clustering with a diversity index of 0.86 for each of the 3 methods. We conclude that flaA typing based on DdeI RFLP of the PCR products is a cheap, rapid, and reliable method for the epidemiological study of C. jejuni isolates of clinical origin in resource-limited regions or in large-scale population surveillance.

  20. 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

  1. Effect of BoLA-DRB3 exon2 polymorphisms on lameness of Chinese Holstein cows.

    Science.gov (United States)

    Sun, Liping; Song, Yapan; Riaz, Hasan; Yang, Liguo

    2013-02-01

    The involvement of bovine lymphocyte antigen (BoLA) in immune system and its role in susceptibility/resistance to infectious diseases has been extensively studied. However, few studies have been conducted to investigate the association between BoLA and gait scores. Our objective was to investigate whether polymorphisms in BoLA gene are associated with susceptibility of lameness in 435 Chinese Holstein cows. Genotyping of the BoLA-DRB3.2 gene was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with three restriction endonucleases (BstUI, BstYI and HaeIII). The relationship between the polymorphisms in BoLA-DRB3.2 gene and gait scores was analyzed by least-squares linear model. The gait score was non-significant among all five BstUI-RFLP and BstYI-RFLP genotypes. However, analysis of seven HaeIII-RFLP genotypes revealed a significantly higher gait score for AB genotype than others. In conclusion, BoLA-DRB3.2 may be a candidate gene for lameness susceptibility in Chinese Holstein cows.

  2. Analysis of Porcine Reproductive and Respiratory Syndrome Virus Isolates from Eastern China with RT-PCR-RFLP%猪繁殖与呼吸综合征病毒华东地区分离株RT-PCR-RFLP分析

    Institute of Scientific and Technical Information of China (English)

    邓雨修; 姜平; 李玉峰; 王先炜; 蒋文明; 汤景元

    2004-01-01

    Porcine reproductive and respiratory syndrome virus(PRRSV) isolates identified in samples from 50 field cases originated from eastern China herds were studied. ORF5 gene of PRRSV isolates was amplified by reverse transcript polymerase chain reaction(RT-PCR) and restriction fragment length polymorphism (RFLP) patterns for enzymes Mlu Ⅰ, Hinc Ⅱ and Sac Ⅱ were determined on these ORF5 genes. The RFLP pattern obtained from 39 isolates was 2-2-1(78%), 3 isolates was 1-1-1(6%) and 8 isolates identified in 2003 was 1-3-1 (16%). The results showed PRRSV strains have variations and existed no less than 3 gene subtypes in the area.

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

    Directory of Open Access Journals (Sweden)

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

    Science.gov (United States)

    Ma, Jian-Gang; Li, Fa-Cai; Zhao, Quan; 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. PMID:27806069

  5. Using T-RFLP data on denitrifier community composition to inform understanding of denitrification in stream sediments (Invited)

    Science.gov (United States)

    Wang, S.; Somers, K.; Sudduth, E.; Hassett, B.; Bernhardt, E. S.; Urban, D. L.

    2010-12-01

    We used terminal restriction fragment length polymorphism (T-RFLP), a molecular fingerprinting method, to characterize denitrifier communities in sediments taken from 48 study streams in North Carolina, USA. In addition to characterizing denitrifier communities, we also used denitrification enzyme activity (DEA) assays to measure potential denitrification rates. Due to differences in watershed land-use, study streams covered a gradient of nitrogen and carbon concentrations, as well as a gradient of contaminant loading from stormwater and sanitary sewers. Nitrogen and carbon (i.e., substrate) concentrations are commonly used to make predictions about denitrification rates in streams. Such models do not take into account denitrifier community composition, which may be an important, independent control of denitrification rates, particularly under stressful conditions (e.g., high contaminant loading) that prevent communities from capitalizing on high substrate availability. Our results indicate that substrate availability by itself was a weak predictor of denitrification rates; the same was also true for denitrifier community composition. However, when both factors were incorporated in a multiple regression model, the percent variation explained increased substantially. These findings suggest that T-RFLP, a relatively cost-effective method, can be used to improve our understanding of controls on denitrification rates in streams with varying watershed land-uses.

  6. Identification of Elymus (Triticeae, Poaceae) and its related genera genomes by RFLP analysis of PCR-amplified Adh genes.

    Science.gov (United States)

    Liu, QuanLan; Zhang, NingNing; Li, Lei; Liu, Jie

    2010-10-01

    Elymus L. is the largest genus in Triticeae, containing about 150 species with four recognized genome donors (St, H, P, and W). Traditionally, the genome compound of this genus is identified based on cytological data. Recently, molecular phylogenetic analysis was used to investigate its genomic combination. Here we describe a restriction fragment length polymorphism (RFLP) assay based on digesting alcohol dehydrogenase (Adh) amplicons with two restriction enzyme combinations, EcoRI-HindIII and EcoRI-PstI, which easily can be used to distinguish Elymus and its closely related genera genomes. The method includes only four steps: (1) amplifying nuclear Adh genes with universal primers; (2) purifying and cloning PCR products; (3) digesting plasmids with restriction enzymes that identify a given genome; (4) running the digested products on an agarose gel and identify the sample based on the restriction profiles. Results showed that: (1) PCR products ranged from 1,200 to 2,000 bp; (2) Adh2 gene was amplified from all the tested genomes; Adh1 gene was amplified from almost all of the tested genomes except the W genome; Adh3 gene was amplified only from the St genome; (3) the EcoRI-HindIII combination was effective to distinguish different Adh gene types (Adh1, Adh2, and Adh3); (4) the Adh2-EcoRI-PstI fragments could be used to distinguish Elymus and its closely related genera genomes. Therefore, This RFLP assay provides an inexpensive and simple means of identifying Elymus genomes.

  7. 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.

  8. Variation in CAG and GGN repeat lengths and CAG/GGN haplotype in androgen receptor gene polymorphism and prostate carcinoma in Nigerians.

    Science.gov (United States)

    Akinloye, O; Gromoll, J; Simoni, M

    2011-01-01

    Prostate cancer has become the most common cancer in Nigerian men. The growth of the prostate gland depends on circulating androgens and intracellular steroid signalling pathways. The effects of androgens are mediated through the androgen receptor (AR), a nuclear transcription factor encoded by the AR gene. The common polymorphisms, CAG and GGN repeats, in exon 1 of this gene have been implicated as possible risk factors. Thus far, existing supporting data are scanty and none are from sub-Saharan African populations. Therefore, this study investigates the possible association between AR polymorphism repeat length (CAG and GGN) and prostate cancer in Nigerians. A total of 261 subjects (70 with prostate cancer, 68 with benign prostate hyperplasia [BPH], 123 age-matched apparently normal subjects as controls) were studied. CAG and GGN repeats length were determined by fragment length analysis using GeneScan. The CAG repeat length in prostate cancer and in BPH compared to the controls was significantly different (P CAG repeats showing a significant odds ratio (OR) in both cases. However, this was not observed in GGN repeat length, which showed no significant difference between cases and controls (P > 0.05). CAG and GGN haplotype variation showed no significant difference between cases and controls (P > 0.05), except that the haplotypes CAG > or =21 and GGN CAG repeat length is a risk factor for prostate cancer, and also suggests an association with BPH.

  9. Genetic variation among isolates of Sarcocystis neurona, the agent of protozoal myeloencephalitis, as revealed by amplified fragment length polymorphism markers.

    Science.gov (United States)

    Elsheikha, H M; Schott, H C; Mansfield, L S

    2006-06-01

    Sarcocystis neurona causes serious neurological disease in horses and other vertebrates in the Americas. Based on epidemiological data, this parasite has recently emerged. Here, the genetic diversity of Sarcocystis neurona was evaluated using the amplified fragment length polymorphism (AFLP) method. Fifteen S. neurona taxa from different regions collected over the last 10 years were used; six isolates were from clinically diseased horses, eight isolates were from wild-caught opossums (Didelphis virginiana), and one isolate was from a cowbird (Molothrus ater). Additionally, four outgroup taxa were also fingerprinted. Nine primer pairs were used to generate AFLP patterns, with a total number of amplified fragments ranging from 30 to 60, depending on the isolate and primers tested. Based on the presence/absence of amplified AFLP fragments and pairwise similarity values, all the S. neurona isolates tested were clustered in one monophyletic group. No significant correlation could be found between genomic similarity and host origin of the S. neurona isolates. AFLP revealed significant intraspecific genetic variations, and S. neurona appeared as a highly variable species. Furthermore, linkage disequilibrium analysis suggested that S. neurona populations within Michigan have an intermediate type of population structure that includes characteristics of both clonal and panamictic population structures. AFLP is a reliable molecular technique that has provided one of the most informative approaches to ascertain phylogenetic relationships in S. neurona and its closest relatives, allowing them to be clustered by relative similarity using band matching and unweighted pair group method with arithmetic mean analysis, which may be applicable to other related protozoal species.

  10. Discrimination of Klebsiella pneumoniae and Klebsiella oxytoca phylogenetic groups and other Klebsiella species by use of amplified fragment length polymorphism.

    Science.gov (United States)

    Jonas, Daniel; Spitzmüller, Bettina; Daschner, Franz D; Verhoef, Jan; Brisse, Sylvain

    2004-01-01

    Bacteria of the genus Klebsiella are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. The two clinically and epidemiologically most important species, Klebsiella pneumoniae and K. oxytoca, have recently been shown to be subdivided into three and two phylogenetic groups, respectively. The aim of this study was an in depth evaluation of the amplified fragment length polymorphism (AFLP) genetic characterization method for epidemiological and phylogenic analyzes of Klebsiella isolates. First, we investigated the variability of AFLP patterns for Klebsiella strains within and between different outbreaks. Second, by use of carefully characterized phylogenetically representative strains, we examined whether different Klebsiella species and phylogenetic groups can be discriminated using AFLP. Twenty-four strains originating from seven presumed outbreaks and 31 non-associated strains were investigated. The AFLP fingerprints of all epidemiologically associated strains showed three or fewer fragment differences, whereas unrelated strains differed by at least four fragments. Cluster analysis of the AFLP data revealed a very high concordance with the phylogenetic assignation of strains based on the gyrA sequence and ribotyping data. The species K. pneumoniae, K. oxytoca, K. terrigena and the possibly synonymous pair K. planticola/K. ornithinolytica each formed a separate cluster. Similarly, strains of the phylogenetic groups of K. pneumoniae and K. oxytoca fell into their corresponding clusters, with only two exceptions. This study provides a preliminary cut-off value for distinguishing epidemiologically non-related Klebsiella isolates based on AFLP data; it confirms the sharp delineation of the recently identified phylogenetic groups, and demonstrates that AFLP is suitable for identification of Klebsiella species and phylogenetic groups.

  11. Genome-wide macrosynteny among Fusarium species in the Gibberella fujikuroi complex revealed by amplified fragment length polymorphisms.

    Science.gov (United States)

    De Vos, Lieschen; Steenkamp, Emma T; Martin, Simon H; Santana, Quentin C; Fourie, Gerda; van der Merwe, Nicolaas A; Wingfield, Michael J; Wingfield, Brenda D

    2014-01-01

    The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.

  12. KARAKTERISTIK GENETIK Kappaphycus alvarezii SEHAT DAN TERINFEKSI PENYAKIT ICE-ICE DENGAN METODE Amplified Fragment Length Polymorphism (AFLP

    Directory of Open Access Journals (Sweden)

    Emma Suryati

    2013-03-01

    Full Text Available Infeksi penyakit ice-ice pada Kappaphycus alvarezii seringkali menyebabkan penurunan produksi yang sangat signifikan. K. alvarezii merupakan alga merah penghasil karaginan yang memiliki nilai ekonomi tinggi dan banyak dimanfaatkan dalam berbagai industri, seperti farmasi, makanan, stabilizer, dan kosmetik. Perbaikan genetik sangat diperlukan untuk meningkatkan produksi. Penelitian ini bertujuan untuk mengetahui karakteristik kemiripan genetik K. alvarezii sehat dan terinfeksi penyakit dari Balai Penelitian dan Pengembangan Budidaya Air Payau (BPPBAP, Maros dengan metode Amplified Fragment Length Polymorphism (AFLP. Pada penelitian ini juga dianalisis K. alvarezii asal Bone (BNE, Gorontalo (GRL, Tambalang (TMB, dan Kendari (KND sebagai kontrol rumput laut sehat. Metode AFLP menggunakan enzim restriksi Psti dan Mset, preamplifikasi dan amplifikasi selektif diawali dengan isolsi DNA, uji genimoc DNA, restriksi dan ligasi. Hasil yang diperoleh menunjukkan penggunaan marker AFLP dengan primer forward P11 dan primer reverse M48, M49 dan M50 terhadap K. alvarezii yang berasal dari Takalar (TKL, dan Mataram (MTR, tanpa infeksi (sehat dan terinfeksi penyakit Takalar ice (TKL+, Mataram ice (MTR+, serta K. alvarezii kontrol (BNE, (GRL, (TMB, dan (KND menghasilkan 519 fragmen dalam 122 lokus dengan ukuran 50 - ~370 pb. Kemiripan genetik K. alvarezii yang terinfeksi penyakit ice-ice lebih rendah jika dibandingkan dengan yang sehat. Kemiripan genetik K. alvarezii dari Takalar sehat (TKL dan terinfeksi ice-ice (TKL+ adalah 0,8176 dan MTR-MTR+ adalah 0,8033.

  13. The Effect of Polymorphisms in DNA Repair Genes and Carcinogen Metabolizers on Leukocyte Telomere Length: A Cohort of Healthy Spanish Smokers.

    Science.gov (United States)

    Verde, Zoraida; Reinoso-Barbero, Luis; Chicharro, Luis; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

    2016-04-01

    Smoking implies exposure to carcinogenic agents that causes DNA damage, which could be suspected to enhance telomere attrition. To protect and deal with DNA damage, cells possess mechanisms that repair and neutralize harmful substances. Polymorphisms altering DNA repair capacity or carcinogen metabolism may lead to synergistic effects with tobacco carcinogen-induced shorter telomere length independently of cancer interaction. The aim of this study was to explore the association between leukocyte telomere length (LTL) and several genetic polymorphisms in DNA repair genes and carcinogen metabolizers in a cohort of healthy smokers. We evaluated the effect of six genetic polymorphisms in cytochrome P1A1 (Ile462Val), XRCC1 (Arg399Gln), APEX1 (Asp148Glu), XRCC3 (Thr241Met), and XPD (Asp312Asn; Lys751Gln) on LTL in a cohort of 145 healthy smokers in addition to smoking habits. Logistic regression analysis showed an association between XRCC1 399Gln allele and shorter telomere length (OR = 5.03, 95% CI = 1.08% to 23.36%). There were not association between the rest of polymorphisms analyzed and LTL. Continuous exposure to tobacco could overwhelm the DNA repair machinery, making the effect of the polymorphisms that reduce repair capacity more pronounced. Analyzing the function of smoking-induced DNA-repair genes and LTL is an important goal in order to identify therapeutic targets to treat smoking-induced diseases. © The Author 2015. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Relationship between a lipoprotein lipase gene polymorphism in placental tissue and insulin resistance in patients with gestational diabetes mellitus.

    Science.gov (United States)

    Li, D D; Su, D Y; Xue, L; Gao, W; Pang, W Y

    2015-07-14

    The aim of this study was to investigate the relationship between a lipoprotein lipase (LPL) gene polymorphism in placental tissue and insulin resistance (IR) in patients with gestational diabetes mellitus. Using polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) analysis, the LPL HindIII RFLP was examined in the placental tissue of 110 patients with gestational diabetes mellitus (observation group) and 110 women with normal gestation (control group). The relationships between fasting plasma glucose (FPG), postprandial plasma glucose (PPG), fasting insulin (FINS), cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), body mass index (BMI), and IR indices and the LPL polymorphism in the two study groups and their offspring were determined. The frequency of the H+ allele was significantly higher in the observation group than in the controls (P diabetes, and was also found to be related to the IR of their offspring.

  15. PCR-restriction fragment length polymorphism assay for detection of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli.

    Science.gov (United States)

    Alonso, Rodrigo; Mateo, Estibaliz; Girbau, Cecilia; Churruca, Estibaliz; Martinez, Irati; Fernández-Astorga, Aurora

    2004-12-01

    A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.

  16. Genotyping of Candida orthopsilosis Clinical Isolates by Amplification Fragment Length Polymorphism Reveals Genetic Diversity among Independent Isolates and Strain Maintenance within Patients▿

    OpenAIRE

    Tavanti, Arianna; Hensgens, Lambert A. M.; Ghelardi, Emilia; Campa, Mario; Senesi, Sonia

    2007-01-01

    Candida parapsilosis former groups II and III have recently been established as independent species named C. orthopsilosis and C. metapsilosis, respectively. In this report, 400 isolates (290 patients) previously classified as C. parapsilosis by conventional laboratory tests were screened by BanI digestion profile analysis of the secondary alcohol dehydrogenase gene fragment and by amplification fragment length polymorphism (AFLP). Thirty-three strains collected from 13 patients were identifi...

  17. GISH, AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and Poncirus trifoliata.

    Science.gov (United States)

    Fu, C H; Chen, C L; Guo, W W; Deng, X X

    2004-11-01

    Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2 n=4 x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.

  18. 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.

  19. 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. PMID:27716792

  20. The V109G polymorphism in the p27 gene is associated with endometriosis.

    Science.gov (United States)

    Camargo-Kosugi, Cíntia M; da Silva, Ismael D C G; Sato, Hélio; D'Amora, Paulo; Carvalho, Cristina V; Nogueira-de-Souza, Naiara C; Girão, Manoel J C B; Schor, Eduardo

    2009-08-01

    To investigate the prevalence of the p27 gene polymorphism in women with endometriosis. Transversal case-control study. Genomic DNA was extracted from cells collected from buccal swabs. The p27 V109G polymorphism was investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in a hospital-based Brazilian population. We analysed the 104 patients and 109 control subjects. The distribution of genotype and allele frequencies of p27 V109G polymorphism was significantly different between the endometriosis cases and healthy women (p=0.016 and 0.002). Women who had at least one mutated allele presented twofold chances for endometriosis development (OR=1.9; 95% CI, 1.120-3.343). The polymorphic variant at codon 109 of the p27 gene seems to be associated with higher risk of endometriosis development.

  1. Analyses of the Genus Aschersonia and Related Genera Using 28S rDNA RFLP Technique%座壳孢及其相近属PCR-RFLP分析

    Institute of Scientific and Technical Information of China (English)

    邱君志; 陈莹; 马慧斐

    2006-01-01

    Eighteen isolates of entomopathogenic fungi, including Aschersonia, Lecanicillium and Beauveria were analyzed by RFLP(restriction fragment length polymorphism) analysis of a 28S rRNA gene fragment. The data obtained by four different endonucleases were used to construct phenograms based on the UPGMA algorithm.Polymorphisms were found between genera and species, with fewer among isolates within species. PCR-RFLP within the 28S rRNA gene was sufficient to distinguish between Aschersonia species. RFLP data showed genetic diversity in Lecanicillium isolates at an infraspecific level. The UPGMA analysis results indicated the closer taxonomic affinity of Lecanicillium to Beauveria compared to Aschersonia.%利用核糖体28S rRNA基因的RFLP对18个昆虫病原真菌(座壳孢、轮枝菌和白僵菌属)菌株进行了分析.用4种限制性内切酶对PCR产物进行了消化并采用UPGMA法对扩增产物的限制性图谱构建了亲缘图.结果表明,属间、种间的多态性高于同种不同菌株间的多态性,并可以清楚地将座壳孢属不同种区分开来,而且轮枝菌不同菌株间存在着遗传差异.轮枝菌与白僵菌间的亲缘关系比其与座壳孢近.

  2. Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

    Science.gov (United States)

    Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

    2010-04-01

    Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

  3. Genetic diversity among wild and cultivated barley as revealed by RFLP

    DEFF Research Database (Denmark)

    Petersen, L.; Østergård, H.; Giese, H.

    1994-01-01

    Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp....... spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns...... an intermediate level. The proportion of gene diversity residing among,geographical groups (F-ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups....

  4. High Degree of Plasmodium vivax Diversity in the Peruvian Amazon Demonstrated by Tandem Repeat Polymorphism Analysis

    Science.gov (United States)

    Kosek, Margaret; Yori, Pablo P.; Gilman, Robert H.; Calderon, Maritza; Zimic, Mirko; Chuquiyauri, Raul; Jeri, Cesar; Pinedo-Cancino, Viviana; Matthias, Michael A.; Llanos-Cuentas, Alejandro; Vinetz, Joseph M.

    2012-01-01

    Molecular tools to distinguish strains of Plasmodium vivax are important for studying the epidemiology of malaria transmission. Two sets of markers—tandem repeat (TR) polymorphisms and MSP3α—were used to study Plasmodium vivax in patients in the Peruvian Amazon region of Iquitos. Of 110 patients, 90 distinct haplotypes were distinguished using 9 TR markers. An MSP3α polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using HhaI and AluI revealed 8 and 9 profiles, respectively, and 36 profiles when analyzed in combination. Combining TR and PCR-RFLP markers, 101 distinct molecular profiles were distinguished among these 110 patients. Nine TR markers arrayed along a 100 kB stretch of a P. vivax chromosome containing the gene for circumsporozoite protein showed non-linear linkage disequilibrium (ISA = 0.03, P = 0.001). These findings demonstrate the potential use of TR markers for molecular epidemiology studies. PMID:22492139

  5. High Degree of Plasmodium vivax Diversity in the Peruvian Amazon Demonstrated by Tandem Repeat Polymorphism Analysis

    OpenAIRE

    Kosek, Margaret; Yori, Pablo P.; Gilman, Robert H.; Calderon, Maritza; Zimic, Mirko; CHUQUIYAURI, RAUL; JERI, CESAR; Pinedo-Cancino, Viviana; Michael A Matthias; Llanos-Cuentas, Alejandro; Vinetz, Joseph M.

    2012-01-01

    Molecular tools to distinguish strains of Plasmodium vivax are important for studying the epidemiology of malaria transmission. Two sets of markers—tandem repeat (TR) polymorphisms and MSP3α—were used to study Plasmodium vivax in patients in the Peruvian Amazon region of Iquitos. Of 110 patients, 90 distinct haplotypes were distinguished using 9 TR markers. An MSP3α polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using HhaI and AluI revealed 8 and 9 profiles, res...

  6. emm Gene Polymorphism among Streptococcus pyogenes Isolated from

    Directory of Open Access Journals (Sweden)

    Mollaii Hamid

    2009-10-01

    Full Text Available DNA sequencing is the most conclusive method for emm (M protein gene typing of Streptococcus pyogenes. This method is not a feasible approach in developing countries where streptococcal infection is widespread among adults and children. Alternatively the PCR-RFLP has the potential for rapid screening of different types of S. pyogenes. To document the emm type distribution of S. pyogenes in a group of patients suffering from pharyngitis, the restriction fragment length polymorphism (RFLP profile of 50 isolates were analyzed. By using Hae III+ HincII (double digestion and Dde I restriction enzymes and based on RFLP, the profile patterns of the isolates were compared. The analysis of data identified 15 distinct RFLP patterns for Hae III+ Hinc II and 13 patterns for Dde I. They differ from each other by at least one band. Although the number of isolates was not sufficient to make any epidemiological conclusion, but the finding demonstrated that the S. pyogenes population among pateints was heterogeneous. Regarding the PCR method, we managed to improve the results by modification of CDC protocol in three different ways. This study was conducted in normal circumstances when pharyngitis was at the peak seasonal incident. However emm amplicon restriction digest analysis is a valuable tool for rapid analysis of S. pyogenes infection in more important situation like outbreaks and in selected type of study like consideration of nosocomial infection.

  7. Identification of Rabbit Myostatin Gene Polymorphisms

    Directory of Open Access Journals (Sweden)

    T. I. Amalianingsih

    2015-08-01

    Full Text Available The existence of selection on the rabbits with potential for meat has only been seen from phenotypic aspects including performance and productivity, while the molecular genetic studies are still very rare. One of the candidate genes for meat production traits in rabbit is myostatin. Totally 50 blood samples of male rabbits from Rex, Satin, Reza (crossing from Rex and Satin, Flemish Giant and FZ3 (crossing from Flemish Giant and Reza breed were used at Indonesian Research Institute for Animal Production (IRIAP. Genetic polymorphism by Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP method used FspBI restriction enzyme. PCR-RFLP data were analyzed by calculating allele and genotype frequencies. Sequencing was performed in rabbit with different genotypes which represents each of the samples. Genotype of AT had two cut points of the FspBI restriction enzyme at the base position of 508 bp and 444 bp. The cut point at the base position of 446 bp was site mutation base T became A. Genotype of TT had one cut point at the base position of 508 bp and no mutation site. Allele T had higher frequency than allele A and just Rex and Reza rabbit breeds had two alleles. The other rabbits (Satin, Flemish Giant and FZ3 only had one allele i.e., allele T. PCR - RFLP analysis of the MSTN|FspBI gene segments was polymorphic in Rex and Reza rabbit breeds. All of rabbit breeds in this study did not have AA genotype.

  8. Comparative Study of Campylobacter spp. Isolated from Children With Gastroenteritis in Bahonar Hospital, Karaj, Using PCR and RFLP

    Directory of Open Access Journals (Sweden)

    Abdi

    2016-05-01

    Full Text Available Background Campylobacter species are responsible for the majority of cases of food-borne gastroenteritis. The sources of the disease outbreaks are often contaminated water or milk, and consumption of undercooked poultry product is the main cause of sporadic campylobacteriosis cases. Objectives The aims of this study were to determine the prevalence of Campylobacter gastroenteritis in children and to differentiate the interfering species using polymerase chain reaction (PCR and restriction fragment length polymorphism (RFLP methods at the Bahonar hospital in Karaj, Iran. Patients and Methods A total of 150 stool samples were collected from children under 10 years old during the summer of 2014. PCR was performed using genus- and species-specific primers and RFLP was done using AluI and TasI enzymes. Results The results showed the amplification of 400 and 491 bp segments and Campylobacter contamination in 30 (20% samples; 5 out of 30 Campylobacter positive samples (16.66% were identified as C. jejuni, 20 (66.66% as C. coli, 3 (10% as C. jejuni and C. coli (mixed infection, and 2 (6.66% were identified as non-jejuni, non-coli Campylobacter using the PCR method. Following the evaluation of RFLP results, 7 positive samples (23.33% showed the electrophoretic pattern of C. jejuni, 21 (70% showed the electrophoretic pattern of C. coli, and 2 (6.6% showed both of the patterns and mixed contamination with jejuni and coli species. The results of digestion with TasI did not show any C. lari or C. upsaliensis patterns. Conclusions The results of this study showed high percentage of Campylobacter contamination in the tested stool samples. The other surprising finding was the high rate of Campylobacter coli positive samples; the difference between the results of PCR using species-specific primers (hipo and asp and the RFLP method (electrophoretic patterns in some of the positive samples confirms the hypothesis of variations in nucleotide sequences of the hipo

  9. Comparative Study of Campylobacter spp. Isolated from Children With Gastroenteritis in Bahonar Hospital, Karaj, Using PCR and RFLP

    Directory of Open Access Journals (Sweden)

    Arefeh Abdi

    2016-05-01

    Full Text Available Background: Campylobacter species are responsible for the majority of cases of food-borne gastroenteritis. The sources of the disease outbreaks are often contaminated water or milk, and consumption of undercooked poultry product is the main cause of sporadic campylobacteriosis cases. Objectives: The aims of this study were to determine the prevalence of Campylobacter gastroenteritis in children and to differentiate the interfering species using polymerase chain reaction (PCR and restriction fragment length polymorphism (RFLP methods at the Bahonar hospital in Karaj, Iran. Patients and Methods: A total of 150 stool samples were collected from children under 10 years old during the summer of 2014. PCR was performed using genus- and species-specific primers and RFLP was done using AluI and TasI enzymes. Results: The results showed the amplification of 400 and 491 bp segments and Campylobacter contamination in 30 (20% samples; 5 out of 30 Campylobacter positive samples (16.66% were identified as C. jejuni, 20 (66.66% as C. coli, 3 (10% as C. jejuni and C. coli (mixed infection, and 2 (6.66% were identified as non-jejuni, non-coli Campylobacter using the PCR method. Following the evaluation of RFLP results, 7 positive samples (23.33% showed the electrophoretic pattern of C. jejuni, 21 (70% showed the electrophoretic pattern of C. coli, and 2 (6.6% showed both of the patterns and mixed contamination with jejuni and coli species. The results of digestion with TasI did not show any C. lari or C. upsaliensis patterns. Conclusions: The results of this study showed high percentage of Campylobacter contamination in the tested stool samples. The other surprising finding was the high rate of Campylobacter coli positive samples; the difference between the results of PCR using species-specific primers (hipo and asp and the RFLP method (electrophoretic patterns in some of the positive samples confirms the hypothesis of variations in nucleotide sequences of the

  10. LD2SNPing: linkage disequilibrium plotter and RFLP enzyme mining for tag SNPs

    Directory of Open Access Journals (Sweden)

    Cheng Yu-Huei

    2009-06-01

    Full Text Available Abstract Background Linkage disequilibrium (LD mapping is commonly used to evaluate markers for genome-wide association studies. Most types of LD software focus strictly on LD analysis and visualization, but lack supporting services for genotyping. Results We developed a freeware called LD2SNPing, which provides a complete package of mining tools for genotyping and LD analysis environments. The software provides SNP ID- and gene-centric online retrievals for SNP information and tag SNP selection from dbSNP/NCBI and HapMap, respectively. Restriction fragment length polymorphism (RFLP enzyme information for SNP genotype is available to all SNP IDs and tag SNPs. Single and multiple SNP inputs are possible in order to perform LD analysis by online retrieval from HapMap and NCBI. An LD statistics section provides D, D', r2, δQ, ρ, and the P values of the Hardy-Weinberg Equilibrium for each SNP marker, and Chi-square and likelihood-ratio tests for the pair-wise association of two SNPs in LD calculation. Finally, 2D and 3D plots, as well as plain-text output of the results, can be selected. Conclusion LD2SNPing thus provides a novel visualization environment for multiple SNP input, which facilitates SNP association studies. The software, user manual, and tutorial are freely available at http://bio.kuas.edu.tw/LD2NPing.

  11. Investigation of Genetic Diversity of Fusarium oxysporum f. sp. fragariae Using PCR-RFLP

    Science.gov (United States)

    Kim, Ji-Su; Kang, Nam Jun; Kwak, Youn-Sig; Lee, Choungkeun

    2017-01-01

    Fusarium wilts of strawberry, caused by Fusarium oxysporum f. sp. fragariae, is a serious soil-borne disease. Fusarium wilt causes dramatic yield losses in commercial strawberry production and it is a very stubborn disease to control. Reliable chemical control of strawberry Fusarium wilt disease is not yet available. Moreover, other well-known F. oxysporum have different genetic information from F. oxysporum f. sp. fragariae. This analysis investigates the genetic diversity of strawberry Fusairum wilt pathogen. In total, 110 pathogens were isolated from three major strawberry production regions, namely Sukok, Hadong, Sancheong in Gyeongnam province in South Korea. The isolates were confirmed using F. oxysporum f. sp. fragariae species-specific primer sets. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses were executed using the internal transcribed spacer, intergenic spacer, translation elongation factor1-α, and β-tubulin genes of the pathogens and four restriction enzymes: AluI, HhaI, HinP1I and HpyCH4V. Regarding results, there were diverse patterns in the three gene regions except for the β-tubulin gene region. Correlation analysis of strawberry cultivation region, cultivation method, variety, and phenotype of isolated pathogen, confirmed that genetic diversity depended on the classification of the cultivated region. PMID:28381961

  12. Growth hormone genotyping by MspI restriction enzyme and PCR-RFLP method in Aceh cattle breed at Indrapuri District, Aceh Province, Indonesia

    Directory of Open Access Journals (Sweden)

    WIDYA PINTAKA BAYU PUTRA

    2014-04-01

    Full Text Available Putra WPB, Hartatik T, Sumadi. 2014. Growth hormone genotyping by MspI restriction enzyme and PCR-RFLP method in Aceh cattle breed at Indrapuri District, Aceh Province, Indonesia. Biodiversitas 15: 1-5. The objective of this research was to identify growth hormone (GH gene in Aceh cattle at Indrapuri’s Breeding and Forage Centre (IBFC of Aceh Cattle. Fourty one cattle consisting of 21 male and 20 female cattle were used in this study. Polymerase Chain Reaction (PCR - Restriction Fragment Length Polymorphism (RFLP and sequencing method was used to detect MspI site on GH gene. Based on the sequencing, it can be concluded that all cattle were monomorphic. The frequency of TT genotype and T allele were 1.00 relatively. The transition of C (cytosine into T (thymine on 1549 position caused the lost of restriction site. The insertion of T and G (guanine on 1542 and 1552 position caused the length of GH gene were 329 bp in Aceh cattle.

  13. 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.

  14. Association of restriction fragment length polymorphism at the atrial natriuretic peptide gene locus with aldosterone responsiveness to angiotensin in aldosterone-producing adenoma.

    Science.gov (United States)

    Tunny, T J; Jonsson, J R; Klemm, S A; Ballantine, D M; Stowasser, M; Gordon, R D

    1994-11-15

    Primary aldosteronism is an important, potentially curable, form of hypertension. We examined the possible association between restriction fragment length polymorphisms in the atrial natriuretic peptide (ANP) gene and responsiveness of aldosterone to angiotensin II in 59 patients with primary aldosteronism due to aldosterone-producing adenoma (APA). Significant differences in the allelic frequencies of the BglI, TaqI and XhoI polymorphic sites at the ANP gene locus (chromosome 1; 1p36) between angiotensin II-unresponsive and angiotensin II-responsive tumors were observed. Variation in the ANP gene between the two groups may result in altered expression of ANP within the adrenal gland, and may contribute to the biochemical regulation of aldosterone production of these two subgroups of patients with APA.

  15. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    Directory of Open Access Journals (Sweden)

    Pooja H. Gupta

    2015-05-01

    Full Text Available Aim: An attempt has been made to study the toll-like receptors 4 (TLR4 gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR, respectively from Kankrej (22 and Triple cross (24 cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05. Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05. Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

  16. Identification and molecular epidemiology of Campylobacter coli isolates from human gastroenteritis, food, and animal sources by amplified fragment length polymorphism analysis and Penner serotyping

    DEFF Research Database (Denmark)

    Siemer, B.L.; Nielsen, Elsa; On, Stephan L.w.

    2005-01-01

    Campylobacter coli is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish...... isolates of diverse origin identified by routine phenotyping as C coli were examined. Molecular tools identified some 12% of field isolates as Campylobacter jejuni, emphasizing the need for improved identification methods in routine laboratories. Cluster analysis of AFLP profiles of 174 confirmed C. coli...

  17. Molecular and functional diversity of PGPR fluorescent Pseudomonads based on 16S rDNA-RFLP and RAPD markers.

    Science.gov (United States)

    Singh, Bhim Pratap

    2015-09-01

    The genetic and functional diversity of plant growth promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with chickpea (Cicer arietinum L.) rhizosphere was analyzed. In total, 34 isolates along with two reference isolates were screened for various plant growth promoting traits (phosphorous solubilization, ACC deaminase, HCN, IAA and siderophore productions) and antagonist activity against four fungal phytopathogens and three bacterial pathogens. Most of the isolates, that showed PGPR activity, also showed antagonistic activity against all the three fungal pathogens. The genetic relationship was assessed by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (16S rDNA-RFLP). Relationship between both the markers was analyzed based on mantel test and a negative correlation was observed. The study concluded that PGPR traits appeared to be strain specific rather than specific to any phylogenetic group. The study also reported that 16S rDNA based profiling differentiated PGPR fluorescent Pseudomonas on the basis of location rather than biological trait. RAPD profiling could be useful to differentiate among the closely related isolates. The genetic and functional diversity of fluorescent pseudomonads, associated with the chickpea rhizosphere, has useful ecological role and potential utilization in sustainable agriculture.

  18. Application of Reverse Transcription-Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method to Identify H1 Genotype of Measles Virus Infection and Measles Vaccine Related Cases%应用限制性片段长度多态性分析方法鉴别H1基因型麻疹野病毒感染病例和麻疹疫苗相关病例

    Institute of Scientific and Technical Information of China (English)

    李立群; 余文; 赵智娴; 丁峥嵘

    2012-01-01

    Objective Application the method to rapid identify the wild measles strain and China measles vaccine strain in Yunnan province. Method To apply method of reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) and sequence analysis method to detect 8 strains of measles virus. Results The RT-PCR-RFLP method for H1 genotype was used for identifying 8 strains of measles virus, the 5 specimens of wild-type, measles viruses RT-PCR products were cut into 2 fragments, 408 bp and 159 bp, the results showed that 5 specimens of wild-type measles viruses were caused by H1 genotype. The RT-PCR-RFLP method for China vaccine strain identification was used for identifying 3 specimen of measles. 1 specimen RT-PCR products were cut into 2 fragments, 287 bp and 151 bp, which showed that this case was caused by China vaccine strain. 2 specimens of measles were non-H1 wild-type measles viruses. Conclusion The RT-PCR-RFLP method can be used to identify H1 genotype of measles virus and measles vaccine strain. The results are same as the method of nucleoprotein gene sequencing.%目的 在云南省应用H1基因型麻疹野病毒鉴定方法,鉴别H1基因型麻疹野病毒感染病例,应用中国麻疹疫苗株病毒鉴定方法,鉴别接种麻疹疫苗后出现的麻疹样病例是否为麻疹疫苗相关病例.方法 采用逆转录-聚合酶链反应-限制性片段长度多态性分析(Reverse Transcription-Polymerase Chain Reaction-Restriction Fragment Length Polymorphism,RT-PCR-RFLP)方法、序列分析方法鉴别8株麻疹病毒.结果 应用H1基因型麻疹野病毒RT-PCR-RFLP方法对8份标本进行鉴定,其中5份标本RT-PCR产物经 Sal I酶切后,被切成两个片段,分别为408个碱基对(base pair,bp)和159bp,显示5个病例是由H1基因型麻疹野病毒引起的.应用中国麻疹疫苗株病毒RT-PCR-RFLP方法对3份标本进行鉴定,其中1份标本RT-PCR产物经Af1Ⅱ酶切,被切成两个片段,分别为287

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

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... Producing meat tenderness that consumers desire is one of the major problems facing ... The calpain system may also affect the number of skele- tal muscle cell ... tube and was transferred to -20oC freezer. Genomic DNA was.

  20. 用等位基因特异性PCR/限制性片段长度多态性策略构建原发性青光眼致病基因CYP1B1的单倍型%Construction of CYP1B1 gene haplotypes predisposing to primary congenital glaucoma through allele-specific PCR/restriction fragment length polymorphism analysis

    Institute of Scientific and Technical Information of China (English)

    张爱平; 李圣杰; 欧阳琦; 汤荔; 王晓蕾; 吉建; 曹文俊

    2015-01-01

    目的 建立等位基因特异性PCR/限制性片段长度多态性(allele-specific PCR/restriction fragment length polymorphism,AS-PCR/RFLP)法检测原发性先天性青光眼(primary congenital glaucoma,PCG)致病基因CYP1B1常见单核苷酸多态性(single nucleotide polymorphisms,SNPs)及其单倍型的方法.方法 收集20例原发性先天性青光眼患者和20名正常对照为研究对象,首先经测序筛查SNP位点后,再分别以PCR-RFLP和AS-PCR/RFLP策略构建CYP1B1基因rs10012(S1)和rs1056827(S2)及rs1056836(S3)和rs1056837(S4)位点的单倍型,并对这两种策略进行评价.结果 测序共发现4个SNP位点,为第2外显子rs10012 G/C(S1)及rs1056827 T/G (S2)、第3外显子rs1056836C/G(S3)及rs1056837T/C(S4).这些位点在PCG患者和正常对照中的分布呈现不同特点,同时存在rs10012 (S1)和rs1056827(S2)位点的PCG患者和正常对照分别为10例(50%)和5人(25%);同时存在rs1056836 (S3)和rs1056837(S4)位点的PCG患者和正常对照分别为5例(25%)和2人(10%);均未发现上述SNP位点单独存在.确定各位点的分布特点后,首先用PCR-RFLP策略构建rs10012(S1)和rs1056827(S2)位点单倍型,显示杂合突变型除出现目的条带外还存在底物条带,虽提示同时存在rs10012(S1)和rs1056827 (S2),但仍不能证实存在位点间的连锁;AS-PCR/RFLP构建的结果显示,AS-PCR扩增rs10012(S1)位点获得阳性结果的同时,针对rs1056827 (S2)位点的特异性RFLP分析也获得阳性结果.AS-PCR/RFLP对位点rs1056836(S3)和rs1056837 (S4)的分析获得了类似的结果.应用AS-PCR/RFLP成功构建了C-G[rs10012(S1)-rs1056827(S2)]和G-C[rs1056836 (S3)-rs1056837 (S4)]两种单倍型.结论 应用AS-PCR/RFLP策略成功构建PCG致病基因CYP1B1单倍型,该方法准确高效特异可用于构建遗传性疾病基因的单倍型.%Objective To develop an allele-specific PCR (AS-PCR)/restriction fragment length polymorphism (RFLP) assay for CYP1B1 gene haplotypes

  1. Polymorphisms detection of intron 20 of Chinese Holstein STAT4 gene using PCR-RFLP method%利用PCR-RFLP方法检测中国荷斯坦牛STAT4基因内含子20多态性

    Institute of Scientific and Technical Information of China (English)

    张磊; 宋雪梅; 姜俊芳; 蒋永清

    2012-01-01

    This study was aimed to establish a simple detective method for polymorphisms of Chinese Holstein STAT4 gene intron 20. Three hundred and fifty-seven Chinese Holstein cows were used in this study. Firstly, the polymorphism of intron 20 of STAT4 gene was detected by PCR-SSCP method combined with DNA sequencing analysis. Then the detective method was transformed from PCR-SSCP to PCR-RFLP according to DNA sequencing results of the mutant SSCP patterns. The results showed that intron 20 of STAT4 gene had a complete linkage haplotype block ( g. 95879 G > A and g. 96013 G > C. A). Sdu I enzyme detective system was established according to the characteristic that the 96013 G→C mutation may be formed as a restriction enzyme Sdu I polymorphism. By this detective system, three different genotypes, GGGG, GAGC and AACC were found, and the corresponding genotype frequencies were 0. 0784, 0. 4538 and 0. 4678. The frequencies of alleles GG and AC were 0. 3053 and 0. 6947, respectively.%以浙江省金华市伊康乳业奶牛场核心群357头中国荷斯坦牛为试验材料,旨在建立一种中国荷斯坦牛STAT4基因内含子20的单核苷酸多态性的简便检测方法.首先采用PCR-SSCP检测,结合DNA测序方法对STAT4基因内含子20的遗传多态性进行分析,并根据突变形式将PCR-SSCP检测方法转化为PCR-RFLP检测方法.结果发现STAT4基因内含子20内存在由g.95879 G>A和g.96013 G>C组成的完全连锁的单倍型模块.根据96013G→C的突变可形成一个SduⅠ酶切多态位点的特性,建立了该单倍型的Sdu Ⅰ酶切检测体系.利用该体系检测到中国荷斯坦牛群体STAT4内含子20内存在GGGG,GAGC和AACC 3种不同的基因型,基因型频率分别为0.0784,0.4538和0.4678,而GG和AC基因频率分别为0.3053和0.6947.

  2. Genetic Mapping of Laminaria japonica and L. longissima Using Amplified Fragment Length Polymorphism Markers in a "Two-Way Pseudo-Testcross" Strategy

    Institute of Scientific and Technical Information of China (English)

    Yuhui Li; Yingxia Yang; Jidong Liu; Xiuliang Wang; Tianxiang Gao; Delin Duan

    2007-01-01

    With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

  3. Genotyping of Candida albicans using length fragment and high-resolution melting analyses together with minisequencing of a polymorphic microsatellite locus.

    Science.gov (United States)

    Costa, Jean-Marc; Garcia-Hermoso, Dea; Olivi, Martine; Cabaret, Odile; Farrugia, Cécile; Lecellier, Gaël; Dromer, Françoise; Bretagne, Stéphane

    2010-03-01

    Microsatellite length polymorphism (MLP) typing is a PCR-based method used for genotyping of the diploid yeast Candida albicans. However, MLP is subject to homoplasia which can hamper the accuracy of the results. We combined fragment length analysis, high-resolution DNA melting (HRM) analysis, and SNaPshot minisequencing after a single amplification of the CDC3 locus to study 95 epidemiologically independent C. albicans isolates. HRM analysis for a given electrophoretic group led to a maximum of three different curves due to the presence of a SNP upstream of the tandem repeat which could be characterized using the SNaPshot assay. The combination of the three methods had a discriminatory index of 0.88 in complete congruence with previous MLP typing (Mantel test R=0.99, P<10(-)(4)). HRM is a useful tool of adding resolving power to MLP genotyping in identifying SNPs.

  4. The relationship between MHC-DRB1 gene second exon polymorphism and hydatidosis resistance of Chinese merino (Sinkiang Junken type), Kazakh and Duolang sheep

    OpenAIRE

    Li R.Y.; Hui W.Q.; Jia B; Shi G.Q.; Zhao Z.S.; Shen H.; Peng Q; Lv L.M.; Zhou Q.W.; Li H.T.

    2011-01-01

    The present study aimed at detecting the association of ovine major histocompatibility complex class II (Ovar II) DRB1 gene second exon and susceptibility or resistance to hydatidosis in three sheep breeds of Sinkiang. The MHC-DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of healthy sheep and sheep with hydatidosis. PCR products were characterized by the restriction fragment length polymorphism (RFLP) technique. Five restriction enzymes, MvaI, HaeIII, SacI...

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Observation of high seasonal variation in community structure of denitrifying bacteria in arable soil receiving artificial fertilizer and cattle manure by determining T-RFLP of nir gene fragments

    DEFF Research Database (Denmark)

    Priemé, Anders; Wolsing, Martin

    2004-01-01

    Temporal and spatial variation of communities of soil denitrifying bacteria at sites receiving mineral fertilizer (60 and 120 kg N ha-1 year-1) and cattle manure (75 and 150 kg N ha-1 year-1) were explored using terminal restriction fragment length polymorphism (T-RFLP) analyses of PCR amplified...... a significant seasonal shift in the community structure of nirK-containing bacteria. Also, sites treated with mineral fertilizer or cattle manure showed different communities of nirK-containing denitrifying bacteria, since the T-RFLP patterns of soils treated with these fertilizers were significantly different...... nitrite reductase (nirK and nirS) gene fragments. The analyses were done three times during the year: in March, July and October. nirK gene fragments could be amplified in all three months, whereas nirS gene fragments could be amplified only in March. Analysis of similarities in T-RFLP patterns revealed...

  7. TNFR 2 M196R Polymorphism and Acne Vulgaris in Han Chinese:A Case-control Study

    Institute of Scientific and Technical Information of China (English)

    田黎明; 谢红付; 杨婷; 胡耀华; 李吉; 王玮蓁

    2010-01-01

    In this case-control study,the relationship between M196R(676 T→G) variant in exon 6 of tumor necrosis factor receptor type 2(TNFR2) gene and genetic susceptibility of acne vulgaris in Han Chinese was investigated.A total of 93 acne vulgaris patients and 90 healthy subjects from Han Chinese ethnic group were enrolled in this study.Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique was adopted to analyze the single nucleotide polymorphisms(SNPs) of TNFR2 M196R gene,and to ...

  8. 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

  9. 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

  10. ASSOCIATION OF PARATHYROID HORMONE GENE POLYMORPHISM WITH BONE MINERAL DENSITY IN CHINESE WOMEN

    Institute of Scientific and Technical Information of China (English)

    李梅; 孟迅吾; 周学瀛; 邢小平; 余卫

    2003-01-01

    Objective. To investigate the distribution frequency of parathyroid hormone(PTH) gene polymorphism in healthy adults from Bejing area and to explore the association of PTH genotypes with bone mineral density (BMD). Methods. PTH gene polymorphism was detected in 270 subjects by polymerase chain reaction (PCR) and PCR/restriction fragment length polymorphism (PCR/RFLP). The digestion products of restriction enzyme Bst B1 were separated on 1% agarose gels. PTH genotypes were confirmed by DNA sequences analysis. BMD was measured by dual-energy X-ray absorptiometry (DEXA, DPX- L, Lunar). Results. Genotype frequencies of BB, Bb, bb were 73.7% , 25.9% and 0.4% respectively in Beijing adults( P0.05). Conclusion. PTH gene polymorphism is not associated with BMD in Chinese women. The further research to explore the genetic risk factors of osteoporosis should be committed.

  11. Research on the relativity between gene polymorphism and children cardiac insufficiency.

    Science.gov (United States)

    He, X-H; Li, C-L; Ling, N; Wang, Q-W; Wang, Z-Z; An, X-J

    2017-08-01

    We analyzed the relationship between Mink-S27 gene polymorphism and children with cardiac insufficiency. From April 2013 to April 2015, we enrolled 73 cases of children with cardiac insufficiency for this study, and all 73 were placed in the observation group. 76 normal cases were selected for the control group. Restriction fragment length polymorphism (RFLP) was used to make polymorphism analysis of the Mink-S27. Our results showed no significant differences in Mink-S27 genotype and allele distribution in both observation and control groups (p>0.05). In lesion samples collected from children with cardiac insufficiency, we detected significant difference in AA, CC genotype frequency and allele frequency between the observation group and the control group (prelatively high. GNAS2 gene polymorphism was associated with the prevalence of cardiac insufficiency in children. And also the patients' condition was correlated to the frequency of different genotypes and alleles.

  12. POLYMORPHISM OF ANGIOTENSIN Ⅱ TYPE 1 RECEPTOR GENE IN ELDERLY PATIENTS WITH ESSENTIAL HYPERTENSION

    Institute of Scientific and Technical Information of China (English)

    方宁远; 张怡; 陆惠华; 郑迪辉; 邬亦贤; 郑道声

    2003-01-01

    Objective To detect the A/C1166 polymorphism of angiotensin Ⅱ type-1 receptor (AT1 R) gene in essential hypertensive elderly.MethodsThe A/C1166 polymorphism of AT1 R gene was assessed by polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) in a case control study of 87 essential hypertensive elders (EH) and 55 normotensive elders (NT).ResultsThe genotype frequencies of AA, AC, CC were 0.805, 0.161, 0.034 in EH group and 0.927, 0.073, 0.000 in NT group respectively. The frequency of C1166 allele was higher in EH group (0.115) than in NT group (0.036)(P<0.05).ConclusionThe results indicate that A/C1166 polymorphism of AT1 R gene may be associated with essential hypertension in elderly.

  13. [AGTR1 A1166C polymorphism is associated with risk of diabetic nephropathy].

    Science.gov (United States)

    Yin, Xueyao; Li, Hong; Xuan, Junli; Chen, Yixin; Li, Lin; Dong, Xuehong

    2013-01-01

    To investigate whether the angiotensin II type I receptor gene (AGTR1) A1166C polymorphism is associated with a high risk of diabetic nephropathy. The allele frequency and the genotype distribution of the AGTR1 A1166C polymorphism were studied in normal controls (157 cases), simple diabetes (141 cases, duration of diabetes >10 years), and diabetic nephropathy (152 cases) by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Patients with diabetic nephropathy had a higher frequency of C allele of the AGTR1 A1166C polymorphism than that of normal controls and simple diabetes (PAGTR1 C allele may be more susceptible to diabetic nephropathy than diabetic patients with A allele.

  14. Comparison of PCR-RFLP Based on Ribosomal Regions and SSR Markers in Genetic Diversity of Pistachio Die-Back Caused by Paecilomyces variotii

    Directory of Open Access Journals (Sweden)

    Rostami

    2015-01-01

    Full Text Available Background In recent years, die-back of pistachio has become one of the most important diseases in Kerman gardens. With regard to the importance of this disease and the lack of comprehensive information regarding the population genetic structure of the pathogen, it is necessary to set an appropriate indicator in the study of genetic diversity. Objectives In the present study, we examined simple sequence repeats (SSRs and restriction fragment length polymorphism (PCR-RFLPs (two PCR-based marker assays to determine Paecilomyces variotti genetic diversity. Materials and Methods The utility of SSRs and PCR-RFLPs was examined to determine genetic diversity using 20 isolates of Paecilomyces variotii. In order to determine the performance of indicators, effective multiplex ratio (EMR, polymorphism information content (PIC, and marker index (MI were calculated. Results Both systems discriminated 20 isolates of P. variotii successfully but were different in the amount of detectable polymorphism. Using cluster analysis of digestion reaction, SSR based on UPGMA algorithm, and Jaccard similarity coefficient, the isolates with 70% similarity level were divided into 7 and 3 groups, respectively. Reviewed indicators were at higher level for PCR-RFLPs marker. Four restriction endonucleases enzymes in RFLP produced 20 loci that 90% of them were polymorphic; and for SSR it was 32 loci that 37.5% were polymorphic. Conclusions This is the first research in comparing two genetic marker systems in P. variotti. We were prompted to explore polymorphisms utility in P. variotti with a look at using germplasm screening mapping of genome and strain improvement programs.

  15. Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP).

    Science.gov (United States)

    Ni, Xiaowei; Huang, Yelin; Wu, Lin; Zhou, Renchao; Deng, Shulin; Wu, Darong; Wang, Bosun; Su, Guohua; Tang, Tian; Shi, Suhua

    2006-05-01

    Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H (E) = 0.220, I (S) = 0.321), and at species level (P = 85.6% of markers polymorphic, H (E) = 0.339, I (S) = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei's genetic diversity analysis (G (st)=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.

  16. PCR and RFLP variation of conserved region of small subunit ribosomal DNA among Acanthamoeba isolates assigned to either A. castellanii or A. polyphaga.

    Science.gov (United States)

    Kong, H H; Chung, D I

    1996-06-01

    Twelve isolates of Acanthamoeba spp. assigned to either A. castellanii or A. polyphaga, and type strains of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were examined by restriction fragment length polymorphism (RFLP) of a conserved region of small subunit ribosomal RNA gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellaii was 9.8% whereas that among the isolates assigned to A. polyphaga 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. castellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. polyphaga was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. castellanii and A. polyphaga (2.6%) which appeared between the Castellani (or CCAP 1501/2 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphaga. It is suggested that taxonomic validity of the isolates assigned to either A. castellanii or A. polyphaga should be reevaluated.

  17. Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica for PCR-RFLP Based Species Identification

    Directory of Open Access Journals (Sweden)

    Chandra Mohan Siddappa

    2013-01-01

    Full Text Available Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species.

  18. 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.

  19. Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR-RFLP.

    Science.gov (United States)

    Zaeemi, Mahdieh; Haddadzadeh, Hamidreza; Khazraiinia, Parvaneh; Kazemi, Bahram; Bandehpour, M

    2011-04-01

    Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions that leads to economic losses in these animals. A nested PCR-restriction fragment length polymorphism (RFLP) was carried out to identification Theileria species in sheep in some area in western half of Iran (Sari, Rasht, Urmia, Ilam, and Ahvaz). Two hundred and fifty blood samples were taken from sheep during tick activating season (summer of 2008). Microscopic examination revealed that 9.2% (23/250) sheep were infected by Theileria spp. piroplasms. Parasitemia ranged from 0.011% to 0.015%. In nested PCR assessment of DNA samples, 32.8% (82/250) sheep were positive. The negative samples were confirmed by amplifying of ovine beta-actin gene as an internal control. The differentiation of Theileria species was based on RFLP patterns using three restriction enzymes: HpaII, Rsa1, and Bsh 1285I. Out of 82 positive samples, 54.8% (45/82) and 40.2% (33/82) were positive for Theileria lestoquardi and Theileria ovis respectively. Mixed infection was detected in 4.8% (4/82) cases. Based on their PCR product digestion pattern with HpaII (1178, 900, 278, and 106 bp), it seemed to be mixture of Theileria annulata and T. lestoquardi. The presence of T. annulata was supported by sequence analysis. This is the first report of naturally infected sheep with T. annulata in Iran. Geographical distribution of Theileria species in sheep is shown according to the result of microscopy and nested PCR and RFLP data.

  20. PCR-RFLP Analysis of 28 SrDNA for Specification of Fasciola gigantica (Cobbold, 1855 in the Infected Lymnaea auricularia (Linnaeus, 1785 Snails from Northwestern Iran.

    Directory of Open Access Journals (Sweden)

    Mohammad Yakhchali

    2014-09-01

    Full Text Available Fasciolosis in livestock is a crucial concern in the globe, mainly due to its impact on human health. The aim of this study was to determine the rate of infection with Fasciola gigantica (Cobbold, 1855 larvae in the field-collected snails of Lymnaea auricularia (Linnaeus, 1785 from northwestern Iran using a molecular approach.A total of 6,759 pond snails were collected from 28 freshwater bodies in West Azarbaijan. PCR was performed to amplify a 618-bp fragment of the nuclear 28 SrRNA gene of Fasciola. The PCR products were digested by AvaII restriction enzyme to create restriction fragment length polymorphism (RFLP patterns specific for the detection of F. gigantica.Of the total collected snails 496 (7.34 % were L. auricularia, among which 4.64% (23 out of 496 were infected with a Fasciola species according to the PCR analysis. Only 2.22% (11 out of 496 of the infected snails were from the mountainous areas. The highest Fasciola infection rate recorded in the snails of a single site was 1.81% (9 out of 496 snails. Based on the RFLP pattern, F. gigantica accounted for 2.42% of the infection rates in the study sites.Application of PCR-RFLP was proven to be a useful approach for valid and robust detection of the infection with F. gigantica in its intermediate host snails. These findings may therefore be applicable for establishment of the control programs against dissemination of the infection in different regions.

  1. Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR.

    Science.gov (United States)

    Quaresma, Patrícia Flávia; Murta, Silvane Maria Fonseca; Ferreira, Eduardo de Castro; da Rocha-Lima, Ana Cristina Vianna Mariano; Xavier, Ana Amélia Prates; Gontijo, Célia Maria Ferreira

    2009-09-01

    The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.

  2. Study of the Genetic Resemblance of tpi Gene in Cat and Human Giardia Using PCR-RFLP Method

    Directory of Open Access Journals (Sweden)

    Ali Pezeshki

    2013-06-01

    Full Text Available Background & Objectives: Giardia duodenalis is the most common intestinal parasite with cosmopolitan distribution. This parasite has been found in the intestine of humans and other mammalian hosts including cats, dogs, cattle, sheep, deer, pigs and muskrats. It is postulated that animals maybe reservoir for human infection and viceversa. In present study, the possible genetic similarity between cat and humans Giardia and its probable zoonosis were investigated.   Methods: Direct examination and formalin-ether concentration techniques were performed on stray and semi stray cat fecal specimens. Gradient sucrose method was applied for collection and purification of cysts and DNA extraction was performed by phenol-chloroform and CTAB (Cetyl trimethyl ammonium bromid ( methods. DNA of cysts could hardly be extracted after repeated freezing and thawing. Polymerase chain reaction (PCR was performed for DNA amplification. In this study triosephosphate isomerase (tpi gene was selected as a molecular marker. Two sets of primers (PM 290 and PM 924 were considered. Two restriction enzymes RsaI and AvaI were also used to determine restriction fragment length polymorphism (RFLP for PCR fragments amplified by both primer sets.   Results: Ten samples were positive for Giardia cysts which were examined for molecular investigation. Four cat isolates were amplified by PM 290. PCR-RFLP patterns were found to be similar to human isolates AC≠AF 069556 (subgroup of AC≠U 57897 with possibility of cross-transmission.   Conclusion: Therefore the similarity of genomic characters of isolates of cat and human Giardia implies possibility of zoonosis and transmission of these protozoa from cat to human and vice versa.

  3. Genetic characterization of Toxoplasma gondii in Yunnan black goats (Capra hircus) in southwest China by PCR-RFLP.

    Science.gov (United States)

    Miao, Qiang; Huang, Si-Yang; Qin, Si-Yuan; Yu, Xin; Yang, Yan; Yang, Jian-Fa; Zhu, Xing-Quan; Zou, Feng-Cai

    2015-01-27

    Toxoplasma gondii is a protozoan parasite that infects almost all warm-blooded animals and human beings. Goats are one of the susceptible animals to T. gondii. However, little is known of genetic diversity of T. gondii in Yunnan black goats in China. The objective of this present study was to determine the genotypes of T. gondii isolates from black goats in Yunnan province, southwest China. Genomic DNA was extracted from liver (n = 403), lung (n = 403) and lymph nodes (n = 250) of Yunnan black goats and assayed for T. gondii infection by semi-nested PCR of B1 gene. Then, the positive DNA samples were typed at 10 genetic markers using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. These markers include 9 nuclear loci, namely, SAG1, SAG2 (5'-SAG2 and 3'-SAG2, alternative SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast locus Apico. Out of 403 tested samples, 20 (4.96%) DNA samples were T. gondii positive by amplification of B1 gene. Among them, 2 isolates were genotyped at all loci, and 6 isolates were genotyped for 8 or more loci. In total, seven samples belong to ToxoDB PCR-RFLP genotype#10 (Type I), and one belongs to genotype ToxoDB #9. To our knowledge, this is the first report of ToxoDB#9 and ToxoDB#10 T. gondii in Yunnan black goats in China. These results revealed a wide distribution of these T. gondii in Yunnan black goats in China, which has important implications for public health.

  4. 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...

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

    Directory of Open Access Journals (Sweden)

    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

  6. Genetic and epigenetic diversity and structure of Phragmites australis from local habitats of the Songnen Prairie using amplified fragment length polymorphism markers.

    Science.gov (United States)

    Qiu, T; Jiang, L L; Yang, Y F

    2016-08-19

    The genetic and epigenetic diversity and structure of naturally occurring Phragmites australis populations occupying two different habitats on a small spatial scale in the Songnen Prairie in northeastern China were investigated by assessing amplified fragment length polymorphisms (AFLPs) and methylation-sensitive amplified polymorphisms (MSAPs) through fluorescent capillary detection. The two groups of P. australis were located in a seasonal waterlogged low-lying and alkalized meadow with a pH of 8-8.5 and in an alkaline patch without accumulated rainwater and with a pH greater than 10. These groups showed high levels of genetic diversity at the habitat level based on the percentage of polymorphic bands (90.32, 82.56%), Nei's gene diversity index (0.262, 0.248), and the Shannon diversity index (0.407, 0.383). Although little is known about the between-habitat genetic differentiation of P. australis on a small spatial scale, our results implied significant genetic differentiation between habitats. Extensive epigenetic diversity within habitats, along with clear differentiation, was found. Specifically, the former habitat (Habitat 1, designated H1) harbored higher levels of genetic and epigenetic diversity than the latter (Habitat 2, designated H2), and population-level diversity was also high. This study represents one of few attempts to predict habitat-based genetic differentiation of reeds on a small scale. These assessments of genetic and epigenetic variation are integral aspects of molecular ecological studies on P. australis. Possible causes for within- and between-habitat genetic and epigenetic variations are discussed.

  7. Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

    Science.gov (United States)

    Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

    2017-01-01

    Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

  8. Genotyping of Candida orthopsilosis clinical isolates by amplification fragment length polymorphism reveals genetic diversity among independent isolates and strain maintenance within patients.

    Science.gov (United States)

    Tavanti, Arianna; Hensgens, Lambert A M; Ghelardi, Emilia; Campa, Mario; Senesi, Sonia

    2007-05-01

    Candida parapsilosis former groups II and III have recently been established as independent species named C. orthopsilosis and C. metapsilosis, respectively. In this report, 400 isolates (290 patients) previously classified as C. parapsilosis by conventional laboratory tests were screened by BanI digestion profile analysis of the secondary alcohol dehydrogenase gene fragment and by amplification fragment length polymorphism (AFLP). Thirty-three strains collected from 13 patients were identified as C. orthopsilosis, thus giving the first retrospective evidence that C. orthopsilosis was responsible for 4.5% of the infections/colonization attributed to C. parapsilosis. AFLP was proven to unambiguously identify C. orthopsilosis at the species level and efficiently delineate intraspecific genetic relatedness. A high percentage of polymorphic AFLP bands was observed for independent isolates collected from each patient. Statistical analysis of the pairwise genetic distances and bootstrapping revealed that clonal reproduction and recombination both contribute to C. orthopsilosis genetic population structure. AFLP patterns of sequential isolates obtained from two patients demonstrated that a successful strain colonization within the same patient occurred, as revealed by strain maintenance in various body sites. No association between AFLP markers and drug resistance was observed, and none of the clinical C. orthopsilosis isolates were found to produce biofilm in vitro.

  9. Female-only sex-linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii.

    Science.gov (United States)

    Jiang, Xue-Hui; Qiu, Gao-Feng

    2013-12-01

    Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex-reversed females (neomales). To provide molecular evidence for the proposed system, novel sex-linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male-specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female-specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female-specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female-linked SCAR markers can be applied for rapid detection of prawn gender. These sex-specific SCAR markers and sex-associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).

  10. A comparison of Peronospora parasitica (Downy mildew) isolates from Arabidopsis thaliana and Brassica oleracea using amplified fragment length polymorphism and internal transcribed spacer 1 sequence analyses.

    Science.gov (United States)

    Rehmany, A P; Lynn, J R; Tör, M; Holub, E B; Beynon, J L

    2000-07-01

    Amplified fragment length polymorphism (AFLP) fingerprints and internal transcribed spacer 1 (ITS1) sequences from 27 Peronospora parasitica isolates (collected from Arabidopsis thaliana or Brassica oleracea), 5 Albugo candida isolates (from the same hosts and from Capsella bursa-pastoris), and 1 Bremia lactucae isolate (from Lactuca sativa) were compared. The AFLP analysis divided the isolates into five groups that correlated with taxonomic species and, in most cases, with host origin. The only exception was a group consisting of A. candida isolates from both B. oleracea and C. bursa-pastoris. ITS1 sequence analysis divided the isolates into the same five groups, demonstrated the divergence between P. parasitica isolates from A. thaliana and B. oleracea, and, using previously published ITS1 sequences, clearly showed the relationship between A. candida isolates from different hosts.

  11. Use of pulsed field gel electrophoresis (PFGE and single-enzyme amplified fragment length polymorphism(SE-AFLP to subtype isolates of Salmonella entericaserotype enteritidis

    Directory of Open Access Journals (Sweden)

    Caterina Mammina

    2005-03-01

    Full Text Available

    Serotype Enteritidis is still the main serotype infecting humans and poultry worldwide. Subtyping of isolates belonging to this serotype is difficult, because of the wide clonal circulation of a few bacterial clones.

    This study presents the results of the characterization of 49 isolates of S. Enteritidis identified at the southern Italy Centre for Enteric Pathogens (CEPIM during the years 2002-2003 by the methods of Pulsed Field Gel Electrophoresis (PFGE and Single-Enzyme Amplified Fragment Length Polymorphism (SE-AFLP.

    Clustering of the strains by SE-AFLP and PFGE is very similar, but the first technique is more rapid and user-friendly and does not require sophisticated equipment. Further work is needed for a more accurate assessment of SEAFLP, but preliminary results suggest it could be a promising support to epidemiological investigations.

  12. Restriction fragment length polymorphism of the 5S-rRNA-NTS region: a rapid and precise method for plant identification.

    Science.gov (United States)

    Bertea, Cinzia Margherita; Gnavi, Giorgio

    2012-01-01

    Molecular genetic methods have several advantages over classical morphological and chemical analyses. The genetic method requires genotype instead than phenotype, therefore PCR-based techniques have been widely used for a rapid identification of plant species, varieties and chemotypes. Recently, the molecular discrimination of some higher plant species has been evaluated using sequences of a 5S-rRNA gene spacer region. The variation in the nontranscribed sequence (NTS) region has been used in a number of plant species for studying intraspecific variation, genome evolution, and phylogenetic reconstruction. Here, we describe a rapid method based on the use of the 5S-rRNA-NTS region as a tool for plant DNA fingerprinting, which combines PCR, sequencing and restriction fragment length polymorphism analyses.

  13. Polymerase chain reaction-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment for authentication of four clam species.

    Science.gov (United States)

    Fernandez, Alicia; García, Teresa; Gonzalez, Isabel; Asensio, Luis; Rodriguez, Miguel Angel; Hernández, Pablo E; Martin, Rosario

    2002-04-01

    Specific identification of four clam species, Ruditapes decussatus (grooved carpet shell), Venerupis pullastra (pullet carpet shell), Ruditapes philippinarum (Japanese carpet shell), and Venerupis rhomboides (yellow carpet shell), was achieved by polymerase chain reaction-restriction fragment length polymorphism analysis of a fragment of the mitochondrial 16S rRNA gene. Amplification of DNA isolated from the foot muscle produced fragments of 511 bp for V. pullastra, 523 bp for R. decussatus, 545 bp for R. philippinarum, and 502 bp for V. rhomboides. The restriction profiles obtained by agarose gel electrophoresis when amplicons were digested with endonucleases BsmAI and BsrI allowed unequivocal identification of the four clam species. This approach would be less costly, simpler, and quicker than conventional sequencing of polymerase chain reaction products followed by detailed comparison of individual sequences, especially when large numbers of samples need to be analyzed.

  14. [Determination of Leishmania species by PCR-RFLP in the smear samples taken from the lesions of cutaneous leishmaniasis cases].

    Science.gov (United States)

    Ertabaklar, Hatice; Ertuğ, Sema; Çalışkan, Serçin Özlem; Bozdoğan, Bülent

    2016-04-01

    The forms of the disease caused by Leishmania species in Turkey as well as in Aegean region are cutaneous and visceral leishmaniasis (CL and VL, respectively), and the agent of CL is commonly L.tropica. However, L.infantum was also reported as being CL agent recently. Direct microscopic examination, serological tests and culture are the conventional methods used for the diagnosis of CL. Since the specificities of these methods are high their sensitivities are variable and identification at species level is not possible. Recently, the use of polymerase chain reaction (PCR)-based molecular methods enabled the rapid and reliable diagnosis and species identification. The aim of this study was to investigate the performance of PCR-restriction fragment length polymorphism (RFLP) method both for the detection and identification of Leishmania species simultaneously in CL patients. A total of 30 smear samples that were positive for Leishmania amastigotes with microscopic examination, obtained from CL-suspected cases admitted to Adnan Menderes University Medical School Hospital, Parasitology Laboratory (located at Aydin, in the Aegean region of Turkey) between 2012-2014 period were included in the study. Ten samples taken from the skin lesions caused by Staphylococcus aureus (n= 5) and Candida albicans (n= 5) were also included as negative controls. DNA extractions from the smears were performed by the use of a commercial kit (Macherey-Nagel NucleoSpin Tissue® Kit, Germany). DNA isolation was also performed from L.major, L.infantum and L.tropica promastigotes that were grown in culture as positive controls. In PCR method LITSR and L5.8S primers targeting to ITS (internal transcribed spacer)-1 region were used. In RFLP method, the amplified PCR products were cleaved by BsuRI (HaeIII) restriction enzyme for the species identification. As a result, restriction profiles of all samples (n= 30) were in accordance with L.tropica restriction profile. No band was observed in the

  15. Effect of the Pro12Ala Polymorphism of the Peroxisome Proliferator-activated Receptor γ2 Gene on Lipid Profile and Adipokines Levels in Obese Subjects.

    Science.gov (United States)

    Becer, E; Çırakoğlu, A

    2017-06-30

    Peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of metabolism, adipokines production and secretion. The aim of this study was to investigate the association between the PPARγ2 gene Pro12Ala polymorphism in obesity in terms of body mass index (BMI), lipid parameters, homeostasis model assessment of insulin resistance (HOMA-IR), serum lipid, leptin, adiponectin, resistin and chemerin levels. The study included 160 obese and 140 non obese subjects. The Pro12Ala polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Serum lipid, leptin, adiponectin, resistin and chemerin levels were measured. No association was found between the Pro12Ala polymorphism and BMI. Strikingly, in the study group, obese subjects with the AA genotype had significantly higher triglycerides (p = 0.046) and resistin (p Pro12Ala polymorphism has no direct association with obesity but does have significant influences on lipid profiles and adipokines levels.

  16. Molecular-level secondary structure, polymorphism, and dynamics of full-length -synuclein fibrils studied by solid-state NMR

    Science.gov (United States)

    Heise, Henrike; Hoyer, Wolfgang; Becker, Stefan; Andronesi, Ovidiu C.; Riedel, Dietmar; Baldus, Marc

    2005-11-01

    The 140-residue protein -synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly 13C/15N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V, were analyzed. 13C and 15N signals exhibited linewidths of HR ALIGN=LEFT WIDTH=50% NOSHADE SIZE=1>

  17. Association between the TCF7L2 rs12255372 (G/T) gene polymorphism and type 2 diabetes mellitus in a Cameroonian population: a pilot study

    OpenAIRE

    Nanfa, Dieudonne; Sobngwi, Eugene; Atogho-Tiedeu, Barbara; Noubiap, Jean Jacques N; Donfack, Olivier Sontsa; Mofo, Edith Pascale Mato; Guewo-Fokeng, Magellan; Nguimmo Metsadjio, Aurelie; Ndonwi Ngwa, Elvis; Pokam Fosso, Priscille; Djahmeni, Eric; Djokam-Dadjeu, Rosine; Evehe, Marie-Solange; Aminkeng, Folefac; Mbacham, Wilfred F

    2015-01-01

    Background To study the relationship between the rs12255372 (G/T) polymorphism of the transcription factor 7-like 2 (TCF7L2) and type 2 diabetes mellitus (T2DM) in a Cameroonian population. Methods This case–control study included 60 T2DM patients and 60 healthy normoglycemic controls, all unrelated and of Cameroonian origin, aged above 40 years (range 40–87). The Restriction Fragment Length Polymorphism - Polymerase Chain Reaction (RFLP-PCR) was used for genotyping. Results The T allele freq...

  18. 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.

  19. 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.

  20. Mitochondrial DNA Polymorphism in Natural Populations of Drosophila albomicans (Ⅰ)——Remarkable mtDNA Polymorphism in the Population of D.albomicans

    Institute of Scientific and Technical Information of China (English)

    王文; 凌发瑶; 施立明

    1994-01-01

    The technique of mtDNA restriction fragments length polymorphism(RFLP)was used tosurvey the population structure of D.albomicans.Remarkable mtDNA polymorphism has been observed inD.albomicans populations.A total of 34 nucleomorphs were detected from 82 isofemale lines assayed by only8 restriction enzymes.The cause and the effect of this phenomenon were discussed.As a result,it is sug-gested that a mechanism which maintains mtDNA diversity exists in this fly,and that the high intra-popula-tional polymorphism could numerically conceal the extent of differentiation between populations.In addition,on the base of restriction maps,it was found that the mtDNA molecule of D.albomicans might be impactedby the selection pressure during its evolution process both on the nucleotide composition and on the function-al regions.

  1. Genetic polymorphism of human glutathione S-transferase A1 gene in mainland Chinese and its association with phenotype

    Institute of Scientific and Technical Information of China (English)

    JiePING; HuiWANG

    2005-01-01

    AIM Human glutathione S-transferase A1 (GSTA1) is an important phase Ⅱ metabolizing enzyme involved in the metabolism of many therapeutic drugs and is responsible for the metabolic detoxification of numerous promutagens and procarcinogens. The genetic polymorphism of GSTA1 has important implications for drug efficacy and cancer susceptibility. In this study, we determined the distribution of GSTA1 genetic polymorphism in Mainland Chinese. And we also investigated whether there exists the potential phenotype alterations caused by the genetic polymorphism in human. METHODS Genomic DNA was ex-tracted from peripheral blood of 140 Chinese people and 16 liver tissues obtained from non-liverish patients who underwent partial hepatectomy. And then the genotypes of human GSTA1 gene were analyzed by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP).

  2. Molecular typing of nosocomial Staphylococcus aureus strains associated to biofilm based on the coagulase and protein A gene polymorphisms

    Science.gov (United States)

    Salehzadeh, Ali; Zamani, Hojjatolah; Langeroudi, Maedeh Keshtkar; Mirzaie, Amir

    2016-01-01

    Objective(s): Staphylococcus aureus is an important bacterial pathogen responsible for a variety numbers of nosocomial and community acquired infections. Biofilm formation is regarded as an important factor in the establishment of S. aureus infection. The contribution of the genetic background of S. aureus to biofilm formation is poorly understood. The aim of the present work was to genotype S. aureus strains associated to biofilm based on the coagulase and protein A genes and to evaluate the association between the genetic background and the biofilm forming ability of clinical S. aureus isolates. Materials and Methods: A total number of 100 S. aureus were isolated from nosocomial infections and biofilm formation capability was investigated using phenotypic assay and molecular detection of biofilm associated genes. The strains were genotyped based on coagulase (coa) and protein A (spa) gene polymorphisms using restriction fragments length polymorphism-polymerase chain reaction (RFLP-PCR). Results: RFLP-PCR of coa gene generated two types and three subtypes. Amplification of spa gene resulted in two banding patterns and their restriction digestion generated three subtypes. The combined coa and spa RFLP patterns generated nine genotypes (G1-G9). The genotypes G4 and G1 were the most prevalent (32.1% and 24.3%, respectively). Conclusion: High clonal diversity of S. aureus strains able to produce biofilm was observed. Biofilm formation correlates with the spa and coa clonal lineage in our population and testing for multiple gene polymorphisms could be employed for local epidemiologic purposes. PMID:28096965

  3. Molecular typing of nosocomial Staphylococcus aureus strains associated to biofilm based on the coagulase and protein A gene polymorphisms

    Directory of Open Access Journals (Sweden)

    Ali Salehzadeh

    2016-12-01

    Full Text Available Objective(s: Staphylococcus aureus is an important bacterial pathogen responsible for a variety numbers of nosocomial and community acquired infections. Biofilm formation is regarded as an important factor in the establishment of S. aureus infection. The contribution of the genetic background of S. aureus to biofilm formation is poorly understood. The aim of the present work was to genotype S. aureus strains associated to biofilm based on the coagulase and protein A genes and to evaluate the association between the genetic background and the biofilm forming ability of clinical S. aureus isolates. Materials and Methods: A total number of 100 S. aureus were isolated from nosocomial infections and biofilm formation capability was investigated using phenotypic assay and molecular detection of biofilm associated genes. The strains were genotyped based on coagulase (coa and protein A (spa gene polymorphisms using restriction fragments length polymorphism-polymerase chain reaction (RFLP-PCR. Results: RFLP-PCR of coa gene generated two types and three subtypes. Amplification of spa gene resulted in two banding patterns and their restriction digestion generated three subtypes. The combined coa and spa RFLP patterns generated nine genotypes (G1-G9. The genotypes G4 and G1 were the most prevalent (32.1% and 24.3%, respectively. Conclusion: High clonal diversity of S. aureus strains able to produce biofilm was observed. Biofilm formation correlates with the spa and coa clonal lineage in our population and testing for multiple gene polymorphisms could be employed for local epidemiologic purposes.

  4. PCR based RFLP genotyping of bovine lymphocyte antigen DRB3.2 in Iranian Holstein population.

    Science.gov (United States)

    Pashmi, Morteza; Qanbari, Saber; Ghorashi, Seyed Ali; Salehi, Abdolreza

    2007-02-01

    Major Histocompatibility Complex (MHC) class II locus DRB3 was investigated by PCR based restriction fragment length polymorphism (PCR-RFLP) assay. A total of 262 Holstein cows participating in the national recording system were sampled from 10 herds. A two-step polymerase chain reaction was carried out in order to amplify a 284 base-pair fragment of exon 2 of the target gene. Second PCR products were treated with three restriction endonucleas enzymes RsaI, BstYI and HaeIII. Digested fragments were analyzed by polyacrylamid gel electrophoresis. Twenty-eight BoLA-DRB3 alleles were identified. Identified alleles are: BoLA-DRB3.2 *3, *6, *7, *8, *9, *10, *11, *12, *13, *14, *15, *16,20, *21, *22, *23, *24, *25, *26, *27, *28, *32, *36, *37, *40, *51, *iaa and *ibb. The BoLA-DRB3.2*40 allele that was observed in this study has not been reported previously. The calculated frequencies were as follows: 2.29, 1.34, 0.19, 14.5, 0.38, 3.05, 12.21, 1.34, 2.29, 1.34, 2.48, 9.16, 0.95, 0.77, 6.68, 9.16, 17.94, 1.15, 0.57, 1.15, 0.95, 0.57, 0.38, 1.91, 0.38, 5.73, 0.19 and 0.95% respectively. The six most frequently observed alleles (BoLA-DRB3.2 *8, *11, *16, *22, *23 and *24) accounted for 69.65% of the alleles in these 10 herds. The results of this study confirm the allelic distribution of six most frequent alleles in Holstein population's worldwide.

  5. Specific detection of benzimidazole resistance in Colletotrichum gloeosporioides from fruit crops by PCR-RFLP.

    Science.gov (United States)

    Chung, Wen-Hsin; Chung, Wen-Chuan; Peng, Mun-Tsu; Yang, Hong-Ren; Huang, Jenn-Wen

    2010-02-28

    Anthracnose diseases, caused by Colletotrichum gloeosporioides, are a worldwide problem and are especially important in Taiwan owing to the severe economic damage they cause to tropical fruits that are grown for local consumption and export. Benzimidazoles are systemic fungicides widely used for controlling these diseases in Taiwan. Thirty-one isolates of C. gloeosporioides from mango and strawberry grown in Taiwan were examined for their sensitivity to benzimidazole fungicides. The responses of the isolates grown on benzimidazole-amended culture media were characterized as sensitive, moderately resistant, resistant or highly resistant. Analysis of point mutations in the beta-tubulin gene by DNA sequencing of PCR-amplified fragments revealed a substitution of GCG for GAG at codon 198 in resistant and highly resistant isolates and a substitution of TAC for TTC at codon 200 in moderately resistant isolates. A set of specific primers, TubGF1 and TubGR, was designed to amplify a portion of the beta-tubulin gene for the detection of benzimidazole-resistant C. gloeosporioides. Bsh1236I restriction maps of the amplified beta-tubulin gene showed that the resistant isolate sequence, but not the sensitive isolate sequence, was cut. The PCR restriction fragment length polymorphism (PCR-RFLP) was validated to detect benzimidazole-resistant and benzimidazole-sensitive C. gloeosporioides isolates recovered from avocado, banana, carambola, dragon fruit, grape, guava, jujube, lychee, papaya, passion fruit and wax apple. This method has the potential to become a valuable tool for monitoring the occurrence of benzimidazole-resistant C. gloeosporioides and for assessment of the need for alternative management practices. Copyright 2009 Elsevier B.V. All rights reserved.

  6. 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

    Directory of Open Access Journals (Sweden)

    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.

  7. Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis

    Science.gov (United States)

    Kerkhof, L.; Santoro, M.; Garland, J.

    2000-01-01

    In this report, we describe an experiment conducted at Kennedy Space Center in the biomass production chamber (BPC) using soybean plants for purification and processing of human hygiene water. Specifically, we tested whether it was possible to detect changes in the root-associated bacterial assemblage of the plants and ultimately to identify the specific microorganism(s) which differed when plants were exposed to hygiene water and other hydroponic media. Plants were grown in hydroponics media corresponding to four different treatments: control (Hoagland's solution), artificial gray water (Hoagland's+surfactant), filtered gray water collected from human subjects on site, and unfiltered gray water. Differences in rhizosphere microbial populations in all experimental treatments were observed when compared to the control treatment using both community level physiological profiles (BIOLOG) and molecular fingerprinting of 16S rRNA genes by terminal restriction fragment length polymorphism analysis (TRFLP). Furthermore, screening of a clonal library of 16S rRNA genes by TRFLP yielded nearly full length SSU genes associated with the various treatments. Most 16S rRNA genes were affiliated with the Klebsiella, Pseudomonas, Variovorax, Burkholderia, Bordetella and Isosphaera groups. This molecular approach demonstrated the ability to rapidly detect and identify microorganisms unique to experimental treatments and provides a means to fingerprint microbial communities in the biosystems being developed at NASA for optimizing advanced life support operations.

  8. Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis

    Science.gov (United States)

    Kerkhof, L.; Santoro, M.; Garland, J.

    2000-01-01

    In this report, we describe an experiment conducted at Kennedy Space Center in the biomass production chamber (BPC) using soybean plants for purification and processing of human hygiene water. Specifically, we tested whether it was possible to detect changes in the root-associated bacterial assemblage of the plants and ultimately to identify the specific microorganism(s) which differed when plants were exposed to hygiene water and other hydroponic media. Plants were grown in hydroponics media corresponding to four different treatments: control (Hoagland's solution), artificial gray water (Hoagland's+surfactant), filtered gray water collected from human subjects on site, and unfiltered gray water. Differences in rhizosphere microbial populations in all experimental treatments were observed when compared to the control treatment using both community level physiological profiles (BIOLOG) and molecular fingerprinting of 16S rRNA genes by terminal restriction fragment length polymorphism analysis (TRFLP). Furthermore, screening of a clonal library of 16S rRNA genes by TRFLP yielded nearly full length SSU genes associated with the various treatments. Most 16S rRNA genes were affiliated with the Klebsiella, Pseudomonas, Variovorax, Burkholderia, Bordetella and Isosphaera groups. This molecular approach demonstrated the ability to rapidly detect and identify microorganisms unique to experimental treatments and provides a means to fingerprint microbial communities in the biosystems being developed at NASA for optimizing advanced life support operations.

  9. Methanotrophic bacteria associated to rice roots: the cultivar effect assessed by T-RFLP and microarray analysis.

    Science.gov (United States)

    Lüke, Claudia; Bodrossy, Levente; Lupotto, Elisabetta; Frenzel, Peter

    2011-10-01

    Rice plants play a key role in regulating methane emissions from paddy fields by affecting both underlying processes: methane production and oxidation. Specific differences were reported for methane oxidation rates; however, studies on the bacterial communities involved are rare. Here, we analysed the methanotrophic community on the roots of 18 different rice cultivars by pmoA-based terminal restriction fragment length polymorphism (T-RFLP) and microarray analysis. Both techniques showed comparable and consistent results revealing a high diversity dominated by type II and type Ib methanotrophs. pmoA microarrays have been successfully used to study methane-oxidizing bacteria in various environments. However, the microarray's full potential resolving community structure has not been exploited yet. Here, we provide an example on how to include this information into multivariate statistics. The analysis revealed a rice cultivar effect on the methanotroph community composition that could be affiliated to the plant genotype. This effect became only significant by including the specific phylogenetic resolution provided by the microarray into the statistical analysis.

  10. Gene polymorphism of alpha-2 macroglobulin in patients with Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    HAO Yi-xin; FU Qiang; GUO Pin-e; ZHANG Jian-rong; SHEN Qian

    2005-01-01

    Objective: To explore the relationship between polymorphism of α2-macroglobulin (A2M)gene and Parkinson's disease (PD)in Han Nationality in Shanghai. Methods:The distributions of A2M gene polymorphism (a Val1000Ile in exon24, V/I)were detected in 66 PD patients and 120 healthy controls using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. Results:The I allelic frequency in A2M exon24 gene of PD patients (90.9 %) was significantly lower than that of the healthy controls(96.3%)(OR=0.39,P=0. 033),so was the I/I genotype(OR=0.32,P=0. 015),especially in the patients more than 60 years old (OR= 0.31 ,P= 0.04). Conclusion :The I allele in exon24 of A2M gene might inhibit the onset of PD in Han Nationality in Shanghai.

  11. Influence of p53 (rs1625895 polymorphism in kidney transplant recipients

    Directory of Open Access Journals (Sweden)

    Negar Azarpira

    2014-01-01

    Full Text Available Reperfusion injury predisposes the kidney allograft to acute rejection. Apoptosis is a mechanism that results in graft injury, and TP53 is an important involved gene. To determine the association between single nucleotide polymorphism (SNP in the pro-apoptotic protein p53 (rs1625895 and acute rejection in renal transplants, we studied 100 recipients of kidney allografts and 100 healthy individuals served as controls. The polymorphism was determined by the polymerase chain reaction restriction-fragment length polymorphism (PCR-RFLP test. Overall, 31 recipients developed rejection. There was no difference in the genotype frequencies between the recipients and the controls. However, we found a difference of genotype and allele frequencies between recipients with and those without rejection. The WW genotype was more frequent in recipients with rejection. Although rejection is a complex immunologic event and functional importance of SNPs has not been confirmed yet, we suggest that wild type p53 may promote apoptosis during inflammation.

  12. An association study between polymorphisms in five genes in glutamate and GABA pathway and paranoid schizophrenia.

    Science.gov (United States)

    Zhang, Boyu; Yuan, Yanbo; Jia, Yanbin; Yu, Xin; Xu, Qi; Shen, Yucun; Shen, Yan

    2005-01-01

    Dysfunctions of glutamatergic and GABAergic neurotransmission are two important hypotheses for the pathogenesis of schizophrenia. Thus, genes in the pathway are candidates for schizophrenia susceptibility. Phosphate-activated glutaminase (GLS), glutamine synthetase (GLUL), glutamic acid decarboxylase (GAD), GABA transaminase (ABAT) and succinic semialdehyde dehydrogenase (ALDH5A1) are five primary enzymes in glutamate and GABA synthetic and degradative pathway. In order to investigate the possible involvement of these genes in the development of paranoid schizophrenia, we genotyped 80 paranoid schizophrenics from northern China and 108 matched controls by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) methods or directly sequencing of PCR product. Seven SNPs were found to be polymorphic in the population investigated. No significant differences in the genotype distributions or allele frequencies between patients and controls were found. Therefore, we conclude the polymorphisms studied in the five genes do not play major roles in pathogenesis of paranoid schizophrenia in the population investigated.

  13. Study on relationship of apolipoprotein E gene polymorphism and genetic susceptibility of stress urinary incontinence

    Institute of Scientific and Technical Information of China (English)

    Tong Jia-li; Lang Jing-he; Zhu lan

    2010-01-01

    Objective: To explore the relationship between apolipoprotein E (ApoE) gene polymorphism and susceptibility of stress urinary incontinence (SUI).Methods: ApoE genotypes were examined by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) technique in 99 patients with SUI and 101 asymptomatic controls. Results: The frequency of allele e3 of ApoE was slightly lower in patients with anatomic SUI than that in controls (79.44% vs. 81.68%), while the frequency of allele e4 of ApoE was slightly higher in patients with anatomic SUI than that in controls (10.00% vs. 9.90%). No significant difference was found in frequency of allele e3 or e4 between SUI patients and controls (χ2=0.523, P=0.770).Conclusion: The gene polymorphism of ApoE is not independently involved in the development of SUI.

  14. PGC-1alpha Gly482Ser polymorphism associates with hypertension among Danish whites

    DEFF Research Database (Denmark)

    Andersen, Gitte; Wegner, Lise; Jensen, Dorit Packert

    2005-01-01

    . The common Gly482Ser polymorphism of PGC-1alpha has previously shown association with arterial hypertension among Austrian men. Thus, we aimed at investigating this relationship in the Danish white population. The Gly482Ser polymorphism was genotyped in a total of 2562 Danish white subjects using polymerase...... chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and a GenoView locked nucleic acid assay (LNA), and the relationships of this variant with blood pressure levels and arterial hypertension were analyzed. Furthermore, we performed a combined analysis of the data from the present study...... in combination with previously published results. The Ser/Ser genotype was significantly associated with a reduced risk of hypertension and with lower systolic, diastolic, and mean arterial blood pressure levels, predominantly among women. Finally, in a combined analysis using data obtained in both sexes...

  15. Diversity of the marine picocyanobacteria Prochlorococcus and Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed spacer sequences Diversidad de las picocianobacterias marinas Prochlorococcus y Synechococcus por medio de polimorfismos de longitud de fragmentos de restricción terminal en secuencias del espaciador transcrito interno del ARNr 16S - 23S

    Directory of Open Access Journals (Sweden)

    PARIS LAVIN

    2008-12-01

    Full Text Available In order to assess the appropriateness of the use of internal transcribed spacer (ITS sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the 16S-23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them by denaturing gradient gel electrophoresis (DGGE and terminal restriction fragment length polymorphisms (T-RFLP. When using the standard application of these techniques, we obtained more than one band or terminal restriction fragment (T-RF per strain or cloned sequence. Reports in literature have suggested that these anomalies can result from the formation of secondary structures. Secondary structures of the ITS sequences of Prochlorococcus and Synechococcus strains were computationally modelled at the different temperatures that were used during the polymerase chain reaction (PCR. Modelling results predicted the existence of hairpin loops that would still be present at the extensión temperature; it is likely that these loops produced incomplete and single stranded PCR products. We modified the standard T-RFLP procedure by adding the labelled ITS primer in the last two cycles of the PCR reaction; this resulted, in most cases, in only one T-RF per ribotype. Application of this technique to a natural picoplankton community in marine waters off northern Chile, showed that it was possible to identify the presence, and determine the relative abundance, of several phylogenetic lineages within the genera Prochlorococcus and Synechococcus inhabiting the euphotic zone. Phylogenetic analysis of ITS sequences obtained by cloning and sequencing DNA from the same sample confirmed the presence of the different genotypes. With the proposed modification, T-RFLP profiles should therefore be suitable for studying the diversity of natural populations of cyanobacteria, and should become an important tool to study the factors influencing the genetic structure and

  16. 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.

  17. Differentiation of Acidithiobacillus ferrooxidans and A. thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis.

    Science.gov (United States)

    Bergamo, Rogério F; Novo, Maria Teresa M; Veríssimo, Ricardo V; Paulino, Luciana C; Stoppe, Nancy C; Sato, Maria Inês Z; Manfio, Gilson P; Prado, Paulo Inácio; Garcia, Oswaldo; Ottoboni, Laura M M

    2004-09-01

    Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli.

  18. 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.

  19. 中国大豆疫霉菌群体遗传结构的RFLP分析%Genetic Diversity of Phytophthora sojae in China Based on RFLP

    Institute of Scientific and Technical Information of China (English)

    肖江涛; 苗苗; 高坤; 向桂林; 杨帅; 董莎萌; 王源超; 王克荣

    2011-01-01

    [目的]分析中国大豆疫霉菌的群体遗传结构,探索不同地区大豆疫霉菌群体间的亲缘关系.[方法]采用RFLP(restriction fragment length polymorphism)技术,对大豆疫霉菌进行群体遗传结构的分析;利用Popgene V1.32软件计算大豆疫霉菌群体间的遗传相似度;利用NTSYSpc V2.10软件估算菌株间的遗传距离,并依据遗传距离构建UPGMA(unweighted pair-group method with arithmetic averages)系统树状图谱.[结果]采用探针PS127558对来自黑龙江、新疆和内蒙古等5个大豆疫霉菌地理群体的133个菌株的遗传多样性进行了分析,共得到25条杂交带,其中多态性条带为24个,占96%.黑龙江分别和新疆、内蒙古群体间遗传相似度较高.相对于黑龙江群体遗传结构的高度复杂性,新疆和内蒙古菌株的群体遗传结构则比较简单,分别有88.2%和100%的菌株分属于同一聚类组,且均有超过58%的菌株和部分黑龙江菌株指纹图谱完全相同.Shannon's多样性指数也表明黑龙江群体的遗传多样性最为丰富.福建和其它群体之间的遗传相似度较低,且分别有45.5%和54.5%的福建群体都属于聚类组E和F,遗传背景较为单一.福建群体的Shannon's多样性指数低于黑龙江和美国.美国和黑龙江群体之间的遗传相似度较高,且部分美国菌株和黑龙江菌株指纹图谱相同.[结论]新疆、内蒙古的大豆疫霉菌起源于黑龙江,福建的大豆疫霉菌可能为一个独立的进化分支或起源于其它地区.此外,中国和美国的大豆疫霉菌群体间存在菌源交流.%[ Objective ] The objective of this study is to explore the genetic diversity among Phytophthora sojae strains from soybean-growing regions in China and America. [Method] Restriction fragment length polymorphism (RFLP) was used to examine the genetic relationships among strains of P. sojae collected from 5 different regions. Nei's genetic distances among the strains of P

  20. An AluI RFLP detected in the human prion protein (PrP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Harris, M.S.; Devine-Gage, E. (New York State Institute for Basic Research in Developmental Disabilities, Staten Island (USA)); Robakis, N.K. (Mt. Sinai School of Medicine, New York, NY (USA))

    1990-01-25

    Probe pEA974 contains a 974 bp fragment present in Prp Clone XIV inserted into pBR322. A PvuII RFLP has been previously described. AluI identifies a two allele polymorphism of either a band at 965 bp, a band at 565 bp, or two bands at 965 bp and 565 bp. The allele frequency was studied in 14 European Caucasians. The PrP gene has been sublocalized to the p arm of chromosome 20. Mendelian pattern of inheritance was shown in an informative family.

  1. A semester-long project for teaching basic techniques in molecular biology such as restriction fragment length polymorphism analysis to undergraduate and graduate students.

    Science.gov (United States)

    DiBartolomeis, Susan M

    2011-01-01

    Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky(73). Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers.

  2. Distribution of genes encoding putative virulence factors and fragment length polymorphisms in the vrrA gene among Brazilian isolates of Bacillus cereus and Bacillus thuringiensis.

    Science.gov (United States)

    Zahner, Viviane; Cabral, Diana Aparecida; Régua-Mangia, Adriana Hamond; Rabinovitch, Leon; Moreau, Gaétan; McIntosh, Douglas

    2005-12-01

    One hundred twenty-one strains of the Bacillus cereus complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (bceT, hblA, nheBC, plc, sph, and vip3A) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the vrrA open reading frame as the target. Amplicons were generated from isolates of B. cereus and Bacillus thuringiensis for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in Bacillus thuringiensis than in B. cereus. The vip3A sequence, which encodes an insecticidal toxin, was detected exclusively in B. thuringiensis. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that B. cereus and B. thuringiensis should be considered to represent a single species.

  3. Genetic alteration of penicillin non-susceptible Streptococcus pneumoniae observed throughout recurrence of acute otitis media detected by amplified fragment length polymorphism analysis.

    Directory of Open Access Journals (Sweden)

    Sugata K

    2001-06-01

    Full Text Available The prevalence of penicillin non-susceptible Streptococcus pneumoniae (PNSSP is increasing among isolates from acute otitis media (AOM. Repeated episodes of antibiotic exposure are a well-known risk factor for the isolation of PNSSP although otitis-prone or recurrent AOM cases frequently require repeated courses of antibiotic treatment. In order to evaluate the chronological alteration of S. pneumoniae during recurrences of AOM, strains of S. pneumoniae were isolated from 11 patients, each of whom had experienced 2-4 episodes of AOM, were examined. Every bacterial specimen obtained from a single episode of recurrent AOM was examined by PCR-based penicillin-binding protein (PBP assay, serotyping, and amplified fragment length polymorphism (AFLP, then compared to other samples from the same case. Two cases (18.2% showed strain diversity during repeated antibiotic treatments by serotyping or PBP-assay. By AFLP analysis, 6 cases (54.5% demonstrated heterogeneous strains during recurrent AOM. Clonal survivors of previous episodes of AOM were not always the cause of subsequent episodes of AOM, even in otitis-prone cases.

  4. DNA sequence analysis and restriction fragment length polymorphisms of the P1 gene of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

    Science.gov (United States)

    Reed, R B; Frost, J B; Kort, K; Myers, S D; Lesse, A J

    1996-09-01

    Brazilian purpuric fever (BPF) is a fulminant pediatric disease caused by specific strains of Haemophilus influenzae biogroup aegyptius. A conserved epitope on the P1 protein of strains of H. influenzae biogroup aegyptius is seen on most virulent isolates. The P1 protein from a Brazilian case-clone strain of H. influenzae biogroup aegyptius was analyzed by cloning and sequencing the gene. Three major variable regions are present within the P1 gene of the BPF clone in an architecture similar to that of the previously sequenced P1 genes from H. influenzae. The DNA sequence data of the P1 gene provided information for restriction fragment length polymorphism analyses among strains of H. influenzae biogroup aegyptius. Using PCR for amplification of the P1 gene, we found that AlwI restriction of this gene allowed for a highly accurate segregation of virulent strains of H. influenzae biogroup aegyptius associated with BPF. The strong association of virulent phenotypes with specific AlwI restriction patterns of the P1 gene provides a basis for the convenient and accurate identification of strains of H. influenzae biogroup aegyptius which cause BPF.

  5. Length polymorphisms for intergenic spacer regions of 16S-23S rDNA in members of the new hydrogen-producing bacteria

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A method based on PCR amplification of the 16S rRNA gene (rDNA) -23S rDNA intergenic spacer regions (ISR) was developed for the identification of species within the novel group hydrogen-producing anaerobes. The sizes of the PCR products varied from 1264 to 398 bp. Strain of isolate Rennanqilyf 3 was characterized as having products of 1262, 398, 638, 437 and 436 bp. The isolate Rennanqilyf 1 had product of 1264 bp. The isolate Rennanqilyf 13 had products of 1261, 579 and 485 bp. Of the 3 species of the novel group hydrogenproducing anaerobes examined, no one was indistinguishable. Two environmental isolates were identified as hydrogen-producing bacteria, which were new species in present taxon. Rennanqilyf 3 could not be associated With any Clostridium sp. Studied. Rennanqilyf 1 could be classified into Clostridium genus. The combination between 16S rDNA equencing and length polymorphisms of IRS in 16S-23S rDNA is a better method for determining species of the hydrogen-producing bacteria.

  6. Identification of Amplified Fragment Length Polymorphism (AFLP Markers Tightly Associated with Drought Stress Gene in Male Sterile and Fertile Salvia miltiorrhiza Bunge

    Directory of Open Access Journals (Sweden)

    Hongbo Guo

    2013-03-01

    Full Text Available Consistent grain yield in drought environment has attracted wide attention due to global climate change. However, the important drought-related traits/genes in crops have been rarely reported. Many near-isogenic lines (NILs of male sterile and fertile Salvia miltiorrhiza have been obtained in our previous work through testcross and backcross in continuous field experiments conducted in 2006–2009. Both segregating sterile and fertile populations were subjected to bulked segregant analysis (BSA and amplified fragment length polymorphism (AFLP with 384 and 170 primer combinations, respectively. One out of 14 AFLP markers (E9/M3246 was identified in treated fertile population as tightly linked to the drought stress gene with a recombination frequency of 6.98% and at a distance of 7.02 cM. One of 15 other markers (E2/M5357 was identified in a treated sterile population that is closely associated with the drought stress gene. It had a recombination frequency of 4.65% and at a distance of 4.66 cM. Interestingly, the E9/M3246 fragment was found to be identical to another AFLP fragment E11/M4208 that was tightly linked to the male sterile gene of S. miltiorrhiza with 95% identity and e-value 4 × 10−93. Blastn analysis suggested that the drought stress gene sequence showed higher identity with nucleotides in Arabidopsis chromosome 1–5.

  7. Amplified fragment length polymorphism and multilocus sequence analysis-based genotypic relatedness among pathogenic variants of Xanthomonas citri pv. citri and Xanthomonas campestris pv. bilvae.

    Science.gov (United States)

    Bui Thi Ngoc, Lan; Vernière, Christian; Jouen, Emmanuel; Ah-You, Nathalie; Lefeuvre, Pierre; Chiroleu, Frédéric; Gagnevin, Lionel; Pruvost, Olivier

    2010-03-01

    Three pathogenic variants (i.e. pathotypes) have been described within Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker. Pathotype A strains naturally infect a wide range of Citrus species and members of some related genera. In contrast, pathotypes A* and A(w) have narrow host ranges within the genus Citrus and have been isolated from Mexican lime (Citrus aurantifolia L.) and from Mexican lime and alemow (Citrus macrophylla L.), respectively. We used amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA) based on four partial housekeeping gene sequences (atpD, dnaK, efp and gyrB ) for the genotypic classification of Xanthomonas citri pv. citri and the poorly characterized citrus pathogen Xanthomonas campestris pv. bilvae. A Mantel test showed that genetic distances derived from AFLP and MLSA were highly correlated. X. campestris pv. bilvae showed a close relatedness to the type strain of X. citri, indicating that this pathovar should be reclassified as X. citri pv. bilvae. All pathotype A* and A(w) strains were most closely related to X. citri pv. citri strains with a wide host range (pathotype A), confirming previous DNA-DNA hybridization data. Pathotype A(w) should be considered a junior synonym of pathotype A* on the basis of pathogenicity tests, AFLP, MLSA and PCR using pathovar-specific primers. Evolutionary genome divergences computed from AFLP data suggested that pathotype A* (including A(w) strains) is a group of strains that shows a wider genetic diversity than pathotype A.

  8. Mapping the carriage of flaA-restriction fragment length polymorphism Campylobacter genotypes on poultry carcasses through the processing chain and comparison to clinical isolates.

    Science.gov (United States)

    Duffy, Lesley L; Blackall, Patrick J; Cobbold, Rowland N; Fegan, Narelle

    2015-06-01

    Poultry are considered a major source for campylobacteriosis in humans. A total of 1866 Campylobacter spp. isolates collected through the poultry processing chain were typed using flaA-restriction fragment length polymorphism to measure the impact of processing on the genotypes present. Temporally related human clinical isolates (n = 497) were also typed. Isolates were obtained from whole chicken carcass rinses of chickens collected before scalding, after scalding, before immersion chilling, after immersion chilling and after packaging as well as from individual caecal samples. A total of 32 genotypes comprising at least four isolates each were recognised. Simpson's Index of Diversity (D) was calculated for each sampling site within each flock, for each flock as a whole and for the clinical isolates. From caecal collection to after packaging samples the D value did not change in two flocks, decreased in one flock and increased in the fourth flock. Dominant genotypes occurred in each flock but their constitutive percentages changed through processing. There were 23 overlapping genotypes between clinical and chicken isolates. The diversity of Campylobacter is flock dependant and may alter through processing. This study confirms that poultry are a source of campylobacteriosis in the Australian population although other sources may contribute. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  9. Comparison of genetic diversity of the invasive weed Rubus alceifolius poir. (Rosaceae) in its native range and in areas of introduction, using amplified fragment length polymorphism (AFLP) markers.

    Science.gov (United States)

    Amsellem, L; Noyer, J L; Le Bourgeois, T; Hossaert-McKey, M

    2000-04-01

    Theory predicts that colonization of new areas will be associated with population bottlenecks that reduce within-population genetic diversity and increase genetic differentiation among populations. This should be especially true for weedy plant species, which are often characterized by self-compatible breeding systems and vegetative propagation. To test this prediction, and to evaluate alternative scenarios for the history of introduction, the genetic diversity of Rubus alceifolius was studied with amplified fragment length polymorphism (AFLP) markers in its native range in southeast Asia and in several areas where this plant has been introduced and is now a serious weed (Indian Ocean islands, Australia). In its native range, R. alceifolius showed great genetic variability within populations and among geographically close populations (populations sampled ranging from northern Vietnam to Java). In Madagascar, genetic variability was somewhat lower than in its native range, but still considerable. Each population sampled in the other Indian Ocean islands (Mayotte, La Réunion, Mauritius) was characterized by a single different genotype of R. alceifolius for the markers studied, and closely related to individuals from Madagascar. Queensland populations also included only a single genotype, identical to that found in Mauritius. These results suggest that R. alceifolius was first introduced into Madagascar, perhaps on multiple occasions, and that Madagascan individuals were the immediate source of plants that colonized other areas of introduction. Successive nested founder events appear to have resulted in cumulative reduction in genetic diversity. Possible explanations for the monoclonality of R. alceifolius in many areas of introduction are discussed.

  10. Development of amplified fragment length polymorphism (AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot.

    Science.gov (United States)

    Casasnovas, F; Fantini, E N; Palazzini, J M; Giaj-Merlera, G; Chulze, S N; Reynoso, M M; Torres, A M

    2013-06-01

    The objective of this work was to design an amplified fragment length polymorphism (AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil. Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non-PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils. The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas. © 2013 The Society for Applied Microbiology.

  11. Rediscovery of historical Vitis vinifera varieties from the South Anatolia region by using amplified fragment length polymorphism and simple sequence repeat DNA fingerprinting methods.

    Science.gov (United States)

    Yilancioglu, Kaan; Cetiner, Selim

    2013-05-01

    Anatolia played an important role in the diversification and spread of economically important Vitis vinifera varieties. Although several biodiversity studies have been conducted with local cultivars in different regions of Anatolia, our aim is to gain a better knowledge on the biodiversity of endangered historical V. vinifera varieties in the northern Adana region of southern Anatolia, particularly those potentially displaying viticulture characteristics. We also demonstrate the genetic relatedness in a selected subset of widely cultivated and commercialized V. vinifera collection cultivars, which were obtained from the National Grapevine Germplasm located at the Institute of Viticulture, Turkey. In the present study, microsatellites were used in narrowing the sample size from 72 accessions down to a collection of 27 varieties. Amplified fragment length polymorphisms were then employed to determine genetic relatedness among this collection and local V. vinifera cultivars. The unweighted pair group method with arithmetic mean cluster and principal component analyses revealed that Saimbeyli local cultivars form a distinct group, which is distantly related to a selected subset of V. vinifera collection varieties from all over Turkey. To our knowledge, this is the first study conducted with these cultivars. Further preservation and use of these potential viticultural varieties will be helpful to avoid genetic erosion and to promote continued agriculture in the region.

  12. Identification of Amplified Fragment Length Polymorphism (AFLP) Markers Tightly Associated with Drought Stress Gene in Male Sterile and Fertile Salvia miltiorrhiza Bunge.

    Science.gov (United States)

    Zhang, Yuejin; Guo, Lijun; Shu, Zhiming; Sun, Yiyue; Chen, Yuanyuan; Liang, Zongsuo; Guo, Hongbo

    2013-03-22

    Consistent grain yield in drought environment has attracted wide attention due to global climate change. However, the important drought-related traits/genes in crops have been rarely reported. Many near-isogenic lines (NILs) of male sterile and fertile Salvia miltiorrhiza have been obtained in our previous work through testcross and backcross in continuous field experiments conducted in 2006-2009. Both segregating sterile and fertile populations were subjected to bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) with 384 and 170 primer combinations, respectively. One out of 14 AFLP markers (E9/M3246) was identified in treated fertile population as tightly linked to the drought stress gene with a recombination frequency of 6.98% and at a distance of 7.02 cM. One of 15 other markers (E2/M5357) was identified in a treated sterile population that is closely associated with the drought stress gene. It had a recombination frequency of 4.65% and at a distance of 4.66 cM. Interestingly, the E9/M3246 fragment was found to be identical to another AFLP fragment E11/M4208 that was tightly linked to the male sterile gene of S. miltiorrhiza with 95% identity and e-value 4 × 10-93. Blastn analysis suggested that the drought stress gene sequence showed higher identity with nucleotides in Arabidopsis chromosome 1-5.

  13. Use of an intron length polymorphism to localize the tropoelastin gene to mouse Chromosome 5 in a region of linkage conservation with human Chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Wydner, K.S.; Passmore, H.C. [Rutgers Univ., Piscataway, NJ (United States); Sechler, J.L.; Boyd, C.D. [UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ (United States)

    1994-09-01

    The complete coding sequence for mouse tropoelastin was obtained from overlapping reverse transcriptase polymerase chain reaction (PCR) amplimers. These cDNA fragments were derived from mouse tropoelastin mRNA using PCR oligomers complementary to conserved domains within rat tropoelastin mRNA. A comparison of coding domains of mouse and rat tropoelastin mRNA revealed a greater than 93% homology at the nucleotide level and over 96% similarity in the predicted amino acid sequence. PCR primers complementary to regions of the mouse tropoelastin mRNA were used to define a novel intron length polymorphism (ILP) within intron 8 of the mouse tropoelastin gene (Eln). This ILP proved to be informative in an intraspecific backcross in which genomic DNA samples from 75 backcross mice were used to map the tropoelastin gene to a position in the distal half of mouse chromosome 5. The linkage and genetic distances between Eln and the closest molecular markers used in this study are centromere-D5Mit95, D5Mit96-6.7 cM-Gus, Eln-4.0 cM-Zp3-telomere.

  14. Genetic stability of strains preserved on LENTICULE discs and by freeze-drying: a comparison using fluorescent amplified fragment length polymorphism analysis.

    Science.gov (United States)

    Desai, Meeta; Russell, Julie Elizabeth; Gharbia, Saheer

    2006-04-01

    Fluorescent amplified fragment length polymorphism (FAFLP) analysis, a high-resolution genome fingerprinting method, was used to ascertain the DNA integrity of bacterial strains during preservation by lenticulation and by traditional freeze-drying into glass ampoules. This was achieved by comparing FAFLP genotypes of a range of paired bacterial isolates recovered from LENTICULE discs (preserved between 1995 and 2004) and from freeze-dried (FD) cultures in glass ampoules (preserved between 1966 and 2000). A choice of two endonuclease combinations EcoRI/MseI or HindIII/HhaI was used for FAFLP analysis of the five different bacterial genera comprising of 10 strains. Each of these 10 strains exhibited unique FAFLP profiles. However, there were no detectable differences between the FAFLP profiles for each of the individual strains, irrespective of their preservation format or their year of preservation. Thus, the FAFLP data suggests that LENTICULE production does not result in any detectable genetic changes during drying onto LENTICULE discs and storage for at least 5 years. The provision of such FD reference cultures on LENTICULE discs rather than FD glass ampoules will provide a cost-effective format that is easier to use.

  15. Polymerase chain reaction-restriction fragment length polymorphism assays to distinguish Liriomyza huidobrensis (Diptera: Agromyzidae) from associated species on lettuce cropping systems in Italy.

    Science.gov (United States)

    Masetti, Antonio; Luchetti, Andrea; Mantovani, Barbara; Burgio, Giovanni

    2006-08-01

    The pea leafminer, Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), is a serious insect pest infesting open field lettuce plantings in northern Italy. In these cropping systems, it coexists with several other agromyzid species that have negligible economic importance on open field vegetables. The rapid detection of L. huidobrensis is crucial for effective management strategies, but the identification of agromyzids to species can be very difficult at adult as well at immature stages. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay is proposed to separate L. huidobrensis from Liriomyza bryoniae (Kaltenbach), Liriomyza trifolii (Burgess), and Chromatomyia horticola (Goureau), which usually occur in the same lettuce plantings. An approximately 1,031-bp region of the mitochondrial genome encompassing the 3' region of cytochrome oxidase I, the whole leucine tRNA, and all of the cytochrome oxidase II was amplified by PCR and digested using the enzymes PvuII and SnaBI separately. Both endonucleases cut the amplicons of L. huidobrensis in two fragments, whereas the original band was not cleaved in the other analyzed species. The presence of Dacnusa spp. DNA does not bias the assay, because the PCR conditions and the primer set here described do not amplify any tract of this endoparasitic wasp genome.

  16. Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes.

    Science.gov (United States)

    Carret, C; Walas, F; Carcy, B; Grande, N; Précigout, E; Moubri, K; Schetters, T P; Gorenflot, A

    1999-01-01

    The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.

  17. Analysis of the bacterial diversity existing on animal hide and wool: development of a preliminary PCR-restriction fragment length polymorphism fingerprint database for identifying isolates.

    Science.gov (United States)

    Chen, Yu; Gao, Hongwei; Zhang, Yanming; Deng, Mingjun; Wu, Zhenxing; Zhu, Laihua; Duan, Qing; Xu, Biao; Liang, Chengzhu; Yue, Zhiqin; Xiao, Xizhi

    2012-01-01

    Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqII, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool.

  18. 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

  19. Relationship between TBX20 gene polymorphism and congenital heart disease.

    Science.gov (United States)

    Yang, X F; Zhang, Y F; Zhao, C F; Liu, M M; Si, J P; Fang, Y F; Xing, W W; Wang, F L

    2016-06-02

    Congenital heart disease in children is a type of birth defect. Previous studies have suggested that the transcription factor, TBX20, is involved in the occurrence and development of congenital heart disease in children; however, the specific regulatory mechanisms are yet to be evaluated. Hence, this study aimed to evaluate the relationship between the TBX20 polymorphism and the occurrence and development of congenital heart disease. The TBX20 gene sequence was obtained from the NCBI database and the polymorphic locus candidate was predicted. Thereafter, the specific gene primers were designed for the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) of DNA extracted from the blood of 80 patients with congenital heart disease and 80 controls. The results of the PCR were subjected to correlation analysis to identify the differences between the amplicons and to determine the relationship between the TBX20 gene polymorphism and congenital heart disease. One of the single nucleotide polymorphic locus was found to be rs3999950: c.774T>C (Ala265Ala). The TC genotype frequency in the patients was higher than that in the controls, similar to that for the C locus. The odds ratio of the TC genotypes was above 1, indicating that the presence of the TC genotype increases the incidence of congenital heart diseases. Thus, rs3999950 may be associated with congenital heart disease, and TBX20 may predispose children to the defect.

  20. Detection of deletion and mutation on pig Mx1 gene (gene resistance to influenza virus with PCR-RFLP Nar I restriction

    Directory of Open Access Journals (Sweden)

    Cece Sumantri

    2001-03-01

    Full Text Available The study was done to detect the incident of deletion and mutation in exon 14th of Mx1 gene in pig. Six hundred base pairs at the position (1937 to 2537 of the 14th exon of the pig Mx1 gene was amplified by polymerase chain reaction (PCR from 15 breed of pig DNA sample. The amplified PCR products were digested by Nar I enzyme that called restriction fragment length polymorphism (PCR-RFLP technique. The results show genetic polymorphism at the 14th exon of pig Mx1 gene. The Nar I digested revealed three phenotyphic variation (C/C, C/N and N/N, designated for Nar I cut homozygote, heterozygote and for Nar I no cut homozygote, respectively. The Nar I (N/N or C/N type is corresponding to (1 the deletion 11 bp at the position 2064 to 2075. This type was observed in Landrace breed. (2 the incidence of two point mutation at the position 2065 Guanine (G change to Thymine (T and at the position 2124 Guanine (G change to Adenine (A.This type was observed in Chinese native pig (Meishan and Vietnamese native pig.

  1. Genetic polymorphism of T6235C mutation in 3 non-coding region of CYP1A1 and GSTM1 genes and lung cancer susceptibility in the Mongolian population

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To estimate the relative risk for lung cancer associated with genetic polymorphism of T6235C mutation in 3' non-coding region(MspⅠ)of cytochrome P450 1A1(CYP1A1)and glutathione S-transferase M1(GSTM1)in the Mongolian population in Inner Mongolian Region of China.Methods Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and multiplex PCR methods were used to analyze blood samples obtained from 263 case subjects and 263 control subjects to determine their genotypes for CYP1...

  2. Introduction of a PCR-RFLP method for the detection of two mutations in VHL gene

    Directory of Open Access Journals (Sweden)

    Antonio Alejandro Esperón

    2013-07-01

    Full Text Available Background: Von Hippel-Lindau disease is an inherited neoplastic disorder caused by germline mutations in the VHL gene. In Cuba, molecular diagnosis is performed by the method of single-strand conformation polymorphism of DNA of the three exons of the gene followed by sequencing. This method is expensive, complicated and time-consuming. Objective: to describe the introduction of the molecular diagnosis of mutations c.362A>G and c.481C> in the VHL gene by PCR-RFLP. Methods: computer software CLC Sequence viewer 6.5.1 was used to identify restriction enzymes with cleavage sites modified by mutation c.362A>G in the exon 2 and c.481C>T in exon 3 in the VHL gene. DNA samples of patients already diagnosed through SSCP-sequencing were used. Such samples were amplified by PCR method followed by the enzymatic digestion with SfaNI and BtgZI restriction enzymes. Amplified fragments were analyzed by agarose gel electrophoresis. Results obtained using both methods were compared. Results: effectiveness of the PRC-RFLP method for the diagnosis of c.362A>G and c.481C>T mutations in the VHL gene was standardized and proved. Conclusions: PCR-RFLP method has advantages over SSCP-sequencing strategy for establishing a fast, reproducible and reliable diagnosis of VHL disease in family cases molecularly characterized.

  3. Aplicação do método PCR-RFLP para tipagem de HPV em infecções cervicais de pacientes atendidas no Lepac, Universidade Estadual de Maringá Application of the method of PCR-RFLP to the knowledge of HPV types in cervical infections from patients assisted in Lepac, State University of Maringá (UEM

    Directory of Open Access Journals (Sweden)

    Maristela Gabriel

    2002-03-01

    Full Text Available O método da Reação em Cadeia da Polimerase, associado com o Polimorfismo de Fragmentos de DNA obtidos por Enzimas de Restrição (PCR-RFLP foi, aplicado em 20 amostras clínicas cervicais de pacientes portadoras de anormalidades colpocitológicas atendidas no Lepac, Universidade Estadual de Maringá (UEM. O DNA e o tipo de HPV foram detectados e determinados em todas as amostras analisadas. A grande maioria das amostras cervicais era portadora de tipos de HPV com alto potencial para o desenvolvimento neoplásico. O método PCR-RFLP demonstrou grande poder discriminatório, pois foi capaz de detectar o DNA e determinar o potencial para o desenvolvimento neoplásico de até quatro tipos de HPV presentes de forma concomitante em uma única amostra cervical. Os resultados obtidos demonstraram que esse método pode ser utilizado rotineiramente com alta sensibilidade e reprodutibilidade, sendo um método de escolha na triagem primária de pacientes com risco de desenvolver neoplasias cervicais, contribuindo com informações que auxiliem num possível controle dessa doença.The method of Polymerase Chain Reaction associated with Restriction Fragment Length Polymorphism (PCR-RFLP was applied in 20 cervical clinical samples from patients with cytologic abnormalities, assisted in LEPAC-UEM. The DNA and the type of HPV were detected and determined in all of the analyzed samples. The great majority of the cervical samples showed HPV types, with high potential for neoplasia development. The method of PCR-RFLP proved great discrimination power, because it was able to detect the DNA and to determine the potential of neoplasia development for up to four different HPV types present in the same cervical sample. The results obtained demonstrated that this method can be used routinely, with high sensibility and reprodutibility, being a choice method in primary screen of patients with risk of developing cervical cancer, contributing with information that help a

  4. RELN gene polymorphisms and susceptibility to autism in Chinese Han population.

    Science.gov (United States)

    Tian, Peichao

    2012-01-01

    Single nucleotide polymorphisms (SNPs) in the Reelin gene (RELN) are likely candidates to confer risk for autism. The objective of the present study is to investigate the association of RELN gene SNPs with autism. A total of 367 Chinese Han subjects were recruited, including 186 autism patients and 181 unrelated healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods were used to detect RELN gene polymorphisms. The association between SNPs and autism was analyzed in this study. The g.333509A>C in intron12 and g.504742G>A in exon60 were detected in the RELN gene and a significant association was found between the g.504742G>A polymorphism and autism. Allele and genotype frequencies for the g.504742G>A polymorphism in autistic patients were significantly different for healthy subjects. There was no significantly difference in g.333509A>C polymorphism and autism in the studied populations. Our findings indicated that g.333509A>C was not significantly associated with autism. The g.504742G>A polymorphic variant in the RELN gene might affect subjects susceptibility toward autism in Chinese Han population.

  5. Correlation between IL-6 gene polymorphisms and sepsis of Chinese Han population in Henan province

    Directory of Open Access Journals (Sweden)

    Meng-xuan YANG

    2011-01-01

    Full Text Available Objective To investigate the correlation between-572G/C and-174G/C polymorphism of IL-6 gene and sepsis of Chinese Han population in Henan province.Methods A population-based case-control study involving 99 patients with sepsis and 260 health volunteers(control was carried out.The-572C/G and-174G/C polymorphism of IL-6 gene was analyzed by polymerase chain reaction and restriction fragment-length polymorphism(PCR-RFLP technique.Results The genotype frequencies of all 359 cases were in Hardy-Weinberg equilibrium(P>0.05.No polymorphism was found in-174 site(GG genotype only,while alleles G,C and genotypes GG,GC,CC were found in-572 site,and no significant difference of allele frequency existed between patients and controls.Unconditional logistic regression analysis showed-G572C polymorphism was related to sepsis,the susceptibility to sepsis of patients with GG genotype was significantly higher than that of patients with CC genotype(OR=2.411,95% CI=1.045-5.562,P=0.039 after age and gender correction.Conclusions The-G572C polymorphism of IL-6 gene associates with sepsis,and the GG is the risk genotype of sepsis.There maybe no polymorphism in-G174C of IL-6 gene of Chinese Han population in Henan province.

  6. Leptin and leptin-receptor polymorphisms in fertile and infertile men.

    Science.gov (United States)

    Khosropour, Saeid; Hamidi, Maryam; Fattahi, Amir; Khodadadi, Iraj; Karami, Manoochehr; Fazilati, Mohammad; Vaisi-Raygani, Asad; Tavilani, Heidar

    2017-02-01

    The association of leptin (LEP) -2548G/A and/or leptin receptor (LEPR) Gln223Arg polymorphisms with male infertility and plasma FSH, LH, and testosterone (T) levels was examined. The genotypes and allele frequency distributions of LEP -2548G/A and LEPR Gln223Arg polymorphisms were investigated in 150 fertile and 150 infertile men by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Also, plasma levels of FSH, LH, and T were measured using commercial ELISA kits. Frequencies of AA, AG and GG genotypes of LEP-2548G/A polymorphism were statistically different in fertile and infertile men (p=0.012). The AG genotype showed a protective effect which could decrease risk of male infertility about 3 fold (p = 0.004). We did not observe any differences in frequencies of LEPR Gln223Arg alleles and genotypes between groups (p > 0.05). Sperm counts from infertile men with the AG and GG genotypes of the LEP polymorphism were significantly higher than AA genotype (p 0.05). Our study suggests that the LEP -2548G/A polymorphism may play a role in male fertility and the AG genotype may have a protective effect through increasing sperm counts. The distribution of genotypes of LEP -2548G/A polymorphism are different in fertile and infertile males and may be a useful tool in evaluation of male infertility. LEP: leptin; LEPR: leptin receptor; T: testosterone; FSH: follicle-stimulating hormone; LH: luteinizing hormone.

  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. 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.

  9. Perilipin Gene 1237 T>C Polymorphism is not Associated with Obesity Risk in Northern Chinese Han Adults

    Institute of Scientific and Technical Information of China (English)

    DONG-SHENG HU; JING XIE; DA-HAI YU; GUO-HENG XU; JIE LU; JIN-XIU YANG; CHUN-YANG LI; YAN-YAN LI

    2009-01-01

    Objective To identify the association between PLIN 1237 polymorphism and obesity in Chinese Han adults. Methods A total of 994 adults (157 obese subjects, 322 overweight subjects, and 515 normal controls) were recruited from two rural communities. PLIN 1237 polymorphism was genotyped by polymerase chain reaction-restriction-fragment-length-polymorphism (PCR-RFLP). Association between PLIN polymorphisms and obesity status was estimated by ordinal logistic regression. Results The three genotypes of PLIN 1237 were detected with a percentage of 54.3%, 37.1%, and 8.6% in TT, TC, and CC genotypes, respectively. For the PLIN 1237 polymorphism locus, the frequency of alleles T and C was 0.73 and 0.27, respectively. The PLIN 1237 polymorphisms were in Hardy-Weinberg equilibrium. PLIN 1237 polymorphism was not associated with obesity. The odds ratio for overweight or obesity for the CC+TC genotype was 0.8 (0.4, 1.4) in women (P=0.4) and 0.6 (0.3, 1.3) in men (P=0.2) after adjustment for age, education, household income and alcohol consumption, smoking, and physical activity. Conclusion Chinese Han adults have a lower frequency of variant-allele C in PLIN 1237. PLIN 1237 T>C polymorphism is not significantly associated with obesity in northern Chinese adults.

  10. Comparison of Variable-Number Tandem-Repeat Markers typing and IS1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins

    Directory of Open Access Journals (Sweden)

    Marttila Harri

    2010-03-01

    Full Text Available Abstract Background Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects. Methods A novel PCR-based genotyping method, variable number tandem repeat (VNTR typing of eight mycobacterial interspersed repetitive units (MIRUs, was evaluated for its ability to characterize Finnish Mycobacterium avium subsp. hominissuis strains isolated from pigs (n = 16 and humans (n = 13 and the results were compared with those obtained by the conventional IS1245 RFLP method. Results The MIRU-VNTR results showed a discriminatory index (DI of 0,92 and the IS1245 RFLP resulted in DI 0,98. The combined DI for both methods was 0,98. The MIRU-VNTR test has the advantages of being simple, reproducible, non-subjective, which makes it suitable for large-scale screening of M. avium strains. Conclusions Both typing methods demonstrated a high degree of similarity between the strains of human and porcine origin. The parallel application of the methods adds epidemiological value to the comparison of the strains and their origins. The present approach and results support the hypothesis that there is a common source of M. avium subsp. hominissuis infection for pigs and humans or alternatively one species may be the infective source to the other.

  11. Distribution Study of Chlamydia trachomatis Serovars among High-Risk Women in China Performed Using PCR-Restriction Fragment Length Polymorphism Genotyping▿

    Science.gov (United States)

    Gao, Xing; Chen, Xiang-Sheng; Yin, Yue-Ping; Zhong, Ming-Ying; Shi, Mei-Qin; Wei, Wan-Hui; Chen, Qiang; Peeling, Rosanna W.; Mabey, David

    2007-01-01

    This was one of the first epidemiological studies in China focused on genital Chlamydia trachomatis serotype distribution in high-risk female populations using omp1 gene-based restriction fragment length polymorphism analysis. One thousand seven hundred seventy cervical swab samples from women attending sexually transmitted disease clinics and female sex workers in six cities in China (Shenzhen and Guangzhou in southern China, Nanjing and Shanghai in eastern China, and Nanning and Chengdu in southwestern China) were subjected to serovar genotyping. The proportion of omp1 genes successfully amplified in 240 C. trachomatis plasmid-positive samples was 94.2% (226/240). Serotypes E (n = 63; 27.9%), F (n = 53; 23.5%), G (n = 28; 12.4%), and D (n = 25; 11.1%) were most prevalent. Though there was no significant difference in the geographic distribution of C. trachomatis, serotype E was predominant in the South (32.1%) and East (27.1%), while serotype F was predominant in the Southwest (28.3%). Serotype F infection was associated with young age and single status. Serovar G was associated with lower abdominal pain; 47.5% of asymptomatic patients were infected with serovar E. These results provide information on distribution of genital C. trachomatis serotypes among high-risk women in China and indicate that high-risk women, including those who are asymptomatic, can be infected with multiple serovars of C. trachomatis, revealing exposure to multiple sources of infection. Although the scope for generalizations is limited by our small sample size, our results showing clinical correlations with genotypes are informative. PMID:17301282

  12. Genotypic identification of some lactic acid bacteria by amplified fragment length polymorphism analysis and investigation of their potential usage as starter culture combinations in Beyaz cheese manufacture.

    Science.gov (United States)

    Karahan, A G; Başyiğit Kiliç, G; Kart, A; Sanlidere Aloğlu, H; Oner, Z; Aydemir, S; Erkuş, O; Harsa, S

    2010-01-01

    In this study, 2 different starter culture combinations were prepared for cheesemaking. Starter culture combinations were formed from 8 strains of lactic acid bacteria. They were identified as Lactococcus lactis ssp. lactis (2 strains), Lactobacillus plantarum (5 strains), and Lactobacillus paraplantarum (1 strain) by amplified fragment length polymorphism analysis. The effects of these combinations on the physicochemical and microbiological properties of Beyaz cheeses were investigated. These cheeses were compared with Beyaz cheeses that were produced with a commercial starter culture containing Lc. lactis ssp. lactis and Lc. lactis ssp. cremoris as control. All cheeses were ripened in brine at 4 degrees C for 90 d. Dry matter, fat in dry matter, titratable acidity, pH, salt in dry matter, total N, water-soluble N, and ripening index were determined. Sodium dodecyl sulfate-PAGE patterns of cheeses showed that alpha(S)-casein and beta-casein degraded slightly during the ripening period. Lactic acid bacteria, total mesophilic aerobic bacteria, yeast, molds, and coliforms were also counted. All analyses were repeated twice during d 7, 30, 60, and 90. The starter culture combinations were found to be significantly different from the control group in pH, salt content, and lactobacilli, lactococci, and total mesophilic aerobic bacteria counts, whereas the cheeses were similar in fat, dry matter content, and coliform, yeast, and mold counts. The sensory analysis of cheeses indicated that textural properties of control cheeses presented somewhat lower scores than those of the test groups. The panelists preferred the tastes of treatment cheeses, whereas cheeses with starter culture combinations and control cheeses had similar scores for appearance and flavor. These results indicated that both starter culture combinations are suitable for Beyaz cheese production.

  13. Fluorescent amplified fragment length polymorphism (FAFLP genotyping demonstrates the role of biofilm-producing methicillin-resistant periocular Staphylococcus epidermidis strains in postoperative endophthalmitis

    Directory of Open Access Journals (Sweden)

    Hasnain Seyed E

    2006-01-01

    Full Text Available Abstract Background An observational case series was used to study the virulence characteristics and genotypes of paired Staphylococcus epidermidis isolates cultured from intraocular samples and from periocular environment of patients with postcataract surgery endophthalmitis. Methods Eight S. epidermidis isolates were obtained from three patients (2 from patients #1 and 2 and 4 from patient #3 whose vitreous and/or anterior chamber (AC specimens and preoperative lid/conjunctiva samples were culture positive. Cultures were identified by API-Staph phenotypic identification system and genotypically characterized by Fluorescent Amplified Fragment Length Polymorphism (FAFLP and checked for their antimicrobial susceptibility. The isolates were tested for biofilm-production and methicillin-resistance (MR by PCR amplification of icaAB and mecA gene respectively. Results Four out of eight S. epidermidis strains showed multiple drug resistance (MDR. All the eight strains were PCR positive for mecA gene whereas seven out of eight strains were positive for icaAB genes. In all three patients FAFLP typing established vitreous isolates of S. epidermidis strains to be indistinguishable from the strains isolated from the patient's conjunctival swabs. However, from patient number three there was one isolate (1030b from lid swab, which appeared to be nonpathogenic and ancestral having minor but significant differences from other three strains from the same patient. This strain also lacked icaAB gene. In silico analysis indicated possible evolution of other strains from this strain in the patient. Conclusion Methicillin-resistant biofilm positive S. epidermidis strains colonizing the conjunctiva and eyelid were responsible for postoperative endophthalmitis (POE.

  14. 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.

  15. PCR-RFLP and AP-PCR of rbcL and ITS of rDNA Show That × Taxodiomeria peizhongii (Taxodium x Cryptomeria) Is not an Intergeneric Hybrid

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    ×Taxodiomeria peizhongiiZ. J. Ye, J. J. Zhang et S. H. Pan was regarded as a new intergeneric hybrid between Taxodium mucronatum Tenore (as the female donor) and Cryptomeria fortunei Hooibrenk ex Otto et Dietr (as the male donor). To confirm the authenticity of the intergeneric hybrid, we analyzed the rbcL gene and the internal transcribed spacer (ITS) of 26S-18S ribosomal RNA gene of the three species using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and arbitrarily primed PCR (AP-PCR), and obtained the following results: i) Taxodiomeria peizhongii had the same RFLP maps of the rbcL gene and the ITS as Taxodium mucronatum, but was different from C. fortunei; ii) a 311-bp PCR amplification product was obtained in C. fortunei by AP-PCR of ITS, but was not found in Taxodiomeria peizhongii. Our results have demonstrated that C. fortunei did not provide any genome for Taxodiomeria peizhongii, implying that T. peizhongii is not an intergeneric hybrid between the two species.

  16. 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.

  17. Identification of Puffer Species by PCR-RFLP Analysis and Chip Bio-analysis System%应用PCR-RFLP和芯片生物分析系统鉴别河豚鱼品种

    Institute of Scientific and Technical Information of China (English)

    陈双雅; 陈文炳; 张津; 陈伟玲; 徐敦明; 周昱

    2012-01-01

    应用聚合酶链式反应-限制性片段长度多态分析和芯片生物分析系统建立5种河豚的分子生物学品种鉴定新方法。首先提取鱼的DNA进行细胞色素b基因的PCR扩增,然后用DdeⅠ、HaeⅢ和NlaⅢ三种限制性内切酶进行酶切,在Agilent DNA1000芯片上对酶切片段进行分离。该方法可成功区分5个河豚鱼品种,是一种快速、简便、有效的鱼类品种鉴定分析手段。%Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis and chip bio-analysis system were used to identify five puffer species.A 464-bp fragment of mitochondrial cytochrome b gene was amplified by PCR and the products were digested with the restriction endonuclease DdeⅠ,Hae Ⅲ and Nla Ⅲ,respectively.The resulting fragments were further resolved on DNA chip.Five puffer species were successfully identified by this method.In conclusion,PCR-RFLP and chip bio-analysis system could provide a fast,easy,automatic and reliable analysis approach.

  18. GENETIC DI VERSITY AN ALYSIS OF THERMOPHILIC BACTERIA FROM C ANDRADIMUKA CR ATER IN CENT RAL J AVA EMPLOYING PCR-RFLP OF 16S- rRNA GEN E

    Directory of Open Access Journals (Sweden)

    EMMY DESILIYARNI

    1999-01-01

    Full Text Available The specific primers for bacteria (63f a nd 1387r were use d to amplify the 16 S-rRNA genes fro m total com munit y genom ic DNA of t herm ophi lic bacteri a. The t otal communit y genomic DNA was obtained from mud s and wate r sa mples of Candrad imuka crater, Dieng P lateau, Centra l Ja va. PCR products were cloned into vector pCR*2.1-TOPO (3.9 kb and transformed into Escherichia coli TOPIC. Tw o tetrameric restriction endonucleases Rsal and Hhal were employed to generate Restric tion Fra gment Length Polymorphisms (RFLP p aterns . T hese enzym es yielded 10 and 9 groups of 16S-rRNA pro files or OTU (Opera tion al Taxonomic Un its from 27 1 6S-rRNA ge ne c lones. Rsal w as fo un d to be mo re d iscriminativ e in diff er entiat in g the cl on es t han Hhal. R sal-RFLP indi cated that OTU 7 and OTU 3 represented the most abundant clones, i.e. 6 and 5 clones respective ly. The distribution of 16S-r RNA gene c lones could indicate relati ve dist ri butio n of specifi c groups of thermophili c bacteria in th ei r natural h abitat. Analysis o f diversity at the DNA level could represent both culturable and unculturabl e bacteria in t he envir onment . Similarity analysis sho wed that at level 0.600 there were 8 diff erent groups from 1 0 RFLP profiles genera te d by Rsal digestion. This study in dicated tha t the re were at leas t 8 grou ps of different thermoph ilic bacter ia occupying Candradimuka crater.

  19. The Recent-Transmission of Mycobacterium tuberculosis Strains among Iranian and Afghan Relapse Cases: a DNA-fingerprinting using RFLP and spoligotyping

    Directory of Open Access Journals (Sweden)

    Zarifi Abolhasan

    2008-08-01

    Full Text Available Abstract Background Relapse of tuberculosis (TB may develop as the result of reactivation of the endogenous primary infection, or as a result of a exogenous reinfection. This survey evaluated the rate of reactivation versus recent transmission among Iranian and Afghan relapse cases. Methods The sputum specimens were digested, examined microscopically for acid-fast bacilli, and inoculated into Löwenstein-Jensen slants by standard procedures. Thereafter, the susceptibility and identification tests were performed on culture positive specimens. Subsequently, the strains that were identified as Mycobacterium tuberculosis (258 isolates were subjected to IS6110 restriction fragment length polymorphism (RFLP and spoligotyping. Additional patient's information was collected for further epidemiological analysis. Patients whose isolates had identical genotyping patterns were considered a cluster with recent transmission episode. Results Out of 258 available isolates, 72(28% had multi-drug resistant (MDR-TB in ratio and 42 (16.2% had other resistant. Notably, 38 of MDR-TB cases (52% were isolated from Afghan patients. By IS6110-RFLP typing method, 65 patients (25% were clustered in 29 clusters. In cluster cases, the intra-community transmissions between Iranian and Afghan patients were 41%. All MDR-TB patients in clusters had either Haarlem I or Beijing characteristic. The risk factors like sex, family history, close contact, living condition, PPD test result and site of TB infection were not associated with clustering. Although, the MDR-TB strains were more frequent in non-cluster cases (31% than cluster one(18% (P M. tuberculosis strains isolated from non-cluster cases were belong to EAI3 (51; 30% and CASI(32;18.6% superfamilies. Conclusion During the studied period, reactivation of a previous infection remain the more probable cause of recurrence. Although, the evidence of intra- community transmission between Iranian and Afghan TB cases, highlighted

  20. Applying Data Mining to Classify Age by Intestinal Microbiota in 92 Healthy Men Using a Combination of Several Restriction Enzymes for T-RFLP Experiments.

    Science.gov (United States)

    Kobayashi, Toshio; Osaki, Takako; Oikawa, Shinya

    2014-01-01

    The composition of the intestinal microbiota was measured following consumption of identical meals for 3 days in 92 Japanese men, and terminal restriction fragment length polymorphism (T-RFLP) was used to analyze their feces. The obtained operational taxonomic units (OTUs) and the subjects' ages were classified by using Data mining (DM) software that compared these data with continuous data and for 5 partitions for age divided at 5 years intervals between the ages of 30 and 50. The DM provided Decision trees in which the selected OTUs were closely related to the ages of the subjects. DM was also used to compare the OTUs from the T-RFLP data with seven restriction enzymes (two enzymes of 516f-BslI and 516f-HaeIII, two enzymes of 27f-MspI and 27f-AluI, three enzymes of 35f-HhaI, 35f-MspI and 35f-AluI) and their various combinations. The OTUs delivered from the five enzyme-digested partitions were analyzed to classify their age clusters. For use in future DM processing, we discussed the enzymes that were effective for accurate classification. We selected two OTUs (HA624 and HA995) that were useful for classifying the subject's ages. Depending on the 16S rRNA sequences of the OTUs, Ruminicoccus obeum clones 1-4 were present in 18 of 36 bacterial candidates in the older age group-related OTU (HA624). On the other hand, Ruminicoccus obeum clones 1-33 were present in 65 of 269 candidates in the younger age group-related OUT (HA995).

  1. Mitochondrial DNA polymorphisms in Phytophthora infestans: new haplotypes are identified and re-defined by PCR.

    Science.gov (United States)

    Yang, Zhi-Hui; Qi, Ming-Xing; Qin, Yu-Xuan; Zhu, Jie-Hua; Gui, Xiu-Mei; Tao, Bu; Xu, Xiao-Hu; Zhang, Fu-Guang

    2013-11-01

    Polymorphisms of mitochondrial DNA (mt-DNA) are particularly useful for monitoring specific pathogen populations like Phytophthora infestans. Basically type I and II of P. infestans mt-DNA were categorized by means of polymorphism lengths caused by an ~2 kb insertion, which can be detected via restriction enzyme digestion. In addition genome sequencing of haplotype Ib has been used as a simple Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method to indirectly identify type I and II alterations through EcoR I restriction enzyme DNA fragment patterns of the genomic P4 area. However, with the common method, wrong mt-DNA typing occurs due to an EcoR I recognition site mutation in the P4 genomic area. Genome sequencing of the four haplotypes (Ia, Ib, IIa, and IIb) allowed us to thoroughly examine mt-DNA polymorphisms and we indentified two hypervariable regions (HVRs) named HVRi and HVRii. The HVRi length polymorphism caused by a 2 kb insertion/deletion was utilized to identify mt-DNA types I and II, while another length polymorphism in the HVRii region is caused by a variable number of tandem repeats (n = 1, 2, or 3) of a 36 bp sized DNA stretch and was further used to determine mt-DNA sub-types, which were described as R(n = 1, 2, or 3). Finally, the P. infestans mt-DNA haplotypes were re-defined as IR(1) or IIR(2) according to PCR derived HVRi and HVRii length polymorphisms. Twenty-three isolates were chosen to verify the feasibility of our new approach for identifying mt-DNA haplotypes and a total of five haplotypes (IR(1), IR(2), IR(3), IIR(2) and IIR(3)) were identified. Additionally, we found that six isolates determined as type I by our method were mistakenly identified as type II by the PCR-RFLP technique. In conclusion, we propose a simple and rapid PCR method for identification of mt-DNA haplotypes based on sequence analyses of the mitochondrial P. infestans genome.

  2. Glucocorticoid receptor gene polymorphisms and potential association to chronic obstructive pulmonary disease susceptibility and severity

    Directory of Open Access Journals (Sweden)

    Schwabe K

    2009-12-01

    Full Text Available Abstract Objective As chronic obstructive pulmonary disease (COPD is known for poor glucocorticoid (GC response, we hypothesized that polymorphic variants of the glucocorticoid receptor (GR gene might predispose for COPD and/or disease severity. Materials and methods Three out of about 50 of the most abundant receptor GR gene polymorphisms were investigated in a case-control study which included 207 patients with chronic bronchitis or COPD (mean FEV1 50.5% predicted, GOLD I-IV and 106 age matched healthy subjects (mean FEV1 101.8% predicted. These were genotyped: a for the N363S (Exon 2; 1220 A > G (I; b the BCLI restriction fragment length polymorphism (Intron 2; 647 C > G (II; and c the ER2223EK (Exon 2; 198, 200 G > A (III, using RT-PCR and PCR-RFLP method on genomic DNA isolated from EDTA blood. Results Genotype distribution between COPD and healthy subjects were alike in all of these three polymorphisms. N363S was found in 0.94% of the healthy and 0% of the COPD subjects. BCLI was detected in 11.3% of the controls and 15.5% of the COPD patients whereas heterozygote frequency was less in the COPD (44.4% group (controls 60.4%. ER2223EK lacks in any of the study subjects. Further, SNPs did not correlate with COPD severity stage (GOLD, exacerbation rates, and clinical course. Conclusion COPD is not linked to gene polymorphisms N363S, BCLI-RFLP, and ER2223EK. Since we analyzed only these 3 receptor gene polymorphisms, this study cannot rule out that other GR gene variants and linkages may be of influence.

  3. Oxytocin Receptor Gene Polymorphisms in Patients With Diabetes

    Directory of Open Access Journals (Sweden)

    Saravani

    2015-04-01

    Full Text Available Background Type 2 Diabetes (T2D is a chronic metabolic disease associated with increased mortality and morbidity. High levels of glucose can damage organs, such as the kidneys, eyes and nerves. Oxytocin (OXT can regulate feeding behavior, energy balance, insulin sensitivity and insulin secretion. The OXT Receptor (OXTR mediates the action of OXT on cells. The role of OXTR polymorphism in carbohydrate metabolism disorders, especially in T2D, is not clear. Objectives The current study aimed to investigate the possible associations between OXTR polymorphism and the risk of developing T2D. Patients and Methods To study genetic polymorphisms, 120 patients with T2D and 120 controls were selected. Genotyping of the OXTR rs53576 and rs2254298 variants was performed using allele-specific Polymerase Chain Reaction (PCR and Restriction Fragment Length Polymorphism (RFLP PCR, respectively. Data were analyzed using Chi-square analysis and logistic regression. Results The logistic regression analysis suggested no significant associations of OXTR Single Nucleotide Polymorphism (SNP rs22542987 in genotypes (OR = 1.054, 95% CI: 0.557 - 1.995, P = 0.871 and alleles of patients with T2D in the study population (OR = 1.004, 95% CI: 0.547 - 1.845, P = 1. The rs53576 polymorphism showed the TT genotype (OR = 0.466, %95CI: 0.22 - 0.94, P = 0.035, as well as T allele (OR = 0.66, %95 CI: (0.46 - 0.95, P = 0.03 in the patients and control group with a significant difference suggesting the protective role this polymorphism plays in T2D. Conclusions Our findings showed that the genotype TT rs53576 OXTR, as well as T allele had significant differences in our population and play a protective role. Therefore, it is suggested to place more interest on these OXTR in large populations and different ethnic groups.

  4. Studies on association between lipoprotein lipase gene polymorphisms of Pvu Ⅱ site and hypertriglyceridemics in Chinese

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To explore whether Pvu Ⅱ restriction fragment length polymorphisms (RFLP) in the lipoprotein lipase (LPL) gene are associated with hypertriglyceridemics (HTG).Methods Pvu Ⅱ restriction fragment length polymorphisms in the lipoprotein lipase gene on a sample of 135 HTG patients and 193 age-matched healthy individuals in Chengdu area were detected with the method of PCR-RFLP.Results The P+P+ genotype frequency and P+ allelic frequency of LPL gene for HTG cases are higher than those for control groups (0.460 vs 0.337, P<0.05; 0.689 vs 0.565, P<0.01). The serum levels of TG, apoC Ⅱ, apoC Ⅲ, apoE and TG/HDL-C in P+P+ genotype are higher than those in P-P- genotype (P<0.05).Conclusion The results suggest that P+P+ genotype in the lipoprotein lipase gene is associated with susceptibility to hypertriglyceridemics in Chinese population.

  5. 18S rRNA基因巢式PCR-RFLP鉴定吉林、大庆地区断奶前奶牛隐孢子虫分离株%Chracterization of Cryptosporidium spp from preweaned calves of Jilin and Daqing area by 18S rRNA gene nested PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    苏艳; 白光彦; 孙喜东; 刘妍; 王春仁; 张静; 宋军澎; 尹继刚

    2011-01-01

    Cryptosporidium SPP isolated from feces of preweaned calves in Jilin and Daqing area were identified by 18S rRNA gene nested PCR-RFLP. The genomic DNA of Cryptosporidium was extracted from fecal samples and amplified by using the 18S rRNA gene nested PCR-RFLP assay,and Blast and MEGA4.0 softwares were used to analyze their homology and phylogeny. Meanwhile, their amplified products were digested with restriction enzymes Ssp Ⅰ ,Vsp Ⅰ and Mbo Ⅱ ,respectively. All the digested products were analyzed with RFLP assay. As demonstrated by 18S rRNA gene analysis, the J ilin isolates included C. bovis, and C. ryanae. While the Daqing isolates included C. bovis,C. ryanae and C. andersoni.%利用18S rRNA巢式聚合酶链反应(Nested PCR)-限制片段长度多态性(Restriction fragment length polymorphism,RFLP)鉴定吉林、大庆地区牛源隐孢子虫分离株.采取吉林、大庆地区断奶前犊牛粪便,提取DNA后经18S rRNA基因巢式PCR扩增,扩增产物测序后用Blast和MEGA4.0软件进行同源性和系统发育树分析.同时扩增产物分别用Ssp Ⅰ、Vsp Ⅰ和Mbo Ⅱ酶切后进行RFLP分析.通过18S rRNA基因PCR-RFLP分析和测序比对分析表明,吉林分离株包括2种隐孢子虫,分别为C.bovis和C.ryanae,大庆分离株包括3种,分别为C.boris、C.ryanae和C.andersoni.

  6. 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

  7. Role of leptin G-2548A polymorphism in age- and gender-specific development of obesity

    Indian Academy of Sciences (India)

    Adeela Shahid; Sobia Rana; Saqib Mahmood; Shahid Saeed

    2015-09-01

    Leptin is involved in the regulation of food intake and energy expenditure, and therefore, is central to adipositysensing pathway. We examined the relationship of the leptin G-2548A polymorphism with obesity and obesityrelated anthropometric and metabolic parameters in a total of 394 (239 obese and 155 non-obese) subjects between 5 and 45 years of age. Body weight, height, waist circumference (WC), hip circumference (HC) and blood pressure (BP) were measured. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated. Levels of fasting blood glucose (FBG), insulin, leptin and leptin receptor were determined, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The LEP G-2548A polymorphism showed association with obesity in children and adolescents (≤18 years of age) but not in adults. However, analysis by gender stratification revealed association with obesity in girls only. In addition, G-2548A polymorphism showed association with BMI, WC, HC, fasting blood glucose and serum leptin levels. This suggests that G-2548A polymorphism may influence the susceptibility to metabolic disturbances and obesity at an early life. Further investigation with a larger sample size is required to validate the effect of LEP G-2548A polymorphism in obese Pakistani girls.

  8. 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

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

    Directory of Open Access Journals (Sweden)

    N Swapna

    2011-01-01

    Full Text Available Background : 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. Aim : The aim of this study was to evaluate association between VDR Fok I polymorphism and genetic susceptibility to essential hypertension. Materials and Methods : 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. Results : The genotype distribution and allele frequencies of Fok I (T/C [rs2228570] VDR polymorphism differed significantly between patients and controls (χ2 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. Conclusions : Our data suggest that VDR gene Fok I polymorphism is associated with the risk of developing essential hypertension

  10. A novel approach for rapid screening of mitochondrial D310 polymorphism

    Directory of Open Access Journals (Sweden)

    Güllüoğlu Bahadır M

    2006-01-01

    Full Text Available Abstract Background Mutations in the mitochondrial DNA (mtDNA have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310 within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphic length variation (6-C to 9-C among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region. Methods 25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data. Results Samples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals. Conclusion In conclusion, BsaXI RFLP analysis is a simple and

  11. Molecular Variability Within and Among Verticillium dahliae Vegetative Compatibility Groups Determined by Fluorescent Amplified Fragment Length Polymorphism and Polymerase Chain Reaction Markers.

    Science.gov (United States)

    Collado-Romero, Melania; Mercado-Blanco, Jesús; Olivares-García, Concepción; Valverde-Corredor, Antonio; Jiménez-Díaz, Rafael M

    2006-05-01

    ABSTRACT A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and

  12. Construction of a linkage map and QTL analysis of horticultural traits for watermelon [Citrullus lanatus (THUNB.) MATSUM & NAKAI] using RAPD, RFLP and ISSR markers.

    Science.gov (United States)

    Hashizume, T; Shimamoto, I; Hirai, M

    2003-03-01

    We have been constructing linkage maps for watermelon ( Citrullus lanatus) on the basis of random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), inter-simple sequence repeats (ISSRs) and isozymes using an F(2) population derived from a crossing between a cultivated inbred line (H-7; C. lanatus) and an African wild form (SA-1; C. lanatus). A total of 120 F(2) plants was used for construction of a linkage map using 477 RAPDs, 53 RFLPs, 23 ISSRs and one isozyme markers. Linkage analysis revealed that 554 loci could be mapped to 11 linkage groups that extended for 2,384 centimorgans (cM). While a BC(1) population [(H-7 x SA-1) x H-7] consisting of 60 individuals was grown and scored for quantitative traits. Another linkage map with a total length of 1,729 cM was constructed in the BC(1) using genetic markers found to segregate in the F(2) population. A QTL analysis was applied by means of interval mapping for locating such agronomic traits as hardness of rind, Brix of flesh juice, flesh color (red and yellow) and rind color. The relative order of markers in the BC(1) map was essentially the same as that on the linkage map in the F(2). A total of five QTLs for four agronomic traits was detected. The QTL for hardness of rind was mapped on group 4. The linkage group 8 contained the QTL for sugar content of the flesh as expressed in Brix of the juice. The QTL for red flesh color was detected on groups 2 and 8. The QTL for rind color mapped on the group 3. The present map and QTL analysis may provide a useful tool for breeders by introducing valuable wild watermelon genes to cultivars.

  13. Gains in QTL detection using an ultra-high density SNP map based on population sequencing relative to traditional RFLP/SSR markers.

    Directory of Open Access Journals (Sweden)

    Huihui Yu

    Full Text Available Huge efforts have been invested in the last two decades to dissect the genetic bases of complex traits including yields of many crop plants, through quantitative trait locus (QTL analyses. However, almost all the studies were based on linkage maps constructed using low-throughput molecular markers, e.g. restriction fragment length polymorphisms (RFLPs and simple sequence repeats (SSRs, thus are mostly of low density and not able to provide precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, we constructed an ultra-high density genetic map based on high quality single nucleotide polymorphisms (SNPs from low-coverage sequences of a recombinant inbred line (RIL population of rice, generated using new sequencing technology. The quality of the map was assessed by validating the positions of several cloned genes including GS3 and GW5/qSW5, two major QTLs for grain length and grain width respectively, and OsC1, a qualitative trait locus for pigmentation. In all the cases the loci could be precisely resolved to the bins where the genes are located, indicating high quality and accuracy of the map. The SNP map was used to perform QTL analysis for yield and three yield-component traits, number of tillers per plant, number of grains per panicle and grain weight, using data from field trials conducted over years, in comparison to QTL mapping based on RFLPs/SSRs. The SNP map detected more QTLs especially for grain weight, with precise map locations, demonstrating advantages in detecting power and resolution relative to the RFLP/SSR map. Thus this study provided an example for ultra-high density map construction using sequencing technology. Moreover, the results obtained are helpful for understanding the genetic bases of the yield traits and for fine mapping and cloning of QTLs.

  14. Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer

    Directory of Open Access Journals (Sweden)

    Teofânia HDA Vidigal

    2001-07-01

    Full Text Available In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found.

  15. Mycobacterium bovis: polymerase chain reaction identification in bovine Lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    Zanini MS

    2001-01-01

    Full Text Available Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54 obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100% that grew in culture, 9 samples (60% that failed to grow in culture, plus 6 (37.5% samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

  16. 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.

  17. 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.

  18. Role of the VDR Bsm I and Apa I polymorphisms in the risk of colorectal cancer in Kashmir.

    Science.gov (United States)

    Rasool, Sabha; Kadla, Showkat A; Rasool, Vamiq; Qazi, Falak; Khan, Tanzeela; Shah, Nisar A; Ganai, Bashir A

    2014-01-01

    A case-control study aiming to evaluate the relationship between Bsm I and Apa I restriction fragment gene polymorphisms and colorectal cancer (CRC) was carried out in Kashmir, including a total of 368 subjects (180 cases and 188 controls). DNA samples extracted from the blood of the subjects were analyzed for 3' untranslated region (3' UTR) Apa I and Bsm I polymorphisms using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). A statistically significant 2.7-fold increased risk was observed in individuals found homozygous for the presence of the 'b' allele, in comparison to subjects homozygous for the 'B' allele (odds ratio (OR) 2.7, 95% confidence interval (CI) 1.49-4.86 (Bsm I)), and a statistically insignificant 2-fold increased risk was found among individuals with the 'aa' genotype, as compared to subjects with the 'AA' genotype (OR 2.017, 95% CI 0.86-4.7). Our study also yielded statistically significant results when the Apa I polymorphism was stratified by age (≤ 50 years) and dwelling area (rural area), and the Bsm I polymorphism by gender (male gender), suggesting a possible role of Apa I and Bsm I polymorphisms in the etiology of CRC in Kashmir. We conclude that Apa I and Bsm I single-nucleotide polymorphisms (SNPs) in the vitamin D receptor gene (VDR) might be associated with susceptibility to CRC among Kashmiris. © 2014 S. Karger GmbH, Freiburg.

  19. [Application of restriction fragment length polymorphism-polymerase chain reaction-flaA and resistotype to identify potential undiagnosed outbreaks of campylobacteriosis in Spain].

    Science.gov (United States)

    Pérez-Boto, David; López-Portolés, José Antonio; Simón, Cristina; Echeita, María Aurora

    2014-01-01

    Outbreaks of campylobacteriosis are infrequent and usually involve a low number of patients, although it is estimated that many more remain undiagnosed. The most successful techniques for outbreak investigation in Campylobacter spp. (PFGE, MLST) have the drawback of being laborious and not available in many laboratories. During the year 2008, 352 isolates of C. jejuni and C. coli from 16 hospitals were received in our laboratory. All strains were genotyped by RFLP-PCR-flaA (flaA type) and phenotyped with their resistotype. It was established that the strains of the same species from the same hospital, isolated over a period of up to 11 days, with MIC values of±1 dilution with the same flaA type could belong to an outbreak. Strains that met these criteria would be later subtyped by KpnI-PFGE and MLST. A total of 23 out of 352 isolates, distributed in 10 groups, met the criteria for being associated with putative undiagnosed outbreaks. The similarity of the PFGE-profiles in 8 groups was greater than 95% among the isolates from each group. In 7 of the groups, the sequence types (MLST) were coincident. The use of 2 easy markers (resistotype and RFLP-PCR-flaA) may detect isolates probably belonging to an undiagnosed outbreak of campylobacteriosis. Accurate diagnosis requires other molecular markers and epidemiological data of each isolate. The study suggests that, as in other countries, the number of outbreaks of campylobacteriosis in Spain is probably underestimated. Copyright © 2013 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  20. Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

    Directory of Open Access Journals (Sweden)

    Patrícia Carvalho de Sequeira

    2005-11-01

    Full Text Available Simple double repetitive element polymerase chain reaction (MaDRE-PCR and Pvu II-IS1245 restriction fragment length polymorphism (RFLP typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

  1. Effects of OATP -C gene polymorphisms and low-dose rifampicin on serum bilirubin level

    Institute of Scientific and Technical Information of China (English)

    WeiZHANG; Yi-jingHE; QingLI; Hong-haoZHOU

    2005-01-01

    AIM OATP-C is a liver-specific organic anion uptake transporter and shows high affinity for bilirubin uptaking. Rifampicin has been identified as a potent inhibitor of OATP-C both in vitro and in vivo. This study was set to determine the allele frequencies of OATP-C*1a', OATP-C*1b, and OATP-C* 15 in Chinese population, and secondly, to quantitate the contribution of the OATP-C gene polymorphisms and low-dose rifampicin adminstration to the serum bilirubin level in vivo. METHODS Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and a novel tetreprimers method were used to identify OATP-C*1a, OATP-C*1b, and OATP-C*15 genotypes.

  2. Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP.

    Science.gov (United States)

    Marty, Esther; Buchs, Jasmin; Eugster-Meier, Elisabeth; Lacroix, Christophe; Meile, Leo

    2012-04-01

    Pathogenic, spoilage, and technologically important microorganisms were monitored in 21 spontaneously fermented Swiss meat products manufactured with meat from wildlife or animals grown in natural habitat. Thereby, PCR-restriction fragment length polymorphism (RFLP) on rpoB and 16S rRNA gene sequences provided a powerful tool for fast and accurate identification of the main microbial population. Lactobacillus sakei and Lactobacillus curvatus dominated in fermented meat products followed by Staphylococcus species, which constituted 88.2% of all Gram-positive, catalase-positive cocci (GCC(+)) with cell counts varying from 2.6 to 7.0 log cfu/g during maturation. Staphylococcus equorum was prevalent in frequency and cell counts during maturation (18.0%; 5.0-7.3 log cfu/g) and in the end products (28.4%; 1.8-6.2 log cfu/g) implicating a new presumptive starter species for meat fermentation. Nine out of 14 end products indicated safety risks to consumers due to the high incidence of Staphylococcus saprophyticus or Staphylococcus epidermidis combined with cell counts of 7.4 and 4.9 log cfu/g, respectively. This fact was supported by the detection of Staphylococcus aureus and Enterobacteriaceae in ready-to-eat products strongly exceeding the tolerable limit of 2 log cfu/g. Spontaneously fermented meat products produced from wildlife or animals grown in natural habitats not only gave rise to hygienic and safety concerns but also provided new presumptive starter strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. 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.

  4. 捻转血矛线虫伊维菌素耐药株P-糖蛋白基因多态性的PCR-RFLP分析%Polymorphism analysis of P-glycoprotein gene in Haemonchus contortus of ivermectin resistance strains by PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    刘鑫远; 杨晓野; 王瑞; 杨莲茹; 罗晓平; 阿拉腾宝力格; 贺志雄

    2012-01-01

    To study the differences of P-glycoprotein gene between ivermectin-resistant(HY) and sus- ceptible strains(HM) of Haemonchus contortus and further clarify the relationship between P-glycoprotein gene and ivermectin resistance in future, the polymorphism of P-glycoprotein gene from ivermectin-resis- rant strain of H. contortus in sheep was analyzed by PCR-restriction fragment length polymorphism. The result showed that the PCR products of HM group were resistive to the restriction enzyme Kpn I , whileHY group were vulnerable to the enzyme. The statistics data showed that 10 nematodes of ivermectin-sus- ceptible strains were all homozygote(BB);in 20 nematodes of ivermectin-resistance strain, there were 6 ivermectin-resistant homozygotes (AA, 30 %), 13 heterozygotes (AB, 65% ) and only 1 ivermectin-suscepti- ble homozygote(BB, 5 %). The heterozygote(AB) was the most dominant genotype, followed by ivermectin- resistant homozygotes(AA),and ivermectin-susceptible homozygote(BB) was the least. The resistance al- lele A was the most dominant gene,and took 62.5%,while allele B was only 37.5% of the samples. The homologous analysis revealed that the P-glycoprotein gene of ivermectin resistance(hcpgp-1) shared low homology(80. 2% to 89. 3%) with the susceptible strains. Besides, mutant sites on the hcpgp-1 gene of ivermectin-resistant strains were also found. This paper could provide a reference for studying the effects of P-glycoprotein gene in ivermectin resistance. And it also could supply basic information for the mechanism study of resistance to common anthelmintics of H. contortus.%为了解P-糖蛋白基因在捻转血矛线虫伊维菌素耐药株和敏感株间的差异,进一步弄清该基因与伊维菌素耐药性产生的关系,用PCR-RFLP方法分析了捻转血矛线虫伊维菌素耐药株P-糖蛋白基因hcpgp-1的多态性,并对所得序列进行测序。结果表明:捻转血矛线虫伊维菌素敏感

  5. Clinical relevance of the glucocorticoid receptor gene polymorphisms in glucocorticoid-induced ocular hypertension and primary open angle glaucoma

    Institute of Scientific and Technical Information of China (English)

    Xiu-Qing; Wang; Zhao-Xia; Duan; Xiang-Ge; He; Xi-Yuan; Zhou

    2015-01-01

    AIM: To avoid the side effects of ocular hypertension of glucocorticoid(GC) usage in eye, we must identify susceptible individuals, which exists in about one-third of all population. Further, the majority of all primary open angle glaucoma(POAG) patients show this phenotype.Glucocorticoid receptor(GR) regulates C responsiveness in trabecular meshwork(TM) cells. In this study, single nucleotide polymorphism(SNP) genotyping was used to determine whether there are differences in the Bcl I(rs41423247) and N363S(rs6195) polymorphisms of the GR gene in healthy and POAG patients, and glucocorticoid-induced ocular hypertension(GIOH)populations.METHODS: Three hundred and twenty-seven unrelated Chinese adults, including 111 normal controls, 117 GIOH subjects and 99 POAG patients, were recruited. DNA samples were prepared and the Bcl I and N363 S polymorphisms were screened using real-time polymerase chain reaction(RT-PCR)-restriction fragment length polymorphism(RFLP) analysis. Frequencies of the Bcl I and N363 S polymorphisms were determined and compared using Fisher’s exact test and the Chi-squared test.RESULTS: Only the Bcl I polymorphism was identified in the Chinese Han population. The frequency of the G allele was 21.6 % in normal controls, 18.3% in GIOH patients, and 13.64% in the POAG patients. There was no significant difference in polymorphism or allele frequency in the 3 groups. Furthermore, no N363 S polymorphism was found in the study subjects.CONCLUSION: The Bcl I polymorphisms in GR gene had no association with GIOH and POAG patients, and N363 S polymorphism might not exist in the Chinese Han population. Therefore, the Bcl I polymorphism might not be responsible for the development of GC-induced ocular hypertension or POAG.

  6. A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

    Directory of Open Access Journals (Sweden)

    Loo Keat

    2012-05-01

    Full Text Available Abstract Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele and 0.11 (T allele; for eNOS +894 G > T, the allele frequencies were 0.58 (G allele and 0.43 (T allele; and for eNOS −786 T > C, the allele

  7. Association between Vitamin D Receptor Polymorphism and Serum Vitamin D Levels in Children with Low-Energy Fractures.

    Science.gov (United States)

    Karpiński, Michal; Galicka, Anna; Milewski, Robert; Popko, Janusz; Badmaev, Vladimir; Stohs, Sidney J

    2017-01-01

    Fractures of bones, especially forearm fractures, are very common in children and their number is increasing. This study was designed to determine the impact of vitamin D serum levels and vitamin D receptor (VDR) polymorphisms on the occurrence of low-energy fractures in children. The study group consisted of 100 children with clinically relevant bone fractures and a control group consisted of 127 children without fractures. Total vitamin D [25(OH)D3 plus 25(OH)D2] serum concentrations were evaluated in every patient. Genotypes for 4 restriction fragment length polymorphisms of the vitamin D receptor gene (FokI, ApaI, TaqI, and BsmI) were determined by standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Differences in concentrations of vitamin D were observed between the group with bone fractures (median = 12 ng/ml) and the control group (median = 16 ng/ml; p = 0.000044). Higher levels of vitamin D reduced the risk of fracture by 1.06 times (p = 0.0005). No impact of particular VDR polymorphism on the occurrence of low-energy fractures in children was detected. However, there were significant differences in the prevalence of FokI polymorphism genotypes between the fracture and control groups (p = 0.05). Furthermore, the recessive "aa" genotype of ApaI polymorphism and the dominant "TT" genotype of TaqI polymorphism were associated with higher levels of vitamin D (p = 0.005 and p = 0.036, respectively). Vitamin D deficiency is an independent risk factor for fractures in children. ApaI polymorphism recessive "aa" and TaqI polymorphism dominant "TT" genotypes are associated with higher levels of vitamin D in serum.

  8. How strong is the association between CAG and GGN repeat length polymorphisms in the androgen receptor gene and prostate cancer risk?

    NARCIS (Netherlands)

    Zeegers, M.P.; Kiemeney, L.A.L.M.; Nieder, A.M.; Ostrer, H.

    2004-01-01

    OBJECTIVE: Although narrative reviews have suggested an association between (CAG)n and (GGN)n polymorphisms in the AR gene and prostate cancer, it has never been quantified systematically. The purpose of this meta-analysis was to provide relative and absolute quantitative summary estimates with suff

  9. Size polymorphism of chicken major histocompatibility complex-encoded B-G molecules is due to length variation in the cytoplasmic heptad repeat region

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1990-01-01

    B-G antigens are cell-surface molecules encoded by a highly polymorphic multigene family located in the chicken major histocompatibility complex (MHC). Rabbit antisera to B-G molecules immunoprecipitate 3-6 bands from iodinated erythrocytes by sodium dodecyl sulfate (SDS) gels under reducing...

  10. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    Science.gov (United States)

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    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. PMID:27242682

  11. PCR-RFLP for the detection and differentiation of the canine piroplasm species and its use with filter paper-based technologies.

    Science.gov (United States)

    Jefferies, R; Ryan, U M; Irwin, P J

    2007-03-15

    Canine piroplasmosis is an emerging disease worldwide, with multiple species of piroplasm now recognised to infect dogs. A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection and differentiation of each of the piroplasm species currently known to infect dogs on the basis of the 18S ribosomal RNA gene. The assay can potentially amplify and discriminate between Theileria annae, Theileria equi, Babesia conradae, Babesia gibsoni, Babesia sp. (Coco) and each of the Babesia canis subspecies. Non-canine piroplasm species can also potentially be detected using the described assay, however amplification of Neospora caninum was also observed. The PCR was found to have a high detection limit, capable of detecting a 2.7x10(-7)% parasitaemia or the equivalent of 1.2 molecules of target DNA when using DNA extracted from whole EDTA blood and detected a parasitaemia of 2.7x10(-5)% using blood applied to both Flinders Technology Associates (FTA) cards and IsoCodetrade mark Stix. The application of blood samples to filter paper may greatly assist in piroplasm identification in regions of the world where local technologies for molecular characterisation are limited. The assay reported here has the potential to be standardised for routine screening of dogs for piroplasmosis.

  12. 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.

  13. River basin-related genetic structuring in an endangered fish species, Chondrostoma lusitanicum, based on mtDNA sequencing and RFLP analysis.

    Science.gov (United States)

    Mesquita, N; Carvalho, G; Shaw, P; Crespo, E; Coelho, M M

    2001-03-01

    Chondrostoma lusitanicum is a Portuguese endemic cyprinid with a restricted distribution and reduced numbers in some basins, justifying its status as a threatened species. We examined genetic population structure using samples from throughout its geographical range in Portugal, using sequencing of b cytochrome and restriction fragment length polymorphism analysis of the NADH subunits 5 and 6. There was reduced within-population genetic variability but considerable among-population differentiation, particularly marked between both the Mira and Arade basins in the extreme south and other populations. These results confirm phylogeographic relationships suggested by previous fragmentary allozyme studies for C. lusitanicum, and are in accordance with allozyme and mitochondrial DNA data on phylogeography of coexisting cyprinid species of the genus Leuciscus. The levels of genetic divergence revealed by sequence and RFLP data showed strongly concordant patterns: geographical genetic structuring, with the definition of three distinct groups, was observed. The high values of nucleotide divergence and pairwise sequence divergence of the Mira and Arade groups, when compared with all other samples, support a distinct taxonomic status probably at the species level. Results are also discussed in relation to conservation of this highly fragmented species, in terms of Evolutionary Significant Units and Management Units.

  14. 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.

  15. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP.