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Sample records for pcr-based genotyping methods

  1. Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping of Clostridium botulinum

    DEFF Research Database (Denmark)

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina

    2013-01-01

    in terms of cost, time, labor, and supplies. Eleven botulinum toxin–producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin–producing clostridia were used to compare 4 DNA extraction procedures: Chelex® 100 matrix, Phenol......Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient...

  2. Internal quality control of PCR-based genotyping methods in research studies and patient diagnostics

    DEFF Research Database (Denmark)

    Bladbjerg, Else-Marie; Gram, Jørgen; Jespersen, Jørgen

    2002-01-01

    Genetic analyses are increasingly integrated in the clinical laboratory, and internal quality control programmes are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n = 500) by ammonium acetate....... Control of data handling revealed 0.1% reading mistakes and 0.5% entry mistakes. Based on our experiences we propose an internal quality control programme for widely used PCR-based haemostasis polymorphism analyses.......-isolation (pre-analytical factors), DNA-amplification, digestion with restriction enzymes, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality (42 microg/ml blood...

  3. A PCR-based genotyping method to distinguish between wild-type and ornamental varieties of Imperata cylindrica.

    Science.gov (United States)

    Cseke, Leland J; Talley, Sharon M

    2012-02-20

    viable wind-dispersed seeds that spread cogongrass over wide distances(5-7). JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass(8-10), and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential(3,7,11). The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

  4. Genotyping of PCR-based polymorphisms and linkage-disequilibrium analysis at the NF1 locus

    Energy Technology Data Exchange (ETDEWEB)

    Purandare, S.M.; Viskochil, D.H.; Cawthon, R. [Univ. of Utah, Salt Lake City, UT (United States)] [and others

    1996-07-01

    Six polymorphism across the NF1 gene have been adapted for genotyping through application of PCR-based assays. Three exon-based polymorphisms - at positions 702, 2034, and 10647 in the NF1 cDNA - were genotyped by mutagenically separated PCR (MS-PCR). A fourth polymorphism, DV1.9, is an L1 insertion element in intron 30, and the other two polymorphisms, GXAlu and EVI-20, are short tandem repeats in intron 27b. All the polymorphisms were evaluated in a cohort of 110 CEPH individuals who previously had been analyzed by use of eight RFLPs at the NF1 locus. Pairwise linkage-disequilibrium analyses with the six PCR-based polymorphisms and their flanking markers demonstrated disequilibrium between all tested loci. Genotypes of the four diallelic polymorphisms (702, 2034, 10647, and DV1.9) were also evaluated in cohorts from the CEPH, African, and Japanese populations. The CEPH and Japanese cohorts showed similar heterozygosities and linkage-disequilibrium coefficients. The African cohort showed a higher degree of heterozygosity and lower linkage-disequilibrium values, compared with the CEPH and Japanese cohorts. 36 refs., 2 figs., 3 tabs.

  5. High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

    Science.gov (United States)

    Tanzi, Elisabetta; Bianchi, Silvia; Fasolo, Maria Michela; Frati, Elena R; Mazza, Francesca; Martinelli, Marianna; Colzani, Daniela; Beretta, Rosangela; Zappa, Alessandra; Orlando, Giovanna

    2013-01-01

    Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs. Copyright © 2012 Wiley Periodicals, Inc.

  6. Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis.

    Science.gov (United States)

    Waterfall, C M; Cobb, B D

    2001-12-01

    Allele-specific amplification (ASA) is a generally applicable technique for the detection of known single nucleotide polymorphisms (SNPs), deletions, insertions and other sequence variations. Conventionally, two reactions are required to determine the zygosity of DNA in a two-allele system, along with significant upstream optimisation to define the specific test conditions. Here, we combine single tube bi-directional ASA with a 'matrix-based' optimisation strategy, speeding up the whole process in a reduced reaction set. We use sickle cell anaemia as our model SNP system, a genetic disease that is currently screened using ASA methods. Discriminatory conditions were rapidly optimised enabling the unambiguous identification of DNA from homozygous sickle cell patients (HbS/S), heterozygous carriers (HbA/S) or normal DNA in a single tube. Simple downstream mathematical analyses based on product yield across the optimisation set allow an insight into the important aspects of priming competition and component interactions in this competitive PCR. This strategy can be applied to any polymorphism, defining specific conditions using a multifactorial approach. The inherent simplicity and low cost of this PCR-based method validates bi-directional ASA as an effective tool in future clinical screening and pharmacogenomic research where more expensive fluorescence-based approaches may not be desirable.

  7. PCR-based identification of eight Lactobacillus species and 18 hr-HPV genotypes in fixed cervical samples of South African women at risk of HIV and BV.

    NARCIS (Netherlands)

    Dols, J.A.M.; Reid, G.; Kort, R.; Schuren, F.H.J.; Tempelman, H.; Bontekoe, T.R.; Korporaal, H.; van der Veer, E.M.; Smit, P.W,; Boon, M.E.

    2012-01-01

    Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant

  8. PCR-based identification of eight lactobacillus species and 18 hr-HPV genotypes in fixed cervical samples of south african women at risk of HIV and BV

    NARCIS (Netherlands)

    Dols, J.A.M.; Reid, G.; Kort, R.; Schuren, F.H.J.; Tempelman, H.; Bontekoe, T.R.; Korporaal, H.; Veer, E.M. van der; Smit, P.W.; Boon, M.E.

    2012-01-01

    Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant

  9. Internal quality control of PCR-based genotyping methods

    DEFF Research Database (Denmark)

    Bladbjerg, Else-Marie; Gram, Jørgen; Jespersen, Jørgen

    2002-01-01

    Internal quality control programmes for genetic analyses are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n = 500) and analysed for 18 polymorphisms by polymerase chain reaction (PCR), i...... because of positive reagent blanks (controls (Control of data handling revealed 0.1% reading mistakes and 0.5% entry mistakes. Based on our experiences, we propose an internal quality control programme......, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality (42 micrograms/ml blood, A260/A280 ratio > 1.75, negative DNAsis tests). Occasionally, results were reanalysed...

  10. Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method

    DEFF Research Database (Denmark)

    Malorny, B.; Hoorfar, Jeffrey; Hugas, M.

    2003-01-01

    A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained...... presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole......-based diagnostic methods and is currently proposed as international standard document....

  11. PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

    Science.gov (United States)

    Singh, Om P; Bali, Prerna; Hemingway, Janet; Subbarao, Sarala K; Dash, Aditya P; Adak, Tridibes

    2009-01-01

    Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F) in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method was refractory to non

  12. PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

    Directory of Open Access Journals (Sweden)

    Dash Aditya P

    2009-07-01

    Full Text Available Abstract Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR, an Amplification Refractory Mutation System (ARMS and Primer Introduced Restriction Analysis-PCR (PIRA-PCR were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method

  13. Rust resistance evaluation of advanced wheat (triticum aestivum l.) genotypes using pcr-based dna markers

    International Nuclear Information System (INIS)

    Rahman, S.U.; Younis, M.; Iqbal, M.Z.; Nawaz, M.

    2014-01-01

    The most effective and environmental friendly approach for the control of wheat rust disease is the use of resistant genotypes. The present study was conducted to explore rust resistance potential of 85 elite wheat genotypes (36 varieties and 49 advanced lines) using various types of DNA markers like STS, SCAR and SSR. DNA markers linked with different genes conferring resistance to rusts (Leaf rust=Lr, Yellow rust=Yr and Stem rust=Sr) were employed in this study. A total of 18 genes, consisting of eleven Lr (lr1, lr10, lr19, lr21, lr28, lr34, lr39, lr46, lr47, lr51 and lr52), four Yr (yr5, yr18, yr26 and yr29) and three Sr genes (sr2, sr29, and sr36) were studied through linked DNA markers. Maximum number of Lr genes was found in 17 advanced lines and 9 varieties, Yr genes in 26 advanced lines and 20 wheat varieties, and Sr genes in 43 advanced lines and 27 varieties. Minimum number of Lr genes was found in advanced line D-97 and variety Kohinoor-83, Yr genes in wheat variety Bwp-97 and Sr genes in 6 advanced lines and 8 varieties. Molecular data revealed that genotypes having same origin, from a specified area showed resistance for similar type of genes. In this study, an average similarity of 84% was recorded among wheat genotypes. Out of 18 loci, 15 were found to be polymorphic. (author)

  14. Comparison of three PCR-based assays for SNP genotyping in sugar beet

    Science.gov (United States)

    Background: PCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved t...

  15. Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples

    DEFF Research Database (Denmark)

    Malorny, B.; Cook, N.; D'Agostino, M.

    2004-01-01

    As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse...... or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic...... specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which...

  16. Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6

    Directory of Open Access Journals (Sweden)

    Gustafsson Rasmus K L

    2012-12-01

    Full Text Available Abstract Background For titer assessment of human herpesvirus 6 (HHV-6, IFA targeting viral proteins or a TCID50 method with ocular inspection for CPE can be used. These methods rely on the subjective decision of the assessor, obstructing the ability to obtain unanimous results. Findings We have developed and validated an alternative TCID50 read-out approach where infection in the titration culture plate is assessed by viral DNA load change by quantitative PCR. A ten time increase in viral DNA load was determined as cut point for infection since that yielded a maximum correlation with viral protein expression (93%. The average intra-assay CV was 9% for quantitative PCR read-out of TCID50 compared to 45% for ocular inspection read-out of TCID50, 14% for IFA read-out of TCID50, and 43% for an infectious units approach using IFA. The average inter-assay CV for quantitative PCR read-out of TCID50 was 73%, compared to 66%, 25% and 77% for the ocular inspection read-out for TCID50, IFA read-out of TCID50 and infectious unit approaches respectively. Conclusions The quantitative PCR based read-out of TCID50 proved to be more robust and easier to interpret than traditional TCID50 assessment approaches for HHV-6, and therefore it might be considered as an alternative method.

  17. Diagnosis of Giardia infections by PCR-based methods in children of an endemic area

    Directory of Open Access Journals (Sweden)

    EB David

    2011-01-01

    Full Text Available The present study was designed to estimate the prevalence of Giardia infection in preschool- and school-aged children living in an endemic area. Fecal samples from 573 children were processed by zinc sulfate centrifugal flotation, centrifugal sedimentation (using a commercial device for fecal concentration - TF-Test kit® and polymerase chain reaction (PCR-based methods. Of the stool samples assessed, 277 (48.3% were positive for intestinal parasites and/or commensal protozoa. Centrifugal flotation presented the highest diagnostic sensitivity for Giardia infections. The kappa index revealed that both coproparasitological techniques closely agreed on the Giardia diagnosis (86% versus satisfactory (72% and poor (35% concordances for commensal protozoan and helminth infections, respectively. Concerning Giardia molecular diagnosis, from the 71 microscopy-positive samples, specific amplification of gdh and tpi fragments was noted in 68 (95.7% and 64 (90% samples, respectively. Amplification of gdh and tpi genes was observed, respectively, in 95.7% and 90% of microscopy-positive Giardia samples. For 144 microscopy-negative samples, gdh and tpi gene amplification products were obtained from 8.3% and 35.9% samples, respectively. The agreement between these genes was about 40%. The centrifuge-flotation based method was the most suitable means of Giardia diagnosis assessed in the present study by combining accuracy and low cost.

  18. Genotyping for DQA1 and PM loci in urine using PCR-based amplification: effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing.

    Science.gov (United States)

    Vu, N T; Chaturvedi, A K; Canfield, D V

    1999-05-31

    Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for individualization of biological samples have reached a new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investigated the influence of storage conditions, sample volume, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based genotyping for DQA1 and PM loci in urine. Urine specimens from male and female volunteers were divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by dialfiltration, using 3000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin method gave comparable typing results. Larger sample volume yielded a higher amount of DNA, but the typing rates were not affected for sample volumes between 1 and 5 ml. The quantifiable amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage samples produced significantly higher success rates than that of frozen samples, while there was only marginal difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of DQA1 + PM genotype was achieved for all samples of fresh urine, independent of gender

  19. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

    Science.gov (United States)

    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing. Here, we evaluated ten of these methods (BacH, BacHum-UCD, B. thetaiotaomic...

  20. A PCR-based method for identification of bifidobacteria from the human alimentary tract at the species level

    NARCIS (Netherlands)

    Venema, K.; Maathuis, A.J.H.

    2003-01-01

    A polymerase chain reaction (PCR)-based method was developed for the identification of isolates of Bifidobacterium at the species level. Using two Bifidobacterium-specific primers directed against the 16S ribosomal gene (Bif164 and Bif662), a PCR product was obtained from the type strains of 12

  1. Development of PCR-based detection methods for the quarantine phytopathogen Synchytrium endobioticum, causal agent of wart disease

    NARCIS (Netherlands)

    Boogert, van den P.H.J.F.; Gent-Pelzer, van M.P.E.; Bonants, P.J.M.; Boer, de S.H.; Wander, J.G.N.; Lévesque, C.A.; Leeuwen, van G.C.M.; Baayen, R.P.

    2005-01-01

    Abstract PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and

  2. Diagnosis of clinical samples spotted on FTA cards using PCR-based methods.

    Science.gov (United States)

    Jamjoom, Manal; Sultan, Amal H

    2009-04-01

    The broad clinical presentation of Leishmaniasis makes the diagnosis of current and past cases of this disease rather difficult. Differential diagnosis is important because diseases caused by other aetiologies and a clinical spectrum similar to that of leishmaniasis (e.g. leprosy, skin cancers and tuberculosis for CL; malaria and schistosomiasis for VL) are often present in endemic areas of endemicity. Presently, a variety of methods have been developed and tested to aid the identification and diagnosis of Leishmania. The advent of the PCR technology has opened new channels for the diagnosis of leishmaniasis in a variety of clinical materials. PCR is a simple, rapid procedure that has been adapted for diagnosis of leishmaniasis. A range of tools is currently available for the diagnosis and identification of leishmaniasis and Leishmania species, respectively. However, none of these diagnostic tools are examined and tested using samples spotted on FTA cards. Three different PCR-based approaches were examined including: kDNA minicircle, Leishmania 18S rRNA gene and PCR-RFLP of Intergenic region of ribosomal protein. PCR primers were designed that sit within the coding sequences of genes (relatively well conserved) but which amplify across the intervening intergenic sequence (relatively variable). These were used in PCR-RFLP on reference isolates of 10 of the most important Leishmania species: L. donovani, L. infantum, L. major & L. tropica. Digestion of PCR products with restriction enzymes produced species-specific restriction patterns allowed discrimination of reference isolates. The kDNA minicircle primers are highly sensitive in diagnosis of both bone marrow and skin smears from FTA cards. Leishmania 18S rRNA gene conserved region is sensitive in identification of bone marrow smear but less sensitive in diagnosing skin smears. The intergenic nested PCR-RFLP using P5 & P6 as well as P1 & P2 newly designed primers showed high level of reproducibility and sensitivity

  3. Validation of a PCR-based method for detection of food-borne thermotolerant Campylobacters in a multicenter collaborative trial

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Cook, N.; D'Agostino, M.

    2004-01-01

    A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for c......% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples....

  4. Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

    OpenAIRE

    Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

    1999-01-01

    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy pr...

  5. A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA

    Directory of Open Access Journals (Sweden)

    M Heydarnezhadi

    2011-09-01

    Full Text Available Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers.Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %.Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.

  6. European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

    Science.gov (United States)

    Gianfranceschi, Monica Virginia; Rodriguez-Lazaro, David; Hernandez, Marta; González-García, Patricia; Comin, Damiano; Gattuso, Antonietta; Delibato, Elisabetta; Sonnessa, Michele; Pasquali, Frederique; Prencipe, Vincenza; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Kozačinski, Lidija; Tomic, Danijela Horvatek; Zdolec, Nevijo; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John Elmerdahl; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Paiusco, Antonella; De Cesare, Alessandra; Manfreda, Gerardo; De Medici, Dario

    2014-08-01

    The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

    Science.gov (United States)

    Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

    2016-01-01

    This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.

  8. HLA-DQA1 typing in Danes by two polymerase chain reaction (PCR) based methods

    DEFF Research Database (Denmark)

    Cowland, J B; Madsen, H O; Morling, N

    1995-01-01

    (ASA) method, which together recognise eight alleles. In 146 unrelated Danish individuals, the HLA-DQA1 alleles were in Hardy-Weinberg equilibrium. For identity testing, the power of discrimination (PD) of HLA-DQA1 was 0.932 with the RDB method and 0.942 with the PCR-RFLP/ASA method. For paternity...

  9. Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

    Science.gov (United States)

    Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

    1999-01-01

    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

  10. Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

    Science.gov (United States)

    Torriani, S; Zapparoli, G; Dellaglio, F

    1999-10-01

    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

  11. A novel PCR-based method to enumerate Salmonella in animal feed

    DEFF Research Database (Denmark)

    Löfström, Charlotta; Andersson, Gunnar; Häggblom, Per

    2010-01-01

    and pellets) only three gave results that differed between the PCR and MSRV methods. Ct values for naturally contaminated samples were higher compared to samples artificially contaminated with low numbers (approx. 2 CFU/25 g feed) of stressed Salmonella. To allow quantification of low numbers of Salmonella...

  12. Inverse PCR-based method for isolating novel SINEs from genome.

    Science.gov (United States)

    Han, Yawei; Chen, Liping; Guan, Lihong; He, Shunping

    2014-04-01

    Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.

  13. Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables.

    Science.gov (United States)

    Vojkovska, H; Kubikova, I; Kralik, P

    2015-03-01

    Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. © 2014

  14. PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.).

    Science.gov (United States)

    Colabella, Fernando; Libkind, Diego

    2016-01-01

    It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (i.e. Phaffia rhodozyma, synonym Xanthophyllomyces dendrorhous) is much greater than previously thought. P. rhodozyma is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. P. rhodozyma was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of P. rhodozyma and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus Phaffia (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  15. A Rapid and Inexpensive PCR-Based STR Genotyping Method for Identifying Forensic Specimens

    Science.gov (United States)

    2006-06-01

    this report) 20. Security Classif. (of this page) 21 . No. of Pages 22. Price Unclassified Unclassified 18 Form DOT F 1700.7 (8-72) Reproduction of...Promega PowerPlex 2.1 system (7) except that the buffer was 1x Amplitaq gold reaction buffer, 0.1% TritonX-100, and 0.2mM each dNTP in a 25µl final...electrophoresis were done using the PowerPlex 16 System (Promega Corp.; Madison, WI). rEsulTs ANd dIsCussION The goal of this study was to develop a simple DNA

  16. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

    Directory of Open Access Journals (Sweden)

    Huiling Qiu

    Full Text Available DNA vector-encoded Tough Decoy (TuD miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer, which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD vector in which only two sets of shorter oligonucleotides (< 60 mer were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324 were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo.

  17. A PCR based method to detect Russula spp. in soil samples and Limodorum abortivum roots in Mediterranean environments

    Directory of Open Access Journals (Sweden)

    Eduardo Larriba

    2015-04-01

    Full Text Available Aim of study: Orchidaceaehas the largest number of species of any family in the plant kingdom. This family is subject to a high risk of extinction in natural environments, such as natural parks and protected areas. Recent studies have shown the prevalence of many species of orchids to be linked to fungal soil diversity, due to their myco-heterotrophic behaviour. Plant communities determine fungal soil diversity, and both generate optimal conditions for orchid development. Area of study: The work was carried out in n the two most important natural parks in Alicante (Font Roja and Sierra Mariola, in South-eastern of Spain. Material and Methods: We designed a molecular tool to monitor the presence of Russula spp. in soil and orchids roots, combined with phytosociological methods. Main results: Using a PCR-based method, we detected the presence in the soil and Limodorum abortivum orchid roots of the mycorrhizal fungi Russula spp. The species with highest coverage was Quercus rotundifolia in areas where the orchid was present. Research highlights: We present a useful tool based on PCR to detect the presence of Russula spp. in a natural environment. These results are consistent with those obtained in different studies that linked the presence of the mycorrhizal fungi Russula spp. in roots of the species Limodorum and the interaction between these fungal species and Quercus ilex trees in Mediterranean forest environments.

  18. Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee.

    Science.gov (United States)

    von Hertwig, Aline Morgan; Sant'Ana, Anderson S; Sartori, Daniele; da Silva, Josué José; Nascimento, Maristela S; Iamanaka, Beatriz Thie; Pelegrinelli Fungaro, Maria Helena; Taniwaki, Marta Hiromi

    2018-05-01

    Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B 2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees.

    Science.gov (United States)

    Guglielmo, F; Bergemann, S E; Gonthier, P; Nicolotti, G; Garbelotto, M

    2007-11-01

    The goal of this research was the development of a PCR-based assay to identify important decay fungi from wood of hardwood tree species in northern temperate regions. Eleven taxon-specific primers were designed for PCR amplification of either nuclear or mitochondrial ribosomal DNA regions of Armillaria spp., Ganoderma spp., Hericium spp., Hypoxylon thouarsianum var. thouarsianum, Inonotus/Phellinus-group, Laetiporus spp., Perenniporia fraxinea, Pleurotus spp., Schizophyllum spp., Stereum spp. and Trametes spp. Multiplex PCR reactions were developed and optimized to detect fungal DNA and identify each taxon with a sensitivity of at least 1 pg of target DNA in the template. This assay correctly identified the agents of decay in 82% of tested wood samples. The development and optimization of multiplex PCRs allowed for reliable identification of wood rotting fungi directly from wood. Early detection of wood decay fungi is crucial for assessment of tree stability in urban landscapes. Furthermore, this method may prove useful for prediction of the severity and the evolution of decay in standing trees.

  20. Comparison of a PCR-Based Method with Culture and Direct Examination for Diagnosis of Acanthamoeba keratitis

    Directory of Open Access Journals (Sweden)

    S Farnia

    2009-05-01

    Full Text Available "nBackground: The aim was to compare three different methods (direct examination, culture and PCR meth­ods for the diagnosis of Acanthamoeba keratitis (AK in corneal scrapes."nMethods: Twenty eight corneal scrapes and contact lenses were collected from keratitis patients and re­ferred to the De­partment of Medical Parasitology and Mycology, School of Public Health, Tehran Univer­sity of Medical Sci­ences. Corneal scrapes were divided in three parts for direct examination, culture on non-nutrient agar and PCR analysis. PCR analysis was also performed using a 18S rRNA gene primer pair (DF3 region. DF3 (Diagnostic frag­ment 3 is a region of the nuclear small subunit ribosomal RNA gene which is specific for detecting Acan­thamoeba strains."nResults:  Acanthamoeba was the causative agent of keratitis in 50% of the patients. Direct smear of all pre­pared corneal scrapes in AK patients was negative and culture was positive in only 14.3% of the isolates. PCR analysis was positive in 71.4% of AK patients. These three methods were negative in corneal scrapes of non-AK patients. The sensitivity and specificity of PCR technique for the detection of Acanthamoeba sp. were calculated as 71.4% and 100%, respectively."nConclusion: According to high sensitivity and specificity of PCR-based method, this study confirmed that PCR using 18S rRNA gene primers (DF3 region is more useful for detecting AK cases compare to culture and direct microscopy methods.

  1. Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform.

    Science.gov (United States)

    Opota, Onya; Brouillet, René; Greub, Gilbert; Jaton, Katia

    2017-01-01

    The advances in molecular biology of the last decades have dramatically improved the field of diagnostic bacteriology. In particular, PCR-based technologies have impacted the diagnosis of infections caused by obligate intracellular bacteria such as pathogens from the Chlamydiacae family. Here, we describe a real-time PCR-based method using the Taqman technology for the diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus infection. The method presented here can be applied to various clinical samples and can be adapted on opened molecular diagnostic platforms.

  2. Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach.

    Directory of Open Access Journals (Sweden)

    Iveta Svobodová

    Full Text Available Detection and characterization of circulating cell-free fetal DNA (cffDNA from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods' performance parameters-standard curve linearity, detection limit and measurement precision-were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438.

  3. Novel One-Step Multiplex PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of -Thalassemia

    Directory of Open Access Journals (Sweden)

    Raquel M. Fernández

    2013-01-01

    Full Text Available Preimplantation genetic diagnosis (PGD of single gene disorders, combined with HLA matching (PGD-HLA, has emerged as a tool for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here, we present a novel one-step multiplex PCR to genotype a spectrum of STRs to simultaneously perform HLA typing and PGD for -thalassemia. This method is being routinely used for PGD-HLA cycles in our department, with a genotyping success rate of 100%. As an example, we present the first successful PGD-HLA typing in Spain, which resulted in the birth of a boy and subsequent successful HSC transplantation to his affected brother, who is doing well 4 years following transplantation. The advantage of our method is that it involves only a round of single PCR for multiple markers amplification (up to 10 markers within the HLA and 6 markers at the -globin loci. This strategy has allowed us to considerably reduce the optimization of the PCR method for each specific PGD-HLA family as well as the time to obtain molecular results in each cycle.

  4. Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing

    Directory of Open Access Journals (Sweden)

    Elizabeth Schaeffer

    2017-01-01

    Full Text Available Our objective was to determine if whole genome amplification (WGA provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3 or trophoblastic cells (Day 5 were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo’s sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p<0.001. Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL was also used to determine sex. AMELY peak’s height was higher and this peak’s presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p<0.001. Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89 and RPL17 for Trisomy 18 (AUC = 0.94. Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

  5. Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database

    Science.gov (United States)

    Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A.; Falkinham, Joseph O.; Williams, Myra D.; Vasireddy, Ravikiran; Wilson, Rebecca W.; Turenne, Christine

    2013-01-01

    Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible. PMID:23175249

  6. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping of mycobacterium intracellulare for strain comparison with establishment of a PCR-based database.

    Science.gov (United States)

    Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A; Falkinham, Joseph O; Williams, Myra D; Vasireddy, Ravikiran; Wilson, Rebecca W; Turenne, Christine; Wallace, Richard J

    2013-02-01

    Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the "gold standard" of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.

  7. Performance of the digene LQ, RH and PS HPVs genotyping systems on clinical samples and comparison with HC2 and PCR-based Linear Array.

    Science.gov (United States)

    Godínez, Jose M; Tous, Sara; Baixeras, Nuria; Moreno-Crespi, Judith; Alejo, María; Lejeune, Marylène; Bravo, Ignacio G; Bosch, F Xavier; de Sanjosé, Silvia

    2011-11-18

    Certain Human Papillomaviruses (HPVs) are the infectious agents involved in cervical cancer development. Detection of HPVs DNA is part of the cervical cancer screening protocols and HPVs genotyping has been proposed for its inclusion in these preventive programs. The aim of this study was to evaluate three novel genotyping tests, namely Qiagen LQ, RH and PS, in clinical samples with and without abnormalities. For this, 305 cervical samples were processed and the results of the evaluated techniques were compared with those obtained in the HPVs diagnostic process in our lab, by using HC2 and Linear Array (LA) technologies. The concordances and kappa statistics (k) for each technique compared with HC2 were 98.69% (k = 0.94) for LQ, 98.03% (k = 0.91) for RH and 91.80% (k = 0.82) for PS. There was a very good agreement in HPVs type-specific concordance for the most prevalent types HPV16 (kappa range = 0.83-0.90), HPV18 (k.r.= 0.74-0.80) and HPV45 (k.r.= 0.82-0.90). The three tests showed an overall good concordance for HPVs detection when compared with HR-HC2 system. LQ and RH rendered lower detection rate for multiple infections than LA genotyping. However, our understanding of the clinical significance of multiple HPVs infections is still incomplete and therefore the relevance of the lower ability to detect multiple infections needs to be evaluated.

  8. Performance of the digene LQ, RH and PS HPVs genotyping systems on clinical samples and comparison with HC2 and PCR-based Linear Array

    Directory of Open Access Journals (Sweden)

    Godínez Jose M

    2011-11-01

    Full Text Available Abstract Background Certain Human Papillomaviruses (HPVs are the infectious agents involved in cervical cancer development. Detection of HPVs DNA is part of the cervical cancer screening protocols and HPVs genotyping has been proposed for its inclusion in these preventive programs. The aim of this study was to evaluate three novel genotyping tests, namely Qiagen LQ, RH and PS, in clinical samples with and without abnormalities. For this, 305 cervical samples were processed and the results of the evaluated techniques were compared with those obtained in the HPVs diagnostic process in our lab, by using HC2 and Linear Array (LA technologies. Results The concordances and kappa statistics (k for each technique compared with HC2 were 98.69% (k = 0.94 for LQ, 98.03% (k = 0.91 for RH and 91.80% (k = 0.82 for PS. There was a very good agreement in HPVs type-specific concordance for the most prevalent types HPV16 (kappa range = 0.83-0.90, HPV18 (k.r.= 0.74-0.80 and HPV45 (k.r.= 0.82-0.90. Conclusions The three tests showed an overall good concordance for HPVs detection when compared with HR-HC2 system. LQ and RH rendered lower detection rate for multiple infections than LA genotyping. However, our understanding of the clinical significance of multiple HPVs infections is still incomplete and therefore the relevance of the lower ability to detect multiple infections needs to be evaluated.

  9. Screening for methicillin-resistant Staphylococcus aureus in clinical swabs using a high-throughput real-time PCR-based method

    DEFF Research Database (Denmark)

    Ornskov, D; Kolmos, B; Bendix Horn, P

    2008-01-01

    2005, all patients and healthcare personnel have been screened for MRSA colonisation, involving analysis of 300-400 samples daily. To deal with this number of samples, a PCR-based method customised for high-throughput analysis and a system for fast reporting of MRSA carrier status were developed. Swab...... samples were incubated overnight in a selective tryptone soya broth and were analysed by PCR the following day. Using this strategy, non-colonised individuals were identified within 24 h, while MRSA-positive samples were analysed further by traditional microbiological methods to determine the resistance...... pattern. This is a cost-effective approach, as the greatest expense in hospitals involves the isolation of patients of unknown MRSA status. The method was evaluated by testing 2194 clinical samples, with a sensitivity and specificity of 100% and 94%, respectively. The analytical sensitivity was 97...

  10. Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Krause, Michael; Hansen, F.

    2007-01-01

    no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mu l improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples...... the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 mu l were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 mu l) had...

  11. Optimization of a Real Time PCR based method for the detection of Listeria monocytogenes in pork meat.

    Science.gov (United States)

    Gattuso, Antonietta; Gianfranceschi, Monica Virginia; Sonnessa, Michele; Delibato, Elisabetta; Marchesan, Massimo; Hernandez, Marta; De Medici, Dario; Rodriguez-Lazaro, David

    2014-08-01

    The aim of this study was to optimize a Real-Time PCR protocol for a rapid detection of Listeria monocytogenes in pork meat, using reduced volumes of primary selective enrichment broth and times of incubation to decrease the cost and time for analysis. Forty-five samples of pork meat were artificially contaminated with two different levels of L. monocytogenes (1-10 CFU per sample and 10-100 CFU per sample), homogenized in three different volumes of Half Fraser Broth (1:3; 1:5 and 1:10) and incubated at 30°C ± 1°C for 5h, 8h and 24h. The detection was conducted in parallel by Real-Time PCR and the ISO standard 11290-1 methods. L. monocytogenes was detected in all the samples after 24h by Real-Time PCR method, also using reduced volumes of Half Fraser Broth. This represents a clear advantage as the time to final detection and the inherent costs were significantly reduced compared to the ISO reference method. All samples artificially contaminated were correctly detected also after 8 of incubation at 30°C ± 1°C in Half Fraser Broth and 24h in Fraser Broth at 37°C ± 1°C using cultural method. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Evaluation and In-House Validation of Five DNA Extraction Methods for PCR-based STR Analysis of Bloodstained Denims

    Directory of Open Access Journals (Sweden)

    Henry Perdigon

    2004-06-01

    Full Text Available One type of crime scene evidence commonly submitted for analysis is bloodstain on denim. However, chemicals (e.g., indigo used to produce denim materials may co-purify with DNA and hence, affect subsequent DNA analysis. The present study compared five methods (e.g., standard organic, organic with hydrogen peroxide (H2O2, modified FTA™, organic/Chelex®-Centricon®, and QIAamp® DNA Mini Kit-based procedures for the isolation of blood DNA from denim. A Short Tandem Repeat (STR-based analysis across two to nine STR markers, namely, HUMvWA, HUMTH01, D8S306, HUMFES/FPS, HUMDHFRP2, HUMF13A01, HUMFGA, HUMTPOX, and HUMCSF1PO, was used to evaluate successful amplification of blood DNA extracted from light indigo, dark indigo, indigo-sulfur, pure indigo, sulfur-top, and sulfur-bottom denim materials. The results of the present study support the utility of organic/Chelex®-Centricon® and QIAamp® Kit procedures in extracting PCR-amplifiable DNA from five different types of denim materials for STR analysis. Furthermore, a solid-based method using FTA™ classic cards was modified to provide a simple, rapid, safe, and cost-effective procedure for extracting blood DNA from light, dark indigo and pure indigo denim materials. However, DNA eluted from bloodstained sulfur-dyed denims (e.g., sulfur-top and sulfur-bottom using FTA™ procedure was not readily amplifiable.

  13. PCR-based methods for identification of potentially zoonotic ascaridoid parasites of the dog, fox and cat.

    Science.gov (United States)

    Jacobs, D E; Zhu, X; Gasser, R B; Chilton, N B

    1997-11-01

    Genomic DNA was extracted from ascaridoid nematodes collected from dogs, foxes and cats. A region spanning the second internal transcribed spacer (ITS-2) of the ribosomal DNA of each sample was amplified by PCR. Representative ITS-2 products for each nematode species (Toxocara canis, Toxocara cati and Toxascaris leonina) were sequenced. Restriction sites were identified for use as genetic markers in a PCR-linked RFLP assay. The three species could be differentiated from each other and from other ascaridoids that may be found in human tissues by use of two endonucleases, HinfI and RsaI. Primers were designed to unique regions of the ITS-2 sequences of the three species for use in diagnostic PCR procedures and primer sets evaluated against panels of homologous and heterologous DNA samples. Results suggest that both methods are good candidates for further development for the detection and/or identification of ascaridoid larvae in human tissues.

  14. A new PCR-based method shows that blue crabs (Callinectes sapidus (Rathbun)) consume winter flounder (Pseudopleuronectes americanus (Walbaum)).

    Science.gov (United States)

    Collier, Jackie L; Fitzgerald, Sean P; Hice, Lyndie A; Frisk, Michael G; McElroy, Anne E

    2014-01-01

    Winter flounder (Pseudopleuronectes americanus) once supported robust commercial and recreational fisheries in the New York (USA) region, but since the 1990s populations have been in decline. Available data show that settlement of young-of-the-year winter flounder has not declined as sharply as adult abundance, suggesting that juveniles are experiencing higher mortality following settlement. The recent increase of blue crab (Callinectes sapidus) abundance in the New York region raises the possibility that new sources of predation may be contributing to juvenile winter flounder mortality. To investigate this possibility we developed and validated a method to specifically detect winter flounder mitochondrial control region DNA sequences in the gut contents of blue crabs. A survey of 55 crabs collected from Shinnecock Bay (along the south shore of Long Island, New York) in July, August, and September of 2011 showed that 12 of 42 blue crabs (28.6%) from which PCR-amplifiable DNA was recovered had consumed winter flounder in the wild, empirically supporting the trophic link between these species that has been widely speculated to exist. This technique overcomes difficulties with visual identification of the often unrecognizable gut contents of decapod crustaceans, and modifications of this approach offer valuable tools to more broadly address their feeding habits on a wide variety of species.

  15. LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

    Science.gov (United States)

    Lehle, Simon; Hildebrand, Dominic G.; Merz, Britta; Malak, Peter N.; Becker, Michael S.; Schmezer, Peter; Essmann, Frank; Schulze-Osthoff, Klaus; Rothfuss, Oliver

    2014-01-01

    DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications. PMID:24371283

  16. A multiplex PCR-based method to identify strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples.

    Science.gov (United States)

    Bisset, S A; Knight, J S; Bouchet, C L G

    2014-02-24

    A multiplex PCR-based method was developed to overcome the limitations of microscopic examination as a means of identifying individual infective larvae from the wide range of strongylid parasite species commonly encountered in sheep in mixed sheep-cattle grazing situations in New Zealand. The strategy employed targets unique species-specific sequence markers in the second internal transcribed spacer (ITS-2) region of ribosomal DNA of the nematodes and utilises individual larval lysates as reaction templates. The basic assay involves two sets of reactions designed to target the ten strongylid species most often encountered in ovine faecal cultures under New Zealand conditions (viz. Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Cooperia curticei, Cooperia oncophora, Nematodirus spathiger, Chabertia ovina, and Oesophagostomum venulosum). Five species-specific primers, together with a pair of "generic" (conserved) primers, are used in each of the reactions. Two products are generally amplified, one by the generic primer pair regardless of species (providing a positive PCR control) and the other (whose size is indicative of the species present) by the appropriate species-specific primer in combination with one or other of the generic primers. If necessary, any larvae not identified by these reactions can subsequently be tested using primers designed specifically to detect those species less frequently encountered in ovine faecal cultures (viz. Ostertagia ostertagi, Ostertagia leptospicularis, Cooperia punctata, Nematodirus filicollis, and Bunostomum trigonocephalum). Results of assays undertaken on >5500 nematode larvae cultured from lambs on 16 different farms distributed throughout New Zealand indicated that positive identifications were initially obtained for 92.8% of them, while a further 4.4% of reactions gave a generic but no visible specific product and 2.8% gave no discernible

  17. Validation of a PCR-based method for the detection of various rendered materials in feedstuffs using a forensic DNA extraction kit.

    Science.gov (United States)

    Myers, Michael J; Yancy, Haile F; Araneta, Michael; Armour, Jennifer; Derr, Janice; Hoostelaere, Lawrence A D; Farmer, Doris; Jackson, Falana; Kiessling, William M; Koch, Henry; Lin, Huahua; Liu, Yan; Mowlds, Gabrielle; Pinero, David; Riter, Ken L; Sedwick, John; Shen, Yuelian; Wetherington, June; Younkins, Ronsha

    2006-01-01

    A method trial was initiated to validate the use of a commercial DNA forensic kit to extract DNA from animal feed as part of a PCR-based method. Four different PCR primer pairs (one bovine pair, one porcine pair, one ovine primer pair, and one multispecies pair) were also evaluated. Each laboratory was required to analyze a total of 120 dairy feed samples either not fortified (control, true negative) or fortified with bovine meat and bone meal, porcine meat and bone meal (PMBM), or lamb meal. Feeds were fortified with the animal meals at a concentration of 0.1% (wt/wt). Ten laboratories participated in this trial, and each laboratory was required to evaluate two different primer pairs, i.e., each PCR primer pair was evaluated by five different laboratories. The method was considered to be validated for a given animal source when three or more laboratories achieved at least 97% accuracy (29 correct of 30 samples for 96.7% accuracy, rounded up to 97%) in detecting the fortified samples for that source. Using this criterion, the method was validated for the bovine primer because three laboratories met the criterion, with an average accuracy of 98.9%. The average false-positive rate was 3.0% in these laboratories. A fourth laboratory was 80% accurate in identifying the samples fortified with bovine meat and bone meal. A fifth laboratory was not able to consistently extract the DNA from the feed samples and did not achieve the criterion for accuracy for either the bovine or multispecies PCR primers. For the porcine primers, the method was validated, with four laboratories meeting the criterion for accuracy with an average accuracy of 99.2%. The fifth laboratory had a 93.3% accuracy outcome for the porcine primer. Collectively, these five laboratories had a 1.3% false-positive rate for the porcine primer. No laboratory was able to meet the criterion for accuracy with the ovine primers, most likely because of problems with the synthesis of the primer pair; none of the

  18. Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

    Science.gov (United States)

    Dreesen, Jos; Destouni, Aspasia; Kourlaba, Georgia; Degn, Birte; Mette, Wulf Christensen; Carvalho, Filipa; Moutou, Celine; Sengupta, Sioban; Dhanjal, Seema; Renwick, Pamela; Davies, Steven; Kanavakis, Emmanouel; Harton, Gary; Traeger-Synodinos, Joanne

    2014-01-01

    Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD. PMID:24301057

  19. Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

    Science.gov (United States)

    Smiljanic, M; Kaase, M; Ahmad-Nejad, P; Ghebremedhin, B

    2017-07-10

    Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. Among the carbapenem-resistant isolates the genes bla KPC , bla VIM , bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC , bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

  20. Detection of HPV-DNA by a PCR-based method in formalin-fixed, paraffin-embedded tissue from rare endocervical carcinoma types.

    Science.gov (United States)

    Nofech-Mozes, Sharon; Khalifa, Mahmoud M; Ismiil, Nadia; Dubé, Valerie; Saad, Reda S; Sun, Peizhu; Seth, Arun; Ghorab, Zeina

    2010-01-01

    High-risk human papilloma virus (HPV) seems to play a role in the pathogenesis of cervical squamous neoplasia and adenocarcinomas of the mucinous and endometrioid cell types. Cervical serous, clear cell, and small cell carcinomas differ from the conventional endocervical adenocarcinoma in their clinical characteristics. The data on the role of HPV in their pathogenesis are limited. In this study, we examined the presence of high-risk HPV-DNA in rare types of cervical carcinoma using polymerase chain reaction-based test. In-house cervical serous, clear cell, and small cell carcinoma cases accessioned between 2000 and 2008 were tested for HPV by polymerase chain reaction amplification of DNA extracted from deparaffinized sections using Roche AMPLICOR HPV Amplification Detection and Control Kits. The kit detects all 13 high-risk HPV-DNA genotypes. The positive cut-off point for AMPLICOR HPV Test was A450 = 0.2. We identified 4 serous, 3 clear cell, 1 mixed clear cell and serous, and 5 small cell carcinomas. High-risk HPV-DNA tested positive in 3 out of 4 serous carcinomas, 2 out of 3 cervical clear cell carcinomas, and all 5 cases of small cell carcinoma and the mixed cell type. Our report documents HPV status in a series of archival unusual types of adenocarcinoma of the uterine cervix. It suggests a robust association between high-risk HPV and these rare subtypes. Despite their unique clinical setting and morphologic appearance, the majority of these tumors likely share a common HPV-mediated carcinogenic pathway. Our observation is particularly significant in cervical cancer prevention as we enter the HPV vaccination era.

  1. Comparison of PCR-Based Diagnosis with Centrifuged-Based Enrichment Method for Detection of Borrelia persica in Animal Blood Samples.

    Science.gov (United States)

    Naddaf, S R; Kishdehi, M; Siavashi, Mr

    2011-01-01

    The mainstay of diagnosis of relapsing fever (RF) is demonstration of the spirochetes in Giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by microscopy. This study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spirochetemia. Animal blood samples with low degrees of spirochetemia were tested with two PCRs and a nested PCR targeting flaB, GlpQ, and rrs genes. Also, a centrifuged-based enrichment method and Giemsa staining were performed on blood samples with various degrees of spirochetemia. The flaB-PCR and nested rrs-PCR turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. The GlpQ-PCR was positive as far as at least one spirochete was seen in 5-10 microscopic fields. The sensitivity of GlpQ-PCR increased when DNA from Buffy Coat Layer (BCL) was used as template. The centrifuged-based enrichment method turned positive with as low concentration as 50 bacteria/ml blood, while Giemsa thick staining detected bacteria with concentrations ≥ 25000 bacteria/ml. Centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some PCR assays. Due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of RF in rural health centers.

  2. Comparison of PCR-Based Diagnosis with Centrifuged-Based Enrichment Method for Detection of Borrelia Persica in Animal Blood Samples

    Directory of Open Access Journals (Sweden)

    SR Naddaf

    2011-06-01

    Full Text Available Background: The mainstay of diagnosis of relapsing fever (RF is demonstration of the spirochetes in Giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by mi­cros­copy. This study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spi­ro­chetemia. Methods: Animal blood samples with low degrees of spirochetemia were tested with two PCRs and a nested PCR tar­get­ing flaB, GlpQ, and rrs genes. Also, a centrifuged-based enrichment method and Giemsa staining were per­formed on blood samples with various degrees of spirochetemia. Results: The flaB-PCR and nested rrs-PCR turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. The GlpQ-PCR was positive as far as at least one spi­ro­chete was seen in 5-10 microscopic fields. The sensitivity of GlpQ-PCR increased when DNA from Buffy Coat Layer (BCL was used as template. The centrifuged-based enrichment method turned positive with as low concentra­tion as 50 bacteria/ml blood, while Giemsa thick staining detected bacteria with concentrations ≥ 25000 bacteria/ml. Conclusion: Centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some PCR assays. Due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of RF in rural health centers.

  3. Comparison of PCR-Based Diagnosis with Centrifuged-Based Enrichment Method for Detection of Borrelia persica in Animal Blood Samples

    Directory of Open Access Journals (Sweden)

    SR Naddaf

    2011-06-01

    Background: The mainstay of diagnosis of relapsing fever (RF is demonstration of the spirochetes in Giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by mi­cros­copy. This study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spi­ro­chetemia. Methods: Animal blood samples with low degrees of spirochetemia were tested with two PCRs and a nested PCR tar­get­ing flaB, GlpQ, and rrs genes. Also, a centrifuged-based enrichment method and Giemsa staining were per­formed on blood samples with various degrees of spirochetemia. Results: The flaB-PCR and nested rrs-PCR turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. The GlpQ-PCR was positive as far as at least one spi­ro­chete was seen in 5-10 microscopic fields. The sensitivity of GlpQ-PCR increased when DNA from Buffy Coat Layer (BCL was used as template. The centrifuged-based enrichment method turned positive with as low concentra­tion as 50 bacteria/ml blood, while Giemsa thick staining detected bacteria with concentrations ≥ 25000 bacteria/ml.  Conclusion: Centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some PCR assays. Due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of RF in rural health centers.  

  4. Combining land use information and small stream sampling with PCR-based methods for better characterization of diffuse sources of human fecal pollution.

    Science.gov (United States)

    Peed, Lindsay A; Nietch, Christopher T; Kelty, Catherine A; Meckes, Mark; Mooney, Thomas; Sivaganesan, Mano; Shanks, Orin C

    2011-07-01

    Diffuse sources of human fecal pollution allow for the direct discharge of waste into receiving waters with minimal or no treatment. Traditional culture-based methods are commonly used to characterize fecal pollution in ambient waters, however these methods do not discern between human and other animal sources of fecal pollution making it difficult to identify diffuse pollution sources. Human-associated quantitative real-time PCR (qPCR) methods in combination with low-order headwatershed sampling, precipitation information, and high-resolution geographic information system land use data can be useful for identifying diffuse source of human fecal pollution in receiving waters. To test this assertion, this study monitored nine headwatersheds over a two-year period potentially impacted by faulty septic systems and leaky sanitary sewer lines. Human fecal pollution was measured using three different human-associated qPCR methods and a positive significant correlation was seen between abundance of human-associated genetic markers and septic systems following wet weather events. In contrast, a negative correlation was observed with sanitary sewer line densities suggesting septic systems are the predominant diffuse source of human fecal pollution in the study area. These results demonstrate the advantages of combining water sampling, climate information, land-use computer-based modeling, and molecular biology disciplines to better characterize diffuse sources of human fecal pollution in environmental waters.

  5. A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model - Towards an international standard

    DEFF Research Database (Denmark)

    D'Agostino, M.; Wagner, M.; Vazquez-Boland, J.A.

    2004-01-01

    the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 It of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR...

  6. Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin.

    Science.gov (United States)

    Srůtková, Dagmar; Spanova, Alena; Spano, Miroslav; Dráb, Vladimír; Schwarzer, Martin; Kozaková, Hana; Rittich, Bohuslav

    2011-10-01

    Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)(5) primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

    Science.gov (United States)

    Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

    2017-10-27

    Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Molecular methods for bacterial genotyping and analyzed gene regions

    Directory of Open Access Journals (Sweden)

    İbrahim Halil Yıldırım1, Seval Cing Yıldırım2, Nadir Koçak3

    2011-06-01

    Full Text Available Bacterial strain typing is an important process for diagnosis, treatment and epidemiological investigations. Current bacterial strain typing methods may be classified into two main categories: phenotyping and genotyping. Phenotypic characters are the reflection of genetic contents. Genotyping, which refers discrimination of bacterial strains based on their genetic content, has recently become widely used for bacterial strain typing. The methods already used in genotypingof bacteria are quite different from each other. In this review we tried to summarize the basic principles of DNA-based methods used in genotyping of bacteria and describe some important DNA regions that are used in genotyping of bacteria. J Microbiol Infect Dis 2011;1(1:42-46.

  9. Multiple centroid method to evaluate the adaptability of alfalfa genotypes

    Directory of Open Access Journals (Sweden)

    Moysés Nascimento

    2015-02-01

    Full Text Available This study aimed to evaluate the efficiency of multiple centroids to study the adaptability of alfalfa genotypes (Medicago sativa L.. In this method, the genotypes are compared with ideotypes defined by the bissegmented regression model, according to the researcher's interest. Thus, genotype classification is carried out as determined by the objective of the researcher and the proposed recommendation strategy. Despite the great potential of the method, it needs to be evaluated under the biological context (with real data. In this context, we used data on the evaluation of dry matter production of 92 alfalfa cultivars, with 20 cuttings, from an experiment in randomized blocks with two repetitions carried out from November 2004 to June 2006. The multiple centroid method proved efficient for classifying alfalfa genotypes. Moreover, it showed no unambiguous indications and provided that ideotypes were defined according to the researcher's interest, facilitating data interpretation.

  10. Comparative analysis of minor histocompatibility antigens genotyping methods

    Directory of Open Access Journals (Sweden)

    A. S. Vdovin

    2016-01-01

    Full Text Available The wide range of techniques could be employed to find mismatches in minor histocompatibility antigens between transplant recipients and their donors. In the current study we compared three genotyping methods based on polymerase chain reaction (PCR for four minor antigens. Three of the tested methods: allele-specific PCR, restriction fragment length polymorphism and real-time PCR with TaqMan probes demonstrated 100% reliability when compared to Sanger sequencing for all of the studied polymorphisms. High resolution melting analysis was unsuitable for genotyping of one of the tested minor antigens (HA-1 as it has linked synonymous polymorphism. Obtained data could be used to select the strategy for large-scale clinical genotyping.

  11. Methods for MHC genotyping in non-model vertebrates.

    Science.gov (United States)

    Babik, W

    2010-03-01

    Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems. © 2009 Blackwell Publishing Ltd.

  12. [Analytic methods for seed models with genotype x environment interactions].

    Science.gov (United States)

    Zhu, J

    1996-01-01

    Genetic models with genotype effect (G) and genotype x environment interaction effect (GE) are proposed for analyzing generation means of seed quantitative traits in crops. The total genetic effect (G) is partitioned into seed direct genetic effect (G0), cytoplasm genetic of effect (C), and maternal plant genetic effect (Gm). Seed direct genetic effect (G0) can be further partitioned into direct additive (A) and direct dominance (D) genetic components. Maternal genetic effect (Gm) can also be partitioned into maternal additive (Am) and maternal dominance (Dm) genetic components. The total genotype x environment interaction effect (GE) can also be partitioned into direct genetic by environment interaction effect (G0E), cytoplasm genetic by environment interaction effect (CE), and maternal genetic by environment interaction effect (GmE). G0E can be partitioned into direct additive by environment interaction (AE) and direct dominance by environment interaction (DE) genetic components. GmE can also be partitioned into maternal additive by environment interaction (AmE) and maternal dominance by environment interaction (DmE) genetic components. Partitions of genetic components are listed for parent, F1, F2 and backcrosses. A set of parents, their reciprocal F1 and F2 seeds is applicable for efficient analysis of seed quantitative traits. MINQUE(0/1) method can be used for estimating variance and covariance components. Unbiased estimation for covariance components between two traits can also be obtained by the MINQUE(0/1) method. Random genetic effects in seed models are predictable by the Adjusted Unbiased Prediction (AUP) approach with MINQUE(0/1) method. The jackknife procedure is suggested for estimation of sampling variances of estimated variance and covariance components and of predicted genetic effects, which can be further used in a t-test for parameter. Unbiasedness and efficiency for estimating variance components and predicting genetic effects are tested by

  13. Methods for discovering and validating relationships among genotyped animals

    Science.gov (United States)

    Genomic selection based on single-nucleotide polymorphisms (SNPs) has led to the collection of genotypes for over 2.2 million animals by the Council on Dairy Cattle Breeding in the United States. To assure that a genotype is assigned to the correct animal and that the animal’s pedigree is correct, t...

  14. Phenotypic and Genotypic Eligible Methods for Salmonella Typhimurium Source Tracking.

    Science.gov (United States)

    Ferrari, Rafaela G; Panzenhagen, Pedro H N; Conte-Junior, Carlos A

    2017-01-01

    Salmonellosis is one of the most common causes of foodborne infection and a leading cause of human gastroenteritis. Throughout the last decade, Salmonella enterica serotype Typhimurium (ST) has shown an increase report with the simultaneous emergence of multidrug-resistant isolates, as phage type DT104. Therefore, to successfully control this microorganism, it is important to attribute salmonellosis to the exact source. Studies of Salmonella source attribution have been performed to determine the main food/food-production animals involved, toward which, control efforts should be correctly directed. Hence, the election of a ST subtyping method depends on the particular problem that efforts must be directed, the resources and the data available. Generally, before choosing a molecular subtyping, phenotyping approaches such as serotyping, phage typing, and antimicrobial resistance profiling are implemented as a screening of an investigation, and the results are computed using frequency-matching models (i.e., Dutch, Hald and Asymmetric Island models). Actually, due to the advancement of molecular tools as PFGE, MLVA, MLST, CRISPR, and WGS more precise results have been obtained, but even with these technologies, there are still gaps to be elucidated. To address this issue, an important question needs to be answered: what are the currently suitable subtyping methods to source attribute ST. This review presents the most frequently applied subtyping methods used to characterize ST, analyses the major available microbial subtyping attribution models and ponders the use of conventional phenotyping methods, as well as, the most applied genotypic tools in the context of their potential applicability to investigates ST source tracking.

  15. Phenotypic and Genotypic Eligible Methods for Salmonella Typhimurium Source Tracking

    Directory of Open Access Journals (Sweden)

    Rafaela G. Ferrari

    2017-12-01

    Full Text Available Salmonellosis is one of the most common causes of foodborne infection and a leading cause of human gastroenteritis. Throughout the last decade, Salmonella enterica serotype Typhimurium (ST has shown an increase report with the simultaneous emergence of multidrug-resistant isolates, as phage type DT104. Therefore, to successfully control this microorganism, it is important to attribute salmonellosis to the exact source. Studies of Salmonella source attribution have been performed to determine the main food/food-production animals involved, toward which, control efforts should be correctly directed. Hence, the election of a ST subtyping method depends on the particular problem that efforts must be directed, the resources and the data available. Generally, before choosing a molecular subtyping, phenotyping approaches such as serotyping, phage typing, and antimicrobial resistance profiling are implemented as a screening of an investigation, and the results are computed using frequency-matching models (i.e., Dutch, Hald and Asymmetric Island models. Actually, due to the advancement of molecular tools as PFGE, MLVA, MLST, CRISPR, and WGS more precise results have been obtained, but even with these technologies, there are still gaps to be elucidated. To address this issue, an important question needs to be answered: what are the currently suitable subtyping methods to source attribute ST. This review presents the most frequently applied subtyping methods used to characterize ST, analyses the major available microbial subtyping attribution models and ponders the use of conventional phenotyping methods, as well as, the most applied genotypic tools in the context of their potential applicability to investigates ST source tracking.

  16. A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1

    DEFF Research Database (Denmark)

    Fry, Norman K.; Alexiou-Daniel, Stella; Bangsborg, Jette Marie

    1999-01-01

    OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded...

  17. Impact of inter-genotypic recombination and probe cross-reactivity on the performance of the Abbott RealTime HCV Genotype II assay for hepatitis C genotyping.

    Science.gov (United States)

    Sridhar, Siddharth; Yip, Cyril C Y; Chan, Jasper F W; To, Kelvin K W; Cheng, Vincent C C; Yuen, Kwok-Yung

    2018-05-01

    The Abbott RealTime HCV Genotype II assay (Abbott-RT-HCV assay) is a real-time PCR based genotyping method for hepatitis C virus (HCV). This study measured the impact of inter-genotypic recombination and probe cross-reactivity on the performance of the Abbott-RT-HCV assay. 517 samples were genotyped using the Abbott-RT-HCV assay over a one-year period, 34 (6.6%) were identified as HCV genotype 1 without further subtype designation raising the possibility of inaccurate genotyping. These samples were subjected to confirmatory sequencing. 27 of these 34 (79%) samples were genotype 1b while five (15%) were genotype 6. One HCV isolate was an inter-genotypic 1a/4o recombinant. This is a novel natural HCV recombinant that has never been reported. Inter-genotypic recombination and probe cross-reactivity can affect the accuracy of the Abbott-RT-HCV assay, both of which have significant implications on antiviral regimen choice. Confirmatory sequencing of ambiguous results is crucial for accurate genotyping. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Sequencing of the Chlamydophila psittaci ompA Gene Reveals a New Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method

    Science.gov (United States)

    Geens, Tom; Desplanques, Ann; Van Loock, Marnix; Bönner, Brigitte M.; Kaleta, Erhard F.; Magnino, Simone; Andersen, Arthur A.; Everett, Karin D. E.; Vanrompay, Daisy

    2005-01-01

    Twenty-one avian Chlamydophila psittaci isolates from different European countries were characterized using ompA restriction fragment length polymorphism, ompA sequencing, and major outer membrane protein serotyping. Results reveal the presence of a new genotype, E/B, in several European countries and stress the need for a discriminatory rapid genotyping method. PMID:15872282

  19. Methods of developing core collections based on the predicted genotypic value of rice ( Oryza sativa L.).

    Science.gov (United States)

    Li, C T; Shi, C H; Wu, J G; Xu, H M; Zhang, H Z; Ren, Y L

    2004-04-01

    The selection of an appropriate sampling strategy and a clustering method is important in the construction of core collections based on predicted genotypic values in order to retain the greatest degree of genetic diversity of the initial collection. In this study, methods of developing rice core collections were evaluated based on the predicted genotypic values for 992 rice varieties with 13 quantitative traits. The genotypic values of the traits were predicted by the adjusted unbiased prediction (AUP) method. Based on the predicted genotypic values, Mahalanobis distances were calculated and employed to measure the genetic similarities among the rice varieties. Six hierarchical clustering methods, including the single linkage, median linkage, centroid, unweighted pair-group average, weighted pair-group average and flexible-beta methods, were combined with random, preferred and deviation sampling to develop 18 core collections of rice germplasm. The results show that the deviation sampling strategy in combination with the unweighted pair-group average method of hierarchical clustering retains the greatest degree of genetic diversities of the initial collection. The core collections sampled using predicted genotypic values had more genetic diversity than those based on phenotypic values.

  20. Genomic Characterization of Flavobacterium psychrophilum Serotypes and Development of a Multiplex PCR-Based Serotyping Scheme

    Directory of Open Access Journals (Sweden)

    Tatiana Rochat

    2017-09-01

    Full Text Available Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997 for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

  1. Comparison of three boosting methods in parent-offspring trios for genotype imputation using simulation study

    Directory of Open Access Journals (Sweden)

    Abbas Mikhchi

    2016-01-01

    Full Text Available Abstract Background Genotype imputation is an important process of predicting unknown genotypes, which uses reference population with dense genotypes to predict missing genotypes for both human and animal genetic variations at a low cost. Machine learning methods specially boosting methods have been used in genetic studies to explore the underlying genetic profile of disease and build models capable of predicting missing values of a marker. Methods In this study strategies and factors affecting the imputation accuracy of parent-offspring trios compared from lower-density SNP panels (5 K to high density (10 K SNP panel using three different Boosting methods namely TotalBoost (TB, LogitBoost (LB and AdaBoost (AB. The methods employed using simulated data to impute the un-typed SNPs in parent-offspring trios. Four different datasets of G1 (100 trios with 5 k SNPs, G2 (100 trios with 10 k SNPs, G3 (500 trios with 5 k SNPs, and G4 (500 trio with 10 k SNPs were simulated. In four datasets all parents were genotyped completely, and offspring genotyped with a lower density panel. Results Comparison of the three methods for imputation showed that the LB outperformed AB and TB for imputation accuracy. The time of computation were different between methods. The AB was the fastest algorithm. The higher SNP densities resulted the increase of the accuracy of imputation. Larger trios (i.e. 500 was better for performance of LB and TB. Conclusions The conclusion is that the three methods do well in terms of imputation accuracy also the dense chip is recommended for imputation of parent-offspring trios.

  2. Reducing false-positive incidental findings with ensemble genotyping and logistic regression based variant filtering methods.

    Science.gov (United States)

    Hwang, Kyu-Baek; Lee, In-Hee; Park, Jin-Ho; Hambuch, Tina; Choe, Yongjoon; Kim, MinHyeok; Lee, Kyungjoon; Song, Taemin; Neu, Matthew B; Gupta, Neha; Kohane, Isaac S; Green, Robert C; Kong, Sek Won

    2014-08-01

    As whole genome sequencing (WGS) uncovers variants associated with rare and common diseases, an immediate challenge is to minimize false-positive findings due to sequencing and variant calling errors. False positives can be reduced by combining results from orthogonal sequencing methods, but costly. Here, we present variant filtering approaches using logistic regression (LR) and ensemble genotyping to minimize false positives without sacrificing sensitivity. We evaluated the methods using paired WGS datasets of an extended family prepared using two sequencing platforms and a validated set of variants in NA12878. Using LR or ensemble genotyping based filtering, false-negative rates were significantly reduced by 1.1- to 17.8-fold at the same levels of false discovery rates (5.4% for heterozygous and 4.5% for homozygous single nucleotide variants (SNVs); 30.0% for heterozygous and 18.7% for homozygous insertions; 25.2% for heterozygous and 16.6% for homozygous deletions) compared to the filtering based on genotype quality scores. Moreover, ensemble genotyping excluded > 98% (105,080 of 107,167) of false positives while retaining > 95% (897 of 937) of true positives in de novo mutation (DNM) discovery in NA12878, and performed better than a consensus method using two sequencing platforms. Our proposed methods were effective in prioritizing phenotype-associated variants, and an ensemble genotyping would be essential to minimize false-positive DNM candidates. © 2014 WILEY PERIODICALS, INC.

  3. Use of a New High Resolution Melting Method for Genotyping Pathogenic Leptospira spp.

    Directory of Open Access Journals (Sweden)

    Florence Naze

    Full Text Available Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis.In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984. This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island.Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures.

  4. Helicobacter pylori in dyspepsia: Phenotypic and genotypic methods of diagnosis

    Directory of Open Access Journals (Sweden)

    Vignesh Shetty

    2017-01-01

    Full Text Available Background: Helicobacter pylori affects almost half of the world's population and therefore is one of the most frequent and persistent bacterial infections worldwide. H. pylori is associated with chronic gastritis, ulcer disease (gastric and duodenal, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Several diagnostic methods exist to detect infection and the option of one method or another depends on various genes, such as availability, advantages and disadvantages of each method, monetary value, and the age of patients. Materials and Methods: Patients with complaints of abdominal pain, discomfort, acidity, and loss of appetite were chosen for endoscopy, detailed history was contained, and a physical examination was conducted before endoscopy. Biopsies (antrum + body were received from each patient and subjected to rapid urease test (RUT, histopathological examination (HPE, polymerase chain reaction (PCR, and culture. Results: Of the total 223 biopsy specimens obtained from dyspeptic patients, 122 (54.7% were positive for H. pylori for HPE, 109 (48.9% by RUT, 65 (29.1% by culture, and 117 (52.5% by PCR. The specificity and sensitivity were as follows: RUT (99% and 88.5%, phosphoglucosamine mutase PCR assay (100% and 95.9%, and culture (100% and 53.3%, respectively. Conclusion: In this study, we compared the various diagnostic methods used to identify H. pylori infection indicating that, in comparison with histology as gold standard for detection of H. pylori infection, culture and PCR showed 100% specificity whereas RUT and PCR showed 99% and 100% sensitivity, respectively.

  5. Genetic Diversity Analysis of Tagetes Species Using PCR Based Molecular Markers

    International Nuclear Information System (INIS)

    Shahzadi, I.; Ahmad, R.; Waheed, U.; Shah, M. F.

    2016-01-01

    Tagetes is a genus of medicinally important wild and cultivated plants containing several chemical compounds. Lack of information on variation at molecular level present in Tagetes species is paramount to understand the genetic basis of medicinally important compounds. Current study aims at finding genetic variability in Tagetes species using random and specific molecular markers. Two primer systems including 25 RAPD and 3 STS (limonene gene) were used to ascertain genetic diversity of 15 Tagetes genotypes belonging to different species. We found that 20 of the 25 tested RAPD primers generated stable band patterns with 167 loci of amplification products. The proportion of polymorphic bands was 95.21 percent for RAPD primers. Three STS primers generated a total of 29 amplification products, of which 96.55 percent were polymorphic. Homology of genotypes was 53.18 percent and 51.11 percent with RAPD and STS primers respectively. The dendrogram obtained revealed that the range of overall genetic distances estimated was 22 percent to 100 percent through RAPD and 9 percent to 100 percent through STS markers. The findings help to establish that PCR-based assay such as RAPD and STS could be used successfully for estimation of genetic diversity of different genotypes of Tagetes that can be used for selection of parents for improvement of the species. (author)

  6. Specific PCR-based detection of Alternaria helianthi

    DEFF Research Database (Denmark)

    Udayashankar, A.C.; Nayaka, S. Chandra; Archana, B.

    2012-01-01

    Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method...... tested. The detection limit of the PCR method was of 10 pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification...

  7. Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus

    Science.gov (United States)

    2012-01-01

    Background The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. Method This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Result Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. Conclusion The molecular typing method presented is one tool that could be incorporated into the forensic

  8. Discriminative power of Campylobacter phenotypic and genotypic typing methods.

    Science.gov (United States)

    Duarte, Alexandra; Seliwiorstow, Tomasz; Miller, William G; De Zutter, Lieven; Uyttendaele, Mieke; Dierick, Katelijne; Botteldoorn, Nadine

    2016-06-01

    The aim of this study was to compare different typing methods, individually and combined, for use in the monitoring of Campylobacter in food. Campylobacter jejuni (n=94) and Campylobacter coli (n=52) isolated from different broiler meat carcasses were characterized using multilocus sequence typing (MLST), flagellin gene A restriction fragment length polymorphism typing (flaA-RFLP), antimicrobial resistance profiling (AMRp), the presence/absence of 5 putative virulence genes; and, exclusively for C. jejuni, the determination of lipooligosaccharide (LOS) class. Discriminatory power was calculated by the Simpson's index of diversity (SID) and the congruence was measured by the adjusted Rand index and adjusted Wallace coefficient. MLST was individually the most discriminative typing method for both C. jejuni (SID=0.981) and C. coli (SID=0.957). The most discriminative combination with a SID of 0.992 for both C. jejuni and C. coli was obtained by combining MLST with flaA-RFLP. The combination of MLST with flaA-RFLP is an easy and feasible typing method for short-term monitoring of Campylobacter in broiler meat carcass. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Stepwise threshold clustering: a new method for genotyping MHC loci using next-generation sequencing technology.

    Directory of Open Access Journals (Sweden)

    William E Stutz

    Full Text Available Genes of the vertebrate major histocompatibility complex (MHC are of great interest to biologists because of their important role in immunity and disease, and their extremely high levels of genetic diversity. Next generation sequencing (NGS technologies are quickly becoming the method of choice for high-throughput genotyping of multi-locus templates like MHC in non-model organisms. Previous approaches to genotyping MHC genes using NGS technologies suffer from two problems:1 a "gray zone" where low frequency alleles and high frequency artifacts can be difficult to disentangle and 2 a similar sequence problem, where very similar alleles can be difficult to distinguish as two distinct alleles. Here were present a new method for genotyping MHC loci--Stepwise Threshold Clustering (STC--that addresses these problems by taking full advantage of the increase in sequence data provided by NGS technologies. Unlike previous approaches for genotyping MHC with NGS data that attempt to classify individual sequences as alleles or artifacts, STC uses a quasi-Dirichlet clustering algorithm to cluster similar sequences at increasing levels of sequence similarity. By applying frequency and similarity based criteria to clusters rather than individual sequences, STC is able to successfully identify clusters of sequences that correspond to individual or similar alleles present in the genomes of individual samples. Furthermore, STC does not require duplicate runs of all samples, increasing the number of samples that can be genotyped in a given project. We show how the STC method works using a single sample library. We then apply STC to 295 threespine stickleback (Gasterosteus aculeatus samples from four populations and show that neighboring populations differ significantly in MHC allele pools. We show that STC is a reliable, accurate, efficient, and flexible method for genotyping MHC that will be of use to biologists interested in a variety of downstream applications.

  10. Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus.

    Science.gov (United States)

    Rogers, Stephanie M; Payton, Mark; Allen, Robert W; Melcher, Ulrich; Carver, Jesse; Fletcher, Jacqueline

    2012-05-17

    The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough

  11. Assessing and comparison of different machine learning methods in parent-offspring trios for genotype imputation.

    Science.gov (United States)

    Mikhchi, Abbas; Honarvar, Mahmood; Kashan, Nasser Emam Jomeh; Aminafshar, Mehdi

    2016-06-21

    Genotype imputation is an important tool for prediction of unknown genotypes for both unrelated individuals and parent-offspring trios. Several imputation methods are available and can either employ universal machine learning methods, or deploy algorithms dedicated to infer missing genotypes. In this research the performance of eight machine learning methods: Support Vector Machine, K-Nearest Neighbors, Extreme Learning Machine, Radial Basis Function, Random Forest, AdaBoost, LogitBoost, and TotalBoost compared in terms of the imputation accuracy, computation time and the factors affecting imputation accuracy. The methods employed using real and simulated datasets to impute the un-typed SNPs in parent-offspring trios. The tested methods show that imputation of parent-offspring trios can be accurate. The Random Forest and Support Vector Machine were more accurate than the other machine learning methods. The TotalBoost performed slightly worse than the other methods.The running times were different between methods. The ELM was always most fast algorithm. In case of increasing the sample size, the RBF requires long imputation time.The tested methods in this research can be an alternative for imputation of un-typed SNPs in low missing rate of data. However, it is recommended that other machine learning methods to be used for imputation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Kernel machine methods for integrative analysis of genome-wide methylation and genotyping studies.

    Science.gov (United States)

    Zhao, Ni; Zhan, Xiang; Huang, Yen-Tsung; Almli, Lynn M; Smith, Alicia; Epstein, Michael P; Conneely, Karen; Wu, Michael C

    2018-03-01

    Many large GWAS consortia are expanding to simultaneously examine the joint role of DNA methylation in addition to genotype in the same subjects. However, integrating information from both data types is challenging. In this paper, we propose a composite kernel machine regression model to test the joint epigenetic and genetic effect. Our approach works at the gene level, which allows for a common unit of analysis across different data types. The model compares the pairwise similarities in the phenotype to the pairwise similarities in the genotype and methylation values; and high correspondence is suggestive of association. A composite kernel is constructed to measure the similarities in the genotype and methylation values between pairs of samples. We demonstrate through simulations and real data applications that the proposed approach can correctly control type I error, and is more robust and powerful than using only the genotype or methylation data in detecting trait-associated genes. We applied our method to investigate the genetic and epigenetic regulation of gene expression in response to stressful life events using data that are collected from the Grady Trauma Project. Within the kernel machine testing framework, our methods allow for heterogeneity in effect sizes, nonlinear, and interactive effects, as well as rapid P-value computation. © 2017 WILEY PERIODICALS, INC.

  13. Optimization of a method for the profiling and quantification of saponins in different green asparagus genotypes.

    Science.gov (United States)

    Vázquez-Castilla, Sara; Jaramillo-Carmona, Sara; Fuentes-Alventosa, Jose María; Jiménez-Araujo, Ana; Rodriguez-Arcos, Rocío; Cermeño-Sacristán, Pedro; Espejo-Calvo, Juan Antonio; Guillén-Bejarano, Rafael

    2013-07-03

    The main goal of this study was the optimization of a HPLC-MS method for the qualitative and quantitative analysis of asparagus saponins. The method includes extraction with aqueous ethanol, cleanup by solid phase extraction, separation by reverse phase chromatography, electrospray ionization, and detection in a single quadrupole mass analyzer. The method was used for the comparison of selected genotypes of Huétor-Tájar asparagus landrace and selected varieties of commercial diploid hybrids of green asparagus. The results showed that while protodioscin was almost the only saponin detected in the commercial hybrids, eight different saponins were detected in the Huétor-Tájar asparagus genotypes. The mass spectra indicated that HT saponins are derived from a furostan type steroidal genin having a single bond between carbons 5 and 6 of the B ring. The total concentration of saponins was found to be higher in triguero asparagus than in commercial hybrids.

  14. PCR-Based EST Mapping in Wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    J. PERRY GUSTAFSON

    2009-04-01

    Full Text Available Mapping expressed sequence tags (ESTs to hexaploid wheat is aimed to reveal the structure and function of the hexaploid wheat genome. Sixty eight ESTs representing 26 genes were mapped into all seven homologous chromosome groups of wheat (Triticum aestivum L using a polymerase chain reaction technique. The majority of the ESTs were mapped to homologous chromosome group 2, and the least were mapped to homologous chromosome group 6. Comparative analysis between the EST map from this study and the EST map based on RFLPs showed 14 genes that have been mapped by both approaches were mapped to the same arm of the same homologous chromosome, which indicated that using PCR-based ESTs was a reliable approach in mapping ESTs in hexaploid wheat.

  15. Prevalence of Hepatitis C Virus Subgenotypes 1a and 1b in Japanese Patients: Ultra-Deep Sequencing Analysis of HCV NS5B Genotype-Specific Region

    Science.gov (United States)

    Wu, Shuang; Kanda, Tatsuo; Nakamoto, Shingo; Jiang, Xia; Miyamura, Tatsuo; Nakatani, Sueli M.; Ono, Suzane Kioko; Takahashi-Nakaguchi, Azusa; Gonoi, Tohru; Yokosuka, Osamu

    2013-01-01

    Background Hepatitis C virus (HCV) subgenotypes 1a and 1b have different impacts on the treatment response to peginterferon plus ribavirin with direct-acting antivirals (DAAs) against patients infected with HCV genotype 1, as the emergence rates of resistance mutations are different between these two subgenotypes. In Japan, almost all of HCV genotype 1 belongs to subgenotype 1b. Methods and Findings To determine HCV subgenotype 1a or 1b in Japanese patients infected with HCV genotype 1, real-time PCR-based method and Sanger method were used for the HCV NS5B region. HCV subgenotypes were determined in 90% by real-time PCR-based method. We also analyzed the specific probe regions for HCV subgenotypes 1a and 1b using ultra-deep sequencing, and uncovered mutations that could not be revealed using direct-sequencing by Sanger method. We estimated the prevalence of HCV subgenotype 1a as 1.2-2.5% of HCV genotype 1 patients in Japan. Conclusions Although real-time PCR-based HCV subgenotyping method seems fair for differentiating HCV subgenotypes 1a and 1b, it may not be sufficient for clinical practice. Ultra-deep sequencing is useful for revealing the resistant strain(s) of HCV before DAA treatment as well as mixed infection with different genotypes or subgenotypes of HCV. PMID:24069214

  16. An accurate and efficient method for large-scale SSR genotyping and applications.

    Science.gov (United States)

    Li, Lun; Fang, Zhiwei; Zhou, Junfei; Chen, Hong; Hu, Zhangfeng; Gao, Lifen; Chen, Lihong; Ren, Sheng; Ma, Hongyu; Lu, Long; Zhang, Weixiong; Peng, Hai

    2017-06-02

    Accurate and efficient genotyping of simple sequence repeats (SSRs) constitutes the basis of SSRs as an effective genetic marker with various applications. However, the existing methods for SSR genotyping suffer from low sensitivity, low accuracy, low efficiency and high cost. In order to fully exploit the potential of SSRs as genetic marker, we developed a novel method for SSR genotyping, named as AmpSeq-SSR, which combines multiplexing polymerase chain reaction (PCR), targeted deep sequencing and comprehensive analysis. AmpSeq-SSR is able to genotype potentially more than a million SSRs at once using the current sequencing techniques. In the current study, we simultaneously genotyped 3105 SSRs in eight rice varieties, which were further validated experimentally. The results showed that the accuracies of AmpSeq-SSR were nearly 100 and 94% with a single base resolution for homozygous and heterozygous samples, respectively. To demonstrate the power of AmpSeq-SSR, we adopted it in two applications. The first was to construct discriminative fingerprints of the rice varieties using 3105 SSRs, which offer much greater discriminative power than the 48 SSRs commonly used for rice. The second was to map Xa21, a gene that confers persistent resistance to rice bacterial blight. We demonstrated that genome-scale fingerprints of an organism can be efficiently constructed and candidate genes, such as Xa21 in rice, can be accurately and efficiently mapped using an innovative strategy consisting of multiplexing PCR, targeted sequencing and computational analysis. While the work we present focused on rice, AmpSeq-SSR can be readily extended to animals and micro-organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. A Multipoint Method for Detecting Genotyping Errors and Mutations in Sibling-Pair Linkage Data

    OpenAIRE

    Douglas, Julie A.; Boehnke, Michael; Lange, Kenneth

    2000-01-01

    The identification of genes contributing to complex diseases and quantitative traits requires genetic data of high fidelity, because undetected errors and mutations can profoundly affect linkage information. The recent emphasis on the use of the sibling-pair design eliminates or decreases the likelihood of detection of genotyping errors and marker mutations through apparent Mendelian incompatibilities or close double recombinants. In this article, we describe a hidden Markov method for detect...

  18. Development of a bead-based multiplex genotyping method for diagnostic characterization of HPV infection.

    Directory of Open Access Journals (Sweden)

    Mee Young Chung

    Full Text Available The accurate genotyping of human papillomavirus (HPV is clinically important because the oncogenic potential of HPV is dependent on specific genotypes. Here, we described the development of a bead-based multiplex HPV genotyping (MPG method which is able to detect 20 types of HPV (15 high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 5 low-risk HPV types 6, 11, 40, 55, 70 and evaluated its accuracy with sequencing. A total of 890 clinical samples were studied. Among these samples, 484 were HPV positive and 406 were HPV negative by consensus primer (PGMY09/11 directed PCR. The genotyping of 484 HPV positive samples was carried out by the bead-based MPG method. The accuracy was 93.5% (95% CI, 91.0-96.0, 80.1% (95% CI, 72.3-87.9 for single and multiple infections, respectively, while a complete type mismatch was observed only in one sample. The MPG method indiscriminately detected dysplasia of several cytological grades including 71.8% (95% CI, 61.5-82.3 of ASCUS (atypical squamous cells of undetermined significance and more specific for high grade lesions. For women with HSIL (high grade squamous intraepithelial lesion and SCC diagnosis, 32 women showed a PPV (positive predictive value of 77.3% (95% CI, 64.8-89.8. Among women >40 years of age, 22 women with histological cervical cancer lesions showed a PPV of 88% (95% CI, 75.3-100. Of the highest risk HPV types including HPV-16, 18 and 31 positive women of the same age groups, 34 women with histological cervical cancer lesions showed a PPV of 77.3% (95% CI, 65.0-89.6. Taken together, the bead-based MPG method could successfully detect high-grade lesions and high-risk HPV types with a high degree of accuracy in clinical samples.

  19. Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia.

    Science.gov (United States)

    Tan, Liping; Yu, Xingang; Abdullahi, Auwalu Yusuf; Wu, Sheng; Zheng, Guochao; Hu, Wei; Song, Meiran; Wang, Zhen; Jiang, Biao; Li, Guoqing

    2015-11-01

    Giardia lamblia is a zoonotic flagellate protozoan in the intestine of human and many mammals including dogs. To assess a threat of dog-derived G. lamblia to humans, the common dog-derived G. lamblia assemblages A, C, and D were genotyped by high-resolution melting (HRM) technology. According to β-giardin gene sequence, the qPCR-HRM primers BG5 and BG7 were designed. A series of experiments on the stability, sensitivity, and accuracy of the HRM method were also tested. Results showed that the primers BG5 and BG7 could distinguish among three assemblages A, C, and D, which Tm value differences were about 1 °C to each other. The melting curves of intra-assay reproducibility were almost coincided, and those of inter-assay reproducibility were much the same shape. The lowest detection concentration was about 5 × 10(-6)-ng/μL sample. The genotyping results from 21 G. lamblia samples by the HRM method were in complete accordance with sequencing results. It is concluded that the HRM genotyping method is rapid, stable, specific, highly sensitive, and suitable for clinical detection and molecular epidemiological survey of dog-derived G. lamblia.

  20. Dry matter genotypes of Cynodon by microwave and conventional oven methods

    Directory of Open Access Journals (Sweden)

    Euclides Reuter de Oliveira

    2013-02-01

    Full Text Available The aimed of this work was to comparing the drying process in a microwave oven and forced air ventilation, as well as their effects on the chemical composition of different genotypes of the genus Cynodon (Tifton 85, Jiggs, Russell, Tifton 68 and Vaquero collected at different ages cutting (28, 48, 63 and 79 days. The experimental design was a randomized block in a split-plot design, with 4 replicates. There was no difference (P>0.05 between the methods analyzed on the chemical composition of the genotypes studied. Increasing age cutoff negatively influenced (P<0.05 the crude protein content of the different plant parts. A significant increase (P<0.05 of dry matter, neutral detergent fiber, acid detergent fiber and dry matter production was observed with increasing age cut. The use of the microwave oven is a quick and precise method obtain the dry matter content of the fodder showing efficiency similar to the method of drying in an oven with forced air circulation. The genotypes showed better chemical composition results when handled at age 28 days.

  1. A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f.sp. tritici, based on DNA extraction from infected leaf and Multiplex PCR genotyping

    Directory of Open Access Journals (Sweden)

    Enjalbert Jérôme

    2011-07-01

    Full Text Available Abstract Background Puccinia striiformis f.sp. tritici (PST, an obligate fungal pathogen causing wheat yellow/stripe rust, a serious disease, has been used to understand the evolution of crop pathogen using molecular markers. However, numerous questions regarding its evolutionary history and recent migration routes still remains to be addressed, which need the genotyping of a large number of isolates, a process that is limited by both DNA extraction and genotyping methods. To address the two issues, we developed here a method for direct DNA extraction from infected leaves combined with optimized SSR multiplexing. Findings We report here an efficient protocol for direct fungal DNA extraction from infected leaves, avoiding the costly and time consuming step of spore multiplication. The genotyping strategy we propose, amplified a total of 20 SSRs in three Multiplex PCR reactions, which were highly polymorphic and were able to differentiate different PST populations with high efficiency and accuracy. Conclusion These two developments enabled a genotyping strategy that could contribute to the development of molecular epidemiology of yellow rust disease, both at a regional or worldwide scale.

  2. HRM and SNaPshot as alternative forensic SNP genotyping methods.

    Science.gov (United States)

    Mehta, Bhavik; Daniel, Runa; McNevin, Dennis

    2017-09-01

    Single nucleotide polymorphisms (SNPs) have been widely used in forensics for prediction of identity, biogeographical ancestry (BGA) and externally visible characteristics (EVCs). Single base extension (SBE) assays, most notably SNaPshot® (Thermo Fisher Scientific), are commonly used for forensic SNP genotyping as they can be employed on standard instrumentation in forensic laboratories (e.g. capillary electrophoresis). High resolution melt (HRM) analysis is an alternative method and is a simple, fast, single tube assay for low throughput SNP typing. This study compares HRM and SNaPshot®. HRM produced reproducible and concordant genotypes at 500 pg, however, difficulties were encountered when genotyping SNPs with high GC content in flanking regions and differentiating variants of symmetrical SNPs. SNaPshot® was reproducible at 100 pg and is less dependent on SNP choice. HRM has a shorter processing time in comparison to SNaPshot®, avoids post PCR contamination risk and has potential as a screening tool for many forensic applications.

  3. Development of a cost-efficient novel method for rapid, concurrent genotyping of five common single nucleotide polymorphisms of the brain derived neurotrophic factor (BDNF) gene by tetra-primer amplification refractory mutation system.

    Science.gov (United States)

    Wang, Cathy K; Xu, Michael S; Ross, Colin J; Lo, Ryan; Procyshyn, Ric M; Vila-Rodriguez, Fidel; White, Randall F; Honer, William G; Barr, Alasdair M

    2015-09-01

    Brain derived neurotrophic factor (BDNF) is a molecular trophic factor that plays a key role in neuronal survival and plasticity. Single nucleotide polymorphisms (SNPs) of the BDNF gene have been associated with specific phenotypic traits in a large number of neuropsychiatric disorders and the response to psychotherapeutic medications in patient populations. Nevertheless, due to study differences and occasionally contrasting findings, substantial further research is required to understand in better detail the association between specific BDNF SNPs and these psychiatric disorders. While considerable progress has been made recently in developing advanced genotyping platforms of SNPs, many high-throughput probe- or array-based detection methods currently available are limited by high costs, slow processing times or access to advanced instrumentation. The polymerase chain reaction (PCR)-based, tetra-primer amplification refractory mutation system (T-ARMS) method is a potential alternative technique for detecting SNP genotypes efficiently, quickly, easily, and cheaply. As a tool in psychopathology research, T-ARMS was shown to be capable of detecting five common SNPs in the BDNF gene (rs6265, rs988748, rs11030104, 11757G/C and rs7103411), which are all SNPs with previously demonstrated clinical relevance to schizophrenia and depression. The present technique therefore represents a suitable protocol for many research laboratories to study the genetic correlates of BDNF in psychiatric disorders. Copyright Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Pyroprinting: a rapid and flexible genotypic fingerprinting method for typing bacterial strains.

    Science.gov (United States)

    Black, Michael W; VanderKelen, Jennifer; Montana, Aldrin; Dekhtyar, Alexander; Neal, Emily; Goodman, Anya; Kitts, Christopher L

    2014-10-01

    Bacterial strain typing is commonly employed in studies involving epidemiology, population ecology, and microbial source tracking to identify sources of fecal contamination. Methods for differentiating strains generally use either a collection of phenotypic traits or rely on some interrogation of the bacterial genotype. This report introduces pyroprinting, a novel genotypic strain typing method that is rapid, inexpensive, and discriminating compared to the most sensitive methods already in use. Pyroprinting relies on the simultaneous pyrosequencing of polymorphic multicopy loci, such as the intergenic transcribed spacer regions of rRNA operons in bacterial genomes. Data generated by sequencing combinations of variable templates are reproducible and intrinsically digitized. The theory and development of pyroprinting in Escherichia coli, including the selection of similarity thresholds to define matches between isolates, are presented. The pyroprint-based strain differentiation limits and phylogenetic relevance compared to other typing methods are also explored. Pyroprinting is unique in its simplicity and, paradoxically, in its intrinsic complexity. This new approach serves as an excellent alternative to more cumbersome or less phylogenetically relevant strain typing methods. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    Science.gov (United States)

    Reinitz, David M.; Yoshino, T.P.; Cole, Rebecca A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  6. Impact of enumeration method on diversity of Escherichia coli genotypes isolated from surface water.

    Science.gov (United States)

    Martin, E C; Gentry, T J

    2016-11-01

    There are numerous regulatory-approved Escherichia coli enumeration methods, but it is not known whether differences in media composition and incubation conditions impact the diversity of E. coli populations detected by these methods. A study was conducted to determine if three standard water quality assessments, Colilert ® , USEPA Method 1603, (modified mTEC) and USEPA Method 1604 (MI), detect different populations of E. coli. Samples were collected from six watersheds and analysed using the three enumeration approaches followed by E. coli isolation and genotyping. Results indicated that the three methods generally produced similar enumeration data across the sites, although there were some differences on a site-by-site basis. The Colilert ® method consistently generated the least diverse collection of E. coli genotypes as compared to modified mTEC and MI, with those two methods being roughly equal to each other. Although the three media assessed in this study were designed to enumerate E. coli, the differences in the media composition, incubation temperature, and growth platform appear to have a strong selective influence on the populations of E. coli isolated. This study suggests that standardized methods of enumeration and isolation may be warranted if researchers intend to obtain individual E. coli isolates for further characterization. This study characterized the impact of three USEPA-approved Escherichia coli enumeration methods on observed E. coli population diversity in surface water samples. Results indicated that these methods produced similar E. coli enumeration data but were more variable in the diversity of E. coli genotypes observed. Although the three methods enumerate the same species, differences in media composition, growth platform, and incubation temperature likely contribute to the selection of different cultivable populations of E. coli, and thus caution should be used when implementing these methods interchangeably for

  7. PCR-based detection of a rare linear DNA in cell culture

    Directory of Open Access Journals (Sweden)

    Saveliev Sergei V.

    2002-01-01

    Full Text Available The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

  8. PCR-based detection of a rare linear DNA in cell culture.

    Science.gov (United States)

    Saveliev, Sergei V.

    2002-11-11

    The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10(7) or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

  9. A Simple PCR Method for Rapid Genotype Analysis of Mycobacterium ulcerans

    Science.gov (United States)

    Stinear, Timothy; Davies, John K.; Jenkin, Grant A.; Portaels, Françoise; Ross, Bruce C.; OppEdIsano, Frances; Purcell, Maria; Hayman, John A.; Johnson, Paul D. R.

    2000-01-01

    Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype. PMID:10747130

  10. PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

    DEFF Research Database (Denmark)

    Liu, Yilan; Yang, Maohua; Yan, Daojiang

    2016-01-01

    Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double...... for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision...... of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli....

  11. Assessment of soy genotype and processing method on quality of soybean tofu.

    Science.gov (United States)

    Stanojevic, Sladjana P; Barac, Miroljub B; Pesic, Mirjana B; Vucelic-Radovic, Biljana V

    2011-07-13

    Protein quality in six soybean varieties, based on subunit composition of their protein, was correlated with quality of the produced tofu. Also, protein changes due to a pilot plant processing method involving high temperature/pressure and commercial rennet as coagulant were assessed. In each soybean variety, glycinin (11S) and β-conglycinin (7S) as well as 11S/7S ratio significantly changed from beans to tofu. Between varieties, the 11S/7S protein ratio in seed indicated genotypic influence on tofu yield and gel hardness (r = 0.91 and r = 0.99, respectively; p soybean β'-subunit of 7S protein negatively influenced tofu hardness (r = -0.91, p Seed protein composition and proportion of 7S protein subunits under the applied production method had an important role in defining tofu quality.

  12. An NS5A single optimized method to determine genotype, subtype and resistance profiles of Hepatitis C strains.

    Directory of Open Access Journals (Sweden)

    Elisabeth Andre-Garnier

    Full Text Available The objective was to develop a method of HCV genome sequencing that allowed simultaneous genotyping and NS5A inhibitor resistance profiling. In order to validate the use of a unique RT-PCR for genotypes 1-5, 142 plasma samples from patients infected with HCV were analysed. The NS4B-NS5A partial region was successfully amplified and sequenced in all samples. In parallel, partial NS3 sequences were analyzed obtained for genotyping. Phylogenetic analysis showed concordance of genotypes and subtypes with a bootstrap >95% for each type cluster. NS5A resistance mutations were analyzed using the Geno2pheno [hcv] v0.92 tool and compared to the list of known Resistant Associated Substitutions recently published. In conclusion, this tool allows determination of HCV genotypes, subtypes and identification of NS5A resistance mutations. This single method can be used to detect pre-existing resistance mutations in NS5A before treatment and to check the emergence of resistant viruses while undergoing treatment in major HCV genotypes (G1-5 in the EU and the US.

  13. A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa.

    Science.gov (United States)

    Boubaker, Ghalia; Marinova, Irina; Gori, Francesca; Hizem, Amani; Müller, Norbert; Casulli, Adriano; Jerez Puebla, Luis Enrique; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2016-08-01

    Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters.

    Science.gov (United States)

    Bergmann, I; Mundt, K; Sontag, M; Baumstark, I; Nettmann, E; Klocke, M

    2010-03-01

    Quantitative real-time PCR (Q-PCR) is commonly applied for the detection of certain microorganisms in environmental samples. However, some environments, like biomass-degrading biogas fermenters, are enriched with PCR-interfering substances. To study the impact of the DNA extraction protocol on the results of Q-PCR-based analysis of the methane-producing archaeal community in biogas fermenters, nine different protocols with varying cell disruption and DNA purification approaches were tested. Differences in the quantities of the isolated DNA and the purity parameters were found, with the best cell lysis efficiencies being obtained by a combined lysozyme/SDS-based lysis. When DNA was purified by sephacryl columns, the amount of DNA decreased by one log cycle but PCR inhibitors were eliminated sufficiently. In the case of detection of methanogenic Archaea, the chosen DNA isolation protocol strongly influenced the Q-PCR-based determination of 16S rDNA copy numbers. For example, with protocols including mechanical cell disruption, the 16S rDNA of Methanobacteriales were predominantly amplified (81-90% of the total 16S rDNA copy numbers), followed by the 16S rDNA of Methanomicrobiales (9-18%). In contrast, when a lysozyme/SDS-based cell lysis was applied, the 16S rDNA copy numbers determined for these two orders were the opposite (Methanomicrobiales 82-95%, Methanobacteriales 4-18%). In extreme cases, the DNA isolation method led to discrimination of some groups of methanogens (e.g. members of the Methanosaetaceae). In conclusion, for extraction of high amounts of microbial DNA with high purity from samples of biogas plants, a combined lysozyme/SDS-based cell lysis followed by a purification step with sephacryl columns is recommended. Copyright 2010 Elsevier GmbH. All rights reserved.

  15. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species.

    Directory of Open Access Journals (Sweden)

    Brant K Peterson

    Full Text Available The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via "SNP chip" microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more of individuals for a range of markers (hundreds to hundreds of thousands. Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms.

  16. Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

    Science.gov (United States)

    Matlawska-Wasowska, Ksenia; Gale, James M; Nickl, Christian K; Khalili, Parisa; Shirley, Brian; Wilson, Bridget S; Vasef, Mohammad A; Winter, Stuart S

    2014-12-01

    Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

  17. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

    Science.gov (United States)

    Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong

    2014-09-19

    The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

  18. Yield and nutritional quality of greenhouse lettuce (Lactuca sativa L. as affected by genotype and production methods

    Directory of Open Access Journals (Sweden)

    Govedarica-Lučić Aleksandra

    2014-01-01

    Full Text Available Greenhouse experiments were conducted in winter growing seasons in order to evaluate the effects of genotype and production methods on yield and nutritional quality of lettuce (Lactuca sativa L.. A three-year (2009-2011 study was conducted by randomized block system in a greenhouse without additional heating. The trial included three genotypes of lettuce (Archimedes RZ, Santoro RZ, Kibou RZ. Each row with these genotypes was exposed to the following variants of covering: control-planting on bare soil, mulching before sowing with PE-black foil, agro textile-covering plants after planting with agro textile (17 g, a combination of mulching + agro textile. Throughout of all the three years of the trial, it was continuously evidenced that the genotype “Santoro RZ” had the biggest heads and the highest yield (15.33 kg 10 m-2, which leads to conclusion that the yield of lettuce is a genotype characteristics. Moreover, the nutritional value (ascorbic acid concentration has shown that, depending on the method of production, in average, the combination of mulching + agro textile (26.77 mg 100 g-1 had the highest content while the control variant had significantly lower vitamin C content (21.10 mg 100 g-1. The three-year researches have shown that the production method and genotype significantly affect the nitrate content. An average nitrate content was 2196.33 mg kg-1 on the control variant, and 2526.24 mg kg-1 on agro textile. Leafy lettuce of genotyp „Kibou RZ“ had lower nitrate content (2176.85 mg kg-1 compared to „Archimedes RZ“ (2843.05 mg kg-1 and „Santoro RZ“ (2221.37 mg kg-1. However nitrate concentration in all treatments remained within the European Union’s permissible levels.

  19. Comparing simple root phenotyping methods on a core set of rice genotypes.

    Science.gov (United States)

    Shrestha, R; Al-Shugeairy, Z; Al-Ogaidi, F; Munasinghe, M; Radermacher, M; Vandenhirtz, J; Price, A H

    2014-05-01

    Interest in belowground plant growth is increasing, especially in relation to arguments that shallow-rooted cultivars are efficient at exploiting soil phosphorus while deep-rooted ones will access water at depth. However, methods for assessing roots in large numbers of plants are diverse and direct comparisons of methods are rare. Three methods for measuring root growth traits were evaluated for utility in discriminating rice cultivars: soil-filled rhizotrons, hydroponics and soil-filled pots whose bottom was sealed with a non-woven fabric (a potential method for assessing root penetration ability). A set of 38 rice genotypes including the OryzaSNP set of 20 cultivars, additional parents of mapping populations and products of marker-assisted selection for root QTLs were assessed. A novel method of image analysis for assessing rooting angles from rhizotron photographs was employed. The non-woven fabric was the easiest yet least discriminatory method, while the rhizotron was highly discriminatory and allowed the most traits to be measured but required more than three times the labour of the other methods. The hydroponics was both easy and discriminatory, allowed temporal measurements, but is most likely to suffer from artefacts. Image analysis of rhizotrons compared favourably to manual methods for discriminating between cultivars. Previous observations that cultivars from the indica subpopulation have shallower rooting angles than aus or japonica cultivars were confirmed in the rhizotrons, and indica and temperate japonicas had lower maximum root lengths in rhizotrons and hydroponics. It is concluded that rhizotrons are the preferred method for root screening, particularly since root angles can be assessed. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  20. Transcriptomic SNP discovery for custom genotyping arrays: impacts of sequence data, SNP calling method and genotyping technology on the probability of validation success.

    Science.gov (United States)

    Humble, Emily; Thorne, Michael A S; Forcada, Jaume; Hoffman, Joseph I

    2016-08-26

    Single nucleotide polymorphism (SNP) discovery is an important goal of many studies. However, the number of 'putative' SNPs discovered from a sequence resource may not provide a reliable indication of the number that will successfully validate with a given genotyping technology. For this it may be necessary to account for factors such as the method used for SNP discovery and the type of sequence data from which it originates, suitability of the SNP flanking sequences for probe design, and genomic context. To explore the relative importance of these and other factors, we used Illumina sequencing to augment an existing Roche 454 transcriptome assembly for the Antarctic fur seal (Arctocephalus gazella). We then mapped the raw Illumina reads to the new hybrid transcriptome using BWA and BOWTIE2 before calling SNPs with GATK. The resulting markers were pooled with two existing sets of SNPs called from the original 454 assembly using NEWBLER and SWAP454. Finally, we explored the extent to which SNPs discovered using these four methods overlapped and predicted the corresponding validation outcomes for both Illumina Infinium iSelect HD and Affymetrix Axiom arrays. Collating markers across all discovery methods resulted in a global list of 34,718 SNPs. However, concordance between the methods was surprisingly poor, with only 51.0 % of SNPs being discovered by more than one method and 13.5 % being called from both the 454 and Illumina datasets. Using a predictive modeling approach, we could also show that SNPs called from the Illumina data were on average more likely to successfully validate, as were SNPs called by more than one method. Above and beyond this pattern, predicted validation outcomes were also consistently better for Affymetrix Axiom arrays. Our results suggest that focusing on SNPs called by more than one method could potentially improve validation outcomes. They also highlight possible differences between alternative genotyping technologies that could be

  1. Molecular Diagnosis of Brettanomyces bruxellensis’ Sulfur Dioxide Sensitivity Through Genotype Specific Method

    Directory of Open Access Journals (Sweden)

    Marta Avramova

    2018-06-01

    Full Text Available The yeast species Brettanomyces bruxellensis is associated with important economic losses due to red wine spoilage. The most common method to prevent and/or control B. bruxellensis spoilage in winemaking is the addition of sulfur dioxide into must and wine. However, recently, it was reported that some B. bruxellensis strains could be tolerant to commonly used doses of SO2. In this work, B. bruxellensis response to SO2 was assessed in order to explore the relationship between SO2 tolerance and genotype. We selected 145 isolates representative of the genetic diversity of the species, and from different fermentation niches (roughly 70% from grape wine fermentation environment, and 30% from beer, ethanol, tequila, kombucha, etc.. These isolates were grown in media harboring increasing sulfite concentrations, from 0 to 0.6 mg.L-1 of molecular SO2. Three behaviors were defined: sensitive strains showed longer lag phase and slower growth rate and/or lower maximum population size in presence of increasing concentrations of SO2. Tolerant strains displayed increased lag phase, but maximal growth rate and maximal population size remained unchanged. Finally, resistant strains showed no growth variation whatever the SO2 concentrations. 36% (52/145 of B. bruxellensis isolates were resistant or tolerant to sulfite, and up to 43% (46/107 when considering only wine isolates. Moreover, most of the resistant/tolerant strains belonged to two specific genetic groups, allowing the use of microsatellite genotyping to predict the risk of sulfur dioxide resistance/tolerance with high reliability (>90%. Such molecular diagnosis could help the winemakers to adjust antimicrobial techniques and efficient spoilage prevention with minimal intervention.

  2. Comparison of Four Human Papillomavirus Genotyping Methods: Next-generation Sequencing, INNO-LiPA, Electrochemical DNA Chip, and Nested-PCR.

    Science.gov (United States)

    Nilyanimit, Pornjarim; Chansaenroj, Jira; Poomipak, Witthaya; Praianantathavorn, Kesmanee; Payungporn, Sunchai; Poovorawan, Yong

    2018-03-01

    Human papillomavirus (HPV) infection causes cervical cancer, thus necessitating early detection by screening. Rapid and accurate HPV genotyping is crucial both for the assessment of patients with HPV infection and for surveillance studies. Fifty-eight cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, electrochemical DNA chip, and next-generation sequencing (NGS). Seven HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or electrochemical DNA chip. Although NGS is relatively expensive and complex, it may serve as a sensitive HPV genotyping method. Because of its highly sensitive detection of multiple HPV genotypes, NGS may serve as an alternative for diagnostic HPV genotyping in certain situations. © The Korean Society for Laboratory Medicine

  3. Detection of HCV genotypes using molecular and radio-isotopic methods

    International Nuclear Information System (INIS)

    Ahmad, N.; Baig, S.M.; Shah, W.A.; Khattak, K.F.; Khan, B.; Qureshi, J.A.

    2004-01-01

    Hepatitis C virus (HCV) accounts for most cases of acute and chronic non-A and non-B liver diseases. Persistent HCV infection may lead to liver cirrhosis and hepatocellular carcinoma. Six major HCV genotypes have been recognized. Infection with different genotypes results in different clinical pictures and responses to antiviral therapy. In the area of Faisalabad (Punjab province of Pakistan), the prevalence and molecular epidemiology of Hepatitis C virus infection had never been investigated before. In this study, we have made an attempt to determine the prevalence, distribution and clinical significance of HCV infection in 1100 suspected patients of liver disease by nested reverse transcriptase polymerase chain reaction (RTPCR) over a period of four years. HCV genotypes of isolates were determined by dot-blot hybridization with genotype specific radiolabeled probes in 337 subjects. The proportion of patients with HCV genotypes 1,2,3 and 4 were 37.38%, 1.86%, 16.16% and 0.29% respectively. Mixed infection of HCV genotype was detected in 120 (35.6%) patients, whereas 31 (9.1%) samples remained unclassified. This study revealed changing epidemiology of hepatitis C virus genotype 1 and 3 in the patients. Multiple infection of HCV genotype in the same patient may be of great clinical and pathological importance and interest. (author)

  4. Similar predictions of etravirine sensitivity regardless of genotypic testing method used: comparison of available scoring systems.

    Science.gov (United States)

    Vingerhoets, Johan; Nijs, Steven; Tambuyzer, Lotke; Hoogstoel, Annemie; Anderson, David; Picchio, Gaston

    2012-01-01

    The aims of this study were to compare various genotypic scoring systems commonly used to predict virological outcome to etravirine, and examine their concordance with etravirine phenotypic susceptibility. Six etravirine genotypic scoring systems were assessed: Tibotec 2010 (based on 20 mutations; TBT 20), Monogram, Stanford HIVdb, ANRS, Rega (based on 37, 30, 27 and 49 mutations, respectively) and virco(®)TYPE HIV-1 (predicted fold change based on genotype). Samples from treatment-experienced patients who participated in the DUET trials and with both genotypic and phenotypic data (n=403) were assessed using each scoring system. Results were retrospectively correlated with virological response in DUET. κ coefficients were calculated to estimate the degree of correlation between the different scoring systems. Correlation between the five scoring systems and the TBT 20 system was approximately 90%. Virological response by etravirine susceptibility was comparable regardless of which scoring system was utilized, with 70-74% of DUET patients determined as susceptible to etravirine by the different scoring systems achieving plasma viral load <50 HIV-1 RNA copies/ml. In samples classed as phenotypically susceptible to etravirine (fold change in 50% effective concentration ≤3), correlations with genotypic score were consistently high across scoring systems (≥70%). In general, the etravirine genotypic scoring systems produced similar results, and genotype-phenotype concordance was high. As such, phenotypic interpretations, and in their absence all genotypic scoring systems investigated, may be used to reliably predict the activity of etravirine.

  5. Mendel Meets CSI: Forensic Genotyping as a Method to Teach Genetics & DNA Science

    Science.gov (United States)

    Kurowski, Scotia; Reiss, Rebecca

    2007-01-01

    This article describes a forensic DNA science laboratory exercise for advanced high school and introductory college level biology courses. Students use a commercial genotyping kit and genetic analyzer or gene sequencer to analyze DNA recovered from a fictitious crime scene. DNA profiling and STR genotyping are outlined. DNA extraction, PCR, and…

  6. A Maximum-Likelihood Method to Correct for Allelic Dropout in Microsatellite Data with No Replicate Genotypes

    Science.gov (United States)

    Wang, Chaolong; Schroeder, Kari B.; Rosenberg, Noah A.

    2012-01-01

    Allelic dropout is a commonly observed source of missing data in microsatellite genotypes, in which one or both allelic copies at a locus fail to be amplified by the polymerase chain reaction. Especially for samples with poor DNA quality, this problem causes a downward bias in estimates of observed heterozygosity and an upward bias in estimates of inbreeding, owing to mistaken classifications of heterozygotes as homozygotes when one of the two copies drops out. One general approach for avoiding allelic dropout involves repeated genotyping of homozygous loci to minimize the effects of experimental error. Existing computational alternatives often require replicate genotyping as well. These approaches, however, are costly and are suitable only when enough DNA is available for repeated genotyping. In this study, we propose a maximum-likelihood approach together with an expectation-maximization algorithm to jointly estimate allelic dropout rates and allele frequencies when only one set of nonreplicated genotypes is available. Our method considers estimates of allelic dropout caused by both sample-specific factors and locus-specific factors, and it allows for deviation from Hardy–Weinberg equilibrium owing to inbreeding. Using the estimated parameters, we correct the bias in the estimation of observed heterozygosity through the use of multiple imputations of alleles in cases where dropout might have occurred. With simulated data, we show that our method can (1) effectively reproduce patterns of missing data and heterozygosity observed in real data; (2) correctly estimate model parameters, including sample-specific dropout rates, locus-specific dropout rates, and the inbreeding coefficient; and (3) successfully correct the downward bias in estimating the observed heterozygosity. We find that our method is fairly robust to violations of model assumptions caused by population structure and by genotyping errors from sources other than allelic dropout. Because the data sets

  7. Evaluation of a high resolution genotyping method for Chlamydia trachomatis using routine clinical samples.

    Directory of Open Access Journals (Sweden)

    Yibing Wang

    2011-02-01

    Full Text Available Genital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV and the genitourinary genotypes D-K. Although these genotypes can be discriminated by outer membrane protein gene (ompA sequencing or multi-locus sequence typing (MLST, neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.We evaluated variable number tandem repeat (VNTR and ompA sequencing (now called multi-locus VNTR analysis and ompA or "MLVA-ompA" to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43% were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59% were fully genotyped by MLVA-ompA. Only one mixed infection (E & D in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types.Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.

  8. Toward fully automated genotyping: Genotyping microsatellite markers by deconvolution

    Energy Technology Data Exchange (ETDEWEB)

    Perlin, M.W.; Lancia, G.; See-Kiong, Ng [Carnegie Mellon Univ., Pittsburgh, PA (United States)

    1995-11-01

    Dense genetic linkage maps have been constructed for the human and mouse genomes, with average densities of 2.9 cM and 0.35 cM, respectively. These genetic maps are crucial for mapping both Mendelian and complex traits and are useful in clinical genetic diagnosis. Current maps are largely comprised of abundant, easily assayed, and highly polymorphic PCR-based microsatellite markers, primarily dinucleotide (CA){sub n} repeats. One key limitation of these length polymorphisms is the PCR stutter (or slippage) artifact that introduces additional stutter bands. With two (or more) closely spaced alleles, the stutter bands overlap, and it is difficult to accurately determine the correct alleles; this stutter phenomenon has all but precluded full automation, since a human must visually inspect the allele data. We describe here novel deconvolution methods for accurate genotyping that mathematically remove PCR stutter artifact from microsatellite markers. These methods overcome the manual interpretation bottleneck and thereby enable full automation of genetic map construction and use. New functionalities, including the pooling of DNAs and the pooling of markers, are described that may greatly reduce the associated experimentation requirements. 32 refs., 5 figs., 3 tabs.

  9. Numerical taxonomy of the genus Pestivirus: new software for genotyping based on the palindromic nucleotide substitutions method.

    Science.gov (United States)

    Giangaspero, Massimo; Apicella, Claudio; Harasawa, Ryô

    2013-09-01

    The genus Pestivirus from the family Flaviviridae is represented by four established species; Bovine viral diarrhea virus 1 (BVDV-1); Bovine viral diarrhea virus 2 (BVDV-2); Border disease virus (BDV); and Classical swine fever virus (CSFV); as well a tentative species from a Giraffe. The palindromic nucleotide substitutions (PNS) in the 5' untranslated region (UTR) of Pestivirus RNA has been described as a new, simple and practical method for genotyping. New software is described, also named PNS, that was prepared specifically for this PNS genotyping procedure. Pestivirus identification using PNS was evaluated on five hundred and forty-three sequences at genus, species and genotype level using this software. The software is freely available at www.pns-software.com. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    Science.gov (United States)

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

  11. Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens.

    Science.gov (United States)

    Wadowsky, R M; Michaels, R H; Libert, T; Kingsley, L A; Ehrlich, G D

    1996-11-01

    A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.

  12. ABO Blood Group Genotyping by Real-time PCR in Kazakh Population

    Directory of Open Access Journals (Sweden)

    Pavel Tarlykov

    2014-12-01

    Full Text Available Introduction. ABO blood group genotyping is a new technology in hematology that helps prevent adverse transfusion reactions in patients. Identification of antigens on the surface of red blood cells is based on serology; however, genotyping employs a different strategy and is aimed directly at genes that determine the surface proteins. ABO blood group genotyping by real-time PCR has several crucial advantages over other PCR-based techniques, such as high rapidity and reliability of analysis. The purpose of this study was to examine nucleotide substitutions differences by blood types using a PCR-based method on Kazakh blood donors.Methods. The study was approved by the Ethics Committee of the National Center for Biotechnology. Venous blood samples from 369 healthy Kazakh blood donors, whose blood types had been determined by serological methods, were collected after obtaining informed consent. The phenotypes of the samples included blood group A (n = 99, B (n = 93, O (n = 132, and AB (n = 45. Genomic DNA was extracted using a salting-out method. PCR products of ABO gene were sequenced on an ABI 3730xl DNA analyzer (Applied Biosystems. The resulting nucleotide sequences were compared and aligned against reference sequence NM_020469.2. Real-time PCR analysis was performed on CFX96 Touch™ Real-Time PCR Detection System (BioRad.Results. Direct sequencing of ABO gene in 369 samples revealed that the vast majority of nucleotide substitutions that change the ABO phenotype were limited to exons 6 and 7 of the ABO gene at positions 261, 467, 657, 796, 803, 930 and 1,060. However, genotyping of only three of them (261, 796 and 803 resulted in identification of major ABO genotypes in the Kazakh population. As a result, TaqMan probe based real-time PCR assay for the specific detection of genotypes 261, 796 and 803 was developed. The assay did not take into account several other mutations that may affect the determination of blood group, because they have a

  13. Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

    Science.gov (United States)

    Yang, Lei; Naylor, Gavin J P

    2016-01-01

    We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

  14. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

    Science.gov (United States)

    Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

    2015-10-01

    Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. Copyright © 2015 Elsevier GmbH. All rights reserved.

  15. A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

    Science.gov (United States)

    Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

    2014-01-01

    Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. A genotypic method for determining HIV-2 coreceptor usage enables epidemiological studies and clinical decision support.

    Science.gov (United States)

    Döring, Matthias; Borrego, Pedro; Büch, Joachim; Martins, Andreia; Friedrich, Georg; Camacho, Ricardo Jorge; Eberle, Josef; Kaiser, Rolf; Lengauer, Thomas; Taveira, Nuno; Pfeifer, Nico

    2016-12-20

    CCR5-coreceptor antagonists can be used for treating HIV-2 infected individuals. Before initiating treatment with coreceptor antagonists, viral coreceptor usage should be determined to ensure that the virus can use only the CCR5 coreceptor (R5) and cannot evade the drug by using the CXCR4 coreceptor (X4-capable). However, until now, no online tool for the genotypic identification of HIV-2 coreceptor usage had been available. Furthermore, there is a lack of knowledge on the determinants of HIV-2 coreceptor usage. Therefore, we developed a data-driven web service for the prediction of HIV-2 coreceptor usage from the V3 loop of the HIV-2 glycoprotein and used the tool to identify novel discriminatory features of X4-capable variants. Using 10 runs of tenfold cross validation, we selected a linear support vector machine (SVM) as the model for geno2pheno[coreceptor-hiv2], because it outperformed the other SVMs with an area under the ROC curve (AUC) of 0.95. We found that SVMs were highly accurate in identifying HIV-2 coreceptor usage, attaining sensitivities of 73.5% and specificities of 96% during tenfold nested cross validation. The predictive performance of SVMs was not significantly different (p value 0.37) from an existing rules-based approach. Moreover, geno2pheno[coreceptor-hiv2] achieved a predictive accuracy of 100% and outperformed the existing approach on an independent data set containing nine new isolates with corresponding phenotypic measurements of coreceptor usage. geno2pheno[coreceptor-hiv2] could not only reproduce the established markers of CXCR4-usage, but also revealed novel markers: the substitutions 27K, 15G, and 8S were significantly predictive of CXCR4 usage. Furthermore, SVMs trained on the amino-acid sequences of the V1 and V2 loops were also quite accurate in predicting coreceptor usage (AUCs of 0.84 and 0.65, respectively). In this study, we developed geno2pheno[coreceptor-hiv2], the first online tool for the prediction of HIV-2 coreceptor

  17. A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites.

    Science.gov (United States)

    Côté, Nathalie M L; Daligault, Julien; Pruvost, Mélanie; Bennett, E Andrew; Gorgé, Olivier; Guimaraes, Silvia; Capelli, Nicolas; Le Bailly, Matthieu; Geigl, Eva-Maria; Grange, Thierry

    2016-01-01

    Human gastrointestinal parasites are good indicators for hygienic conditions and health status of past and present individuals and communities. While microscopic analysis of eggs in sediments of archeological sites often allows their taxonomic identification, this method is rarely effective at the species level, and requires both the survival of intact eggs and their proper identification. Genotyping via PCR-based approaches has the potential to achieve a precise species-level taxonomic determination. However, so far it has mostly been applied to individual eggs isolated from archeological samples. To increase the throughput and taxonomic accuracy, as well as reduce costs of genotyping methods, we adapted a PCR-based approach coupled with next-generation sequencing to perform precise taxonomic identification of parasitic helminths directly from archeological sediments. Our study of twenty-five 100 to 7,200 year-old archeological samples proved this to be a powerful, reliable and efficient approach for species determination even in the absence of preserved eggs, either as a stand-alone method or as a complement to microscopic studies.

  18. Relatedness of Indian flax genotypes (Linum usitatissimum L.): an inter-simple sequence repeat (ISSR) primer assay.

    Science.gov (United States)

    Rajwade, Ashwini V; Arora, Ritu S; Kadoo, Narendra Y; Harsulkar, Abhay M; Ghorpade, Prakash B; Gupta, Vidya S

    2010-06-01

    The objective of this study was to analyze the genetic relationships, using PCR-based ISSR markers, among 70 Indian flax (Linum usitatissimum L.) genotypes actively utilized in flax breeding programs. Twelve ISSR primers were used for the analysis yielding 136 loci, of which 87 were polymorphic. The average number of amplified loci and the average number of polymorphic loci per primer were 11.3 and 7.25, respectively, while the percent loci polymorphism ranged from 11.1 to 81.8 with an average of 63.9 across all the genotypes. The range of polymorphism information content scores was 0.03-0.49, with an average of 0.18. A dendrogram was generated based on the similarity matrix by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), wherein the flax genotypes were grouped in five clusters. The Jaccard's similarity coefficient among the genotypes ranged from 0.60 to 0.97. When the omega-3 alpha linolenic acid (ALA) contents of the individual genotypes were correlated with the clusters in the dendrogram, the high ALA containing genotypes were grouped in two clusters. This study identified SLS 50, Ayogi, and Sheetal to be the most diverse genotypes and suggested their use in breeding programs and for developing mapping populations.

  19. Development and evaluation of new primers for PCR-based identification of Prevotella intermedia.

    Science.gov (United States)

    Zhou, Yanbin; Liu, Dali; Wang, Yiwei; Zhu, Cailian; Liang, Jingping; Shu, Rong

    2014-08-01

    The aim of this study was to develop new Prevotella intermedia-specific PCR primers based on the 16S rRNA. The new primer set, Pi-192 and Pi-468, increased the accuracy of PCR-based P. intermedia identification and could be useful in the detection of P. intermedia as well as epidemiological studies on periodontal disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

    Directory of Open Access Journals (Sweden)

    Devidas N Chaturbhuj

    Full Text Available OBJECTIVES: Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples. DESIGN: The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity. METHODS: The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88 with known viral loads ranges from 1000-1.8 million RNA copies/ml. RESULTS: Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$, thus making it cost effective. CONCLUSIONS: The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.

  1. PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

    Science.gov (United States)

    Cassell, Gail H.; Lefkowitz, Elliot J.; Glass, John I.

    1995-01-01

    The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be

  2. A Novel Method for Assessing Sex-Specific and Genotype-Specific Response to Injury in Astrocyte Culture

    Science.gov (United States)

    Liu, Mingyue; Oyarzabal, Esteban; Yang, Rui; Murphy, Stephanie J; Hurn, Patricia D.

    2008-01-01

    Female astrocytes sustain less cell death from oxygen-glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1−3 day-old male and female, WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences. PMID:18436308

  3. An Accurate Method for Inferring Relatedness in Large Datasets of Unphased Genotypes via an Embedded Likelihood-Ratio Test

    KAUST Repository

    Rodriguez, Jesse M.

    2013-01-01

    Studies that map disease genes rely on accurate annotations that indicate whether individuals in the studied cohorts are related to each other or not. For example, in genome-wide association studies, the cohort members are assumed to be unrelated to one another. Investigators can correct for individuals in a cohort with previously-unknown shared familial descent by detecting genomic segments that are shared between them, which are considered to be identical by descent (IBD). Alternatively, elevated frequencies of IBD segments near a particular locus among affected individuals can be indicative of a disease-associated gene. As genotyping studies grow to use increasingly large sample sizes and meta-analyses begin to include many data sets, accurate and efficient detection of hidden relatedness becomes a challenge. To enable disease-mapping studies of increasingly large cohorts, a fast and accurate method to detect IBD segments is required. We present PARENTE, a novel method for detecting related pairs of individuals and shared haplotypic segments within these pairs. PARENTE is a computationally-efficient method based on an embedded likelihood ratio test. As demonstrated by the results of our simulations, our method exhibits better accuracy than the current state of the art, and can be used for the analysis of large genotyped cohorts. PARENTE\\'s higher accuracy becomes even more significant in more challenging scenarios, such as detecting shorter IBD segments or when an extremely low false-positive rate is required. PARENTE is publicly and freely available at http://parente.stanford.edu/. © 2013 Springer-Verlag.

  4. A method for genotyping elite breeding stocks of leaf chicory (Cichorium intybus L.) by assaying mapped microsatellite marker loci.

    Science.gov (United States)

    Ghedina, Andrea; Galla, Giulio; Cadalen, Thierry; Hilbert, Jean-Louis; Caenazzo, Silvano Tiozzo; Barcaccia, Gianni

    2015-12-30

    Leaf chicory (Cichorium intybus subsp. intybus var. foliosum L.) is a diploid plant species (2n = 18) of the Asteraceae family. The term "chicory" specifies at least two types of cultivated plants: a leafy vegetable, which is highly differentiated with respect to several cultural types, and a root crop, whose current industrial utilization primarily addresses the extraction of inulin or the production of a coffee substitute. The populations grown are generally represented by local varieties (i.e., landraces) with high variation and adaptation to the natural and anthropological environment where they originated, and have been yearly selected and multiplied by farmers. Currently, molecular genetics and biotechnology are widely utilized in marker-assisted breeding programs in this species. In particular, molecular markers are becoming essential tools for developing parental lines with traits of interest and for assessing the specific combining ability of these lines to breed F1 hybrids. The present research deals with the implementation of an efficient method for genotyping elite breeding stocks developed from old landraces of leaf chicory, Radicchio of Chioggia, which are locally dominant in the Veneto region, using 27 microsatellite (SSR) marker loci scattered throughout the linkage groups. Information on the genetic diversity across molecular markers and plant accessions was successfully assessed along with descriptive statistics over all marker loci and inbred lines. Our overall data support an efficient method for assessing a multi-locus genotype of plant individuals and lineages that is useful for the selection of new varieties and the certification of local products derived from Radicchio of Chioggia. This method proved to be useful for assessing the observed degree of homozygosity of the inbred lines as a measure of their genetic stability; plus it allowed an estimate of the specific combining ability (SCA) between maternal and paternal inbred lines on the

  5. Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

    Science.gov (United States)

    Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (pDNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

  6. Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

    Science.gov (United States)

    Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

    2017-07-25

    Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

  7. A PCR-based protocol to accurately size C9orf72 intermediate-length alleles.

    Science.gov (United States)

    Biasiotto, Giorgio; Archetti, Silvana; Di Lorenzo, Diego; Merola, Francesca; Paiardi, Giulia; Borroni, Barbara; Alberici, Antonella; Padovani, Alessandro; Filosto, Massimiliano; Bonvicini, Cristian; Caimi, Luigi; Zanella, Isabella

    2017-04-01

    Although large expansions of the non-coding GGGGCC repeat in C9orf72 gene are clearly defined as pathogenic for Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), intermediate-length expansions have also been associated with those and other neurodegenerative diseases. Intermediate-length allele sizing is complicated by intrinsic properties of current PCR-based methodologies, in that somatic mosaicism could be suspected. We designed a protocol that allows the exact sizing of intermediate-length alleles, as well as the identification of large expansions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. A rapid and direct real time PCR-based method for identification of Salmonella spp

    DEFF Research Database (Denmark)

    Rodriguez-Lazaro, D.; Hernández, Marta; Esteve, T.

    2003-01-01

    The aim of this work was the validation of a rapid, real-time PCR assay based on TaqMan((R)) technology for the unequivocal identification of Salmonella spp. to be used directly on an agar-grown colony. A real-time PCR system targeting at the Salmonella spp. invA gene was optimized and validated ...

  9. Common genotypes of hepatitis B virus

    International Nuclear Information System (INIS)

    Idrees, M.; Khan, S.; Riazuddin, S.

    2004-01-01

    Objective: To find out the frequency of common genotypes of hepatitis-B virus (HBV). Subjects and Methods: HBV genotypes were determined in 112 HBV DNA positive sera by a simple and precise molecular genotyping system base on PCR using type-specific primers for the determination of genotypes of HBV A through H. Results: Four genotypes (A,B,C and D) out of total eight reported genotypes so far were identified. Genotypes A, B and C were predominant. HBV genotype C was the most predominant in this collection, appearing in 46 samples (41.7%). However, the genotypes of a total of 5 (4.46%) samples could not be determined with the present genotyping system. Mixed genotypes were seen in 8(7.14% HBV) isolates. Five of these were infected with genotypes A/D whereas two were with genotypes C/D. One patient was infected with 4 genotypes (A/B/C/D). Genotype A (68%) was predominant in Sindh genotype C was most predominant in North West Frontier Province (NWFP) (68.96) whereas genotype C and B were dominant in Punjab (39.65% and 25.86% respectively). Conclusion: All the four common genotypes of HBV found worldwide (A,B,C and D) were isolated. Genotype C is the predominant Genotypes B and C are predominant in Punjab and N.W.F.P. whereas genotype A is predominant in Sindh. (author)

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs...

  11. Validation of the DNATyper™15 PCR Genotyping System for Forensic Application

    Directory of Open Access Journals (Sweden)

    Jian Ye

    2015-01-01

    Full Text Available We describe the optimization and validation of the DNATyper™15 multiplex polymerase chain reaction (PCR genotyping system for autosomal short tandem repeat (STR amplification at 14 autosomal loci (D6S1043, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, D13S317, D8S1179, D16S539, Penta E, D5S818, vWA, D18S51, and FGA and  amelogenin, a sex-determining locus. Several DNATyper™15 assay variables were optimized, including hot start Taq polymerase concentration, Taq polymerase activation time, magnesium concentration, primer concentration, annealing temperature, reaction volume, and cycle number. The performance of the assay was validated with respect to species specificity, sensitivity to template concentration, stability, accuracy, influence of the DNA extraction methods, and the ability to genotype the mixture samples. The performance of the DNATyper™15 system on casework samples was compared with that of two widely used STR amplification kits, Identifiler™ (Applied Biosystems, Carlsbad, CA, USA and PowerPlex 16 ® (Promega, Madison, WI, USA. The conditions for PCR-based DNATyper™15 genotyping were optimized. Contamination from forensically relevant nonhuman DNA was not found to impact genotyping results, and full profiles were generated for all the reactions containing ≥ 0.125 ng of DNA template. No significant difference in performance was observed even after the DNATyper™15 assay components were subjected to 20 freeze-thaw cycles. The performances of DNATyper™15, Identifiler™, and PowerPlex 16 ® were comparable in terms of sensitivity and the ability to genotype the mixed samples and case-type samples, with the assays giving the same genotyping results for all the shared loci. The DNA extraction methods did not affect the performance of any of the systems. Our results demonstrate that the DNATyper™15 system is suitable for genotyping in both forensic DNA database work and case-type samples.

  12. A fast and easy real-time PCR genotyping method for the HLA-G 14-bp insertion/deletion polymorphism in the 3' untranslated region

    DEFF Research Database (Denmark)

    Djurisic, S; Sørensen, A E; Hviid, T V F

    2012-01-01

    and reliable method to screen for the HLA-G 14-bp insertion/deletion polymorphism using an optimized real-time polymerase chain reaction protocol. The genotyping assay has been validated by comparison with conventional methods. As results can be obtained within a few hours, the assay will have a potential...

  13. Use of Sequenom sample ID Plus® SNP genotyping in identification of FFPE tumor samples.

    Directory of Open Access Journals (Sweden)

    Jessica K Miller

    Full Text Available Short tandem repeat (STR analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76-139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS. The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.

  14. Comparative study using phenotypic, genotypic and proteomics methods for identification of coagulase-negative staphylococci

    NARCIS (Netherlands)

    Dr. P.F.G. Wolffs; Ing M. Valkenburg; Dr. A.J.C. van den Brule, van den; M.Sc. A. Jansz; Drs A.J.M. Loonen; Ing J.N.B. Bergland

    2012-01-01

    Five methods were compared to determine the best technique for accurate identification of coagulase-negative staphylococci (CoNS) (n=142 strains). MALDI-TOF MS showed the best results for rapid and accurate CoNS differentiation (correct identity in 99.3%). An alternative to this approach could be

  15. Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods

    Directory of Open Access Journals (Sweden)

    Sahar Kianipour

    2018-01-01

    Full Text Available Background: Candida dubliniensis is a newly diagnosed species very similar to Candida albicans phenotypically and first discovered in the mouth of people with AIDS in 1995. Among the different phenotypic and genotypic methods, a cost-effective method should be selected which makes it possible to differentiate these similar species. Materials and Methods: Polymerase chain reaction (PCR-restriction fragment length polymorphism with MspI enzyme and the Duplex-PCR method were done by DNA extraction using boiling. The sequencing of the amplified ribosomal region was used to confirm the C. dubliniensis species. Direct examination and colony count of the yeasts were applied for bronchoalveolar lavage (BAL samples and the growth rate of the yeasts were studied at 45°C. To understand the ability formation of chlamydoconidia in yeast isolates, they were separately cultured on the sunflower seed agar, wheat flour agar, and corn meal agar media. Results: Fifty-nine (49.2% yeast colonies were identified from the total of 120 BAL specimens. Twenty-nine isolated yeasts; including 17 (58.6% of C. albicans/dubliniensis complex and 12 (41.4% of nonalbicans isolates produced pseudohypha or blastoconidia in direct smear with a mean colony count of 42000 CFU/mL. C. albicans with the frequency of 15 (42.9% were the most common isolated yeasts, whereas C. dubliniensis was identified in two nonHIV patients. Conclusion: Sequencing of the replicated gene fragment is the best method for identifying the yeasts, but the determination of the species by phenotypic methods such as the creation of chlamydoconidia in sunflower seeds agar and wheat flour agar media can be cost-effective, have sensitivity and acceptable quality.

  16. Multiplexed genome engineering and genotyping methods applications for synthetic biology and metabolic engineering.

    Science.gov (United States)

    Wang, Harris H; Church, George M

    2011-01-01

    Engineering at the scale of whole genomes requires fundamentally new molecular biology tools. Recent advances in recombineering using synthetic oligonucleotides enable the rapid generation of mutants at high efficiency and specificity and can be implemented at the genome scale. With these techniques, libraries of mutants can be generated, from which individuals with functionally useful phenotypes can be isolated. Furthermore, populations of cells can be evolved in situ by directed evolution using complex pools of oligonucleotides. Here, we discuss ways to utilize these multiplexed genome engineering methods, with special emphasis on experimental design and implementation. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Native nucleic acid electrophoresis as an efficient alternative for genotyping method of influenza virus.

    Science.gov (United States)

    Pajak, Beata; Lepek, Krzysztof

    2014-01-01

    Influenza viruses are the worldwide major causative agents of human and animal acute respiratory infections. Some of the influenza subtypes have caused epidemics and pandemics among humans. The varieties of methods are available for the rapid isolation and identification of influenza viruses in clinical and environmental samples. Since nucleic acids amplification techniques such as RT-PCR have been adapted, fast and sensitive influenza type and subtype determination is possible. However, in some ambiguous cases other, more detailed assay might be desired. The genetic material of influenza virus is highly unstable and constantly mutates. It is known that single nucleotide polymorphisms (SNPs) results in resistance to commercially available anti-viral drugs. The genetic drift of the virus could also result in weakening of immune response to infection. Finally, in a substantial number of patients co-infection with various virus strains or types has been confirmed. Although the detection of co-infection or presence of minor genetic variants within flu-infected patients is not a routine procedure, a rapid and wide spectrum diagnostics of influenza virus infections could reveal an accurate picture of the disease and more importantly, is crucial for choosing the appropriate therapeutics and virus monitoring. Herein we present the evidences that native gel electrophoresis and MSSCP--a method based on multitemperature single strand conformation polymorphism could furnish a useful technique for minor variants, which escape discovery by conventional diagnostic assays.

  18. Detection of Hepatitis B and C Viruses in Almost All Hepatocytes by Modified PCR-Based In Situ Hybridization▿

    Science.gov (United States)

    Nuriya, Hideko; Inoue, Kazuaki; Tanaka, Takeshi; Hayashi, Yukiko; Hishima, Tsunekazu; Funata, Nobuaki; Kaji, Kyosuke; Hayashi, Seishu; Kaneko, Shuichi; Kohara, Michinori

    2010-01-01

    Although PCR-based in situ hybridization (PCR-ISH) can be used to determine the distribution and localization of pathogens in tissues, this approach is hampered by its low specificity. Therefore, we used a highly specific and sensitive PCR-ISH method to reveal the lobular distribution and intracellular localization of hepatitis B virus (HBV) and HCV in chronic liver disease and to clarify the state of persistent HBV and HCV infection in the liver. HBV genomic DNA was detected in almost all hepatocytes, whereas HBV RNA or protein was differentially distributed only in a subset of the HBV DNA-positive region. Further, HCV genomic RNA was detected in almost all hepatocytes and was localized to the cytoplasm. HCV RNA was also detected in the epithelium of the large bile duct but not in endothelial cells, portal tracts, or sinusoidal lymphocytes. In patients with HBV and HCV coinfection, HCV RNA was localized to the noncancerous tissue, whereas HBV DNA was found only in the cancerous tissue. Using this novel PCR-ISH method, we could visualize the staining pattern of HBV and HCV in liver sections, and we obtained results consistent with those of real-time detection (RTD)-PCR analysis. In conclusion, almost all hepatocytes are infected with HBV or HCV in chronic liver disease; this finding implies that the viruses spreads throughout the liver in the chronic stage. PMID:20739486

  19. A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi.

    Directory of Open Access Journals (Sweden)

    Kanako Komaki-Yasuda

    Full Text Available A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient's blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a "fast PCR enzyme". In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the "fast PCR enzyme", with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

  20. Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods.

    Science.gov (United States)

    Kianipour, Sahar; Ardestani, Mohammad Emami; Dehghan, Parvin

    2018-01-01

    Candida dubliniensis is a newly diagnosed species very similar to Candida albicans phenotypically and first discovered in the mouth of people with AIDS in 1995. Among the different phenotypic and genotypic methods, a cost-effective method should be selected which makes it possible to differentiate these similar species. Polymerase chain reaction (PCR)-restriction fragment length polymorphism with MspI enzyme and the Duplex-PCR method were done by DNA extraction using boiling. The sequencing of the amplified ribosomal region was used to confirm the C. dubliniensis species. Direct examination and colony count of the yeasts were applied for bronchoalveolar lavage (BAL) samples and the growth rate of the yeasts were studied at 45°C. To understand the ability formation of chlamydoconidia in yeast isolates, they were separately cultured on the sunflower seed agar, wheat flour agar, and corn meal agar media. Fifty-nine (49.2%) yeast colonies were identified from the total of 120 BAL specimens. Twenty-nine isolated yeasts; including 17 (58.6%) of C. albicans / dubliniensis complex and 12 (41.4%) of nonalbicans isolates produced pseudohypha or blastoconidia in direct smear with a mean colony count of 42000 CFU/mL. C. albicans with the frequency of 15 (42.9%) were the most common isolated yeasts, whereas C. dubliniensis was identified in two nonHIV patients. Sequencing of the replicated gene fragment is the best method for identifying the yeasts, but the determination of the species by phenotypic methods such as the creation of chlamydoconidia in sunflower seeds agar and wheat flour agar media can be cost-effective, have sensitivity and acceptable quality.

  1. PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (Triticum aestivum L.).

    Science.gov (United States)

    Zhang, Xiaofei; Liu, Dongcheng; Jiang, Wei; Guo, Xiaoli; Yang, Wenlong; Sun, Jiazhu; Ling, Hongqing; Zhang, Aimin

    2011-12-01

    Low-molecular-weight glutenin subunits (LMW-GSs) are encoded by a multi-gene family and are essential for determining the quality of wheat flour products, such as bread and noodles. However, the exact role or contribution of individual LMW-GS genes to wheat quality remains unclear. This is, at least in part, due to the difficulty in characterizing complete sequences of all LMW-GS gene family members in bread wheat. To identify full-length LMW-GS genes, a polymerase chain reaction (PCR)-based method was established, consisting of newly designed conserved primers and the previously developed LMW-GS gene molecular marker system. Using the PCR-based method, 17 LMW-GS genes were identified and characterized in Xiaoyan 54, of which 12 contained full-length sequences. Sequence alignments showed that 13 LMW-GS genes were identical to those found in Xiaoyan 54 using the genomic DNA library screening, and the other four full-length LMW-GS genes were first isolated from Xiaoyan 54. In Chinese Spring, 16 unique LMW-GS genes were isolated, and 13 of them contained full-length coding sequences. Additionally, 16 and 17 LMW-GS genes in Dongnong 101 and Lvhan 328 (chosen from the micro-core collections of Chinese germplasm), respectively, were also identified. Sequence alignments revealed that at least 15 LMW-GS genes were common in the four wheat varieties, and allelic variants of each gene shared high sequence identities (>95%) but exhibited length polymorphism in repetitive regions. This study provides a PCR-based method for efficiently identifying LMW-GS genes in bread wheat, which will improve the characterization of complex members of the LMW-GS gene family and facilitate the understanding of their contributions to wheat quality.

  2. First detection of multiple knockdown resistance (kdr)-like mutations in voltage-gated sodium channel using three new genotyping methods in Anopheles sinensis from Guangxi Province, China.

    Science.gov (United States)

    Tan, Wei L; Li, Chun X; Wang, Zhong M; Liu, Mei D; Dong, Yan D; Feng, Xiang Y; Wu, Zhi M; Guo, Xiao X; Xing, Dan; Zhang, Ying M; Wang, Zhong C; Zhao, Tong Y

    2012-09-01

    To investigate knockdown resistance (kdr)-like mutations associated with pyrethroid resistance in Anopheles sinensis (Wiedemann, 1828), from Guangxi province, southwest China, a segment of a sodium channel gene was sequenced and genotyped using three new genotyping assays. Direct sequencing revealed the presence of TTG-to-TCG and TG-to-TTT mutations at allele position L1014, which led to L1014S and L1014F substitutions in a few individual and two novel substitutions of N1013S and L1014W in two DNA templates. A low frequency of the kdr allele mostly in the heterozygous state of L1014S and L1014F was observed in this mosquito population. In this study, the genotyping of An. sinensis using three polymerase chain reaction-based methods generated consistent results, which agreed with the results of DNA sequencing. In total, 52 mosquitoes were genotyped using a direct sequencing assay. The number of mosquitoes and their genotypes were as follows: L/L = 24, L/S = 19, L/F = 8, and F/W = 1. The allelic frequency of L1014, 1014S, and 1014F were 72, 18, and 9%, respectively.

  3. Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid

    Directory of Open Access Journals (Sweden)

    Johnson Sandra

    2010-10-01

    Full Text Available Abstract Background Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR-based methods. To address this problem a simple protocol for prior use of multiple displacement amplification (MDA as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids. Findings A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability. Conclusion MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.

  4. Detection of Fetomaternal Genotype Associations in Early-Onset Disorders: Evaluation of Different Methods and Their Application to Childhood Leukemia

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    Jasmine Healy

    2010-01-01

    Full Text Available Several designs and analytical approaches have been proposed to dissect offspring from maternal genetic contributions to early-onset diseases. However, lack of parental controls halts the direct verification of the assumption of mating symmetry (MS required to assess maternally-mediated effects. In this study, we used simulations to investigate the performance of existing methods under mating asymmetry (MA when parents of controls are missing. Our results show that the log-linear, likelihood-based framework using a case-triad/case-control hybrid design provides valid tests for maternal genetic effects even under MA. Using this approach, we examined fetomaternal associations between 29 SNPs in 12 cell-cycle genes and childhood pre-B acute lymphoblastic leukemia (ALL. We identified putative fetomaternal effects at loci CDKN2A rs36228834 (P=.017 and CDKN2B rs36229158 (P=.022 that modulate the risk of childhood ALL. These data further corroborate the importance of the mother's genotype on the susceptibility to early-onset diseases.

  5. PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: diagnostic value of immunoglobulin kappa and lambda light chain gene rearrangement investigation.

    Science.gov (United States)

    Amara, Khaled; Trimeche, Mounir; Ziadi, Sonia; Sriha, Badreddine; Mokni, Moncef; Korbi, Sadok

    2006-01-01

    Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Igkappa) and lambda (Iglambda) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Igkappa, and Iglambda PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.

  6. Improving genetic evaluation of litter size and piglet mortality for both genotyped and nongenotyped individuals using a single-step method.

    Science.gov (United States)

    Guo, X; Christensen, O F; Ostersen, T; Wang, Y; Lund, M S; Su, G

    2015-02-01

    A single-step method allows genetic evaluation using information of phenotypes, pedigree, and markers from genotyped and nongenotyped individuals simultaneously. This paper compared genomic predictions obtained from a single-step BLUP (SSBLUP) method, a genomic BLUP (GBLUP) method, a selection index blending (SELIND) method, and a traditional pedigree-based method (BLUP) for total number of piglets born (TNB), litter size at d 5 after birth (LS5), and mortality rate before d 5 (Mort; including stillbirth) in Danish Landrace and Yorkshire pigs. Data sets of 778,095 litters from 309,362 Landrace sows and 472,001 litters from 190,760 Yorkshire sows were used for the analysis. There were 332,795 Landrace and 207,255 Yorkshire animals in the pedigree data, among which 3,445 Landrace pigs (1,366 boars and 2,079 sows) and 3,372 Yorkshire pigs (1,241 boars and 2,131 sows) were genotyped with the Illumina PorcineSNP60 BeadChip. The results showed that the 3 methods with marker information (SSBLUP, GBLUP, and SELIND) produced more accurate predictions for genotyped animals than the pedigree-based method. For genotyped animals, the average of reliabilities for all traits in both breeds using traditional BLUP was 0.091, which increased to 0.171 w+hen using GBLUP and to 0.179 when using SELIND and further increased to 0.209 when using SSBLUP. Furthermore, the average reliability of EBV for nongenotyped animals was increased from 0.091 for traditional BLUP to 0.105 for the SSBLUP. The results indicate that the SSBLUP is a good approach to practical genomic prediction of litter size and piglet mortality in Danish Landrace and Yorkshire populations.

  7. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

    Science.gov (United States)

    Smith, Cindy J; Osborn, A Mark

    2009-01-01

    Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

  8. PCR-based Approaches for the Detection of Clinical Methicillin-resistant Staphylococcus aureus

    Science.gov (United States)

    Liu, Ying; Zhang, Jiang; Ji, Yinduo

    2016-01-01

    Staphylococcus aureus is an important pathogen that can cause a variety of infections, including superficial and systematic infections, in humans and animals. The persistent emergence of multidrug resistant S. aureus, particularly methicillin-resistant S. aureus, has caused dramatically economic burden and concerns in the public health due to limited options of treatment of MRSA infections. In order to make a correct choice of treatment for physicians and understand the prevalence of MRSA, it is extremely critical to precisely and timely diagnose the pathogen that induces a specific infection of patients and to reveal the antibiotic resistant profile of the pathogen. In this review, we outlined different PCR-based approaches that have been successfully utilized for the rapid detection of S. aureus, including MRSA and MSSA, directly from various clinical specimens. The sensitivity and specificity of detections were pointed out. Both advantages and disadvantages of listed approaches were discussed. Importantly, an alternative approach is necessary to further confirm the detection results from the molecular diagnostic assays. PMID:27335617

  9. Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

    Directory of Open Access Journals (Sweden)

    Zhenming Yu

    Full Text Available Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

  10. A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi

    Science.gov (United States)

    Komaki-Yasuda, Kanako; Vincent, Jeanne Perpétue; Nakatsu, Masami; Kato, Yasuyuki; Ohmagari, Norio

    2018-01-01

    A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient’s blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a “fast PCR enzyme”. In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the “fast PCR enzyme”, with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses. PMID:29370297

  11. Estimating the numbers of malaria infections in blood samples using high-resolution genotyping data.

    Directory of Open Access Journals (Sweden)

    Amanda Ross

    Full Text Available People living in endemic areas often habour several malaria infections at once. High-resolution genotyping can distinguish between infections by detecting the presence of different alleles at a polymorphic locus. However the number of infections may not be accurately counted since parasites from multiple infections may carry the same allele. We use simulation to determine the circumstances under which the number of observed genotypes are likely to be substantially less than the number of infections present and investigate the performance of two methods for estimating the numbers of infections from high-resolution genotyping data. The simulations suggest that the problem is not substantial in most datasets: the disparity between the mean numbers of infections and of observed genotypes was small when there was 20 or more alleles, 20 or more blood samples, a mean number of infections of 6 or less and where the frequency of the most common allele was no greater than 20%. The issue of multiple infections carrying the same allele is unlikely to be a major component of the errors in PCR-based genotyping. Simulations also showed that, with heterogeneity in allele frequencies, the observed frequencies are not a good approximation of the true allele frequencies. The first method that we proposed to estimate the numbers of infections assumes that they are a good approximation and hence did poorly in the presence of heterogeneity. In contrast, the second method by Li et al estimates both the numbers of infections and the true allele frequencies simultaneously and produced accurate estimates of the mean number of infections.

  12. Isolation and characterization of new Leptospira genotypes from patients in Mayotte (Indian Ocean.

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    Pascale Bourhy

    Full Text Available BACKGROUND: Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean. To date, Leptospira isolates have never been isolated in this endemic region. METHODS AND FINDINGS: Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA, and pulsed field gel electrophoresis (PFGE. Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars. CONCLUSIONS: Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features.

  13. Desmanthus GENOTYPES

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    JOSÉ HENRIQUE DE ALBUQUERQUE RANGEL

    2015-01-01

    Full Text Available Desmanthus is a genus of forage legumes with potential to improve pastures and livestock produc-tion on clay soils of dry tropical and subtropical regions such as the existing in Brazil and Australia. Despite this patterns of natural or enforced after-ripening of Desmanthus seeds have not been well established. Four year old seed banks of nine Desmanthus genotypes at James Cook University were accessed for their patterns of seed softe-ning in response to a range of temperatures. Persistent seed banks were found to exist under all of the studied ge-notypes. The largest seeds banks were found in the genotypes CPI 78373 and CPI 78382 and the smallest in the genotypes CPI’s 37143, 67643, and 83563. An increase in the percentage of softened seeds was correlated with higher temperatures, in two patterns of response: in some accessions seeds were not significantly affected by tempe-ratures below 80º C; and in others, seeds become soft when temperature rose to as little as 60 ºC. At 80 °C the heat started to depress germination. High seed production of Desmanthus associated with dependence of seeds on eleva-ted temperatures to softening can be a very important strategy for plants to survive in dry tropical regions.

  14. Development of a rapid multiplex SSR genotyping method to study populations of the fun-gal plant pathogen Zymoseptoria tritici

    NARCIS (Netherlands)

    Gautier, A.; Marcel, T.C.; Confais, J.; Crane, C.; Kema, G.H.J.; Suffert, F.; Walker, A.S.

    2014-01-01

    Background Zymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are known to be highly diverse and poorly differentiated from each other. However, a genotyping tool is needed to address further

  15. Clinical Assessment of a Nocardia PCR-Based Assay for Diagnosis of Nocardiosis.

    Science.gov (United States)

    Rouzaud, Claire; Rodriguez-Nava, Véronica; Catherinot, Emilie; Méchaï, Frédéric; Bergeron, Emmanuelle; Farfour, Eric; Scemla, Anne; Poirée, Sylvain; Delavaud, Christophe; Mathieu, Daniel; Durupt, Stéphane; Larosa, Fabrice; Lengelé, Jean-Philippe; Christophe, Jean-Louis; Suarez, Felipe; Lortholary, Olivier; Lebeaux, David

    2018-06-01

    The diagnosis of nocardiosis, a severe opportunistic infection, is challenging. We assessed the specificity and sensitivity of a 16S rRNA Nocardia PCR-based assay performed on clinical samples. In this multicenter study (January 2014 to April 2015), patients who were admitted to three hospitals and had an underlying condition favoring nocardiosis, clinical and radiological signs consistent with nocardiosis, and a Nocardia PCR assay result for a clinical sample were included. Patients were classified as negative control (NC) (negative Nocardia culture results and proven alternative diagnosis or improvement at 6 months without anti- Nocardia treatment), positive control (PC) (positive Nocardia culture results), or probable nocardiosis (positive Nocardia PCR results, negative Nocardia culture results, and no alternative diagnosis). Sixty-eight patients were included; 47 were classified as NC, 8 as PC, and 13 as probable nocardiosis. PCR results were negative for 35/47 NC patients (74%). For the 12 NC patients with positive PCR results, the PCR assay had been performed with respiratory samples. These NC patients had chronic bronchopulmonary disease more frequently than did the NC patients with negative PCR results (8/12 patients [67%] versus 11/35 patients [31%]; P = 0.044). PCR results were positive for 7/8 PC patients (88%). There were 13 cases of probable nocardiosis, diagnosed solely using the PCR results; 9 of those patients (69%) had lung involvement (consolidation or nodule). Nocardia PCR testing had a specificity of 74% and a sensitivity of 88% for the diagnosis of nocardiosis. Nocardia PCR testing may be helpful for the diagnosis of nocardiosis in immunocompromised patients but interpretation of PCR results from respiratory samples is difficult, because the PCR assay may also detect colonization. Copyright © 2018 American Society for Microbiology.

  16. PCR-based techniques for leprosy diagnosis: from the laboratory to the clinic.

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    Alejandra Nóbrega Martinez

    2014-04-01

    Full Text Available In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL, paucibacillary (PB, and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.

  17. Bacterial Diversity in the Digestive Tracts of Four Indian Air-Breathing Fish Species Investigated by PCR Based Denaturing Gradient Gel Electrophoresis

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    Suxu He

    Full Text Available ABSTRACT An investigation was conducted to identify the allochthonous microbiota (entire intestine and the autochthonous microbiota in proximal intestine (PI and distal intestine (DI of four species of Indian air-breathing fish (climbing perch; Anabas testudineus, murrel; Channa punctatus, walking catfish; Clarias batrachus and stinging catfish; Heteropneustes fossilis by PCR based denaturing gradient gel electrophoresis (DGGE. High similarities of the allochthonous microbiota were observed between climbing perch and murrel, walking catfish and stinging catfish, indicating similar food behavior. The autochthonous microbiota of PI and DI from climbing perch and murrel revealed more similarity, than the result obtained from walking catfish and stinging catfish. The autochthonous microbiota of climbing perch and murrel were similar with regard to the allochthonous microbiota, but no such similarity was observed in case of walking catfish and stinging catfish. The fish genotype and intestinal bacteria are well matched and show co-evolutionary relationship. Three fish species has its unique bacteria; autochthonous Enterobacter cloacae, Edwardsiella tarda and Sphingobium sp. in DI of climbing perch, Pseudomonas sp.; allochthonous and autochthonous in PI of walking catfish and uncultured bacterium (EU697160.1, uncultured bacterium (JF018065.1 and uncultured bacterium (EU697160.1 for stinging catfish. In murrel, no unique bacteria were detected.

  18. High prevalence of ACE DD genotype among north Indian end stage renal disease patients

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    Pandirikkal Baburajan Vinod

    2006-10-01

    Full Text Available Abstract Background The Renin-Angiotensin system (RAS is a key regulator of both blood pressure and kidney functions and their interaction. In such a situation, genetic variability in the genes of different components of RAS is likely to contribute for its heterogeneous association in the renal disease patients. Angiotensin converting enzyme-1 (ACE-1 is an important component of RAS which determines the vasoactive peptide Angiotensin-II. Methods In the present study, we have investigated 127 ESRD patients and 150 normal healthy controls from north India to deduce the association between ACE gene polymorphism and ESRD. The inclusion criteria for patients included a constantly elevated serum creatinine level above normal range (ranging from 3.4 to 15.8 and further the patients were recommended for renal transplantation. A total of 150 normal healthy controls were also genotyped for ACE I/D polymorphism. The criterion of defining control sample as normal was totally based on the absence of any kidney disease determined from the serum creatinin level. Genotyping of ACE I/D were assayed by polymerase chain reaction (PCR based DNA amplification using specific flanking primers Based on the method described elsewhere. Results The difference of DD and II genotypes was found highly significant among the two groups (p = 0.025; OR = 3.524; 95%CI = 1.54-8.07. The combined genotype DD v/s ID+II comparison validated that DD genotype is a high risk genotype for ESRD (p = 0.001; OR = 5.74; 95%CI limit = 3.4-8.5. However, no correlation was obtained for different biochemical parameters of lipid profile and renal function among DD and non DD genotype. Interestingly, ~87% of the DD ESRD patients were found hypertensive in comparison to the 65% patients of non DD genotype Conclusion Based on these observations we conclude that ACE DD genotype implicate a strong possible role in the hypertensive state and in renal damage among north Indians. The study will help in

  19. [Rapid, simple genotyping method by the variable numbers of tandem repeats (VNTR) for Mycobacterium tuberculosis isolates in Japan--analytical procedure of JATA (12)-VNTR].

    Science.gov (United States)

    Maeda, Shinji; Murase, Yoshiro; Mitarai, Satoshi; Sugawara, Isamu; Kato, Seiya

    2008-10-01

    The discriminatory power of each locus in variable numbers of tandem repeats (VNTR) analyses was evaluated for development of the genotyping method of Mycobacterium tuberculosis (TB) in Japan. By using 325 TB strains collected from whole Japan and 24 mass infection cases (74 isolates), IS6110 restriction fragment length polymorphism (RFLP), spoligotyping and VNTR (35 loci) were analyzed. We excluded 4 loci (VNTRs 2163a, 3232, 3820, and 4120) and selected in top 12 loci (VNTRs 0424, 0960, 1955, 2074, 2163b, 2372, 2996, 3155, 3192, 3336, 4052, and 4156). The cluster rate of IS6110 RFLP was higher than that of 12-locus [Japan Anti-Tuberculosis Association (JATA)] VNTR. And in comparison of the discriminatory power of 12-locus JATA VNTR and that of Supply (15)-VNTR, the JATA (12)-VNTR was superior, even though less loci analyses. Therefore, this JATA (12)-VNTR could be used for TB genotyping in areas where Beijing strains are prevalent.

  20. Stem Cuttings as a Quick In Vitro Screening Method of Sodium Chloride Tolerance in Potato (Solanum) Genotypes

    International Nuclear Information System (INIS)

    Elhag, A. Z.; Mix-Wagnar, G.; Elbassam, N.; Horst, W.

    2008-01-01

    This study was conducted to find how far in vitro explants stem cuttings technique could be suitable for quick screening of NaCl tolerance solanum genotypes and to identify some aspects of their NaCl tolerance. Fifteen solanum genotypes were tested on four NaCl concentrations both in vitro and in vivo, two-node stem cuttings of in vitro produced explants were grown on Murashige and Skoog (MS) salts supplemented with four NaCl concentrations (0,40,80 and 120 mM) for six weeks in vitro. The other part of the in vitro grown explants were transplanted in Kick- Brauck- Manns pots containing sandy loam soil supplemented also with four NaCl concentration (0, 0.1, 0.2 and 0.3 NaCl, w/w) and grown further either for eight weeks or till harvest in a green house. Both experiments were in a completely randomized design with four replicates. The main stem length, shoot dry matter and tuber yield as well as mineral element (Na''+, K + , Ca''2''+ and Cl''-) were measured. The growth of all genotypes was affected by increasing of NaCl. There was a close correlation between growth response (length of explant main stem) in vitro and shoot dry matter and tuber yield in vivo (r=0.81** for dry matter and 0.72** for tuber yield. Na''+ and Cl''- concentrations in shoots wee inversely correlated with the vegetative growth (r=-0.73** for both in vitro and r=-0.89** and r=-0.88** in vivo, respectively). The genotypes showed varied ability to reduce the transport of Na''+ and Cl''- to the shoots, where by NaCl tolerant genotypes showed lower content of both elements than the sensitive ones. K''+ and Ca''2''+ concentrations were decreased with increasing NaCl concentration. The responses for mineral element (Na''+ and Cl - ) accumulation or restriction of explants in vitro and intact plants in vivo were also closely correlated (r=0.79** and 0.71**, respectively) especially at the medium NaCl concentrations (80 mM and 0.2% NaCl). The similar response of the explant and the intact plant

  1. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    OpenAIRE

    Ram, S.; Vimalin, J.M.; Jambulingam, M.; Tiru, V.; Gopalakrishnan, R.K.; Madhavan, H.N.

    2012-01-01

    Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septi...

  2. Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA.

    Science.gov (United States)

    Kim, Sung-Su; Choi, Hyun-Jeung; Kim, Jin Ju; Kim, M Sun; Lee, In-Seon; Byun, Bohyun; Jia, Lina; Oh, Myung Ryurl; Moon, Youngho; Park, Sarah; Choi, Joon-Seok; Chae, Seoung Wan; Nam, Byung-Ho; Kim, Jin-Soo; Kim, Jihun; Min, Byung Soh; Lee, Jae Seok; Won, Jae-Kyung; Cho, Soo Youn; Choi, Yoon-La; Shin, Young Kee

    2018-01-11

    In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.

  3. Development and Validation of a Multiplex PCR-Based Assay for the Upper Respiratory Tract Bacterial Pathogens Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis.

    Science.gov (United States)

    Post; White; Aul; Zavoral; Wadowsky; Zhang; Preston; Ehrlich

    1996-06-01

    Background: Conventional simplex polymerase chain reaction (PCR)-based assays are limited in that they only provide for the detection of a single infectious agent. Many clinical diseases, however, present in a nonspecific, or syndromic, fashion, thereby necessitating the simultaneous assessment of multiple pathogens. Panel-based molecular diagnostic testing can be accomplished by the development of multiplex PCR-based assays, which can detect, individually or severally, different pathogens that are associated with syndromic illness. As part of a larger program of panel development, an assay that can simultaneously detect Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was developed. These organisms were chosen as they are the most common bacterial pathogens associated with both the acute and chronic forms of otitis media; they are also responsible for a high percentage of sinus infections in both children and adults. In addition, H. influenzae and S. pneumoniae are commonly associated with septic meningitits. Methods and Results: Multiple individual PCR-based assays were developed for each of the three target organisms which were then evaluated for sensitivity and specificity. Utilizing the simplex assays that met our designated performance criteria, a matrix style approach was used to develop a duplex H. influenzae-S. pneumoniae assay. The duplex assay was then used as a single component in the development of a triplex assay, wherein the various M. catarrhalis primer-probe sets were tested for compatibility with the existing assay. A single-step PCR protocol, with species-specific primers for each of the three target organisms and a liquid hybridization-gel retardation amplimer detection system, was developed, which amplifies and then discriminates among each of the amplification products according to size. This assay is able to detect all three organisms in a specific manner, either individually or severally. Dilutional experiments

  4. Rapid and sensitive method to identify Mycobacterium avium subsp. paratuberculosis in cow's milk by DNA methylase genotyping.

    Science.gov (United States)

    Mundo, Silvia Leonor; Gilardoni, Liliana Rosa; Hoffman, Federico José; Lopez, Osvaldo Jorge

    2013-03-01

    Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.

  5. PCR based diagnostic assay targeting the beta tubulin gene for the detection of Trichomonas vaginalis infection in vaginal swab samples of symptomatic and asymptomatic women in India

    Directory of Open Access Journals (Sweden)

    Surya Prakash Dwivedi

    2012-10-01

    Full Text Available Objective: To develop an in-house PCR based diagnostic assay for identification of strains isolated from symptomatic and asymptomatic subjects of India, targeting the 毬 -tubulin gene using specific primers. Methods: In the present study a primer set is designed to target a well-conserved region in the beta-tubulin gene of Trichomonas vaginalis (T. vaginalis. All strains of T. vaginalis were tested and successfully detected by PCR yielding a single predicted product of 198 bp in gel electrophoresis, while there was negative response with DNA from Giardia lamblia, Toxoplasma gondii, Leishmania donovani and Entamoeba histolytica. The sensitivity and specificity for a single T. vaginalis cell per PCR was achieved. Axenic Culture, performed with long term axenized T. vaginalis culture system, was routinely examined to identify T. vaginalis. Results: The PCR based investigations with 498 vaginal swab samples from women attending OPD clinics of Halberg Hospital Moradabad and Queen Mary ’s Hospital, Lucknow, India and 17 long term axenic cultures maintained at PGIMER, Chandigarh, India using primer set BTUB 1 & BTUB 2 showed sensitivity and specificity response of 98% and 100%, respectively, while wet preparation in clinically isolated samples responded up to 62.5%. The PCR product sequencing result of symptomatic strains (SS1 of T. vaginalis (744 bp long was submitted to NCBI (Accession No: JF513200. It shows maximum identity 98 % with XM_001284521 Trichomonas vaginalis G-3 beta-tubulin (btub putative partial mRNA. Conclusions: The data gathered in the present study entail that the diagnosis of T. vaginalis infection by PCR may be established as a sensitive and specific protocol, to be incorporated into a joint strategy for the screening of multiple STDs by employing molecular amplification technique. The merits and precautions of the protocol have been discussed.

  6. [Establishment of a novel HLA genotyping method for preimplantation genetic diagnonis using multiple displacement amplification-polymerase chain reaction-sequencing based technique].

    Science.gov (United States)

    Zhang, Yinfeng; Luo, Haining; Zhang, Yunshan

    2015-12-01

    To establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT). Peripheral blood samples and 76 1PN, 2PN, 3PN discarded embryos from 9 couples were collected. The alleles of HLA-A, B, DR loci were detected from the MDA product with the PCR-SBT method. The HLA genotypes of the parental peripheral blood samples were analyzed with the same protocol. The genotypes of specific HLA region were evaluated for distinguishing the segregation of haplotypes among the family members, and primary HLA matching was performed between the embryos. The 76 embryos were subjected to MDA and 74 (97.4%) were successfully amplified. For the 34 embryos from the single blastomere group, the amplification rate was 94.1%, and for the 40 embryos in the two blastomeres group, the rate was 100%. The dropout rates for DQ allele and DR allele were 1.3% and 0, respectively. The positive rate for MDA in the single blastomere group was 100%, with the dropout rates for DQ allele and DR allele being 1.5% and 0, respectively. The positive rate of MDA for the two blastomere group was 100%, with the dropout rates for both DQ and DR alleles being 0. The recombination rate of fetal HLA was 20.2% (30/148). Due to the improper classification and abnormal fertilized embryos, the proportion of matched embryos HLA was 20.3% (15/74),which was lower than the theoretical value of 25%. PGD with HLA matching can facilitate creation of a HLA-identical donor (saviour child) for umbilical cord blood or bone marrow stem cells for its affected sibling with a genetic disease. Therefore, preimplantation HLA matching may provide a tool for couples desiring to conceive a potential donor progeny for transplantation for its sibling with a life-threatening disorder.

  7. Application of a high-throughput genotyping method for loci exclusion in non-consanguineous Australian pedigrees with autosomal recessive retinitis pigmentosa.

    Science.gov (United States)

    Paterson, Rachel L; De Roach, John N; McLaren, Terri L; Hewitt, Alex W; Hoffmann, Ling; Lamey, Tina M

    2012-01-01

    Retinitis pigmentosa (RP) is the most common form of inherited blindness, caused by progressive degeneration of photoreceptor cells in the retina, and affects approximately 1 in 3,000 people. Over the past decade, significant progress has been made in gene therapy for RP and related diseases, making genetic characterization increasingly important. Recently, high-throughput technologies have provided an option for reasonably fast, cost-effective genetic characterization of autosomal recessive RP (arRP). The current study used a single nucleotide polymorphism (SNP) genotyping method to exclude up to 28 possible disease-causing genes in 31 non-consanguineous Australian families affected by arRP. DNA samples were collected from 59 individuals affected with arRP and 74 unaffected family members from 31 Australian families. Five to six SNPs were genotyped for 28 genes known to cause arRP or the related disease Leber congenital amaurosis (LCA). Cosegregation analyses were used to exclude possible causative genes from each of the 31 families. Bidirectional sequencing was used to identify disease-causing mutations in prioritized genes that were not excluded with cosegregation analyses. Two families were excluded from analysis due to identification of false paternity. An average of 28.9% of genes were excluded per family when only one affected individual was available, in contrast to an average of 71.4% or 89.8% of genes when either two, or three or more affected individuals were analyzed, respectively. A statistically significant relationship between the proportion of genes excluded and the number of affected individuals analyzed was identified using a multivariate regression model (pA) and USH2A in two families (c.2276 G>T). This study has shown that SNP genotyping cosegregation analysis can be successfully used to refine and expedite the genetic characterization of arRP in a non-consanguineous population; however, this method is effective only when DNA samples are

  8. PCR-based diagnosis, molecular characterization and detection of atypical strains of avian Chlamydia psittaci in companion and wild birds.

    Science.gov (United States)

    Madani, S A; Peighambari, S M

    2013-02-01

    Chlamydiosis is one of the most important infectious diseases of birds. In this study, 253 clinical samples were taken from 27 bird species belonging to seven orders. Thirty-two (12.6%) samples were positive for Chlamydia psittaci major outer membrane gene (ompA) DNA by a nested polymerase chain reaction (PCR). Twelve nested PCR-positive specimens were typed by ompA gene-based PCR-restricted fragment length polymorphism, using CTU/CTL primers and AluI restriction enzyme. Four restriction patterns were identified, including genotype A (two specimens from an African grey parrot [Psittacus erithacus] and a lorikeet [Trichoglossus haematodus]), genotype B (two specimens from a rock dove [Columbia livia] and a canary [Serinus canaria]), a third new restriction pattern (six specimens from African grey parrots), and a fourth new restriction pattern (two specimens from a ring-necked parakeet [Psittacula krameri] and an Alexandrine parakeet [Psittacula eupatria]). The third and the fourth restriction patterns are suggested to be provisional genotypes I and J, respectively. Partial sequencing of the ompA gene of seven specimens completely correlated with the results of PCR-restricted fragment length polymorphism and confirmed the presence of genotypes A and B and the two new provisional genotypes I and J. The two new genotypes have the closest identity with C. psittaci genotype F and Chlamydia abortus, respectively. From an evolutionary perspective, both new genotypes, particularly genotype J, are intermediate between the two species, C. psittaci and C. abortus.

  9. Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant.

    Directory of Open Access Journals (Sweden)

    Velusamy Srinivasan

    Full Text Available We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA. We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis.

  10. Real-time PCR-based genotyping from whole blood using Taq DNA polymerase and a buffer supplemented with 1,2-propanediol and trehalose

    Czech Academy of Sciences Publication Activity Database

    Utekal, Pavol; Kocanda, Lukáš; Matoušek, P.; Wagner, P.; Bugajev, Viktor; Dráber, Petr

    2015-01-01

    Roč. 416, January (2015), s. 178-182 ISSN 0022-1759 R&D Projects: GA ČR(CZ) GBP302/12/G101; GA MPO FR-TI3/067; GA ČR(CZ) GA14-09807S; GA ČR(CZ) GA14-00703S Institutional support: RVO:68378050 Keywords : 1,2-Propanediol * real-time PCR * SYBR Green I * Taq DNA polymerase * trehalose * Unseparated blood Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.858, year: 2015

  11. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

    OpenAIRE

    Aljanabi, S M; Martinez, I

    1997-01-01

    A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even i...

  12. A TaqMan real-time PCR-based assay for the identification of Fasciola spp.

    Science.gov (United States)

    Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Jowers, Michael J; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

    2011-06-30

    Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

    Science.gov (United States)

    Banjo, Masaya; Iguchi, Atsushi; Seto, Kazuko; Kikuchi, Taisei; Harada, Tetsuya; Scheutz, Flemming; Iyoda, Sunao

    2018-06-01

    In Escherichia coli , more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli . Copyright © 2018 American Society for Microbiology.

  14. An Accurate Method for Inferring Relatedness in Large Datasets of Unphased Genotypes via an Embedded Likelihood-Ratio Test

    KAUST Repository

    Rodriguez, Jesse M.; Batzoglou, Serafim; Bercovici, Sivan

    2013-01-01

    , accurate and efficient detection of hidden relatedness becomes a challenge. To enable disease-mapping studies of increasingly large cohorts, a fast and accurate method to detect IBD segments is required. We present PARENTE, a novel method for detecting

  15. A rapid PCR-based approach for molecular identification of filamentous fungi.

    Science.gov (United States)

    Chen, Yuanyuan; Prior, Bernard A; Shi, Guiyang; Wang, Zhengxiang

    2011-08-01

    In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates.

  16. A MITE-based genotyping method to reveal hundreds of DNA polymorphisms in an animal genome after a few generations of artificial selection

    Directory of Open Access Journals (Sweden)

    Tetreau Guillaume

    2008-10-01

    Full Text Available Abstract Background For most organisms, developing hundreds of genetic markers spanning the whole genome still requires excessive if not unrealistic efforts. In this context, there is an obvious need for methodologies allowing the low-cost, fast and high-throughput genotyping of virtually any species, such as the Diversity Arrays Technology (DArT. One of the crucial steps of the DArT technique is the genome complexity reduction, which allows obtaining a genomic representation characteristic of the studied DNA sample and necessary for subsequent genotyping. In this article, using the mosquito Aedes aegypti as a study model, we describe a new genome complexity reduction method taking advantage of the abundance of miniature inverted repeat transposable elements (MITEs in the genome of this species. Results Ae. aegypti genomic representations were produced following a two-step procedure: (1 restriction digestion of the genomic DNA and simultaneous ligation of a specific adaptor to compatible ends, and (2 amplification of restriction fragments containing a particular MITE element called Pony using two primers, one annealing to the adaptor sequence and one annealing to a conserved sequence motif of the Pony element. Using this protocol, we constructed a library comprising more than 6,000 DArT clones, of which at least 5.70% were highly reliable polymorphic markers for two closely related mosquito strains separated by only a few generations of artificial selection. Within this dataset, linkage disequilibrium was low, and marker redundancy was evaluated at 2.86% only. Most of the detected genetic variability was observed between the two studied mosquito strains, but individuals of the same strain could still be clearly distinguished. Conclusion The new complexity reduction method was particularly efficient to reveal genetic polymorphisms in Ae. egypti. Overall, our results testify of the flexibility of the DArT genotyping technique and open new

  17. The PCR-Based Diagnosis of Central Nervous System Tuberculosis: Up to Date

    Directory of Open Access Journals (Sweden)

    Teruyuki Takahashi

    2012-01-01

    Full Text Available Central nervous system (CNS tuberculosis, particularly tuberculous meningitis (TBM, is the severest form of Mycobacterium tuberculosis (M.Tb infection, causing death or severe neurological defects in more than half of those affected, in spite of recent advancements in available anti-tuberculosis treatment. The definitive diagnosis of CNS tuberculosis depends upon the detection of M.Tb bacilli in the cerebrospinal fluid (CSF. At present, the diagnosis of CNS tuberculosis remains a complex issue because the most widely used conventional “gold standard” based on bacteriological detection methods, such as direct smear and culture identification, cannot rapidly detect M.Tb in CSF specimens with sufficient sensitivity in the acute phase of TBM. Recently, instead of the conventional “gold standard”, the various molecular-based methods including nucleic acid amplification (NAA assay technique, particularly polymerase chain reaction (PCR assay, has emerged as a promising new method for the diagnosis of CNS tuberculosis because of its rapidity, sensitivity and specificity. In addition, the innovation of nested PCR assay technique is worthy of note given its contribution to improve the diagnosis of CNS tuberculosis. In this review, an overview of recent progress of the NAA methods, mainly highlighting the PCR assay technique, was presented.

  18. Engineered XcmI cassette-containing vector for PCR-based ...

    Indian Academy of Sciences (India)

    Unknown

    A simple and general method is described to construct a new vector bearing a synthetic XcmI cassette for direct cloning of PCR-amplified genes of interest. Cleavage of the vector with XcmI generates a linearized molecule with a single thymidine (T) overhang at the 3′ ends (T-vector) that facilitates TA cloning of PCR ...

  19. PCR-based karyotyping of Anopheles gambiae inversion 2Rj identifies the BAMAKO chromosomal form

    Directory of Open Access Journals (Sweden)

    Conway David J

    2007-10-01

    Full Text Available Abstract Background The malaria vector Anopheles gambiae is polymorphic for chromosomal inversions on the right arm of chromosome 2 that segregate nonrandomly between assortatively mating populations in West Africa. One such inversion, 2Rj, is associated with the BAMAKO chromosomal form endemic to southern Mali and northern Guinea Conakry near the Niger River. Although it exploits a unique ecology and both molecular and chromosomal data suggest reduced gene flow between BAMAKO and other A. gambiae populations, no molecular markers exist to identify this form. Methods To facilitate study of the BAMAKO form, a PCR assay for molecular karyotyping of 2Rj was developed based on sequences at the breakpoint junctions. The assay was extensively validated using more than 700 field specimens whose karyotypes were determined in parallel by cytogenetic and molecular methods. As inversion 2Rj also occurs in SAVANNA populations outside the geographic range of BAMAKO, samples were tested from Senegal, Cameroon and western Guinea Conakry as well as from Mali. Results In southern Mali, where 2Rj polymorphism in SAVANNA populations was very low and most of the 2Rj homozygotes were found in BAMAKO karyotypes, the molecular and cytogenetic methods were almost perfectly congruent. Elsewhere agreement between the methods was much poorer, as the molecular assay frequently misclassified 2Rj heterozygotes as 2R+j standard homozygotes. Conclusion Molecular karyotyping of 2Rj is robust and accurate on 2R+j standard and 2Rj inverted homozygotes. Therefore, the proposed approach overcomes the lack of a rapid tool for identifying the BAMAKO form across developmental stages and sexes, and opens new perspectives for the study of BAMAKO ecology and behaviour. On the other hand, the method should not be applied for molecular karyotyping of j-carriers within the SAVANNA chromosomal form.

  20. Evaluation of phenotypic and genotypic methods for epidemiological typing of Staphylococcus aureus isolates from bovine mastitis in Denmark

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Wegener, H. C.; Rosdahl, V. T.

    1995-01-01

    The value of five different typing methods (antibiogram typing, biotyping, phage typing, plasmid profiling and restriction fragment length polymorphism of the gene encoding 16S and 23S ribosomal RNA (ribotyping)), in discriminating 105 Staphylococcus aureus strains from bovine milk samples obtained....... The combination of phage, bio- or ribotyping or all three methods in combination are considered to be an efficient combination of typing methods for epidemiological investigation of S. aureus mastitis....

  1. Determination of antimicrobial resistance of Enterococcus strains isolated from pigs and their genotypic characterization by method of amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting).

    Science.gov (United States)

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Trościańczyk, Aleksandra; Zięba, Przemysław; Gnat, Sebastian

    2017-03-01

    In this study, we analysed phenotypic resistance profiles and their reflection in the genomic profiles of Enterococcus spp. strains isolated from pigs raised on different farms. Samples were collected from five pig farms (n=90 animals) and tested for Enterococcus. MICs of 12 antimicrobials were determined using the broth microdilution method, and epidemiological molecular analysis of strains belonging to selected species (faecalis, faecium and hirae) was performed using the ADSRRS-fingerprinting (amplification of DNA fragments surrounding rare restriction sites) method with a few modifications. The highest percentage of strains was resistant to tetracycline (73.4 %), erythromycin and tylosin (42.5 %) and rifampin (25.2 %), and a large number of strains exhibited high-level resistance to both kanamycin (25.2 %) and streptomycin (27.6 %). The strains of E. faecalis, E. faecium and E. hirae (n=184) revealed varied phenotypic resistance profiles, among which as many as seven met the criteria for multidrug resistance (30.4 % of strains tested). ADSRRS-fingerprinting analysis produced 17 genotypic profiles of individual strains which were correlated with their phenotypic resistance profiles. Only E. hirae strains susceptible to all of the chemotherapeutics tested had two different ADSRRS profiles. Moreover, eight animals were carriers of more than one genotype belonging to the same Enterococcus spp., mainly E. faecalis. Given the possibility of transmission to humans of the high-resistance/multidrug resistance enterococci and the significant role of pigs as food animals in this process, it is necessary to introduce a multilevel control strategy by carrying out research on the resistance and molecular characteristics of indicator bacterial strains isolated from animals on individual farms.

  2. A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Baggesen, Dorte Lau; Porting, P.H.

    1999-01-01

    The purpose of the present study was to investigate the application of ready-to-go Salmonella PCR tests, based on dry chemistry, for final identification of rough presumptive Salmonella isolates. The results were compared with two different biotyping methods performed at two different laboratories......, which did not result in any DNA band. A total of 32 out of the 36 rough presumptive isolates were positive in the PCR. All but one isolate were also identified as Salmonella by the two biochemical methods. All 80 Salmonella strains were also tested in the two multiplex serogroup tests based on PCR beads....... The sensitivity of the BAX Salmonella PCR test was assessed by testing a total of 80 Salmonella isolates, covering most serogroups, which correctly identified all the Salmonella strains by resulting in one 800-bp band in the sample tubes. The specificity of the PCR was assessed using 20 non-Salmonella strains...

  3. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

    Science.gov (United States)

    Gryson, Nicolas

    2010-03-01

    The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

  4. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    Science.gov (United States)

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  5. A PCR-based epidemiological survey of Hepatozoon canis in dogs in Nigeria.

    Science.gov (United States)

    Sasaki, Mizuki; Omobowale, Olutayo; Ohta, Kaisaku; Tozuka, Morito; Matsuu, Aya; Hirata, Haruyuki; Nottidge, Helen Oyebukola; Ikadai, Hiromi; Oyamada, Takashi

    2008-07-01

    The prevalence of Hepatozoon canis infections in dogs in Nigeria was surveyed using molecular methods. DNA was extracted from blood samples obtained from 400 dogs. A primer set that amplified the Babesia canis 18S rRNA gene, which has high similarity to the H. canis 18S rRNA gene, was used for the PCR. As a result, samples from 81 dogs (20.3%) produced 757 bp bands, which differed from the 698 bp band that corresponded to B. canis infection. The sequence of the PCR products of 10 samples were determined, all of which corresponded with the H. canis sequence.

  6. A simple RT-PCR-based strategy for screening connexin identity

    Directory of Open Access Journals (Sweden)

    M. Urban

    1999-08-01

    Full Text Available Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

  7. Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach

    DEFF Research Database (Denmark)

    Nielsen, Morten Thrane; Nielsen, Jakob Blæsbjerg; Anyaogu, Dianna Chinyere

    2013-01-01

    was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to ransformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were...... of solid methodology for genetic manipulation of most species severely hampers pathway haracterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus...... successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor...

  8. Molecular analysis of commercial date palm cultivars in Lybia using ISSR and SRAP PCR-based markers

    Directory of Open Access Journals (Sweden)

    Khalifa Noha S.

    2016-01-01

    Full Text Available Little is known about the molecular structure of the date palm (Phoenix dactylifera L. despite its importance as invaluable drought tolerant crop. Intervarietal variation and cultivar identification are crucial for breeding and gene bank conservation of this plant worldwide. In this work, two PCR based marker systems (ISSR and SRAP were applied on top quality eight commercial cultivars in Libya (Umfetity, Bekrary, Alhamraya, Sufeer Genab, Alsaeedy Show, Farag Barameel, Majhool Alheelo and Alkhadraya. DNA variations were explored using eleven ISSR and nine combinations of SRAP markers. All markers used generated polymorphic bands among the different cultivars that can be used as molecular markers for their differentiation. The genetic distance between cultivars was also estimated from banding patterns. Our results indicate that ISSR and SRAP systems can efficiently identify and differentiate between the selected cultivars. This work can be used as a model to establish a road map for all date palm cultivars worldwide.

  9. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely...... on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S...... targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European...

  10. Comparison of different methods for imputing genome-wide marker genotypes in Swedish and Finnish Red Cattle

    DEFF Research Database (Denmark)

    Ma, Peipei; Brøndum, Rasmus Froberg; Qin, Zahng

    2013-01-01

    This study investigated the imputation accuracy of different methods, considering both the minor allele frequency and relatedness between individuals in the reference and test data sets. Two data sets from the combined population of Swedish and Finnish Red Cattle were used to test the influence...... coefficient was lower when the minor allele frequency was lower. The results indicate that Beagle and IMPUTE2 provide the most robust and accurate imputation accuracies, but considering computing time and memory usage, FImpute is another alternative method....

  11. PCR Based Detection of Genetically Modified Soy in Processed Foods Commercially Available in Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Ibrahim Abdullah Alaraidh

    2009-06-01

    Full Text Available In this research, PCR (polymerase chain reaction technique was applied to detect the presence of GMO sold in the Saudi Arabian market. This method was applied to detect genetically modified soy (GM-soy in particular the roundup ready soy (RRS. To confirm the presence of soy, samples were first tested for the existence of the soy specific lectin gene.  A total of eighty samples were tested out of which two samples tested positive as GM-soy. Not surprisingly, the findings showed the existence of GM-soy in food products in Saudi. This supports the necessity of developing precise quantitative and qualitative ways for routine analyses and detection of GMO products in the Saudi Arabian market. With the discovery of GM products in the Saudi Arabian market it would be of no surprise that other Middle Eastern nations also knowingly or unknowingly import GM crops.

  12. PCR-based identification of selected pathogens associated with endodontic infections in deciduous and permanent teeth.

    Science.gov (United States)

    Cogulu, Dilsah; Uzel, Atac; Oncag, Ozant; Eronat, Cemal

    2008-09-01

    The aim of the present study was to evaluate the presence of the selected pathogens in samples from deciduous and permanent tooth root canals by using PCR method and to determine the association of these organisms with clinical symptoms. A total of 145 children, 5 to 13 years old, were involved in this study. The presence of selected pathogens (Actinomyces israelii, Candida albicans, Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus intermedius, Treponema denticola, Parvimonas micra, Tannerella forsythensis, Enterococcus faecium, Prevotella melaninogenica) in infected root canals was studied using PCR. T. denticola (P = .012, .02) and E. faecalis (P = .012, .04) were highly associated with periapical radiolucency and previous pain, while P. gingivalis was associated with tenderness to percussion in both deciduous and permanent teeth (P = .01, .015). The results of the present study confirm that certain species of microorganisms are associated with clinical signs and symptoms of endodontic disease in both deciduous and permanent teeth.

  13. Review: Diagnostic accuracy of PCR-based detection tests for Helicobacter Pylori in stool samples.

    Science.gov (United States)

    Khadangi, Fatemeh; Yassi, Maryam; Kerachian, Mohammad Amin

    2017-12-01

    Although different methods have been established to detect Helicobacter pylori (H. pylori) infection, identifying infected patients is an ongoing challenge. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for stool PCR test in the diagnosis of H. pylori infection. In this study, a systematic review and meta-analysis were carried out on various sources, including MEDLINE, Web of Sciences, and the Cochrane Library from April 1, 1999, to May 1, 2016. This meta-analysis adheres to the guidelines provided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses report (PRISMA Statement). The clinical value of DNA stool PCR test was based on the pooled false positive, false negative, true positive, and true negative of different genes. Twenty-six of 328 studies identified met the eligibility criteria. Stool PCR test had a performance of 71% (95% CI: 68-73) sensitivity, 96% (95% CI: 94-97) specificity, and 65.6 (95% CI: 30.2-142.5) diagnostic odds ratio (DOR) in diagnosis of H. pylori. The DOR of genes which showed the highest performance of stool PCR tests was as follows: 23S rRNA 152.5 (95% CI: 55.5-418.9), 16S rRNA 67.9 (95%CI: 6.4-714.3), and glmM 68.1 (95%CI: 20.1-231.7). The sensitivity and specificity of stool PCR test are relatively in the same spectrum of other diagnostic methods for the detection of H. pylori infection. In descending order of significance, the most diagnostic candidate genes using PCR detection were 23S rRNA, 16S rRNA, and glmM. PCR for 23S rRNA gene which has the highest performance could be applicable to detect H. pylori infection. © 2017 John Wiley & Sons Ltd.

  14. Evaluation of the Abbott Real Time HCV genotype II assay for Hepatitis C virus genotyping.

    Science.gov (United States)

    Sariguzel, Fatma Mutlu; Berk, Elife; Gokahmetoglu, Selma; Ercal, Baris Derya; Celik, Ilhami

    2015-01-01

    The determination of HCV genotypes and subtypes is very important for the selection of antiviral therapy and epidemiological studies. The aim of this study was to evaluate the performance of Abbott Real Time HCV Genotype II assay in HCV genotyping of HCV infected patients in Kayseri, Turkey. One hundred patients with chronic hepatitis C admitted to our hospital were evaluated between June 2012 and December 2012, HCV RNA levels were determined by the COBAS® AmpliPrep/COBAS® TaqMan® 48 HCV test. HCV genotyping was investigated by the Abbott Real Time HCV Genotype II assay. With the exception of genotype 1, subtypes of HCV genotypes could not be determined by Abbott assay. Sequencing analysis was used as the reference method. Genotypes 1, 2, 3 and 4 were observed in 70, 4, 2 and 24 of the 100 patients, respectively, by two methods. The concordance between the two systems to determine HCV major genotypes was 100%. Of 70 patients with genotype 1, 66 showed infection with subtype 1b and 4 with subtype 1a by Abbott Real Time HCV Genotype II assay. Using sequence analysis, 61 showed infection with subtype 1b and 9 with subtype 1a. In determining of HCV genotype 1 subtypes, the difference between the two methods was not statistically significant (P>0.05). HCV genotype 4 and 3 samples were found to be subtype 4d and 3a, respectively, by sequence analysis. There were four patients with genotype 2. Sequence analysis revealed that two of these patients had type 2a and the other two had type 2b. The Abbott Real Time HCV Genotype II assay yielded results consistent with sequence analysis. However, further optimization of the Abbott Real Time HCV Genotype II assay for subtype identification of HCV is required.

  15. Determination of Molecular Genotyping of Ureaplasma SPP in Women with Genital Infections by 16S–23S rDNA PCR-RFLP Method

    Directory of Open Access Journals (Sweden)

    R. Mirnejad

    2011-04-01

    Full Text Available Introduction & Objective: So far, despite the wide range of methods such as analytic methods used for differentiation of Mycoplasma, the diagnosis of Mycoplasma species is still difficult. Generally the low-level discriminatory power of serological methods because of the rapid changes in size and phase of the dominant antigens in the immune cell surface of Mycoplasmas greatly limits their applicability to the typing of Mycoplasmas. On the contrary,molecular methods do not suffer from these drawbacks and can be used for typing of Mycoplasmas. The aim of this investigation was molecular identification and genotyping of ureaplasma SPP in women with genital infections by 16S–23S rDNA PCR-RFLP.Materials & Methods: Genital swabs were taken from 210 patients who referred to gynecology clinic of Rasool hospital in Tehran, Iran during December 2007 until June 2008. The swabs suspended in PBS, were immediately transferred to laboratory .Following DNA extraction, PCR assay was performed using a genus specific primer pair. These primer sets amplified a 559 bp fragment for Ureaplasma Spp. Samples containing bands of the expected size for Ureaplasma strains were subjected to digestion with different restriction endonuclease enzymes (AluI, Taq I, CacI8, BbsI, EcoRI. Results: Of the 210 samples, Ureaplasma Spp was isolated from 93 patients (44.3% by PCR and 69 samples by culture. In the present study only Biovar 1 (Ureaplasma parvum was isolated from clinical specimens and the results were confirmed using a cutting enzyme TaqI (enzyme specific species of ureaplasma SPP. The results of this analysis using PCR-RFLP and sequencing showed that all had the same genotype and shared identical sequence with the genome sequence of serovar 3 Ureaplasma parvum.Conclusion: Ureaplasma parvum is generally isolated from the genital samples. In this study all isolates were identical and no difference was found among the enzyme patterns of the bacteria after PCR-RFLP .So

  16. PCR-based approach to SINE isolation: simple and complex SINEs.

    Science.gov (United States)

    Borodulina, Olga R; Kramerov, Dmitri A

    2005-04-11

    Highly repeated copies of short interspersed elements (SINEs) occur in eukaryotic genomes. The distribution of each SINE family is usually restricted to some genera, families, or orders. SINEs have an RNA polymerase III internal promoter, which is composed of boxes A and B. Here we propose a method for isolation of novel SINE families based on genomic DNA PCR with oligonucleotide identical to box A as a primer. Cloning of the size-heterogeneous PCR-products and sequencing of their terminal regions allow determination of SINE structure. Using this approach, two novel SINE families, Rhin-1 and Das-1, from the genomes of great horseshoe bat (Rhinolophus ferrumequinum) and nine-banded armadillo (Dasypus novemcinctus), respectively, were isolated and studied. The distribution of Rhin-1 is restricted to two of six bat families tested. Copies of this SINE are characterized by frequent internal insertions and significant length (200-270 bp). Das-1 being only 90 bp in length is one of the shortest SINEs known. Most of Das-1 nucleotide sequences demonstrate significant similarity to alanine tRNA which appears to be an evolutionary progenitor of this SINE. Together with three other known SINEs (ID, Vic-1, and CYN), Das-1 constitutes a group of simple SINEs. Interestingly, three SINE families of this group are alanine tRNA-derived. Most probably, this tRNA gave rise to short and simple but successful SINEs several times during mammalian evolution.

  17. Identification and epidemiological typing of Campylobacter hyointestinalis subspecies by phenotypic and genotypic methods and description of novel subgroups

    DEFF Research Database (Denmark)

    On, Stephan L.W.; Vandamme, P.

    1997-01-01

    of this taxon. Two novel, distinct groups of C. hyointestinalis-like bacteria, originally isolated from the cloacae of Canada geese and human diarrhoeic stools, were also identified by each of the methods used. This appears to be the first report confirming the presence of C. hyointestinalis-like strains from...

  18. Detection of MPL mutations by a novel allele-specific PCR-based strategy.

    Science.gov (United States)

    Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2013-11-01

    MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  19. A comparison of genotyping-by-sequencing analysis methods on low-coverage crop datasets shows advantages of a new workflow, GB-eaSy.

    Science.gov (United States)

    Wickland, Daniel P; Battu, Gopal; Hudson, Karen A; Diers, Brian W; Hudson, Matthew E

    2017-12-28

    Genotyping-by-sequencing (GBS), a method to identify genetic variants and quickly genotype samples, reduces genome complexity by using restriction enzymes to divide the genome into fragments whose ends are sequenced on short-read sequencing platforms. While cost-effective, this method produces extensive missing data and requires complex bioinformatics analysis. GBS is most commonly used on crop plant genomes, and because crop plants have highly variable ploidy and repeat content, the performance of GBS analysis software can vary by target organism. Here we focus our analysis on soybean, a polyploid crop with a highly duplicated genome, relatively little public GBS data and few dedicated tools. We compared the performance of five GBS pipelines using low-coverage Illumina sequence data from three soybean populations. To address issues identified with existing methods, we developed GB-eaSy, a GBS bioinformatics workflow that incorporates widely used genomics tools, parallelization and automation to increase the accuracy and accessibility of GBS data analysis. Compared to other GBS pipelines, GB-eaSy rapidly and accurately identified the greatest number of SNPs, with SNP calls closely concordant with whole-genome sequencing of selected lines. Across all five GBS analysis platforms, SNP calls showed unexpectedly low convergence but generally high accuracy, indicating that the workflows arrived at largely complementary sets of valid SNP calls on the low-coverage data analyzed. We show that GB-eaSy is approximately as good as, or better than, other leading software solutions in the accuracy, yield and missing data fraction of variant calling, as tested on low-coverage genomic data from soybean. It also performs well relative to other solutions in terms of the run time and disk space required. In addition, GB-eaSy is built from existing open-source, modular software packages that are regularly updated and commonly used, making it straightforward to install and maintain

  20. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    International Nuclear Information System (INIS)

    Jackson, Christopher B.; Gallati, Sabina; Schaller, André

    2012-01-01

    Highlights: ► Serial qPCR accurately determines fragmentation state of any given DNA sample. ► Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. ► Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. ► Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze–thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λ nDNA ) and mtDNA (λ mtDNA ) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two

  1. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

  2. In-cell PCR method for specific genotyping of genomic DNA from one individual in a mixture of cells from two individuals: a model study with specific relevance to prenatal diagnosis based on fetal cells in maternal blood

    DEFF Research Database (Denmark)

    Hviid, T Vauvert

    2002-01-01

    only in the male cells, leading to the correct HLA-DPB1 genotyping of the male by DNA sequencing of a nested, linked TSPY-HLA-DPB1 PCR product. CONCLUSION: This approach might be usable on mixed cell populations of fetal and maternal cells obtained after conventional cell-sorting techniques on maternal...... maternal blood samples, the use of such an approach for genotyping by molecular biology techniques in a more routine setting has been hampered by the large contamination of maternal nucleated blood cells in the cell isolates. Therefore, a new method based on in-cell PCR is described, which may overcome...... this problem. Methods and Results: Mixtures of cells from two different individuals were fixed and permeabilized in suspension. After coamplification of a DNA sequence specific for one of the individuals and the DNA sequence to be genotyped, the two PCR products were linked together in the fixed cells positive...

  3. Comparison of the optimized conditions for genotyping of ACE ID ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... variants (Koyama et al., 2008). This method removed the chances of mistyping and gave 100% accuracy in the confirmation of ID heterozygotes but expenses of chromatography did not prove it to be cost effective. Normally, genomic DNA is used as template DNA for. PCR based studies. It is very difficult to ...

  4. Molecular typing of methicillin-resistant Staphylococcus aureus: Comparison of PCR-based open reading frame typing, multilocus sequence typing, and Staphylococcus protein A gene typing.

    Science.gov (United States)

    Ogihara, Shinji; Saito, Ryoichi; Sawabe, Etsuko; Kozakai, Takahiro; Shima, Mari; Aiso, Yoshibumi; Fujie, Toshihide; Nukui, Yoko; Koike, Ryuji; Hagihara, Michio; Tohda, Shuji

    2018-04-01

    The recently developed PCR-based open reading frame typing (POT) method is a useful molecular typing tool. Here, we evaluated the performance of POT for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) isolates and compared its performance to those of multilocus sequence typing (MLST) and Staphylococcus protein A gene typing (spa typing). Thirty-seven MRSA isolates were collected between July 2012 and May 2015. MLST, spa typing, and POT were performed, and their discriminatory powers were evaluated using Simpson's index analysis. The MRSA isolates were classified into 11, 18, and 33 types by MLST, spa typing, and POT, respectively. The predominant strains identified by MLST, spa typing, and POT were ST8 and ST764, t002, and 93-191-127, respectively. The discriminatory power of MLST, spa typing, and POT was 0.853, 0.875, and 0.992, respectively, indicating that POT had the highest discriminatory power. Moreover, the results of MLST and spa were available after 2 days, whereas that of POT was available in 5 h. Furthermore, POT is rapid and easy to perform and interpret. Therefore, POT is a superior molecular typing tool for monitoring nosocomial transmission of MRSA. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  5. Comparative performance of PCR-based assay versus microscopy and culture for the direct detection of Mycobacterium tuberculosis in clinical respiratory specimens in Lebanon.

    Science.gov (United States)

    Araj, G F; Talhouk, R S; Itani, L Y; Jaber, W; Jamaleddine, G W

    2000-09-01

    American University of Beirut Medical Center, Lebanon. To assess the performance of a polymerase chain reaction (PCR) using primers that flank 542 bp within IS6110 in Mycobacterium tuberculosis (TB) vs. microscopy and BACTEC culture, in the diagnosis of tuberculosis. A total of 82 clinical respiratory pulmonary specimens and 73 samples from BACTEC vials were tested by the three methods. Of 24 smear-positive culture-positive (SP-CP) and 11 smear-negative culture-positive (SN-CP) TB specimens, PCR detected 83% and 64%, respectively. Among 17 specimens yielding mycobacteria other than tuberculosis (MOTT), the PCR was positive in 33% SP-CP and 14% SN-CP specimens. Among the 73 BACTEC vials, PCR was positive in 36 of 38 (95%) yielding culture-positive TB, and in one of 20 (5%) yielding culture positive MOTT. None of the 30 smear-negative culture-negative (SN-CN) clinical specimens and 15 of the CN vials were positive by PCR. The overall sensitivity of PCR was 77% and 95% for TB detection in respiratory specimens and BACTEC vials, respectively, and the specificity was 94% in both. Because a substantial number of TB cases are missed, especially in SN-CP specimens, a PCR-based assay utilizing these primers cannot be used reliably, alone, in clinical laboratory diagnosis of mycobacterial respiratory infections.

  6. Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.

    Science.gov (United States)

    Woo, Nain; Kim, Su-Kang; Sun, Yucheng; Kang, Seong Ho

    2018-01-01

    Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. A Fast Method for DEFB1-44C/G SNP Genotyping in Brazilian Patients with Periodontitis

    Directory of Open Access Journals (Sweden)

    Rafael Rafael Amorim Cavalcanti de Siqueira

    2014-01-01

    Full Text Available Aim: Defensins are cationic antimicrobial peptides expressed in epithelial cells. Such peptides exhibit antibacterial, antifungal and antiviral properties, and are a component of the innate immune response. It has been suggested that they have a protective role in the oral cavity. This study evaluated the DEFB1 polymorphism in diabetic patients with or without periodontitis in comparison to healthy controls. Material and Methods: We used Hairpin-Shaped Primer (HP assay to study the distribution of the -44 C/G SNP (rs1800972 in 119 human DNAs obtained from diabetic patients and healthy control patients. Results: The results indicate that there are no differences in distribution between groups and that in diabetic periodontitis patients the homozygous mutant could be found more frequently. Conclusion: Further studies are necessary in order to investigate the role of DEFB1 polymorphisms in diabetic periodontitis patients and the influence of the peptide in periodontal pathogens.

  8. Genotyping of friesian horses to detect a hydrocephalus-associated c.1423C>T mutation in B3GALNT2 using PCR-RFLP and PCR-PIRA methods: Frequency in stallion horses in México.

    Science.gov (United States)

    Ayala-Valdovinos, Miguel Angel; Galindo-García, Jorge; Sánchez-Chiprés, David; Duifhuis-Rivera, Theodor

    2017-04-01

    Hydrocephalus in Friesian horses is an autosomal recessive hereditary disease that can result in an abortion, a stillbirth, or euthanization of a newborn foal. Here, the hydrocephalus-associated c.1423C > T mutation in B3GALNT2 gene was detected with PCR-RFLP and PCR-PIRA methods for horse genotyping. A preliminary genotyping survey was performed on 83 randomly selected Friesian stallion horses to determine the current allele frequency in Mexico. The frequency of the mutant T allele was 9.6%. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods.

    Directory of Open Access Journals (Sweden)

    Agnieszka E Laudy

    Full Text Available Knowledge of the prevalence of ESBL enzymes among P. aeruginosa strains compared to the Enterobacteraiceae family is limited. The phenotypic tests recommended by EUCAST for the detection of ESBL-producing Enterobacteriaceae are not always suited for P. aeruginosa strains. This is mainly due to the presence of other families of ESBLs in P. aeruginosa isolates more often than in Enterobacteriaceae, production of natural AmpC cephalosporinase and its overexpression, and co-production of metallo-β-lactamases. The aim of this study was to determine the occurrence of ESBLs in P. aeruginosa isolated from patients from hospitals in Warsaw, to evaluate the ESBL production of these isolates using currently available phenotypic tests, their modifications, multiplex PCR and molecular typing of ESBL-positive isolates by PFGE. Clinical isolates of P. aeruginosa were collected in 2000-2014 from four Warsaw hospitals. Based on the data obtained in this study, we suggest using three DDST methods with inhibitors, such as clavulanic acid, sulbactam and imipenem, to detect ESBL-producing P. aeruginosa strains. Depending on the appearance of the plates, we suggest a reduction in the distance between discs with antibiotics to 15 mm and the addition of boronic acid at 0.4 mg per disc. The analysed isolates carried genes encoding ESBL from the families VEB (69 isolates with VEB-9, GES (6 with GES-1, 1 GES-5, 5 GES-13 and 2 with GES-15, OXA-2 (12 with OXA-15, 1 OXA-141, 1 OXA-210, 1 OXA-543 and 1 with OXA-544 and OXA-10 (5 isolates with OXA-74 and one with OXA-142. The most important result of this study was the discovery of three new genes, blaGES-15, blaOXA-141 and blaOXA-142; their nucleotide sequences have been submitted to the NCBI GenBank. It is also very important to note that this is the first report on the epidemiological problem of VEB-9-producing bacterial strains, not only in Poland but also worldwide.

  10. Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab.

    Science.gov (United States)

    Wadowsky, R M; Laus, S; Libert, T; States, S J; Ehrlich, G D

    1994-04-01

    A PCR-based assay for Bordetella pertussis was inhibited by using a calcium alginate fiber-tipped swab with an aluminum shaft but not by using a Dacron fiber-tipped swab with a plastic shaft. The calcium alginate fiber component inhibited the assay following storage for less than 1 min in a suspension of 10(3) CFU of B. pertussis per ml, whereas the aluminum shaft component required storage for at least 48 h in order to cause inhibition. We recommend the Dacron swab over the calcium alginate swab for collecting specimens for testing in PCR-based assays.

  11. Deep sequencing analysis of HBV genotype shift and correlation with antiviral efficiency during adefovir dipivoxil therapy.

    Directory of Open Access Journals (Sweden)

    Yuwei Wang

    Full Text Available Viral genotype shift in chronic hepatitis B (CHB patients during antiviral therapy has been reported, but the underlying mechanism remains elusive.38 CHB patients treated with ADV for one year were selected for studying genotype shift by both deep sequencing and Sanger sequencing method.Sanger sequencing method found that 7.9% patients showed mixed genotype before ADV therapy. In contrast, all 38 patients showed mixed genotype before ADV treatment by deep sequencing. 95.5% mixed genotype rate was also obtained from additional 200 treatment-naïve CHB patients. Of the 13 patients with genotype shift, the fraction of the minor genotype in 5 patients (38% increased gradually during the course of ADV treatment. Furthermore, responses to ADV and HBeAg seroconversion were associated with the high rate of genotype shift, suggesting drug and immune pressure may be key factors to induce genotype shift. Interestingly, patients with genotype C had a significantly higher rate of genotype shift than genotype B. In genotype shift group, ADV treatment induced a marked enhancement of genotype B ratio accompanied by a reduction of genotype C ratio, suggesting genotype C may be more sensitive to ADV than genotype B. Moreover, patients with dominant genotype C may have a better therapeutic effect. Finally, genotype shifts was correlated with clinical improvement in terms of ALT.Our findings provided a rational explanation for genotype shift among ADV-treated CHB patients. The genotype and genotype shift might be associated with antiviral efficiency.

  12. Identification of strains with phenotypes similar to those of Staphylococcus aureus isolated from table chicken eggs using MALDI-TOF MS and genotyping methods

    Directory of Open Access Journals (Sweden)

    Marek Agnieszka

    2015-06-01

    Full Text Available The aim of the study was to identify the affinity of 10 Staphylococcus strains isolated from table chicken eggs to specific species. Preliminary analysis performed by API ID32 Staph test identified these strains as S. aureus, but they exhibited a negative reaction in the tube coagulase test. Thus, the analysed strains were initially characterised as Staphylococcus aureus-like (SAL. Further characterisation was performed by genotypic methods, using restriction fragment length polymorphism (RFLP of the coagulase gene (coa and sequencing of the gene rpoB. An attempt was also made to identify the isolated Staphylococcus strains by MALDI-TOF mass spectrometry. The results indicated that none of the strains tested belonged to the species S. aureus. The rpoB sequences of five isolates showed the highest sequence similarity to S. haemolyticus, three isolates to S. chromogenes, and one isolate to S. epidermidis. One strain (SAL4 remained unidentified in this analysis. The results obtained using mass spectrometry were comparable to those based on gene sequence analysis. Strain SAL4, which could not be identified by sequencing, was identified by MALDI-TOF as Staphylococcus chromogenes.

  13. Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment.

    Science.gov (United States)

    Wilson, Heather L; Tran, Thomas; Druce, Julian; Dupont-Rouzeyrol, Myrielle; Catton, Michael

    2017-10-01

    The global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand. Copyright © 2017 American Society for Microbiology.

  14. Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.

    Directory of Open Access Journals (Sweden)

    Morten Thrane Nielsen

    Full Text Available Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC encodes a polyketide synthase, ATEG_08453 (gedR encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.

  15. A novel PCR-based marker for identifying Ns chromosomes in wheat-Psathyrostachys huashanica Keng derivative lines

    Directory of Open Access Journals (Sweden)

    J. Wang

    2013-10-01

    Full Text Available Psathyrostachys huashanica Keng is an endangered species that is endemic to China, which provides an important gene pool for wheat improvement. We developed a quick and reliable PCR-based diagnostic assay to accurately and efficiently detect P. huashanica DNA sequences from introgression lines, which was based on a species-specific marker derived from genomic DNA. The 900-bp PCR-amplified band used as a P. huashanica-specific RAPD marker was tested with 21 different plant species and was converted into a sequence-characterized amplified region (SCAR marker by cloning and sequencing the selected fragments (pHs11. This SCAR marker, which was designated as RHS23, could clearly distinguish the presence of P. huashanica DNA repetitive sequences in wheat-P. huashanica derivative lines. The specificity of the marker was validated using 21 different plant species and a complete set of wheat-P. huashanica disomic addition lines (1Ns–7Ns, 2n=44=22II. This specific sequence targeted the Ns genome of P. huashanica and it was present in all the seven P. huashanica chromosomes. Therefore, this SCAR marker is specific for P. huashanica chromosomes and may be used in the identification of alien repetitive sequences in large gene pools. This diagnostic PCR assay for screening the target genetic material may play a key role in marker-assisted selective breeding programs.

  16. Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism.

    Science.gov (United States)

    Huang, Suzhen; Xue, Tingli; Wang, Zhiquan; Ma, Yuanyuan; He, Xueting; Hong, Jiefang; Zou, Shaolan; Song, Hao; Zhang, Minhua

    2018-04-01

    Furfural-tolerant strain is essential for the fermentative production of biofuels or chemicals from lignocellulosic biomass. In this study, Zymomonas mobilis CP4 was for the first time subjected to error-prone PCR-based whole genome shuffling, and the resulting mutants F211 and F27 that could tolerate 3 g/L furfural were obtained. The mutant F211 under various furfural stress conditions could rapidly grow when the furfural concentration reduced to 1 g/L. Meanwhile, the two mutants also showed higher tolerance to high concentration of glucose than the control strain CP4. Genome resequencing revealed that the F211 and F27 had 12 and 13 single-nucleotide polymorphisms. The activity assay demonstrated that the activity of NADH-dependent furfural reductase in mutant F211 and CP4 was all increased under furfural stress, and the activity peaked earlier in mutant than in control. Also, furfural level in the culture of F211 was also more rapidly decreased. These indicate that the increase in furfural tolerance of the mutants may be resulted from the enhanced NADH-dependent furfural reductase activity during early log phase, which could lead to an accelerated furfural detoxification process in mutants. In all, we obtained Z. mobilis mutants with enhanced furfural and high concentration of glucose tolerance, and provided valuable clues for the mechanism of furfural tolerance and strain development.

  17. A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

    Science.gov (United States)

    Lopez-Jimena, B; Cherif, N; Garcia-Rosado, E; Infante, C; Cano, I; Castro, D; Hammami, S; Borrego, J J; Alonso, M C

    2010-10-01

    To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  18. CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping

    Directory of Open Access Journals (Sweden)

    Amanda K Riffel

    2016-01-01

    regions can impact the performance of PCR-based genotype assays, including TaqMan. Regardless of the test platform used, it is prudent to confirm rare allele calls by an independent method.

  19. Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

    Science.gov (United States)

    Ueno, Tomohiro; Niimi, Hideki; Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

    2015-01-01

    Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and

  20. VacA and cagA genotypes status and antimicrobial resistance properties of Helicobacter pylori strains isolated from meat products in Isfahan province, Iran.

    Science.gov (United States)

    Gilani, A; Razavilar, V; Rokni, N; Rahimi, E

    2017-01-01

    Although Helicobacter pylori has a significant impact on the occurrence of severe clinical syndromes, its exact ways of transmission and origin have not been identified. According to the results of some previously published articles, foods with animal origins play a substantial role in the transmission of H. pylori to humans. The present investigation was carried out to study the vacuolating cytotoxin A ( vacA ) and cytotoxin associated gene A ( cagA ) genotypes status and antibiotic resistance properties of H. pylori strains recovered from minced-meat and hamburger samples. A total of 150 meat product samples were collected from supermarkets. All samples were cultured and the susceptive colonies were then subjected to nested-PCR, PCR-based genotyping and disk diffusion methods. 11 out of 150 samples (7.33%) were positive for H. pylori . All the isolates were further identified using the nested-PCR assay. Prevalence of H. pylori in hamburger and minced-meat samples was 1.42% and 12.5%, respectively. S1a , m1a and cagA were the most commonly detected genotypes. The most commonly detected combined genotypes in the H. pylori strains of minced-meat were s1am1a (10%), s1am1b (10%) and s2m1a (10%). Helicobacter pylori strains of meat products harbored the highest levels of resistance against ampicillin (90.90%), erythromycin (72.72%), amoxicillin (72.72%), trimethoprim (63.63%), tetracycline (63.63%), and clarithromycin (63.63%). Hamburger and minced-meat samples may be the sources of virulent and resistant strains of H. pylori . Meat products are possible sources of resistant and virulent strains of H. pylori similar to those vacA and cagA genotypes. Using healthy raw materials and observation of personal hygiene can reduce the risk of H. pylori in meat products.

  1. Behavior of durum wheat genotypes under normal irrigation and ...

    African Journals Online (AJOL)

    Behavior of durum wheat genotypes under normal irrigation and drought stress conditions in the greenhouse. ... African Journal of Biotechnology ... Genotypes were grouped in cluster analysis (using Ward's method) based on Yp, Ys and ...

  2. magnitude of genotype x environment interaction for bacterial leaf

    African Journals Online (AJOL)

    ACSS

    African Crop Science Journal, Vol. ... effects of treatments into genotype, environment, and genotype x environment (G x E) interactions. Results .... method is economically effective (Niño-Liu et al., ..... This phenomenon indicated differences in.

  3. Comparison of Modified Impedance Whole Blood Platelet Aggregation Method Detecting Platelet Function in ACS Patients with Different CYP2C19 Genotypes.

    Science.gov (United States)

    Cui, Chanjuan; Qiao, Rui; Zhang, Jie

    2016-01-01

    A reliable laboratory test to monitor onclopidogrel platelet reactivity (PR) is very necessary. In addition, genetic factors also play an important part in onclopidogrel PR. This study aimed to modify the original impedance whole blood platelet aggregation assay associated with the release assay to monitor onclopidogrel PR and assess their relationship with genotype. We adjusted the concentration of calcium in the in vitro reaction system of platelet aggregation to modify the original impedance whole blood platelet aggregation assay. Meanwhile, chronolume, which quantified the adenosine triphosphate (ATP) released from platelet dense granules, is added to this reaction system to reflect the platelet release function. In the modified assay, platelet magnified activation time (MAT) and the maximal platelet ATP release value (RV) were used to reflect platelet function parameters. In the original assay, the electrical resistance (omega) and RV were used to reflect platelet function parameters. Onclopidogrel PR was detected by the original impedance whole blood platelet aggregation assay, modified assay, and flow cytometric vasodilator stimulated phosphoprotein (VASP) assay in 168 patients with acute coronary syndromes (ACS). CYP2C19*2 and CYP2C19*3 polymorphisms were also detected in all of these patients. This modified method showed that when 12.5 microL CaCl2 (0.2 mmol/L) was added to the reaction system, MAT was appropriate (93 +/- 23 seconds). The CVs for the modified impedance assay and release assay were 9.31% and 6.13%, respectively. The mean VASP-PRI in the patient group treated with clopidogrel was significantly lower than that in the control group without antiplatelet therapy (54.88 +/- 16.81% vs. 79.86 +/- 10.24%, p 50% group were shorter than that in the PRI 50% group were higher than that in the PRI omega) and RV of the original method showed no differences between the two groups [0 (0-2) vs. 0 (0-1.25), 0.05 (0-0.25) vs. 0.08 (0-0.24); p > 0.05, p > 0

  4. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    Directory of Open Access Journals (Sweden)

    Ram, S.

    2012-01-01

    Full Text Available Aim: Isolation, dark field detection and microscopic agglutination test (MAT are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207 blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21% out of 100 culture positive blood samples, three (2.8% out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001. A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001 signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

  5. Molecular characterization of varicella-zoster virus clinical isolates from 2006 to 2008 in a tertiary care hospital, Dublin, Ireland, using different genotyping methods.

    LENUS (Irish Health Repository)

    Roycroft, Emma

    2012-10-01

    Varicella-zoster virus (VZV), a herpesvirus, is a ubiquitous organism that causes considerable morbidity worldwide and can cause severe complications on reactivation. Phylogenetic analysis was performed on 19 clinical VZV isolates (16 zoster and 3 varicella) found in Ireland, between December 2006 and November 2008, in order to determine whether previously reported viral heterogeneity was still present and whether viral recombination was evident. Open reading-frames (ORFs) from genes 1, 21, 50, and 54, were sequenced. Clades 1, 2, 3, and 5 were identified. Four putative recombinant isolates were detected (three clade 3\\/1 and one clade 5\\/3\\/1). Further sequencing and examination of ORF 22 and 21\\/50, did not elucidate the putative recombinant genotypes further. These two previously published genotyping schemes were examined in light of the new consensus genotyping scheme proposed in 2010. Remarkable VZV heterogeneity remains prevalent in Ireland. This is the first evidence of putative VZV recombination found in Ireland.

  6. DNA elution from buccal cells stored on Whatman FTA Classic Cards using a modified methanol fixation method.

    Science.gov (United States)

    Johanson, Helene C; Hyland, Valentine; Wicking, Carol; Sturm, Richard A

    2009-04-01

    We describe here a method for DNA elution from buccal cells and whole blood both collected onto Whatman FTA technology, using methanol fixation followed by an elution PCR program. Extracted DNA is comparable in quality to published Whatman FTA protocols, as judged by PCR-based genotyping. Elution of DNA from the dried sample is a known rate-limiting step in the published Whatman FTA protocol; this method enables the use of each 3-mm punch of sample for several PCR reactions instead of the standard, one PCR reaction per sample punch. This optimized protocol therefore extends the usefulness and cost effectiveness of each buccal swab sample collected, when used for nucleic acid PCR and genotyping.

  7. A comparison of PCR-based markers for the molecular identification of Sphagnum species of the section Acutifolia

    Directory of Open Access Journals (Sweden)

    Jakub Sawicki

    2011-07-01

    Full Text Available RAPDs, ISJs, ISSRs, ITS and katGs were applied to determine genetic relationships between common Sphagnum species of the section Acutifolia. Twenty populations were genotyped using ten ISJ primers, 12 pairs of katG primers, 10 ISSR and 10 RAPD primers, and a restriction analysis of ITS1 and ITS2. ISSR and katG markers revealed the greatest number of species-specific bands. An analysis of ITS1 and ITS2 regions with restriction enzymes also proved to be a highly effective tool for species identification.

  8. Core Gene Expression and Association of Genotypes with Viral ...

    African Journals Online (AJOL)

    Purpose: To determine genotypic distribution, ribonucleic acid (RNA) RNA viral load and express core gene from Hepatitis C Virus (HCV) infected patients in Punjab, Pakistan. Methods: A total of 1690 HCV RNA positive patients were included in the study. HCV genotyping was tested by type-specific genotyping assay, viral ...

  9. Alteração no método centroide de avaliação da adaptabilidade genotípica Alteration of the centroid method to evaluate genotypic adaptability

    Directory of Open Access Journals (Sweden)

    Moysés Nascimento

    2009-03-01

    Full Text Available O objetivo deste trabalho foi alterar o método centroide de avaliação da adaptabilidade e estabilidade fenotípica de genótipos, para deixá-lo com maior sentido biológico e melhorar aspectos quantitativos e qualitativos de sua análise. A alteração se deu pela adição de mais três ideótipos, definidos de acordo com valores médios dos genótipos nos ambientes. Foram utilizados dados provenientes de um experimento sobre produção de matéria seca de 92 genótipos de alfafa (Medicago sativa realizado em blocos ao acaso, com duas repetições. Os genótipos foram submetidos a 20 cortes, no período de novembro de 2004 a junho de 2006. Cada corte foi considerado um ambiente. A inclusão dos ideótipos de maior sentido biológico (valores médios nos ambientes resultou em uma dispersão gráfica em forma de uma seta voltada para a direita, na qual os genótipos mais produtivos ficaram próximos à ponta da seta. Com a alteração, apenas cinco genótipos foram classificados nas mesmas classes do método centroide original. A figura em forma de seta proporciona uma comparação direta dos genótipos, por meio da formação de um gradiente de produtividade. A alteração no método mantém a facilidade de interpretação dos resultados para a recomendação dos genótipos presente no método original e não permite duplicidade de interpretação dos resultados.ABSTRACT The objective of this work was to modify the centroid method of evaluation of phenotypic adaptability and the phenotype stability of genotypes in order for the method to make greater biological sense and improve its quantitative and qualitative performance. The method was modified by means of the inclusion of three additional ideotypes defined in accordance with the genotypes' average yield in the environments tested. The alfalfa (Medicago sativa L. forage yield of 92 genotypes was used. The trial had a randomized block design, with two replicates, and the data were used to

  10. Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal.

    Science.gov (United States)

    Lobo, M L; Xiao, L; Antunes, F; Matos, O

    2009-06-01

    Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal. The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46.3%) were positive for Cryptosporidium and 67 (38.3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21.7%) and 9 (5.1%) water samples, respectively. C. parvum was the most common species (78.9%), followed by C. hominis (13.2%), C. andersoni (5.3%), and C. muris (2.6%). Subtype IdA15 was identified in all C. hominis-positive water samples. Subtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified. The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples. Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.

  11. Genomic evaluations with many more genotypes

    Directory of Open Access Journals (Sweden)

    Wiggans George R

    2011-03-01

    Full Text Available Abstract Background Genomic evaluations in Holstein dairy cattle have quickly become more reliable over the last two years in many countries as more animals have been genotyped for 50,000 markers. Evaluations can also include animals genotyped with more or fewer markers using new tools such as the 777,000 or 2,900 marker chips recently introduced for cattle. Gains from more markers can be predicted using simulation, whereas strategies to use fewer markers have been compared using subsets of actual genotypes. The overall cost of selection is reduced by genotyping most animals at less than the highest density and imputing their missing genotypes using haplotypes. Algorithms to combine different densities need to be efficient because numbers of genotyped animals and markers may continue to grow quickly. Methods Genotypes for 500,000 markers were simulated for the 33,414 Holsteins that had 50,000 marker genotypes in the North American database. Another 86,465 non-genotyped ancestors were included in the pedigree file, and linkage disequilibrium was generated directly in the base population. Mixed density datasets were created by keeping 50,000 (every tenth of the markers for most animals. Missing genotypes were imputed using a combination of population haplotyping and pedigree haplotyping. Reliabilities of genomic evaluations using linear and nonlinear methods were compared. Results Differing marker sets for a large population were combined with just a few hours of computation. About 95% of paternal alleles were determined correctly, and > 95% of missing genotypes were called correctly. Reliability of breeding values was already high (84.4% with 50,000 simulated markers. The gain in reliability from increasing the number of markers to 500,000 was only 1.6%, but more than half of that gain resulted from genotyping just 1,406 young bulls at higher density. Linear genomic evaluations had reliabilities 1.5% lower than the nonlinear evaluations with 50

  12. APOE Genotyping, Cardiovascular Disease

    Science.gov (United States)

    ... Resources For Health Professionals Subscribe Search APOE Genotyping, Cardiovascular Disease Send Us Your Feedback Choose Topic At a ... help understand the role of genetic factors in cardiovascular disease . However, the testing is sometimes used in clinical ...

  13. Radiosensitivity of fingermillet genotypes

    Energy Technology Data Exchange (ETDEWEB)

    Raveendran, T S; Nagarajan, C; Appadurai, R; Prasad, M N; Sundaresan, N [Tamil Nadu Agricultural Univ., Coimbatore (India)

    1984-07-01

    Varietal differences in radiosensitivity were observed in a study involving 4 genotypes of fingermillet (Eleusine coracana (Linn.) Gaertn.) subjected to gamma-irradiation. Harder seeds were found to tolerate a higher dose of the mutagen.

  14. A high-throughput multiplex method adapted for GMO detection.

    Science.gov (United States)

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  15. Correlation between Group B Streptococcal Genotypes, Their Antimicrobial Resistance Profiles, and Virulence Genes among Pregnant Women in Lebanon

    Directory of Open Access Journals (Sweden)

    Antoine Hannoun

    2009-01-01

    Full Text Available The antimicrobial susceptibility profiles of 76 Streptococcus agalactiae (Group B Streptococci [GBS] isolates from vaginal specimens of pregnant women near term were correlated to their genotypes generated by Random Amplified Polymorphic DNA analysis and their virulence factors encoding genes cylE, lmb, scpB, rib, and bca by PCR. Based on the distribution of the susceptibility patterns, six profiles were generated. RAPD analysis detected 7 clusters of genotypes. The cylE gene was present in 99% of the isolates, the lmb in 96%, scpB in 94.7%, rib in 33%, and bca in 56.5% of isolates. The isolates demonstrated a significant correlation between antimicrobial resistance and genotype clusters denoting the distribution of particular clones with different antimicrobial resistance profiles, entailing the practice of caution in therapeutic options. All virulence factors encoding genes were detected in all seven genotypic clusters with rib and bca not coexisting in the same genome.

  16. Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab.

    OpenAIRE

    Wadowsky, R M; Laus, S; Libert, T; States, S J; Ehrlich, G D

    1994-01-01

    A PCR-based assay for Bordetella pertussis was inhibited by using a calcium alginate fiber-tipped swab with an aluminum shaft but not by using a Dacron fiber-tipped swab with a plastic shaft. The calcium alginate fiber component inhibited the assay following storage for less than 1 min in a suspension of 10(3) CFU of B. pertussis per ml, whereas the aluminum shaft component required storage for at least 48 h in order to cause inhibition. We recommend the Dacron swab over the calcium alginate ...

  17. Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

    DEFF Research Database (Denmark)

    Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno

    2016-01-01

    , was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE...

  18. Analysis of performance of a PCR-based assay to detect DNA of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical samples.

    Science.gov (United States)

    Bernal-Martínez, Leticia; Gago, Sara; Buitrago, María J; Gomez-Lopez, Alicia; Rodríguez-Tudela, Juan L; Cuenca-Estrella, Manuel

    2011-10-01

    The performance of a real-time PCR-based assay was retrospectively analyzed (according to European Organization for Research and Treatment of Cancer/Mycosis Study Group criteria) in the samples of patients with invasive aspergillosis. A total of 711 serial samples (356 whole-blood and 355 serum samples) from 38 adult patients were analyzed. The Aspergillus fumigatus PCR assay results were positive for 89 of 356 (25%) whole-blood samples and 90 of 355 (25.35%) serum samples. Positive PCR results were seen in 29 of 31 (93.5%) patients for which serum was analyzed and in 31 of 33 (93.9%) cases with whole-blood specimens. Both blood and serum samples were available in 26 cases, and significant differences were not observed in this subgroup of cases. The average number of threshold cycles (C(T)) for positive blood samples was 37.6, and the average C(T) for serum was 37.4. The DNA concentration ranged between 2 and 50 fg per μl of sample, with average DNA concentrations of 10.2 and 11.7 fg in positive blood and serum samples, respectively (P > 0.01). The performance of this PCR-based quantitative assay was similar for both serum and blood samples. We recommend serum samples as the most convenient hematological sample to use for Aspergillus DNA quantification when serial determinations are done.

  19. Popcorn genotypes resistance to fall armyworm

    Directory of Open Access Journals (Sweden)

    Nádia Cristina de Oliveira

    2018-02-01

    Full Text Available ABSTRACT: The aim of this study was to evaluate popcorn genotypes for resistance to the fall armyworm, Spodoptera frugiperda. The experiment used a completely randomized design with 30 replicates. The popcorn genotypes Aelton, Arzm 05 083, Beija-Flor, Colombiana, Composto Chico, Composto Gaúcha, Márcia, Mateus, Ufvm Barão Viçosa, Vanin, and Viviane were evaluated,along with the common maize variety Zapalote Chico. Newly hatched fall armyworm larvae were individually assessed with regard to biological development and consumption of food. The data were subjected to multivariate analyses of variance and genetic divergence among genotypes was evaluated through the clustering methods of Tocher based on generalized Mahalanobis distances and canonical variable analyses. Seven popcorn genotypes, namely, Aelton, Arzm 05 083, Composto Chico, Composto Gaúcha, Márcia, Mateus, and Viviane,were shown to form a cluster (cluster I that had antibiosis as the mechanism of resistance to the pest. Cluster I genotypes and the Zapalote Chico genotype could be used for stacking genes for antibiosis and non-preference resistance.

  20. Relationship of some upland rice genotype after gamma irradiation

    Science.gov (United States)

    Suliartini, N. W. S.; Wijayanto, T.; Madiki, A.; Boer, D.; Muhidin; Juniawan

    2018-02-01

    The objective of the research was to group local upland rice genotypes after being treated with gamma irradiation. The research materials were upland rice genotypes resulted from mutation of the second generation and two parents: Pae Loilo (K3D0) and Pae Pongasi (K2D0) Cultivars. The research was conducted at the Indonesian Sweetener and Fiber Crops Research Institute, Malang Regency, and used the augmented design method. Research data were analyzed with R Program. Eight hundred and seventy one genotypes were selected with the selection criteria were based on yields on the average parents added 1.5 standard deviation. Based on the selection, eighty genotypes were analyzed with cluster analyses. Nine observation variables were used to develop cluster dendrogram using average linked method. Genetic distance was measured by euclidean distance. The results of cluster dendrogram showed that tested genotypes were divided into eight groups. Group 1, 2, 7, and 8 each had one genotype, group 3 and 6 each had two genotypes, group 4 had 25 genotypes, and group 5 had 51 genotypes. Check genotypes formed a separate group. Group 6 had the highest yield per plant of 126.11 gram, followed by groups 5 and 4 of 97.63 and 94.08 gram, respectively.

  1. Specificity of the Linear Array HPV Genotyping Test for detecting human papillomavirus genotype 52 (HPV-52)

    OpenAIRE

    Kocjan, Boštjan; Poljak, Mario; Oštrbenk, Anja

    2015-01-01

    Introduction: HPV-52 is one of the most frequent human papillomavirus (HPV) genotypes causing significant cervical pathology. The most widely used HPV genotyping assay, the Roche Linear Array HPV Genotyping Test (Linear Array), is unable to identify HPV- 52 status in samples containing HPV-33, HPV-35, and/or HPV-58. Methods: Linear Array HPV-52 analytical specificity was established by testing 100 specimens reactive with the Linear Array HPV- 33/35/52/58 cross-reactive probe, but not with the...

  2. IS1111 insertion sequences of Coxiella burnetii: characterization and use for repetitive element PCR-based differentiation of Coxiella burnetii isolates

    Directory of Open Access Journals (Sweden)

    Massung Robert F

    2007-10-01

    Full Text Available Abstract Background Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method. Results Isolates Nine Mile phase II, Nine Mile RSA 514, Nine Mile Baca, Scottish, Ohio, Australian QD, Henzerling phase I, Henzerling phase II, M44, KAV, PAV, Q238, Q195 and WAV were tested by PCR and compared to 9Mi/I. Sequencing was used to determine the exact differences in isolates which lacked specific IS elements or produced PCR products of differing size. From this data, an algorithm was created utilizing four primer pairs that allows for differentiation of unknown isolates into five genomic groups. Additional isolates (Priscilla Q177, Idaho Q, Qiyi, Poker Cat, Q229 and Q172 and nine veterinary samples were characterized using the algorithm which resulted in their placement into three distinct genomic groups. Conclusion Through this study significant differences, including missing elements and sequence alterations within and near IS element coding regions, were found between the isolates tested. Further, a method for differentiation of C. burnetii isolates into one of five genomic groups was created. This algorithm may ultimately help to determine the relatedness between known and unknown isolates of C. burnetii.

  3. Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

    DEFF Research Database (Denmark)

    Liu, Yilan; Chen, Jinjin; Thygesen, Anders

    2018-01-01

    Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric...... intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products...

  4. Identification of Actinobacillus pleuropneumoniae serotypes 1, 7, and 12 by multiplex PCR based on genes involved in encapsulation

    DEFF Research Database (Denmark)

    Angen, Øystein; Jessing, Stine Graakjær; Ahrens, Peter

    2005-01-01

    . However, a number of isolates show cross-reaction between serotypes and this has urged the development of quick, serotype specific DNA-based methods necessary. Serotype specific tests have until now been described for the serotypes 2, 5, and 6 (Jessing et al, 2003) and serotypes 1, 2 and 8 (Schuchert et...

  5. Detection of cyclospora in captive chimpanzees and macaques by a quantitative PCR-based mutation scanning approach

    OpenAIRE

    Marangi, M.; Koehler, A.V.; Zanzani, S.A.; Manfredi, M.T.; Brianti, E.; Giangaspero, A.; Gasser, R.B.

    2015-01-01

    Background Cyclospora is a protistan parasite that causes enteritis in several species of animals including humans. The aim of this study was to investigate the presence of Cyclospora in captive non-human primates. Methods A total of 119 faecal samples from Pan troglodytes, Macaca sylvanus, Cercopithecus cephus, Erythrocebus patas, Chlorocebus aethiops and Macaca fascicularis from a wildlife animal rescue center as well as from Macaca fascicularis from an experimental primate research center ...

  6. PCR-Based Molecular Characterization of Toxocara spp. Using Feces of Stray Cats: A Study from Southwest Iran

    OpenAIRE

    Khademvatan, Shahram; Rahim, Fakher; Tavalla, Mahdi; Abdizadeh, Rahman; Hashemitabar, Mahmoud

    2013-01-01

    Feces of stray cat are potential sources of gastrointestinal parasites and play a crucial role in spreading and transmitting parasite eggs, larvae, and oocysts through contamination of soil, food, or water. In this study, we investigated the prevalence of Toxocara spp. infection in stray cats in Ahvaz city, southwest Iran. Eggs of Toxocara spp. in feces of stray cats were detected by the sucrose flotation method, and identification was conducted by polymerase chain reaction (PCR) and DNA sequ...

  7. Development of an aptamer-based concentration method for the detection of Trypanosoma cruzi in blood.

    Directory of Open Access Journals (Sweden)

    Rana Nagarkatti

    Full Text Available Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8-25 nM range. The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.

  8. Identification and molecular epidemiology of dermatophyte isolates by repetitive-sequence-PCR-based DNA fingerprinting using the DiversiLab system in Turkey.

    Science.gov (United States)

    Koc, A Nedret; Atalay, Mustafa A; Inci, Melek; Sariguzel, Fatma M; Sav, Hafize

    2017-05-01

    Dermatophyte species, isolation and identification in clinical samples are still difficult and take a long time. The identification and molecular epidemiology of dermatophytes commonly isolated in a clinical laboratory in Turkey by repetitive sequence-based PCR (rep-PCR) were assessed by comparing the results with those of reference identification. A total of 44 dermatophytes isolated from various clinical specimens of 20 patients with superficial mycoses in Kayseri and 24 patients in Hatay were studied. The identification of dermatophyte isolates was based on the reference identification and rep-PCR using the DiversiLab System (BioMerieux). The genotyping of dermatophyte isolates from different patients was determined by rep-PCR. In the identification of dermatophyte isolates, agreement between rep-PCR and conventional methods was 87.8 % ( 36 of 41). The dermatophyte strains belonged to four clones (A -D) which were determined by the use of rep-PCR. The dermatophyte strains in Clone B, D showed identical patterns with respect to the region. In conclusion, rep-PCR appears to be useful for evaluation of the identification and clonal relationships between Trichophyton rubrum species complex and Trichophyton mentagrophytes species complex isolates. The similarity and diversity of these isolates may be assessed according to different regions by rep-PCR. © 2017 Blackwell Verlag GmbH.

  9. Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental samples.

    Science.gov (United States)

    Saingam, Prakit; Li, Bo; Yan, Tao

    2018-06-01

    DNA-based molecular detection of microbial pathogens in complex environments is still plagued by sensitivity, specificity and robustness issues. We propose to address these issues by viewing them as inadvertent consequences of requiring specific and adequate amplification (SAA) of target DNA molecules by current PCR methods. Using the invA gene of Salmonella as the model system, we investigated if next generation sequencing (NGS) can be used to directly detect target sequences in false-negative PCR reaction (PCR-NGS) in order to remove the SAA requirement from PCR. False-negative PCR and qPCR reactions were first created using serial dilutions of laboratory-prepared Salmonella genomic DNA and then analyzed directly by NGS. Target invA sequences were detected in all false-negative PCR and qPCR reactions, which lowered the method detection limits near the theoretical minimum of single gene copy detection. The capability of the PCR-NGS approach in correcting false negativity was further tested and confirmed under more environmentally relevant conditions using Salmonella-spiked stream water and sediment samples. Finally, the PCR-NGS approach was applied to ten urban stream water samples and detected invA sequences in eight samples that would be otherwise deemed Salmonella negative. Analysis of the non-target sequences in the false-negative reactions helped to identify primer dime-like short sequences as the main cause of the false negativity. Together, the results demonstrated that the PCR-NGS approach can significantly improve method sensitivity, correct false-negative detections, and enable sequence-based analysis for failure diagnostics in complex environmental samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. PCR-based molecular characterization of Toxocara spp. using feces of stray cats: a study from Southwest Iran.

    Science.gov (United States)

    Khademvatan, Shahram; Rahim, Fakher; Tavalla, Mahdi; Abdizadeh, Rahman; Hashemitabar, Mahmoud

    2013-01-01

    Feces of stray cat are potential sources of gastrointestinal parasites and play a crucial role in spreading and transmitting parasite eggs, larvae, and oocysts through contamination of soil, food, or water. In this study, we investigated the prevalence of Toxocara spp. infection in stray cats in Ahvaz city, southwest Iran. Eggs of Toxocara spp. in feces of stray cats were detected by the sucrose flotation method, and identification was conducted by polymerase chain reaction (PCR) and DNA sequencing. Of the 140 fecal samples that were randomly collected from public environments during the months of January to May 2012, 45% were found to harbour Toxocara spp. eggs. The highest prevalence of Toxocara spp. eggs was found in the central area of Ahvaz city (28.6%). T. canis eggs were found in 4 (6.34%) of the 63 positive samples. Stray cats are found in parks, playgrounds, and other public places and may be a potential contamination risk. Identification of Toxocara spp. using molecular methods is sufficiently sensitive to detect low levels of parasites and identify the different Toxocara spp. in feces. The relatively high prevalence of Toxocara spp. infection may continue to increase due to lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, identify, and control this infection as well as stray cats in the region.

  11. PCR-based molecular characterization of Toxocara spp. using feces of stray cats: a study from Southwest Iran.

    Directory of Open Access Journals (Sweden)

    Shahram Khademvatan

    Full Text Available Feces of stray cat are potential sources of gastrointestinal parasites and play a crucial role in spreading and transmitting parasite eggs, larvae, and oocysts through contamination of soil, food, or water. In this study, we investigated the prevalence of Toxocara spp. infection in stray cats in Ahvaz city, southwest Iran. Eggs of Toxocara spp. in feces of stray cats were detected by the sucrose flotation method, and identification was conducted by polymerase chain reaction (PCR and DNA sequencing. Of the 140 fecal samples that were randomly collected from public environments during the months of January to May 2012, 45% were found to harbour Toxocara spp. eggs. The highest prevalence of Toxocara spp. eggs was found in the central area of Ahvaz city (28.6%. T. canis eggs were found in 4 (6.34% of the 63 positive samples. Stray cats are found in parks, playgrounds, and other public places and may be a potential contamination risk. Identification of Toxocara spp. using molecular methods is sufficiently sensitive to detect low levels of parasites and identify the different Toxocara spp. in feces. The relatively high prevalence of Toxocara spp. infection may continue to increase due to lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, identify, and control this infection as well as stray cats in the region.

  12. Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

    Science.gov (United States)

    Abdeldaim, Guma; Svensson, Erik; Blomberg, Jonas; Herrmann, Björn

    2016-11-01

    A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  13. Genotyping of Coxiella burnetii from domestic ruminants in northern Spain

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    Astobiza Ianire

    2012-12-01

    Full Text Available Abstract Background Information on the genotypic diversity of Coxiella burnetii isolates from infected domestic ruminants in Spain is limited. The aim of this study was to identify the C. burnetii genotypes infecting livestock in Northern Spain and compare them to other European genotypes. A commercial real-time PCR targeting the IS1111a insertion element was used to detect the presence of C. burnetii DNA in domestic ruminants from Spain. Genotypes were determined by a 6-loci Multiple Locus Variable number tandem repeat analysis (MLVA panel and Multispacer Sequence Typing (MST. Results A total of 45 samples from 4 goat herds (placentas, N = 4, 12 dairy cattle herds (vaginal mucus, individual milk, bulk tank milk, aerosols, N = 20 and 5 sheep flocks (placenta, vaginal swabs, faeces, air samples, dust, N = 21 were included in the study. Samples from goats and sheep were obtained from herds which had suffered abortions suspected to be caused by C. burnetii, whereas cattle samples were obtained from animals with reproductive problems compatible with C. burnetii infection, or consisted of bulk tank milk (BTM samples from a Q fever surveillance programme. C. burnetii genotypes identified in ruminants from Spain were compared to those detected in other countries. Three MLVA genotypes were found in 4 goat farms, 7 MLVA genotypes were identified in 12 cattle herds and 4 MLVA genotypes were identified in 5 sheep flocks. Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between most MLVA genotypes. Overall 11 different MLVA genotypes were obtained corresponding to 4 different MST genotypes: MST genotype 13, identified in goat, sheep and cattle from Spain; MST genotype 18, only identified in goats; and, MST genotypes 8 and 20, identified in small ruminants and cattle, respectively. All these genotypes had been previously identified in animal and human clinical samples from several

  14. Development of Nested PCR-Based Specific Markers for Detection of Peach Rosette Mosaic Virus in Plant Quarantine.

    Science.gov (United States)

    Lee, S; Kim, C S; Shin, Y G; Kim, J H; Kim, Y S; Jheong, W H

    2016-03-01

    The Peach rosette mosaic virus (PRMV) is a plant pathogen of the genus Nepovirus, and has been designated as a controlled quarantine virus in Korea. In this study, a specific reverse transcription (RT)-PCR marker set, nested PCR marker set, and modified-plasmid positive control were developed to promptly and accurately diagnose PRMV at plant-quarantine sites. The final selected PRMV-specific RT-PCR marker was PRMV-N10/C70 (967 bp), and the nested PCR product of 419 bp was finally amplified. The modified-plasmid positive control, in which the SalI restriction-enzyme region (GTCGAC) was inserted, verified PRMV contamination in a comparison with the control, enabling a more accurate diagnosis. It is expected that the developed method will continuously contribute to the plant-quarantine process in Korea.

  15. Cloning of the koi herpesvirus (KHV gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

    Directory of Open Access Journals (Sweden)

    Gilad Oren

    2005-03-01

    Full Text Available Abstract Background Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV. Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. Results A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV and the channel catfish virus (CCV. The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. Conclusion The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

  16. Genetic characterization of Toxoplasma gondii from Brazilian wildlife revealed abundant new genotypes.

    Science.gov (United States)

    Vitaliano, S N; Soares, H S; Minervino, A H H; Santos, A L Q; Werther, K; Marvulo, M F V; Siqueira, D B; Pena, H F J; Soares, R M; Su, C; Gennari, S M

    2014-12-01

    This study aimed to isolate and genotype T. gondii from Brazilian wildlife. For this purpose, 226 samples were submitted to mice bioassay and screened by PCR based on 18S rRNA sequences. A total of 15 T. gondii isolates were obtained, including samples from four armadillos (three Dasypus novemcinctus, one Euphractus sexcinctus), three collared anteaters (Tamandua tetradactyla), three whited-lipped peccaries (Tayassu pecari), one spotted paca (Cuniculus paca), one oncilla (Leopardus tigrinus), one hoary fox (Pseudalopex vetulus), one lineated woodpecker (Dryocopus lineatus) and one maned wolf (Chrysocyon brachyurus). DNA from the isolates, originated from mice bioassay, and from the tissues of the wild animal, designated as "primary samples", were genotyped by PCR-restriction fragment length polymorphism (PCR/RFLP), using 12 genetic markers (SAG1, SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L258, PK1, CS3 and Apico). A total of 17 genotypes were identified, with 13 identified for the first time and four already reported in published literature. Results herein obtained corroborate previous studies in Brazil, confirming high diversity and revealing unique genotypes in this region. Given most of genotypes here identified are different from previous studies in domestic animals, future studies on T. gondii from wildlife is of interest to understand population genetics and structure of this parasite.

  17. Genetic characterization of Toxoplasma gondii from Brazilian wildlife revealed abundant new genotypes

    Directory of Open Access Journals (Sweden)

    S.N. Vitaliano

    2014-12-01

    Full Text Available This study aimed to isolate and genotype T. gondii from Brazilian wildlife. For this purpose, 226 samples were submitted to mice bioassay and screened by PCR based on 18S rRNA sequences. A total of 15 T. gondii isolates were obtained, including samples from four armadillos (three Dasypus novemcinctus, one Euphractus sexcinctus, three collared anteaters (Tamandua tetradactyla, three whited-lipped peccaries (Tayassu pecari, one spotted paca (Cuniculus paca, one oncilla (Leopardus tigrinus, one hoary fox (Pseudalopex vetulus, one lineated woodpecker (Dryocopus lineatus and one maned wolf (Chrysocyon brachyurus. DNA from the isolates, originated from mice bioassay, and from the tissues of the wild animal, designated as “primary samples”, were genotyped by PCR–restriction fragment length polymorphism (PCR/RFLP, using 12 genetic markers (SAG1, SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L258, PK1, CS3 and Apico. A total of 17 genotypes were identified, with 13 identified for the first time and four already reported in published literature. Results herein obtained corroborate previous studies in Brazil, confirming high diversity and revealing unique genotypes in this region. Given most of genotypes here identified are different from previous studies in domestic animals, future studies on T. gondii from wildlife is of interest to understand population genetics and structure of this parasite.

  18. The potential of plant viruses to promote genotypic diversity via genotype x environment interactions

    DEFF Research Database (Denmark)

    van Mölken, Tamara; Stuefer, Josef F.

    2011-01-01

    † Background and Aims Genotype by environment (G × E) interactions are important for the long-term persistence of plant species in heterogeneous environments. It has often been suggested that disease is a key factor for the maintenance of genotypic diversity in plant populations. However, empirical...... and the G × E interactions were examined with respect to genotypespecific plant responses to WClMV infection. Thus, the environment is defined as the presence or absence of the virus. † Key Results WClMV had a negative effect on plant performance as shown by a decrease in biomass and number of ramets...... evidence for this contention is scarce. Here virus infection is proposed as a possible candidate for maintaining genotypic diversity in their host plants. † Methods The effects of White clover mosaic virus (WClMV) on the performance and development of different Trifolium repens genotypes were analysed...

  19. A qPCR-Based Tool to Diagnose the Presence of Harmful Cyanobacteria and Cyanotoxins in Drinking Water Sources

    Directory of Open Access Journals (Sweden)

    Yi-Ting Chiu

    2017-05-01

    Full Text Available Harmful cyanobacteria have been an important concern for drinking water quality for quite some time, as they may produce cyanotoxins and odorants. Microcystis and Cylindrospermopsis are two common harmful cyanobacterial genera detected in freshwater lakes and reservoirs, with microcystins (MCs and cylindrospermopsin (CYN as their important metabolites, respectively. In this study, two sets of duplex qPCR systems were developed, one for quantifying potentially-toxigenic Microcystis and Microcystis, and the other one for cylindrospermopsin-producing cyanobacteria and Cylindrospermopsis. The duplex qPCR systems were developed and validated in the laboratory by using 338 samples collected from 29 reservoirs in Taiwan and her offshore islands. Results show that cell numbers of Microcystis and Cylindorspermopsis enumerated with microscopy, and MCs and CYN concentrations measured with the enzyme-linked immuno-sorbent assay method, correlated well with their corresponding gene copies determined with the qPCR systems (range of coefficients of determination R2 = 0.392−0.740. The developed qPCR approach may serve as a useful tool for the water industry to diagnose the presence of harmful cyanobacteria and the potential presence of cyanotoxins in source waters.

  20. A qPCR-Based Tool to Diagnose the Presence of Harmful Cyanobacteria and Cyanotoxins in Drinking Water Sources.

    Science.gov (United States)

    Chiu, Yi-Ting; Chen, Yi-Hsuan; Wang, Ting-Shaun; Yen, Hung-Kai; Lin, Tsair-Fuh

    2017-05-20

    Harmful cyanobacteria have been an important concern for drinking water quality for quite some time, as they may produce cyanotoxins and odorants. Microcystis and Cylindrospermopsis are two common harmful cyanobacterial genera detected in freshwater lakes and reservoirs, with microcystins (MCs) and cylindrospermopsin (CYN) as their important metabolites, respectively. In this study, two sets of duplex qPCR systems were developed, one for quantifying potentially-toxigenic Microcystis and Microcystis , and the other one for cylindrospermopsin-producing cyanobacteria and Cylindrospermopsis . The duplex qPCR systems were developed and validated in the laboratory by using 338 samples collected from 29 reservoirs in Taiwan and her offshore islands. Results show that cell numbers of Microcystis and Cylindorspermopsis enumerated with microscopy, and MCs and CYN concentrations measured with the enzyme-linked immuno-sorbent assay method, correlated well with their corresponding gene copies determined with the qPCR systems (range of coefficients of determination R² = 0.392-0.740). The developed qPCR approach may serve as a useful tool for the water industry to diagnose the presence of harmful cyanobacteria and the potential presence of cyanotoxins in source waters.

  1. Pulmonary toxoplasmosis in immunocompromised patients with interstitial pneumonia: a single-centre prospective study assessing PCR-based diagnosis.

    Science.gov (United States)

    Desoubeaux, Guillaume; Cabanne, Églantine; Franck-Martel, Claire; Gombert, Martin; Gyan, Emmanuel; Lissandre, Séverine; Renaud, Marc; Monjanel, Hélène; Dartigeas, Caroline; Bailly, Éric; Van Langendonck, Nathalie; Chandenier, Jacques

    2016-08-01

    Pulmonary toxoplasmosis has become a very rare parasitic infection since the advent of highly active antiretroviral therapies. It is generally diagnosed by the direct microscopic observation of Toxoplasma gondii tachyzoites in bronchoalveolar lavage fluid (BALF). The aim of this study was to assess possible improvements in diagnostic performance associated with the use of real-time PCR. This prospective study was carried out on BALFs obtained from immunocompromised patients over a 2-year period. We systematically compared the results of conventional staining with those of molecular detection. Two cases of pulmonary toxoplasmosis were diagnosed for a total of 336 samples. PCR did not detect any additional cases and was more time-consuming than conventional staining. Conventional staining is a reliable technique and is probably the most appropriate method for experienced microbiology laboratories, whereas T. gondii-specific PCR may be useful for laboratories with less experience in parasitology. 2015_030, May 27th 2015. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  2. Application of PCR-Based Tools to Explore Strongyloides Infection in People in Parts of Northern Australia

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    Gemma J. Robertson

    2017-12-01

    Full Text Available Strongyloidiasis, which is caused by infection with the nematode Strongyloides stercoralis, is endemic to areas of northern Australia. Diagnosis in this region remains difficult due to the distances between endemic communities and diagnostic laboratories, leading to lengthy delays in stool processing for microscopy and culture. PCR represents a viable solution to this difficulty, having potential for high sensitivity detection of S. stercoralis, even in older, unpreserved faecal samples. We prospectively collected 695 faecal specimens that were submitted to The Townsville Hospital Microbiology Laboratory from the North Queensland region for routine parasitological examination, and subjected them to a Strongyloides sp. real-time (qPCR. Results were confirmed with a novel nested conventional PCR assay targeting the 18S rRNA gene, followed by single-strand conformation polymorphism analysis (SSCP. Of the 695 specimens tested, S. stercoralis was detected in three specimens (0.4% by classical parasitological methods (direct microscopy and formyl-ether acetate concentration, whereas 42 positives were detected by qPCR (6.0%. Conventional PCR confirmed the real-time PCR results in 24 of the samples (3.5%. Several apparent false-positive results occurred at higher cycle times (Ct in the qPCR. Use of real-time PCR in these populations is promising for the enhanced detection of disease and to support eradication efforts.

  3. Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear

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    Gholamreza HASSANPOUR

    2016-12-01

    Full Text Available Background: We aimed to quicken and simplify the detection of Plasmodium in blood samples by developing and testing a pan-Plasmodium real-time PCR for accurate screening of individuals suspected of malaria.Methods: A single primer/probe set for pan-species Plasmodium-specific real time PCR targeting a conserved region of the small subunit 18S ribosomal DNA was designed and evaluated for rapid diagnosis and screening of malaria infections using dried blood smears. FTA cards were used for rapid and simple DNA extraction.Results: The primers and probes showed a positive response with the DNA extracted from bloods infected with P. falciparum and P. vivax but not with DNA extracted from various smears from uninfected blood samples. Seven positive cases positive by both microscopy and nested PCR were found among 280 blood samples taken from in South and Southeast Iran. Five samples were identified as positive for P. vivax and two as positive for P. falciparum. All positive samples were positive by real-time PCR. Furthermore, all 38-blood samples positive by microscopy were positive by real-time PCR. No microscopy-negative samples were positive by real-time PCR.Conclusion: By using a simple FTA card for DNA extraction and by application of the real-time PCR developed in this study, sensitivity similar to nested-PCR and microscopy was achieved. This format simplifies the detection of Plasmodium in large numbers of samples.

  4. Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

    Directory of Open Access Journals (Sweden)

    Grazielle Cardoso da Graça

    2012-08-01

    Full Text Available In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70 regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL. A total of 70 Leishmania strains were analysed, including seven reference strains (RS and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP. This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region, internal transcribed spacer (ITS1 and glucose-6-phosphate dehydrogenase (G6pd. A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

  5. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

    Science.gov (United States)

    Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

    2017-06-01

    A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Precise genotyping and recombination detection of Enterovirus

    Science.gov (United States)

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  7. Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.

    Science.gov (United States)

    Martinez-Serra, Jordi; Robles, Juan; Nicolàs, Antoni; Gutierrez, Antonio; Ros, Teresa; Amat, Juan Carlos; Alemany, Regina; Vögler, Oliver; Abelló, Aina; Noguera, Aina; Besalduch, Joan

    2014-01-01

    Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler(®) 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5×10(6) WBC/mL) and purified DNA (20 ng/μL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/μL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.

  8. A novel multiplex RT-qPCR method based on dual-labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus

    DEFF Research Database (Denmark)

    Vázquez, D.; López-Vázquez, C.; Skall, Helle Frank

    2016-01-01

    , resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV...

  9. Hepatitis C virus genotypes in Bahawalpur

    International Nuclear Information System (INIS)

    Qazi, M.A.; Fayyaz, M.; Chaudhry, G.M.D.; Jamil, A.

    2006-01-01

    This study was conducted at Medical Unit-II Bahawal Victoria Hospital / Quaid-e-Azam Medical College Bahawalpur from May 1st , 2005 to December 31st 2005. The objective of this study was to determine hepatitis C virus (HCV) genotypes in Bahawalpur, Pakistan. In consecutive 105 anti-HCV (ELISA-3) positive patients, complete history and physical examination was performed. Liver function tests, complete blood counts and platelet count, blood sugar fasting and 2 hours after breakfast, prothrombin time, serum albumin, serum globulin and abdominal ultrasound were carried out in all the patients. Tru cut biopsy was performed on 17 patients. We studied HCV RNA in all these patients by Nested PCR method. HCV RNA was detected in 98 patients and geno typing assay was done by genotype specific PCR. Among total of 105 anti-HCV positive patients, HCV-RNA was detected in 98 patients. Out of these 98 patients there were 57 (58.2%) males and 41 (42.8%) females. Their age range was 18-75 years. The age 18-29 years 26 (26.5%), 30-39 years 35 (35.7%) and 40-75 37 (37.8%), while 10 (10.2%) patients were diabetics and 34 (34.7%) patients were obese. Liver cirrhosis was present in 10 (10.2%) patients. Forty two (43.9%) patients were symptomatic while 56 (57.1%) were asymptomatic. Out of 98 patients 11 (11.2%) were un type-able and 87 (88.8%) were type able. 70/98 (71.4%) were genotype 3; 10/98 (10.2%) were genotype 1; 03/98 (3.1%) were genotype 2; 03/98 (3.1%) were mixed genotype 2 and 3; 01/98 (1%) were mixed genotype 3a and 3b. Genotype 3 is the most common HCV virus in our area which shows that both virological and biochemical response will be better. Because HCV genotype 3 is more frequent among the drug users which points towards unsafe injection practices in our area. (author)

  10. Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin

    Czech Academy of Sciences Publication Activity Database

    Šrůtková, Dagmar; Španová, A.; Špano, M.; Dráb, V.; Schwarzer, Martin; Kozáková, Hana; Rittich, B.

    2011-01-01

    Roč. 87, č. 1 (2011), s. 10-16 ISSN 0167-7012 R&D Projects: GA MŠk 2B06053 Institutional research plan: CEZ:AV0Z50200510 Keywords : Bifidobacterium longum * ARDRA * pcr Subject RIV: EC - Immunology Impact factor: 2.086, year: 2011

  11. Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa

    DEFF Research Database (Denmark)

    Ito, T.; Olesen, Niels Jørgen; Skall, Helle Frank

    2010-01-01

    of the spread of genotypes to new geographical areas. A monoclonal antibody (MAb) against VHSV genotype IVa was produced, with the aim of providing a simple method of discriminating this genotype from the other VHSV genotypes (I, II, III and IVb). Balb/c mice were injected with purified VHSV-JF00Ehil (genotype...... IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected...

  12. PCR-Based Analysis of ColE1 Plasmids in Clinical Isolates and Metagenomic Samples Reveals Their Importance as Gene Capture Platforms

    Directory of Open Access Journals (Sweden)

    Manuel Ares-Arroyo

    2018-03-01

    Full Text Available ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance

  13. Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

    Science.gov (United States)

    Sang, Fuming; Yang, Yang; Yuan, Lin; Ren, Jicun; Zhang, Zhizhou

    2015-09-01

    Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour

  14. Assessment of the microbiota of a mixed infection of the tongue using phenotypic and genotypic methods simultaneously and a review of the literature

    NARCIS (Netherlands)

    Veloo, A. C. M.; Schepers, R. H.; Welling, G. W.; Degener, J. E.

    We assessed the microbiota of a tongue abscess in which twelve different aerobic and anaerobic bacteria were identified using fluorescent in situ hybridisation (FISH), sequencing of the 16S rRNA gene and phenotypic methods. By applying the 16S rRNA based probes directly on the clinical material, a

  15. Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.

    Science.gov (United States)

    Liang, Te; Liu, Xiaoxiao; Cui, Shulin; Qu, Shenghua; Wang, Dan; Liu, Ning; Wang, Fumin; Ning, Kang; Zhang, Bing; Zhang, Dabing

    2016-02-02

    Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

    Science.gov (United States)

    Hsiao, K M; Lin, H M; Pan, H; Li, T C; Chen, S S; Jou, S B; Chiu, Y L; Wu, M F; Lin, C C; Li, S Y

    1999-01-01

    Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

  17. Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Reis Patricia P

    2010-06-01

    Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

  18. Choosing tree genotypes for phytoremediation of landfill leachate using phyto-recurrent selection

    Science.gov (United States)

    Jill A. Zalesny; Ronald S., Jr. Zalesny; Adam H. Wiese; Richard B. Hall

    2007-01-01

    Information about the response of poplar (Populus spp.) genotypes to landfill leachate irrigation is needed, along with efficient methods for choosing genotypes based on leachate composition. Poplar clones were irrigated during three cycles of phyto-recurrent selection to test whether genotypes responded differently to leachate and water, and to test...

  19. Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias

    Directory of Open Access Journals (Sweden)

    Didion John P

    2012-01-01

    Full Text Available Abstract Background High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. Results We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. Conclusion The problems of ascertainment bias and missing

  20. HBV genotypic variability in Cuba.

    Directory of Open Access Journals (Sweden)

    Carmen L Loureiro

    Full Text Available The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%, mainly A2 (149, 60% but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%, with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7. Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions.

  1. HBV Genotypic Variability in Cuba

    Science.gov (United States)

    Loureiro, Carmen L.; Aguilar, Julio C.; Aguiar, Jorge; Muzio, Verena; Pentón, Eduardo; Garcia, Daymir; Guillen, Gerardo; Pujol, Flor H.

    2015-01-01

    The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%), mainly A2 (149, 60%) but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%), with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7). Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions. PMID:25742179

  2. Evaluation of 14 winter bread wheat genotypes in normal irrigation ...

    African Journals Online (AJOL)

    Evaluation of 14 winter bread wheat genotypes in normal irrigation and stress conditions after anthesis stage. ... African Journal of Biotechnology ... Using biplot graphic method, comparison of indices amounts and mean rating of indices for ...

  3. Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE

    Directory of Open Access Journals (Sweden)

    Martinez-Serra J

    2014-06-01

    Full Text Available Jordi Martinez-Serra,1 Juan Robles,2 Antoni Nicolàs,3 Antonio Gutierrez,1 Teresa Ros,1 Juan Carlos Amat,1 Regina Alemany,4 Oliver Vögler,4 Aina Abelló,2 Aina Noguera,2 Joan Besalduch1 1Department of Hematology, 2Department of Clinical Analysis, Hospital Universitary Son Espases, Palma de Mallorca, Spain; 3ECOGEN, Barcelona, 4Department of Cell Biology, University of the Balearic Islands, Palma de Mallorca, Spain Abstract: Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC. In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler® 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE. After testing 34 samples comparing the real-time crossing point (CP values between WBC (5×106 WBC/mL and purified DNA (20 ng/µL, the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×106 WBC/mL instead of DNA (20 ng/µL, we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification

  4. Effect of different home-cooking methods on textural and nutritional properties of sweet potato genotypes grown in temperate climate conditions.

    Science.gov (United States)

    Nicoletto, Carlo; Vianello, Fabio; Sambo, Paolo

    2018-01-01

    The European Union (EU) market for sweet potato is small but is growing considerably and and has increased by 100% over the last 5 years. The cultivation of sweet potato in temperate climate conditions has not considered extensively and could be a new opportunity for the EU market. Healthy and qualitative traits of different sweet potato cultivars grown in temperate climate conditions were evaluated in accordance with four cooking methods. Traditional cultivars showed high hardness and adhesiveness values. The highest concentrations of sugars (especially maltose) and phenolic acids (caffeic and chlorogenic) were found in samples treated by boiling and steaming. High antioxidant activity was found in fried potatoes. Qualitative traits of sweet potatoes treated by microwaves did not report any significant variation compared to the control. Traditional and new sweet potato cultivars can be cultivated in temperate climate conditions and show interesting qualitative properties, especially as a result of the presence of antioxidant compounds. Concerning global quality, colored varieties expressed a better profile than traditional Italian ones and they are suitable for the European market, giving new opportunities for consumers and producers. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. A fast and cost-effective method for apolipoprotein E isotyping as an alternative to APOE genotyping for patient screening and stratification.

    Science.gov (United States)

    Calero, Olga; García-Albert, Luis; Rodríguez-Martín, Andrés; Veiga, Sergio; Calero, Miguel

    2018-04-13

    Apolipoprotein E (apoE) is a 34 kDa glycoprotein involved in lipid metabolism. The human APOE gene encodes for three different apoE protein isoforms: E2, E3 and E4. The interest in apoE isoforms is high for epidemiological research, patient stratification and identification of those at increased risk for clinical trials and prevention. The isoform apoE4 is associated with increased risk for coronary heart and Alzheimer's diseases. This paper describes a method for specifically detecting the apoE4 isoform from biological fluids by taking advantage of the capacity of apoE to bind "specifically" to polystyrene surfaces as capture and a specific anti-apoE4 monoclonal antibody as reporter. Our results indicate that the apoE-polystyrene binding interaction is highly stable, resistant to detergents and acid and basic washes. The methodology here described is accurate, easily implementable, fast and cost-effective. Although at present, our technique is unable to discriminate homozygous APOE ε4/ε4 from APOE ε3/ε4 and ε2/ε4 heterozygous, it opens new avenues for the development of inexpensive, yet effective, tests for the detection of apoE4 for patients' stratification. Preliminary results indicated that this methodology is also adaptable into turbidimetric platforms, which make it a good candidate for clinical implementation through its translation to the clinical analysis routine.

  6. Identification of polymorphic inversions from genotypes

    Directory of Open Access Journals (Sweden)

    Cáceres Alejandro

    2012-02-01

    Full Text Available Abstract Background Polymorphic inversions are a source of genetic variability with a direct impact on recombination frequencies. Given the difficulty of their experimental study, computational methods have been developed to infer their existence in a large number of individuals using genome-wide data of nucleotide variation. Methods based on haplotype tagging of known inversions attempt to classify individuals as having a normal or inverted allele. Other methods that measure differences between linkage disequilibrium attempt to identify regions with inversions but unable to classify subjects accurately, an essential requirement for association studies. Results We present a novel method to both identify polymorphic inversions from genome-wide genotype data and classify individuals as containing a normal or inverted allele. Our method, a generalization of a published method for haplotype data 1, utilizes linkage between groups of SNPs to partition a set of individuals into normal and inverted subpopulations. We employ a sliding window scan to identify regions likely to have an inversion, and accumulation of evidence from neighboring SNPs is used to accurately determine the inversion status of each subject. Further, our approach detects inversions directly from genotype data, thus increasing its usability to current genome-wide association studies (GWAS. Conclusions We demonstrate the accuracy of our method to detect inversions and classify individuals on principled-simulated genotypes, produced by the evolution of an inversion event within a coalescent model 2. We applied our method to real genotype data from HapMap Phase III to characterize the inversion status of two known inversions within the regions 17q21 and 8p23 across 1184 individuals. Finally, we scan the full genomes of the European Origin (CEU and Yoruba (YRI HapMap samples. We find population-based evidence for 9 out of 15 well-established autosomic inversions, and for 52 regions

  7. The discrimination of d-tartrate positive and d-tartrate negative S. enterica subsp. enterica serovar Paratyphi B isolated in Malaysia by phenotypic and genotypic methods.

    Science.gov (United States)

    Ahmad, Norazah; Hoon, Shirley Tang Gee; Ghani, Mohamed Kamel Abd; Tee, Koh Yin

    2012-06-01

    Serotyping is not sufficient to differentiate between Salmonella species that cause paratyphoid fever from the strains that cause milder gastroenteritis as these organisms share the same serotype Salmonella Paratyphi B (S. Paratyphi B). Strains causing paratyphoid fever do not ferment d-tartrate and this key feature was used in this study to determine the prevalence of these strains among the collection of S. Paratyphi B strains isolated from patients in Malaysia. A total of 105 isolates of S. Paratyphi B were discriminated into d-tartrate positive (dT+) and d-tartrate negative (dT) variants by two lead acetate test protocols and multiplex PCR. The lead acetate test protocol 1 differed from protocol 2 by a lower inoculum size and different incubation conditions while the multiplex PCR utilized 2 sets of primers targeting the ATG start codon of the gene STM3356. Lead acetate protocol 1 discriminated 97.1% of the isolates as S. Paratyphi B dT+ and 2.9% as dT while test protocol 2 discriminated all the isolates as S. Paratyphi B dT+. The multiplex PCR test identified all 105 isolates as S. Paratyphi B dT+ strains. The concordance of the lead acetate test relative to that of multiplex PCR was 97.7% and 100% for protocol 1 and 2 respectively. This study showed that S. Paratyphi B dT+ is a common causative agent of gastroenteritis in Malaysia while paratyphoid fever appears to be relatively uncommon. Multiplex PCR was shown to be a simpler, more rapid and reliable method to discriminate S. Paratyphi B than the phenotypic lead acetate test.

  8. Establishment of a novel two-probe real-time PCR for simultaneously quantification of hepatitis B virus DNA and distinguishing genotype B from non-B genotypes.

    Science.gov (United States)

    Wang, Wei; Liang, Hongpin; Zeng, Yongbin; Lin, Jinpiao; Liu, Can; Jiang, Ling; Yang, Bin; Ou, Qishui

    2014-11-01

    Establishment of a simple, rapid and economical method for quantification and genotyping of hepatitis B virus (HBV) is of great importance for clinical diagnosis and treatment of chronic hepatitis B patients. We hereby aim to develop a novel two-probe real-time PCR for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes. Conserved primers and TaqMan probes for genotype B and non-B genotypes were designed. The linear range, detection sensitivity, specificity and repeatability of the method were assessed. 539 serum samples from HBV-infected patients were assayed, and the results were compared with commercial HBV quantification and HBV genotyping kits. The detection sensitivity of the two-probe real-time PCR was 500IU/ml; the linear range was 10(3)-10(9)IU/ml, and the intra-assay CVs and inter-assay CVs were between 0.84% and 2.80%. No cross-reaction was observed between genotypes B and non-B. Of the 539 detected samples, 509 samples were HBV DNA positive. The results showed that 54.0% (275/509) of the samples were genotype B, 39.5% (201/509) were genotype non-B and 6.5% (33/509) were mixed genotype. The coincidence rate between the method and a commercial HBV DNA genotyping kit was 95.9% (488/509, kappa=0.923, PDNA qPCR kit were achieved. A novel two-probe real-time PCR method for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes was established. The assay was sensitive, specific and reproducible which can be applied to areas prevalent with HBV genotypes B and C, especially in China. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Factor Analysis, AMMI Stability Value (ASV Parameter and GGE Bi-Plot Graphical Method of Quantitative and Qualitative Traits in Potato Genotypes

    Directory of Open Access Journals (Sweden)

    Davood Hassanpanah

    2016-10-01

    Full Text Available Quantitative and qualitative traits and stability of marketable tuber yield of 14 promising potato clones, along with three commercial cultivars (Agria, Marfona and Savalan as checks, were evaluated at the Ardabil Agricultural and Natural Resources Research Station during 2013 and 2014. The experiment was based on a randomized complete block design with four replications. During growing period and after harvest, traits like main stem number per plant, plant height, tuber number and weight per plant, total and marketable tuber yield, dry matter percentage, baking type, hollow heart, tuber inner ring and discoloration of raw tuber flesh after 24 hours were measured. Combined ANONA for quantitative traits showed that there were significant differences among promising clones as to total and marketable tuber yield, tuber number and weight per plant, plant height, tuber mean weight, main stem number per plant and dry matter percentage and their interactions with year in total and marketable tuber yield. The clone 9 (397078-3 with the least amount of marketable tuber yield had significant difference with clones 4 (397045-13, 1 (397031-16, 3 (397031-11, 6 (397009-8 and 12 (397067-6 in 2013 and with clone 4 (397045-13 and Agria cultivar in 2014. The clones 4(397045-13, 1 (397031-16 and 12 (397067-6 had uniform tuber, yellow to dark-yellow skin and light-yellow to yellow flesh color, tuber shape of oval round and round, shallow to mid shallow eyes, no tuber inner ring, hollow heart and tuber inner crack and mid-late maturity. They were selected for home consumption of chips, french-fries and frying. Based on the results of factor analysis, "tuber yield", "number of tuber" and "plant structural and quality "were named as first, second and third quality determining factors respectively. In this experiment, GGE Bi-plot model and AMMI Stability Value (ASV parameter, were acceptable methods for the selection of marketable tuber yield stability which found to

  10. PCR-based detection of bacterial DNA after antimicrobial treatment is indicative of persistent, viable bacteria in the chinchilla model of otitis media.

    Science.gov (United States)

    Post, J C; Aul, J J; White, G J; Wadowsky, R M; Zavoral, T; Tabari, R; Kerber, B; Doyle, W J; Ehrlich, G D

    1996-01-01

    Bacterial deoxyribonucleic acid (DNA) has been previously detected by polymerase chain reactions (PCR) in a significant percentage of culturally-sterile pediatric middle-ear effusions. The current study was designed to determine whether this represents the existence of viable bacteria or the persistence of residual DNA in the middle-ear cleft. The middle-ear cavities of two sets of chinchillas were inoculated with either: 1) 100 colony-forming units (CFU) of live Haemophilus influenzae, 2.2 x 10(6) CFU of pasteurized Moraxella catarrhalis, and 1000 ng of DNA (>10(8) genomic equivalents) from Streptococcus pneumoniae; or 2) 100 CFU of live S pneumoniae, 2.2 x 10(6) CFU of pasteurized M catarrhalis and 1000 ng of purified DNA from H influenzae. Animals were treated with ampicillin for 5 days beginning on day 3. A single-point longitudinal study design was used for sampling to eliminate the possibility of contamination. No DNA was detectable from the heat-killed bacteria or the purified DNA after day 3. However, DNA from the live bacteria persisted through day 21, even though all specimens were culture-negative following the initiation of antimicrobial therapy. These findings indicate that purified DNA and DNA from intact but nonviable bacteria do not persist in the middle-ear cleft in the presence of an effusion, even following high copy inoculation. In contrast, antibiotic-treated bacteria persist in some viable state for weeks as evidenced by the differential ability of the PCR-based assay systems to detect the live bacteria, but not detect the heat-killed organisms.

  11. Genotyping of Hepatitis C virus isolated from hepatitis patients in Southeast of Iran by taqman realtime PCR

    International Nuclear Information System (INIS)

    Farivar, T.N.; Johari, P.

    2011-01-01

    Objectives: To check TaqMan Realtime PCR in detecting genotypes of hepatitis C virus in Iran. Methods: From July 2007 to April 2009, HCV genotyping was done on 52 patients who were referred to Research Centre for infectious Disease and Tropical Medicine, in Bou-Ali Hospital, Zahedan University of Medical Sciences. All these patients had proven hepatitis C infection. Results: Out of 52 anti HCV positive samples, 28(53.84%) had genotype 1, 2 cases (3.88 %) had genotype 2 , 12 (23.08 %) had genotype 3 and 7 (13.4 %) had genotype 4 . Mixed infection with genotypes 1 and 3 was seen in 3 cases (5.77 %). Conclusion: TaqMan probes for detecting genotyping of HCV were successful in picking genotyping of HCV infection especially those with mixed genotypes. (author)

  12. Distribution of Hepatitis C Virus Genotypes in the South Marmara Region

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    Harun Agca

    2014-03-01

    Full Text Available Aim: Hepatitis C virus (HCV is an important caustive agent of hepatitis, cirrhosis and hepatocellular carcinoma both in our country and the world. Prognosis and response to treatment is related with the genotype of HCV which has six genotypes and over a hundred quasispecies. Knowing the HCV genotype is also important for epidemiological data. In this study we aimed to investigate the HCV genotypes of samples sent to Uludag University Hospital Microbiology Laboratory which is the reference centre in the South Marmara Region. Material and Method: This study was done retrospectively to analyse the HCV patients%u2019 sera sent to our laboratory between July 2010and December 2012 for HCV genotyping. Artus HCV QS-RGQ PCR kit (Qiagene,Hilden, Germany was used in Rotor-Gene Q (Qiagene, Hilden Germany for detection of HCV RNA. HCV RNA positive samples of patients%u2019 sera were were used for genotyping by the Linear Array HCV genotyping test (Roche, NJ, USA.Results: 214 (92.6 % of total 231 patients included in the study were genotype 1, one (0.4 % was genotype 2, nine (3.9 % were genotype 3 and, seven (3.4 % were found genotype 4. Three of genotype 3 patients were of foreign nationality, two were born abroad and one of the genotype 4 patients were born abroad. Discussion: Concordant with our country data the most frequent genotype was 1, genotype 2 was seen in patients especially related with foreign countries and genotype 4 was seen rare. The importance of genotype 1, which is seen more frequent in our country and region is; resistance to antiviral treatment and prolonged treatment duration in chronic hepatitis C patients.

  13. Novel approach for CES1 genotyping

    DEFF Research Database (Denmark)

    Bjerre, Ditte; Berg Rasmussen, Henrik

    2018-01-01

    AIM: Development of a specific procedure for genotyping of CES1A1 (CES1) and CES1A2, a hybrid of CES1A1 and the pseudogene CES1P1. MATERIALS & METHODS: The number of CES1A1 and CES1A2 copies and that of CES1P1 were determined using real-time PCR. Long range PCRs followed by secondary PCRs allowed...

  14. Echinococcus granulosus genotypes in Iran

    Science.gov (United States)

    Sharafi, Seyedeh Maryam; Rostami-Nejad, Mohammad; Moazeni, Mohammad; Yousefi, Morteza; Saneie, Behnam; Hosseini-Safa, Ahmad

    2014-01-01

    Hydatidosis, caused by Echinococcus granulosus is one of the most important zoonotic diseases, throughout most parts of the world. Hydatidosis is endemic in Iran and responsible for approximately 1% of admission to surgical wards. There are extensive genetic variations within E. granulosus and 10 different genotypes (G1–G10) within this parasite have been reported. Identification of strains is important for improvement of control and prevention of the disease. No new review article presented the situation of Echinococcus granulosus genotypes in Iran in the recent years; therefore in this paper we reviewed the different studies regarding Echinococcus granulosus genotypes in Iran. PMID:24834298

  15. Rotavirus genotype shifts among Swedish children and adults-Application of a real-time PCR genotyping.

    Science.gov (United States)

    Andersson, Maria; Lindh, Magnus

    2017-11-01

    It is well known that human rotavirus group A is the most important cause of severe diarrhoea in infants and young children. Less is known about rotavirus infections in other age groups, and about how rotavirus genotypes change over time in different age groups. Develop a real-time PCR to easily genotype rotavirus strains in order to monitor the pattern of circulating genotypes. In this study, rotavirus strains in clinical samples from children and adults in Western Sweden during 2010-2014 were retrospectively genotyped by using specific amplification of VP 4 and VP 7 genes with a new developed real-rime PCR. A genotype was identified in 97% of 775 rotavirus strains. G1P[8] was the most common genotype representing 34.9%, followed by G2P[4] (28.3%), G9P[8] (11.5%), G3P[8] (8.1%), and G4P[8] (7.9%) The genotype distribution changed over time, from predominance of G1P[8] in 2010-2012 to predominance of G2P[4] in 2013-2014. There were also age-related differences, with G1P[8] being the most common genotype in children under 2 years (47.6%), and G2P[4] the most common in those over 70 years of age (46.1%.). The shift to G2P[4] in 2013-2014 was associated with a change in the age distribution, with a greater number of rotavirus positive cases in elderly than in children. By using a new real-time PCR method for genotyping we found that genotype distribution was age related and changed over time with a decreasing proportion of G1P[8]. Copyright © 2017. Published by Elsevier B.V.

  16. Direct maximum parsimony phylogeny reconstruction from genotype data.

    Science.gov (United States)

    Sridhar, Srinath; Lam, Fumei; Blelloch, Guy E; Ravi, R; Schwartz, Russell

    2007-12-05

    Maximum parsimony phylogenetic tree reconstruction from genetic variation data is a fundamental problem in computational genetics with many practical applications in population genetics, whole genome analysis, and the search for genetic predictors of disease. Efficient methods are available for reconstruction of maximum parsimony trees from haplotype data, but such data are difficult to determine directly for autosomal DNA. Data more commonly is available in the form of genotypes, which consist of conflated combinations of pairs of haplotypes from homologous chromosomes. Currently, there are no general algorithms for the direct reconstruction of maximum parsimony phylogenies from genotype data. Hence phylogenetic applications for autosomal data must therefore rely on other methods for first computationally inferring haplotypes from genotypes. In this work, we develop the first practical method for computing maximum parsimony phylogenies directly from genotype data. We show that the standard practice of first inferring haplotypes from genotypes and then reconstructing a phylogeny on the haplotypes often substantially overestimates phylogeny size. As an immediate application, our method can be used to determine the minimum number of mutations required to explain a given set of observed genotypes. Phylogeny reconstruction directly from unphased data is computationally feasible for moderate-sized problem instances and can lead to substantially more accurate tree size inferences than the standard practice of treating phasing and phylogeny construction as two separate analysis stages. The difference between the approaches is particularly important for downstream applications that require a lower-bound on the number of mutations that the genetic region has undergone.

  17. NOS3 Glu298Asp genotype and blood pressure response to endurance training: the HERITAGE family study.

    Science.gov (United States)

    Rankinen, T; Rice, T; Pérusse, L; Chagnon, Y C; Gagnon, J; Leon, A S; Skinner, J S; Wilmore, J H; Rao, D C; Bouchard, C

    2000-11-01

    Endothelium-dependent vasodilation is a mechanism that may affect blood pressure response to endurance training. Because NO plays a central role in this process, the endothelial NO synthase gene is a good candidate for the regulation of exercise blood pressure. We investigated the associations between an endothelial NO synthase gene polymorphism (Glu298Asp) and endurance training-induced changes in resting and submaximal exercise blood pressure in 471 white subjects of the HERITAGE Family Study. Two submaximal exercise tests at 50 W were conducted both before and after a 20-week endurance training program. Steady-state exercise blood pressure was measured twice in each test with an automated unit. The Glu298Asp polymorphism was typed with a PCR-based method and digestion with BAN:II. Both systolic and diastolic blood pressure at 50 W decreased in response to the training program, whereas resting blood pressure remained unchanged. The decrease in diastolic blood pressure at 50 W was greater (P=0.0005, adjusted for age, gender, baseline body mass index, and baseline diastolic blood pressure at 50 W) in the Glu/Glu homozygotes (4.4 [SEM 0.4] mm Hg, n=187) than in the heterozygotes (3.1 [0.4] mm Hg, n=213) and the Asp/Asp homozygotes (1.3 [0.7] mm Hg, n=71). The genotype accounted for 2.3% of the variance in diastolic blood pressure at 50 W training response. Both the Glu298 homozygotes and the heterozygotes had a greater (P=0.013) training-induced reduction in rate-pressure product at 50 W than the Asp298 homozygotes. These data suggest that DNA sequence variation in the endothelial NO synthase gene locus is associated with the endurance training-induced decreases in submaximal exercise diastolic blood pressure and rate-pressure product in sedentary normotensive white subjects.

  18. Porphyromonas gingivalis Fim-A genotype distribution among Colombians

    Science.gov (United States)

    Jaramillo, Adriana; Parra, Beatriz; Botero, Javier Enrique; Contreras, Adolfo

    2015-01-01

    Introduction: Porphyromonas gingivalis is associated with periodontitis and exhibit a wide array of virulence factors, including fimbriae which is encoded by the FimA gene representing six known genotypes. Objetive: To identify FimA genotypes of P. gingivalis in subjects from Cali-Colombia, including the co-infection with Aggregatibacter actinomycetemcomitans, Treponema denticola, and Tannerella forsythia. Methods: Subgingival samples were collected from 151 people exhibiting diverse periodontal condition. The occurrence of P. gingivalis, FimA genotypes and other bacteria was determined by PCR. Results: P. gingivalis was positive in 85 patients. Genotype FimA II was more prevalent without reach significant differences among study groups (54.3%), FimA IV was also prevalent in gingivitis (13.0%). A high correlation (p= 0.000) was found among P. gingivalis, T. denticola, and T. forsythia co-infection. The FimA II genotype correlated with concomitant detection of T. denticola and T. forsythia. Conclusions: Porphyromonas gingivalis was high even in the healthy group at the study population. A trend toward a greater frequency of FimA II genotype in patients with moderate and severe periodontitis was determined. The FimA II genotype was also associated with increased pocket depth, greater loss of attachment level, and patients co-infected with T. denticola and T. forsythia. PMID:26600627

  19. Genetic similarity of soybean genotypes revealed by seed protein

    Directory of Open Access Journals (Sweden)

    Nikolić Ana

    2005-01-01

    Full Text Available More accurate and complete descriptions of genotypes could help determinate future breeding strategies and facilitate introgression of new genotypes in current soybean genetic pool. The objective of this study was to characterize 20 soybean genotypes from the Maize Research Institute "Zemun Polje" collection, which have good agronomic performances, high yield, lodging and drought resistance, and low shuttering by seed proteins as biochemical markers. Seed proteins were isolated and separated by PAA electrophoresis. On the basis of the presence/absence of protein fractions coefficients of similarity were calculated as Dice and Roger and Tanamoto coefficient between pairs of genotypes. The similarity matrix was submitted for hierarchical cluster analysis of un weighted pair group using arithmetic average (UPGMA method and necessary computation were performed using NTSYS-pc program. Protein seed analysis confirmed low level of genetic diversity in soybean. The highest genetic similarity was between genotypes P9272 and Kador. According to obtained results, soybean genotypes were assigned in two larger groups and coefficients of similarity showed similar results. Because of the lack of pedigree data for analyzed genotypes, correspondence with marker data could not be determined. In plant with a narrow genetic base in their gene pool, such as soybean, protein markers may not be sufficient for characterization and study of genetic diversity.

  20. Comparative evaluation of GenoType MTBDRplus line probe assay with solid culture method in early diagnosis of multidrug resistant tuberculosis (MDR-TB at a tertiary care centre in India.

    Directory of Open Access Journals (Sweden)

    Raj N Yadav

    Full Text Available The objectives of the study were to compare the performance of line probe assay (GenoType MTBDRplus with solid culture method for an early diagnosis of multidrug resistant tuberculosis (MDR-TB, and to study the mutation patterns associated with rpoB, katG and inhA genes at a tertiary care centre in north India.In this cross-sectional study, 269 previously treated sputum-smear acid-fast bacilli (AFB positive MDR-TB suspects were enrolled from January to September 2012 at the All India Institute of Medical Sciences hospital, New Delhi. Line probe assay (LPA was performed directly on the sputum specimens and the results were compared with that of conventional drug susceptibility testing (DST on solid media [Lowenstein Jensen (LJ method].DST results by LPA and LJ methods were compared in 242 MDR-TB suspects. The LPA detected rifampicin (RIF resistance in 70 of 71 cases, isoniazid (INH resistance in 86 of 93 cases, and MDR-TB in 66 of 68 cases as compared to the conventional method. Overall (rifampicin, isoniazid and MDR-TB concordance of the LPA with the conventional DST was 96%. Sensitivity and specificity were 98% and 99% respectively for detection of RIF resistance; 92% and 99% respectively for detection of INH resistance; 97% and 100% respectively for detection of MDR-TB. Frequencies of katG gene, inhA gene and combined katG and inhA gene mutations conferring all INH resistance were 72/87 (83%, 10/87 (11% and 5/87 (6% respectively. The turnaround time of the LPA test was 48 hours.The LPA test provides an early diagnosis of monoresistance to isoniazid and rifampicin and is highly sensitive and specific for an early diagnosis of MDR-TB. Based on these findings, it is concluded that the LPA test can be useful in early diagnosis of drug resistant TB in high TB burden countries.

  1. Efficient genome-wide genotyping strategies and data integration in crop plants.

    Science.gov (United States)

    Torkamaneh, Davoud; Boyle, Brian; Belzile, François

    2018-03-01

    Next-generation sequencing (NGS) has revolutionized plant and animal research by providing powerful genotyping methods. This review describes and discusses the advantages, challenges and, most importantly, solutions to facilitate data processing, the handling of missing data, and cross-platform data integration. Next-generation sequencing technologies provide powerful and flexible genotyping methods to plant breeders and researchers. These methods offer a wide range of applications from genome-wide analysis to routine screening with a high level of accuracy and reproducibility. Furthermore, they provide a straightforward workflow to identify, validate, and screen genetic variants in a short time with a low cost. NGS-based genotyping methods include whole-genome re-sequencing, SNP arrays, and reduced representation sequencing, which are widely applied in crops. The main challenges facing breeders and geneticists today is how to choose an appropriate genotyping method and how to integrate genotyping data sets obtained from various sources. Here, we review and discuss the advantages and challenges of several NGS methods for genome-wide genetic marker development and genotyping in crop plants. We also discuss how imputation methods can be used to both fill in missing data in genotypic data sets and to integrate data sets obtained using different genotyping tools. It is our hope that this synthetic view of genotyping methods will help geneticists and breeders to integrate these NGS-based methods in crop plant breeding and research.

  2. SPATIAL ANALYSIS TO SUPPORT GEOGRAPHIC TARGETING OF GENOTYPES TO ENVIRONMENTS

    Directory of Open Access Journals (Sweden)

    Glenn eHyman

    2013-03-01

    Full Text Available Crop improvement efforts have benefited greatly from advances in available data, computing technology and methods for targeting genotypes to environments. These advances support the analysis of genotype by environment interactions to understand how well a genotype adapts to environmental conditions. This paper reviews the use of spatial analysis to support crop improvement research aimed at matching genotypes to their most appropriate environmental niches. Better data sets are now available on soils, weather and climate, elevation, vegetation, crop distribution and local conditions where genotypes are tested in experimental trial sites. The improved data are now combined with spatial analysis methods to compare environmental conditions across sites, create agro-ecological region maps and assess environment change. Climate, elevation and vegetation data sets are now widely available, supporting analyses that were much more difficult even five or ten years ago. While detailed soil data for many parts of the world remains difficult to acquire for crop improvement studies, new advances in digital soil mapping are likely to improve our capacity. Site analysis and matching and regional targeting methods have advanced in parallel to data and technology improvements. All these developments have increased our capacity to link genotype to phenotype and point to a vast potential to improve crop adaptation efforts.

  3. A novel rapid genotyping technique for Collie eye anomaly: SYBR Green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on Flinders Technology Associates filter paper.

    Science.gov (United States)

    Chang, Hye-Sook; Mizukami, Keijiro; Yabuki, Akira; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Arai, Toshiro; Yamato, Osamu

    2010-09-01

    Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

  4. Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.

    Directory of Open Access Journals (Sweden)

    Brian O'Farrell

    Full Text Available Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of 200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost.

  5. [Modern methods application of genotyping of infectious diseases pathogens in the context of operational work of specialized anti-epidemic team during the XXII Olympic Winter Games and XI Paralympic Winter Games].

    Science.gov (United States)

    Kuzkin, B P; Kulichenko, A N; Volynkina, A S; Efremenko, D V; Kuznetsova, I V; Kotenev, E S; Lyamkin, G I; Kartsev, N N; Klindukhov, V P

    2015-01-01

    This paper considers the experience of genotyping and sequencing technologies in laboratories of specialized anti-epidemic team (SAET) during the XXII Olympic Winter Games and XI Paralympic Winter Games of 2014 in Sochi. The work carried out during the pre-Olympic period on performance of readiness by SAET for these studies is analyzed. The results of genotyping strains of pathogens during the Olympic Games are presented. A conclusion about the effectiveness of the use of molecular genetic techniques in terms of SAET is made.

  6. A field trial of a PCR-based Mansonella ozzardi diagnosis assay detects high-levels of submicroscopic M. ozzardi infections in both venous blood samples and FTA card dried blood spots.

    Science.gov (United States)

    Medeiros, Jansen Fernandes; Almeida, Tatiana Amaral Pires; Silva, Lucyane Bastos Tavares; Rubio, Jose Miguel; Crainey, James Lee; Pessoa, Felipe Arley Costa; Luz, Sergio Luiz Bessa

    2015-05-20

    Mansonella ozzardi is a poorly understood human filarial parasite with a broad distribution throughout Latin America. Most of what is known about its parasitism has come from epidemiological studies that have estimated parasite incidence using light microscopy. Light microscopy can, however, miss lighter, submicroscopic, infections. In this study we have compared M. ozzardi incidence estimates made using light microscopy, with estimates made using PCR. 214 DNA extracts made from Large Volume Venous Blood Samples (LVVBS) were taken from volunteers from two study sites in the Rio Solimões region: Codajás [n = 109] and Tefé [n = 105] and were subsequently assayed for M. ozzardi parasitism using a diagnostic PCR (Mo-dPCR). Peripheral finger-prick blood samples were taken from the same individuals and used for microscopic examination. Finger-prick blood, taken from individuals from Tefé, was also used for the creation of FTAcard dried blood spots (DBS) that were subsequently subjected to Mo-dPCR. Overall M. ozzardi incidence estimates made with LVVBS PCRs were 1.8 times higher than those made using microscopy (44.9% [96/214] compared with 24.3% [52/214]) and 1.5 times higher than the PCR estimates made from FTAcard DBS (48/105 versus 31/105). PCR-based detection of FTAcard DBS proved 1.3 times more sensitive at diagnosing infections from peripheral blood samples than light microscopy did: detecting 24/105 compared with 31/105. PCR of LVVBS reported the fewest number of false negatives, detecting: 44 of 52 (84.6%) individuals diagnosed by microscopy; 27 of 31 (87.1%) of those diagnosed positive from DBSs and 17 out of 18 (94.4%) of those diagnosed as positive by both alternative methodologies. In this study, Mo-dPCR of LVVBS was by far the most sensitive method of detecting M. ozzardi infections and detected submicroscopic infections. Mo-dPCR FTAcard DBS also provided a more sensitive test for M. ozzardi diagnosis than light microscopy based diagnosis did and

  7. Screening cotton genotypes for seedling drought tolerance

    Directory of Open Access Journals (Sweden)

    Penna Julio C. Viglioni

    1998-01-01

    Full Text Available The objectives of this study were to adapt a screening method previously used to assess seedling drought tolerance in cereals for use in cotton (Gossypium hirsutum L. and to identify tolerant accessions among a wide range of genotypes. Ninety genotypes were screened in seven growth chamber experiments. Fifteen-day-old seedlings were subjected to four 4-day drought cycles, and plant survival was evaluated after each cycle. Three cycles are probably the minimum required in cotton work. Significant differences (at the 0.05 level or lower among entries were obtained in four of the seven experiments. A "confirmation test" with entries previously evaluated as "tolerant" (high survival and "susceptible" (low survival was run. A number of entries duplicated their earlier performance, but others did not, which indicates the need to reevaluate selections. Germplasms considered tolerant included: `IAC-13-1', `IAC-RM4-SM5', `Minas Sertaneja', `Acala 1517E-1' and `4521'. In general, the technique is simple, though time-consuming, with practical value for screening a large number of genotypes. Results from the screening tests generally agreed with field information. The screening procedure is suitable to select tolerant accessions from among a large number of entries in germplasm collections as a preliminary step in breeding for drought tolerance. This research also demonstrated the need to characterize the internal lack of uniformity in growth chambers to allow for adequate designs of experiments.

  8. Sofosbuvir based treatment of chronic hepatitis C genotype 3 infections

    DEFF Research Database (Denmark)

    Dalgard, Olav; Weiland, Ola; Noraberg, Geir

    2017-01-01

    BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) genotype 3 infection with advanced liver disease has emerged as the most challenging to treat. We retrospectively assessed the treatment outcome of sofosbuvir (SOF) based regimes for treatment of HCV genotype 3 infections in a real life setting...... in Scandinavia. METHODS: Consecutive patients with chronic HCV genotype 3 infection were enrolled at 16 treatment centers in Denmark, Sweden, Norway and Finland. Patients who had received a SOF containing regimen were included. The fibrosis stage was evaluated by liver biopsy or transient liver elastography...... was similar for all treatment regimens, but lower in men (p = 0.042), and in patients with decompensated liver disease (p = 0.004). CONCLUSION: We found that sofosbuvir based treatment in a real-life setting could offer SVR rates exceeding 90% in patients with HCV genotype 3 infection and advanced liver...

  9. Giardia and Cryptosporidium species and genotypes in coyotes (Canis latrans).

    Science.gov (United States)

    Trout, James M; Santín, Mónica; Fayer, Ronald

    2006-06-01

    Feces and duodenal scrapings were collected from 22 coyotes (Canis latrans) killed in managed hunts in northeastern Pennsylvania. Polymerase chain reaction (PCR) methods were used to detect Giardia and Cryptosporidium spp. PCR-amplified fragments of Giardia and Cryptosporidium spp. SSU-rRNA genes were subjected to DNA sequence analysis for species/genotype determination. Seven coyotes (32%) were positive for G. duodenalis: three assemblage C, three assemblage D, and one assemblage B. Six coyotes (27%) were positive for Cryptosporidium spp. One isolate shared 99.7% homology with C. muris, whereas five others (23%) shared 100% homology with C. canis, coyote genotype. This is the first report on multiple genotypes of Giardia spp. in coyotes and on the prevalence of Cryptosporidium spp. genotypes in coyotes.

  10. Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

    DEFF Research Database (Denmark)

    Kaczmarczyk, Agnieszka Ewa; Bowra, Steve; Elek, Zoltan

    2012-01-01

    expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging. Results We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and gamma......-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families...... and its subgroups. More over the results indicate the genotypic specific gene expression. Conclusions Quantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal...

  11. Genotyping panel for assessing response to cancer chemotherapy

    Directory of Open Access Journals (Sweden)

    Hampel Heather

    2008-06-01

    Full Text Available Abstract Background Variants in numerous genes are thought to affect the success or failure of cancer chemotherapy. Interindividual variability can result from genes involved in drug metabolism and transport, drug targets (receptors, enzymes, etc, and proteins relevant to cell survival (e.g., cell cycle, DNA repair, and apoptosis. The purpose of the current study is to establish a flexible, cost-effective, high-throughput genotyping platform for candidate genes involved in chemoresistance and -sensitivity, and treatment outcomes. Methods We have adopted SNPlex for genotyping 432 single nucleotide polymorphisms (SNPs in 160 candidate genes implicated in response to anticancer chemotherapy. Results The genotyping panels were applied to 39 patients with chronic lymphocytic leukemia undergoing flavopiridol chemotherapy, and 90 patients with colorectal cancer. 408 SNPs (94% produced successful genotyping results. Additional genotyping methods were established for polymorphisms undetectable by SNPlex, including multiplexed SNaPshot for CYP2D6 SNPs, and PCR amplification with fluorescently labeled primers for the UGT1A1 promoter (TAnTAA repeat polymorphism. Conclusion This genotyping panel is useful for supporting clinical anticancer drug trials to identify polymorphisms that contribute to interindividual variability in drug response. Availability of population genetic data across multiple studies has the potential to yield genetic biomarkers for optimizing anticancer therapy.

  12. An epidemiologic survey of methicillin-resistant Staphylococcus aureus by combined use of mec-HVR genotyping and toxin genotyping in a university hospital in Japan.

    Science.gov (United States)

    Nishi, Junichiro; Yoshinaga, Masao; Miyanohara, Hiroaki; Kawahara, Motoshi; Kawabata, Masaharu; Motoya, Toshiro; Owaki, Tetsuhiro; Oiso, Shigeru; Kawakami, Masayuki; Kamewari, Shigeko; Koyama, Yumiko; Wakimoto, Naoko; Tokuda, Koichi; Manago, Kunihiro; Maruyama, Ikuro

    2002-09-01

    To evaluate the usefulness of an assay using two polymerase chain reaction-based genotyping methods in the practical surveillance of methicillin-resistant Staphylococcus aureus (MRSA). Nosocomial infection and colonization were surveyed monthly in a university hospital in Japan for 20 months. Genotyping with mec-HVR is based on the size of the mec-associated hypervariable region amplified by polymerase chain reaction. Toxin genotyping uses a multiplex polymerase chain reaction method to amplify eight staphylococcal toxin genes. Eight hundred nine MRSA isolates were classified into 49 genotypes. We observed differing prevalences of genotypes for different hospital wards, and could rapidly demonstrate the similarity of genotype for outbreak isolates. The incidence of genotype D: SEC/TSST1 was significantly higher in isolates causing nosocomial infections (49.5%; 48 of 97) than in nasal isolates (31.4%; 54 of 172) (P = .004), suggesting that this genotype may represent the nosocomial strains. The combined use of these two genotyping methods resulted in improved discriminatory ability and should be further investigated.

  13. Discrepancy between Hepatitis C Virus Genotypes and NS4-Based Serotypes: Association with Their Subgenomic Sequences

    Directory of Open Access Journals (Sweden)

    Nan Nwe Win

    2017-01-01

    Full Text Available Determination of hepatitis C virus (HCV genotypes plays an important role in the direct-acting agent era. Discrepancies between HCV genotyping and serotyping assays are occasionally observed. Eighteen samples with discrepant results between genotyping and serotyping methods were analyzed. HCV serotyping and genotyping were based on the HCV nonstructural 4 (NS4 region and 5′-untranslated region (5′-UTR, respectively. HCV core and NS4 regions were chosen to be sequenced and were compared with the genotyping and serotyping results. Deep sequencing was also performed for the corresponding HCV NS4 regions. Seventeen out of 18 discrepant samples could be sequenced by the Sanger method. Both HCV core and NS4 sequences were concordant with that of genotyping in the 5′-UTR in all 17 samples. In cloning analysis of the HCV NS4 region, there were several amino acid variations, but each sequence was much closer to the peptide with the same genotype. Deep sequencing revealed that minor clones with different subgenotypes existed in two of the 17 samples. Genotyping by genome amplification showed high consistency, while several false reactions were detected by serotyping. The deep sequencing method also provides accurate genotyping results and may be useful for analyzing discrepant cases. HCV genotyping should be correctly determined before antiviral treatment.

  14. Comparison of DNA Quantification Methods for Next Generation Sequencing.

    Science.gov (United States)

    Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

    2016-04-06

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

  15. Haplotype-Based Genotyping in Polyploids

    Directory of Open Access Journals (Sweden)

    Josh P. Clevenger

    2018-04-01

    Full Text Available Accurate identification of polymorphisms from sequence data is crucial to unlocking the potential of high throughput sequencing for genomics. Single nucleotide polymorphisms (SNPs are difficult to accurately identify in polyploid crops due to the duplicative nature of polyploid genomes leading to low confidence in the true alignment of short reads. Implementing a haplotype-based method in contrasting subgenome-specific sequences leads to higher accuracy of SNP identification in polyploids. To test this method, a large-scale 48K SNP array (Axiom Arachis2 was developed for Arachis hypogaea (peanut, an allotetraploid, in which 1,674 haplotype-based SNPs were included. Results of the array show that 74% of the haplotype-based SNP markers could be validated, which is considerably higher than previous methods used for peanut. The haplotype method has been implemented in a standalone program, HAPLOSWEEP, which takes as input bam files and a vcf file and identifies haplotype-based markers. Haplotype discovery can be made within single reads or span paired reads, and can leverage long read technology by targeting any length of haplotype. Haplotype-based genotyping is applicable in all allopolyploid genomes and provides confidence in marker identification and in silico-based genotyping for polyploid genomics.

  16. Genotyping Sleep Disorders Patients

    OpenAIRE

    Kripke, Daniel F.; Shadan, Farhad F.; Dawson, Arthur; Cronin, John W.; Jamil, Shazia M.; Grizas, Alexandra P.; Koziol, James A.; Kline, Lawrence E.

    2010-01-01

    Objective The genetic susceptibility factors underlying sleep disorders might help us predict prognoses and responses to treatment. Several candidate polymorphisms for sleep disorders have been proposed, but there has as yet inadequate replication or validation that the candidates may be useful in the clinical setting. Methods To assess the validity of several candidate associations, we obtained saliva deoxyribonucleic acid (DNA) samples and clinical information from 360 consenting research p...

  17. Evaluation of allelopathic potential of safflower genotypes (Carthamus tinctorius L.

    Directory of Open Access Journals (Sweden)

    Motamedi Marzieh

    2016-12-01

    Full Text Available Forty safflower genotypes were grown under normal irrigation and drought stress. In the first experiment, the allelopathic potential of shoot residues was evaluated using the sandwich method. Each genotype residue (0.4 g was placed in a sterile Petri dish and two layers of agar were poured on that. Radish seeds were placed on agar medium. The radish seeds were cultivated without safflower residues as the controls. The length of the radicle, hypocotyl, and fresh biomass weight and seed germination percentages were measured. A pot experiment was also done on two genotypes with the highest and two with the lowest allelopathic activity selected after screening genotypes in the first experiment. Before entering the reproductive phase, irrigation treatments (normal irrigation and drought stress were applied. Shoots were harvested, dried, milled and mixed with the topsoil of new pots and then radish seeds were sown. The pots with safflower genotypes were used to evaluate the effect of root residue allelopathy. The shoot length, fresh biomass weight, and germination percentage were measured. Different safflower genotypes showed varied allelopathic potential. The results of the first experiment showed that Egypt and Iran-Khorasan genotypes caused maximum inhibitory responses and Australia and Iran-Kerman genotypes resulted in minimum inhibitory responses on radish seedling growth. Fresh biomass weight had the most sensitivity to safflower residues. The results of the pot experiment were consistent with the results of in vitro experiments. Residues produced under drought stress had more inhibitory effects on the measured traits. Safflower root residue may have a higher level of allelochemicals or different allelochemicals than shoot residue.

  18. Microsatellite genotyping and genome-wide single nucleotide polymorphism-based indices of Plasmodium falciparum diversity within clinical infections.

    Science.gov (United States)

    Murray, Lee; Mobegi, Victor A; Duffy, Craig W; Assefa, Samuel A; Kwiatkowski, Dominic P; Laman, Eugene; Loua, Kovana M; Conway, David J

    2016-05-12

    In regions where malaria is endemic, individuals are often infected with multiple distinct parasite genotypes, a situation that may impact on evolution of parasite virulence and drug resistance. Most approaches to studying genotypic diversity have involved analysis of a modest number of polymorphic loci, although whole genome sequencing enables a broader characterisation of samples. PCR-based microsatellite typing of a panel of ten loci was performed on Plasmodium falciparum in 95 clinical isolates from a highly endemic area in the Republic of Guinea, to characterize within-isolate genetic diversity. Separately, single nucleotide polymorphism (SNP) data from genome-wide short-read sequences of the same samples were used to derive within-isolate fixation indices (F ws), an inverse measure of diversity within each isolate compared to overall local genetic diversity. The latter indices were compared with the microsatellite results, and also with indices derived by randomly sampling modest numbers of SNPs. As expected, the number of microsatellite loci with more than one allele in each isolate was highly significantly inversely correlated with the genome-wide F ws fixation index (r = -0.88, P 10 % had high correlation (r > 0.90) with the index derived using all SNPs. Different types of data give highly correlated indices of within-infection diversity, although PCR-based analysis detects low-level minority genotypes not apparent in bulk sequence analysis. When whole-genome data are not obtainable, quantitative assay of ten or more SNPs can yield a reasonably accurate estimate of the within-infection fixation index (F ws).

  19. FTO genotype and weight loss

    DEFF Research Database (Denmark)

    Livingstone, Katherine M; Celis-Morales, Carlos; Papandonatos, George D

    2016-01-01

    : Ovid Medline, Scopus, Embase, and Cochrane from inception to November 2015. ELIGIBILITY CRITERIA FOR STUDY SELECTION: Randomised controlled trials in overweight or obese adults reporting reduction in body mass index, body weight, or waist circumference by FTO genotype (rs9939609 or a proxy) after...

  20. FTO genotype and weight loss

    DEFF Research Database (Denmark)

    Livingstone, Katherine M; Celis-Morales, Carlos; Papandonatos, George D

    2016-01-01

    OBJECTIVE: To assess the effect of the FTO genotype on weight loss after dietary, physical activity, or drug based interventions in randomised controlled trials. DESIGN: Systematic review and random effects meta-analysis of individual participant data from randomised controlled trials. DATA SOURC...

  1. 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.

  2. Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples.

    Science.gov (United States)

    Schmidt, H; Taniwaki, M H; Vogel, R F; Niessen, L

    2004-01-01

    The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.

  3. Histomorphological changes in hepatitis C non-responders with respect to viral genotypes

    International Nuclear Information System (INIS)

    Adnan, U.; Mirza, T.; Naz, E.; Aziz, S.

    2013-01-01

    Objective: To evaluate the distinct histopathological changes of chronic hepatitis C (CHC) non-responders in association with viral genotypes. Methods: This cross-sectional study was conducted at the histopathology section of the Dow Diagnostic Research and Reference Laboratory, Dow University of Health Sciences in collaboration with Sarwar Zuberi Liver Centre, Civil Hospital, Karachi from September 2009 to August 2011. Seventy-five non-responders (end-treatment-response [ETR] positive patients) from a consecutive series of viral-RNA positive CHC patients with known genotypes were selected. Their genotypes and pertinent clinical history was recorded. They were subjected to liver biopsies which were assessed for grade, stage, steatosis, stainable iron and characteristic histological lesions. Results: Majority of the patients (63, 84%) had genotype 3 while 12(16%) cases had genotype 1. The genotype 1 patients had significantly higher scores of inflammation (p<0.03) and fibrosis (p<0.04) as compared to genotype 3. Steatosis was significantly present in all genotype 3 patients in higher scores (p<0.001) compared to genotype 1. Stainable iron scores were generally low in the patients in this study, however, it was more commonly seen in genotype 3. The distribution of characteristic histological lesions was noteworthy in both the groups, irrespective of genotype. Conclusion: In this series, the predominant genotype was 3. However, genotype 1 patients were more prone to the aggressive nature of the disease with significantly higher scores of inflammation and fibrosis. Steatosis was characteristically observed in genotype 3 group. Stainable iron could not be attributed as a cause of non-response. (author)

  4. Improved Ancestry Estimation for both Genotyping and Sequencing Data using Projection Procrustes Analysis and Genotype Imputation

    Science.gov (United States)

    Wang, Chaolong; Zhan, Xiaowei; Liang, Liming; Abecasis, Gonçalo R.; Lin, Xihong

    2015-01-01

    Accurate estimation of individual ancestry is important in genetic association studies, especially when a large number of samples are collected from multiple sources. However, existing approaches developed for genome-wide SNP data do not work well with modest amounts of genetic data, such as in targeted sequencing or exome chip genotyping experiments. We propose a statistical framework to estimate individual ancestry in a principal component ancestry map generated by a reference set of individuals. This framework extends and improves upon our previous method for estimating ancestry using low-coverage sequence reads (LASER 1.0) to analyze either genotyping or sequencing data. In particular, we introduce a projection Procrustes analysis approach that uses high-dimensional principal components to estimate ancestry in a low-dimensional reference space. Using extensive simulations and empirical data examples, we show that our new method (LASER 2.0), combined with genotype imputation on the reference individuals, can substantially outperform LASER 1.0 in estimating fine-scale genetic ancestry. Specifically, LASER 2.0 can accurately estimate fine-scale ancestry within Europe using either exome chip genotypes or targeted sequencing data with off-target coverage as low as 0.05×. Under the framework of LASER 2.0, we can estimate individual ancestry in a shared reference space for samples assayed at different loci or by different techniques. Therefore, our ancestry estimation method will accelerate discovery in disease association studies not only by helping model ancestry within individual studies but also by facilitating combined analysis of genetic data from multiple sources. PMID:26027497

  5. Antioxidant capacity of anthocyanins from acerola genotypes

    Directory of Open Access Journals (Sweden)

    Vera Lúcia Arroxelas Galvão De Lima

    2011-03-01

    Full Text Available Anthocyanins from 12 acerola genotypes cultivated at the Active Germplasm Bank at Federal Rural University of Pernambuco were isolated for antioxidant potential evaluation. The antioxidant activity and radical scavenging capacity of the anthocyanin isolates were measured according to the β-carotene bleaching method and 1,1-diphenyl-2-picrylhydrazyl (DPPH free radical scavenging assay, respectively. The antioxidant activity varied from 25.58 to 47.04% at 0.2 mg.mL-1, and it was measured using the β-carotene bleaching method. The free radical scavenging capacity increased according to the increase in concentration and reaction time by the DPPH assay. At 16.7 μg.mL-1 concentration and after 5 minutes and 2 hours reaction time, the percentage of scavenged radicals varied from 36.97 to 63.92% and 73.27 to 94.54%, respectively. Therefore, the antioxidant capacity of acerola anthocyanins varied amongst acerola genotypes and methods used. The anthocyanins present in this fruit may supply substantial dietary source of antioxidant which may promote health and produce disease prevention effects.

  6. In vitro screening of potato genotypes for osmotic stress tolerance

    Directory of Open Access Journals (Sweden)

    Gelmesa Dandena

    2017-02-01

    Full Text Available Potato (Solanum tuberosum L. is a cool season crop which is susceptible to both drought and heat stresses. Lack of suitable varieties of the crop adapted to drought-prone areas of the lowland tropics deprives farmers living in such areas the opportunity to produce and use the crop as a source of food and income. As a step towards developing such varieties, the present research was conducted to evaluate different potato genotypes for osmotic stress tolerance under in vitro conditions and identify drought tolerant genotypes for future field evaluation. The experiment was carried out at the Leibniz University of Hannover, Germany, by inducing osmotic stress using sorbitol at two concentrations (0.1 and 0.2 M in the culture medium. A total of 43 genotypes collected from different sources (27 advanced clones from CIP, nine improved varieties, and seven farmers’ cultivars were used in a completely randomized design with four replications in two rounds. Data were collected on root and shoot growth. The results revealed that the main effects of genotype, sorbitol treatment, and their interactions significantly (P < 0.01 influenced root and shoot growthrelated traits. Under osmotic stress, all the measured root and shoot growth traits were significantly correlated. The dendrogram obtained from the unweighted pair group method with arithmetic mean allowed grouping of the genotypes into tolerant, moderately tolerant, and susceptible ones to a sorbitol concentration of 0.2 M in the culture medium. Five advanced clones (CIP304350.100, CIP304405.47, CIP392745.7, CIP388676.1, and CIP388615.22 produced shoots and rooted earlier than all other genotypes, with higher root numbers, root length, shoot and root mass under osmotic stress conditions induced by sorbitol. Some of these genotypes had been previously identified as drought-tolerant under field conditions, suggesting the capacity of the in vitro evaluation method to predict drought stress tolerant

  7. Epidemiological manifestations of hepatitis C virus genotypes and its association with potential risk factors among Libyan patients

    Directory of Open Access Journals (Sweden)

    Daw Mohamed A

    2010-11-01

    Full Text Available Abstract Background The information on hepatitis C virus genotypes and subtypes among Libyan population and its association with various risk factors is not known. The objectives of this study were to determine the epidemiological manifestations of HCV genotypes among Libyan patients and their association with certain potential risk factors. Methods A total of 1240 of HCV infected patients registered at Tripoli Medical Centre were studied in five years period from January 2005 to October 2009. The information were reviewed and the data were collected. A sample from each patient (785 male; 455 female was analysed for genotyping and sub-typing using specific genotyping assay. The information was correlated with the risk factors studied and the statistical data were analyzed using SPSS version 11.5. Results Off the total patients studied, four different genotypes were reported, including genotypes 1, 2, 3, and 4. Genotype4 was the commonest (35.7%, followed by genotype1 (32.6%. According to subtypes 28% were unclassified genotype 4, 14.6% were genotype 1b and some patients infected with more than one subtype (2.3% genotype 4c/d, 1% genotype 2a/c. Genotypes 1 was the commonest among males, while genotype 4 among females. According to the risk factors studied, Genotype1 and genotype 4 were found with most of the risk factors. Though they were particularly evident surgical intervention, dental procedures and blood transfusion while genotype 1 was only followed by genotype 3 mainly which mainly associated with certain risk groups such as intravenous drug abusers. Conclusion Here in we report on a detailed description of HCV genotype among Libyans. The most common genotype was type 4 followed by genotype 1, other genotypes were also reported at a low rate. The distribution of such genotypes were also variable according to gender and age. The commonly prevalent genotypes found to be attributable to the medical -related transmission of HCV, such as blood

  8. Hepatitis C Virus: Virology and Genotypes

    KAUST Repository

    Abdelaziz, Ahmed

    2017-01-01

    Hepatitis C virus (HCV) is a major causative agent of chronic liver disease worldwide. HCV is characterized by genetic heterogeneity, with at least six genotypes identified. The geographic distribution of genotypes has shown variations in different

  9. Study of Various HCV Genotypes in Patients Managing by Referral Clinic in Yazd Province

    Directory of Open Access Journals (Sweden)

    M Pedarzadeh

    2012-02-01

    Full Text Available Introduction: Determining virus genotype is a major factor for initiation of treatment because various kinds of genotypes need different antiviral drugs. Distribution of hepatitis C genotype in the word is variable in each country or even in each province. So we need to determine distribution pattern of hepatitis C genotype in our region. This study was performed in referral clinic of Yazd province. Methods: This was a descriptive study conducted between 2007 and 2010 on patients who were observed by Yazd referral clinic (the clinic for evaluating and management of patients with high risk behaviors. Ninety two patients who had positive RIBA test for hepatitis C infection were randomly selected and entered the study. Genotyping was performed using RT-PCR method. The primer was "universal primer HCV". Prevalence of various genotypes was analyzed according to gender, addiction and co- existence of HCV-HIV infection. Personal information and laboratory results were analyzed using SPSS. Results: The most common genotype in our study was genotype 3a (65% of cases, followed by 1a (35%. Globally 83% of patients were IV drug addict. Genotype distribution in these patients was similar to others. Fifteen patients had co-infection of HCV-HIV, and 47% of them were contaminated by genotype 1a and 53% with 3a. We could not find any patient contaminated with genotypes 2 or 4. No other genotypes except 1 & 3 or mixed genotype infection could be determined in our patients. Twenty three percent of patients had negative PCR despite positive RIBA test. This indicates that self improvement from acute hepatitis C infection in IV drug addict patients is similar to other people. Conclusion: According to the results of our study, about 2/3 of patients were infected by genotype 3a. This kind of chronic hepatitis C shows a better response to treatment comparing genotype 1a (or 1b with shorter duration and lower cost drugs. But despite higher incidence of genotype 3a, we

  10. Genomic prediction when some animals are not genotyped

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    Lund Mogens S

    2010-01-01

    Full Text Available Abstract Background The use of genomic selection in breeding programs may increase the rate of genetic improvement, reduce the generation time, and provide higher accuracy of estimated breeding values (EBVs. A number of different methods have been developed for genomic prediction of breeding values, but many of them assume that all animals have been genotyped. In practice, not all animals are genotyped, and the methods have to be adapted to this situation. Results In this paper we provide an extension of a linear mixed model method for genomic prediction to the situation with non-genotyped animals. The model specifies that a breeding value is the sum of a genomic and a polygenic genetic random effect, where genomic genetic random effects are correlated with a genomic relationship matrix constructed from markers and the polygenic genetic random effects are correlated with the usual relationship matrix. The extension of the model to non-genotyped animals is made by using the pedigree to derive an extension of the genomic relationship matrix to non-genotyped animals. As a result, in the extended model the estimated breeding values are obtained by blending the information used to compute traditional EBVs and the information used to compute purely genomic EBVs. Parameters in the model are estimated using average information REML and estimated breeding values are best linear unbiased predictions (BLUPs. The method is illustrated using a simulated data set. Conclusions The extension of the method to non-genotyped animals presented in this paper makes it possible to integrate all the genomic, pedigree and phenotype information into a one-step procedure for genomic prediction. Such a one-step procedure results in more accurate estimated breeding values and has the potential to become the standard tool for genomic prediction of breeding values in future practical evaluations in pig and cattle breeding.

  11. Genotype x environment interaction and optimum resource ...

    African Journals Online (AJOL)

    ... x E) interaction and to determine the optimum resource allocation for cassava yield trials. The effects of environment, genotype and G x E interaction were highly significant for all yield traits. Variations due to G x E interaction were greater than those due to genotypic differences for all yield traits. Genotype x location x year ...

  12. Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009-2010.

    Science.gov (United States)

    Shak, Emma B; France, Anne Marie; Cowan, Lauren; Starks, Angela M; Grant, Juliana

    2015-01-01

    Genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control through detection of possible outbreaks. However, 20% of U.S. cases do not have an isolate for testing, and 10% of cases with isolates do not have a genotype reported. TB outbreaks in populations with incomplete genotyping data might be missed by genotyping-based outbreak detection. Therefore, we assessed the representativeness of TB genotyping data by comparing characteristics of cases reported during January 1, 2009-December 31, 2010, that had a genotype result with those cases that did not. Of 22,476 cases, 14,922 (66%) had a genotype result. Cases without genotype results were more likely to be patients <19 years of age, with unknown HIV status, of female sex, U.S.-born, and with no recent history of homelessness or substance abuse. Although cases with a genotype result are largely representative of all reported U.S. TB cases, outbreak detection methods that rely solely on genotyping data may underestimate TB transmission among certain groups.

  13. Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009–2010

    Science.gov (United States)

    Shak, Emma B.; Cowan, Lauren; Starks, Angela M.; Grant, Juliana

    2015-01-01

    Genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control through detection of possible outbreaks. However, 20% of U.S. cases do not have an isolate for testing, and 10% of cases with isolates do not have a genotype reported. TB outbreaks in populations with incomplete genotyping data might be missed by genotyping-based outbreak detection. Therefore, we assessed the representativeness of TB genotyping data by comparing characteristics of cases reported during January 1, 2009–December 31, 2010, that had a genotype result with those cases that did not. Of 22,476 cases, 14,922 (66%) had a genotype result. Cases without genotype results were more likely to be patients <19 years of age, with unknown HIV status, of female sex, U.S.-born, and with no recent history of homelessness or substance abuse. Although cases with a genotype result are largely representative of all reported U.S. TB cases, outbreak detection methods that rely solely on genotyping data may underestimate TB transmission among certain groups. PMID:26556930

  14. Hepatitis C virus genotypes: A plausible association with viral loads

    Directory of Open Access Journals (Sweden)

    Salma Ghulam Nabi

    2013-01-01

    Full Text Available Background and Aim: The basic aim of this study was to find out the association of genotypes with host age, gender and viral load. Material and Methods: The present study was conducted at Social Security Hospital, Pakistan. This study included 320 patients with chronic hepatitis C virus (HCV infection who were referred to the hospital between November 2011 and July 2012. HCV viral detection and genotyping was performed and the association was seen between genotypes and host age, gender and viral load. Results : The analysis revealed the presence of genotypes 1 and 3 with further subtypes 1a, 1b, 3a, 3b and mixed genotypes 1b + 3a, 1b + 3b and 3a + 3b. Viral load quantification was carried out in all 151 HCV ribonucleic acid (RNA positive patients. The genotype 3a was observed in 124 (82.12% patients, 3b was found in 21 (13.91%, 1a was seen in 2 (1.32%, 1b in 1 (0.66%, mixed infection with 1b + 3a in 1 (0.66%, 1b + 3b in 1 (0.66% and 3a + 3b was also found in 1 (0.66% patient. Viral load quantification was carried out in all 151 HCV RNA positive patients and was compared between the various genotypes. The mean viral load in patients infected with genotype 1a was 2.75 × 10 6 , 1b 3.9 × 10 6 , 3a 2.65 × 10 6 , 3b 2.51 × 10 6 , 1b + 3a 3.4 × 106, 1b + 3b 2.7 × 106 and 3a + 3b 3.5 × 10 6 . An association between different types of genotypes and viral load was observed. Conclusion : Further studies should be carried out to determine the association of viral load with different genotypes so that sufficient data is available and can be used to determine the type and duration of therapy needed and predict disease outcome.

  15. Direct maximum parsimony phylogeny reconstruction from genotype data

    Directory of Open Access Journals (Sweden)

    Ravi R

    2007-12-01

    Full Text Available Abstract Background Maximum parsimony phylogenetic tree reconstruction from genetic variation data is a fundamental problem in computational genetics with many practical applications in population genetics, whole genome analysis, and the search for genetic predictors of disease. Efficient methods are available for reconstruction of maximum parsimony trees from haplotype data, but such data are difficult to determine directly for autosomal DNA. Data more commonly is available in the form of genotypes, which consist of conflated combinations of pairs of haplotypes from homologous chromosomes. Currently, there are no general algorithms for the direct reconstruction of maximum parsimony phylogenies from genotype data. Hence phylogenetic applications for autosomal data must therefore rely on other methods for first computationally inferring haplotypes from genotypes. Results In this work, we develop the first practical method for computing maximum parsimony phylogenies directly from genotype data. We show that the standard practice of first inferring haplotypes from genotypes and then reconstructing a phylogeny on the haplotypes often substantially overestimates phylogeny size. As an immediate application, our method can be used to determine the minimum number of mutations required to explain a given set of observed genotypes. Conclusion Phylogeny reconstruction directly from unphased data is computationally feasible for moderate-sized problem instances and can lead to substantially more accurate tree size inferences than the standard practice of treating phasing and phylogeny construction as two separate analysis stages. The difference between the approaches is particularly important for downstream applications that require a lower-bound on the number of mutations that the genetic region has undergone.

  16. Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing.

    Science.gov (United States)

    Mallory, Melanie A; Lucic, Danijela X; Sears, Mitchell T; Cloherty, Gavin A; Hillyard, David R

    2014-05-01

    HCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. To evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5'UTR, core and NS5B. The Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5'UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. Among routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5'UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5'UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. The Abbott assay is an automated HCV genotyping method with improved accuracy over 5'UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Genetic Divergence in Sugarcane Genotypes

    OpenAIRE

    Tahir, Mohammad; Rahman, Hidayatur; Gul, Rahmani; Ali, Amjad; Khalid, Muhammad

    2012-01-01

    To assess genetic divergence of sugarcane germplasm, an experiment comprising 25 sugarcane genotypes was conducted at Sugar Crops Research Institute (SCRI), Mardan, Khyber Pakhtunkhwa, Pakistan, in quadruple lattice design during 2008-09. Among the 14 parameters evaluated, majority exhibited significant differences while some showed nonsignificant mean squares. The initial correlation matrix revealed medium to high correlations. Principal Component Analysis (PCA) showed that there were two pr...

  18. [A study on genotype of 271 mycobacterium tuberculosis isolates in 6 prefectures in Yunnan Province].

    Science.gov (United States)

    Chen, L Y; Yang, X; Ru, H H; Yang, H J; Yan, S Q; Ma, L; Chen, J O; Yang, R; Xu, L

    2018-01-06

    Objective: To understand the characteristics of genotypes of Mycobacterium tuberculosis isolates in Yunnan province, and provide the molecular epidemiological evidence for prevention and control of tuberculosis in Yunnan Province. Methods: Mycobacterium Tuberculosis isolates were collected from 6 prefectures of Yunnan province in 2014 and their Genetypes of Mycobacterium tuberculosis isolates were obtained using spoligotyping and multiple locus variable numbers of tandem repeats analysis (MLVA). The results of spoligotyping were entered into the SITVITWEB database to obtain the Spoligotyping International Type (SIT) patterns and the sublineages of MTB isolates. The genoyping patterns were clustered with BioNumerics (version 5.0). Results: A total of 271 MTB isolates represented patients were collected from six prefectures in Yunnan province. Out of these patients, 196 (72.3%) were male. The mean age of the patients was (41.9±15.1) years. The most MTB isolates were from Puer, totally 94 iusolates(34.69%). Spoligotyping analysis revealed that 151 (55.72%) MTB isolates belonged to the Beijing genotype, while the other 120 (44.28%) were from non-Beijing genotype; 40 genotypes were consisted of 24 unique genotypes and 16 clusters. The 271 isolates were differentiated into 30 clusters (2 to 17 isolates per cluster) and 177 unique genotypes, showing a clustering rate of 23.62%. Beijing genotype strains showed higher clustering rate than non-Beijing genotype strains (29.14% vs 16.67%). The HGI of 12-locus VNTR in total MTB strains, Beijing genotype strains and non-Beijing genotype was 0.993, 0.982 and 0.995 respectively. Conclusion: The Beijing genotype was the predominant genotype in Yunnan Province, the characteristics of Mycobacterium tuberculosis showed high genetic diversity. The genotyping data reflect the potential recent ongoing transmission in some area, which highlights the urgent need for early diagnosis and treatment of the infectious TB cases, to cut off the

  19. The Phenotype/Genotype Correlation of Lactase Persistence among Omani Adults

    Directory of Open Access Journals (Sweden)

    Abdulrahim Al-Abri

    2013-09-01

    Full Text Available Objective: To examine the correlation of lactase persistence phenotype with genotype in Omani adults.Methods: Lactase persistence phenotype was tested by hydrogen breath test in 52 Omani Adults using the Micro H2 analyzer. Results were checked against genotyping using direct DNA sequencing.Results: Forty one individuals with C/C-13910 and T/T-13915 genotypes had positive breath tests (≥20 ppm; while eight of nine individuals with T/C-13910 or T/G-13915 genotypes had negative breath tests (<20 ppm and two subjects were non-hydrogen producers. The agreement between phenotype and genotype using Kappa value was very good (0.93.Conclusion: Genotyping both T/C-13910 and T/G-13915 alleles can be used to assist diagnosis and predict lactose intolerance in the Omani population.

  20. Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus

    Science.gov (United States)

    Kell, Alison M.; Wargo, Andrew R.; Kurath, Gael

    2014-01-01

    Viral genotype displacement events are characterized by the replacement of a previously dominant virus genotype by a novel genotype of the same virus species in a given geographic region. We examine here the fitness of three pairs of infectious hematopoietic necrosis virus (IHNV) genotypes involved in three major genotype displacement events in Washington state over the last 30 years to determine whether increased virus fitness correlates with displacement. Fitness was assessed using in vivo assays to measure viral replication in single infection, simultaneous co-infection, and sequential superinfection in the natural host, steelhead trout. In addition, virion stability of each genotype was measured in freshwater and seawater environments at various temperatures. By these methods, we found no correlation between increased viral fitness and displacement in the field. These results suggest that other pressures likely exist in the field with important consequences for IHNV evolution.

  1. Is incidence of multiple HPV genotypes rising in genital infections?

    Directory of Open Access Journals (Sweden)

    Amir Sohrabi

    2017-11-01

    Full Text Available Frequency of cervical cancer related to Human Papilloma Virus (HPV has increased remarkably in less-developed countries. Hence, applying capable diagnostic methods is urgently needed, as is having a therapeutic strategy as an effective step for cervical cancer prevention. The aim of this study was to investigate the prevalence of various multi-type HPV infection patterns and their possible rising incidence in women with genital infections.This descriptive study was conducted on women who attended referral clinical laboratories in Tehran for genital infections from January 2012 until December 2013. A total of 1387 archival cervical scraping and lesion specimens were collected from referred women. HPV genotyping was performed using approved HPV commercial diagnostic technologies with either INNO-LiPA HPV or Geno Array Test kits.HPV was positive in 563 cases (40.59% with mean age of 32.35 ± 9.96. Single, multiple HPV genotypes and untypable cases were detected in 398 (70.69%, 160 (28.42% and 5 (0.89% cases, respectively. Multiple HPV infections were detected in 92 (57.5%, 42 (26.2%, 17 (10.6% and 9 (5.7% cases as two, three, four and five or more genotypes, respectively. The prevalence of 32 HPV genotypes was determined one by one. Seventeen HPV genotypes were identified in 95.78% of all positive infections. Five dominant genotypes, HPV6, 16, 53, 11 and 31, were identified in a total of 52.35%of the HPV positive cases.In the present study, we were able to evaluate the rate of multiple HPV types in genital infections. Nevertheless, it is necessary to evaluate the role of the dominant HPV low-risk types and the new probably high-risk genotypes, such as HPV53, in the increasing incidences of genital infections. Keywords: Multiple HPV Types, Incidence, Genital infection, Cervical cancer, Iran

  2. The JP2 genotype of Aggregatibacter actinomycetemcomitans and marginal periodontitis in the mixed dentition

    DEFF Research Database (Denmark)

    Jensen, Anne Birkeholm; Ennibi, Oum Keltoum; Ismaili, Zouheir

    2016-01-01

    AIM: To perform a cross-sectional study on the carrier frequency of JP2 and non-JP2 genotypes of A. actinomycetemcomitans in Moroccan schoolchildren and relate the presence of these genotypes to the periodontal status in the mixed dentition. MATERIAL AND METHODS: A plaque sample from 513 children...... the JP2 genotype and 186 (36.3%) were positive for non-JP2 genotypes, whereas A. actinomycetemcomitans could not be detected in the remaining 281 subjects. Among 75 subjects with mixed dentition and selected for clinical examination, clinical attachment loss (CAL) ≥3 mm at two or more periodontal sites...

  3. Occupational Tuberculosis in Denmark through 21 Years Analysed by Nationwide Genotyping

    DEFF Research Database (Denmark)

    Pedersen, Mathias Klok; Andersen, Aase Bengaard; Andersen, Peter Henrik

    2016-01-01

    Tuberculosis (TB) is a well-known occupational hazard. Based on more than two decades (1992-2012) of centralized nationwide genotyping of all Mycobacterium tuberculosis culture-positive TB patients in Denmark, we compared M. tuberculosis genotypes from all cases notified as presumed occupational (N...... = 130) with M. tuberculosis genotypes from all TB cases present in the country (N = 7,127). From 1992 through 2006, the IS6110 Restriction Fragment Length Polymorphism (RFLP) method was used for genotyping, whereas from 2005 to present, the 24-locus-based Mycobacterial Interspersed Repetitive Unit...

  4. Nephele: genotyping via complete composition vectors and MapReduce

    Directory of Open Access Journals (Sweden)

    Mardis Scott

    2011-08-01

    Full Text Available Abstract Background Current sequencing technology makes it practical to sequence many samples of a given organism, raising new challenges for the processing and interpretation of large genomics data sets with associated metadata. Traditional computational phylogenetic methods are ideal for studying the evolution of gene/protein families and using those to infer the evolution of an organism, but are less than ideal for the study of the whole organism mainly due to the presence of insertions/deletions/rearrangements. These methods provide the researcher with the ability to group a set of samples into distinct genotypic groups based on sequence similarity, which can then be associated with metadata, such as host information, pathogenicity, and time or location of occurrence. Genotyping is critical to understanding, at a genomic level, the origin and spread of infectious diseases. Increasingly, genotyping is coming into use for disease surveillance activities, as well as for microbial forensics. The classic genotyping approach has been based on phylogenetic analysis, starting with a multiple sequence alignment. Genotypes are then established by expert examination of phylogenetic trees. However, these traditional single-processor methods are suboptimal for rapidly growing sequence datasets being generated by next-generation DNA sequencing machines, because they increase in computational complexity quickly with the number of sequences. Results Nephele is a suite of tools that uses the complete composition vector algorithm to represent each sequence in the dataset as a vector derived from its constituent k-mers by passing the need for multiple sequence alignment, and affinity propagation clustering to group the sequences into genotypes based on a distance measure over the vectors. Our methods produce results that correlate well with expert-defined clades or genotypes, at a fraction of the computational cost of traditional phylogenetic methods run on

  5. Genotypic and phenotypic characterization of Chikungunya virus of different genotypes from Malaysia.

    Directory of Open Access Journals (Sweden)

    I-Ching Sam

    Full Text Available BACKGROUND: Mosquito-borne Chikungunya virus (CHIKV has recently re-emerged globally. The epidemic East/Central/South African (ECSA strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia. METHODS AND FINDINGS: CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36. Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates. CONCLUSIONS: The greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.

  6. Possible Synergistic Interactions Among Multiple HPV Genotypes in Women Suffering from Genital Neoplasia

    Science.gov (United States)

    Hajia, Massoud; Sohrabi, Amir

    2018-03-27

    Objective: Persistence of HPV infection is the true cause of cervical disorders. It is reported that competition may exist among HPV genotypes for colonization. This survey was designed to establish the multiple HPV genotype status in our community and the probability of multiple HPV infections involvement. Methods: All multiple HPV infections were selected for investigation in women suffering from genital infections referred to private laboratories in Tehran, Iran. A total of 160 multi HPV positive specimens from cervical scraping were identified by the HPV genotyping methods, "INNO-LiPA and Geno Array". Result: In present study, HPV 6 (LR), 16 (HR), 53 (pHR), 31 (HR) and 11 (LR) were included in 48.8% of detected infections as the most five dominant genotypes. HPV 16 was detected at the highest rate with genotypes 53, 31 and 52, while HPV 53 appeared linked with HPV 16, 51 and 56 in concurrent infections. It appears that HPV 16 and 53 may have significant tendencies to associate with each other rather than with other genotypes. Analysis of the data revealed there may be some synergistic interactions with a few particular genotypes such as "HPV 53". Conclusion: Multiple HPV genotypes appear more likely to be linked with development of cervical abnormalities especially in patients with genital infections. Since, there are various patterns of dominant HPV genotypes in different regions of world, more investigations of this type should be performed for careHPV programs in individual countries. Creative Commons Attribution License

  7. Haemoglobin genotype of children with severe malaria seen at the ...

    African Journals Online (AJOL)

    Prof Ezechukwu

    2011-10-23

    Oct 23, 2011 ... malaria seen in University of Benin. Teaching Hospital (UBTH), Benin. City. Patients and methods: ... gested to play crucial role in the defense of host against malaria infection and reduce susceptibility to severe .... Binary logistic regression model using Hb genotype status (abnormal Hb versus HbAA) as the ...

  8. Interaction between genotype and climates for Holstein milk ...

    African Journals Online (AJOL)

    This study was designed to investigate the interaction between genotype and climate for milk and fat production traits of Iranian Holstein dairy herds. Milk and fat production data were grouped in 5 climates, on the basis of Extended De Martonne method. (Co)Variance components and genetic parameters of first lactation ...

  9. Early-onset stargardt disease: phenotypic and genotypic characteristics

    NARCIS (Netherlands)

    Lambertus, S.; Huet, R.A.C. van; Bax, N.M.; Hoefsloot, L.H.; Cremers, F.P.M.; Boon, C.J.F.; Klevering, B.J.; Hoyng, C.B.

    2015-01-01

    OBJECTIVE: To describe the phenotype and genotype of patients with early-onset Stargardt disease. DESIGN: Retrospective cohort study. PARTICIPANTS: Fifty-one Stargardt patients with age at onset METHODS: We reviewed patient medical records for age at onset, medical history, initial

  10. Comparison and suitability of genotype by environment analysis ...

    African Journals Online (AJOL)

    Pearl millet (Pennisetum glaucum (L.) R. Br.) is an important food security and income crop for households living in semi-arid zones in Uganda. However, the genotype by environment interaction, in addition to the several methods used for its assessment, complicates selection of varieties adapted to such semi-arid areas.

  11. detection of the predominant microcystin-producing genotype of ...

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... genotypes of MC-producing cyanobacteria in Mozambique. Polymerase chain ...... 1(7): 359-366. Pan H, Song L, Liu Y, Börner T (2002). ... Miles CO (2009). A convenient and cost-effective method for monitoring marine algal ...

  12. Protein landmarks for diversity assessment in wheat genotypes ...

    African Journals Online (AJOL)

    Grain proteins from 20 Indian wheat genotypes were evaluated for diversity assessment based seed storage protein profiling on sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Genetic diversity was evaluated using Nei's index, Shannon index and Unweighted pair group method with arithmetic ...

  13. Serotonin transporter genotype, salivary cortisol, neuroticism and life events

    DEFF Research Database (Denmark)

    Vinberg, Maj; Miskowiak, Kamilla; Kessing, Lars Vedel

    2014-01-01

    OBJECTIVE: To investigate if cortisol alone or in interaction with other risk factors (familial risk, the serotonin transporter genotype, neuroticism and life events (LEs)) predicts onset of psychiatric disorder in healthy individuals at heritable risk. MATRIAL AND METHODS: In a high-risk study...

  14. Reliable Single Chip Genotyping with Semi-Parametric Log-Concave Mixtures

    NARCIS (Netherlands)

    R.C.A. Rippe (Ralph); J.J. Meulman (Jacqueline); P.H.C. Eilers (Paul)

    2012-01-01

    textabstractThe common approach to SNP genotyping is to use (model-based) clustering per individual SNP, on a set of arrays. Genotyping all SNPs on a single array is much more attractive, in terms of flexibility, stability and applicability, when developing new chips. A new semi-parametric method,

  15. Towards a database for genotype-phenotype association research: mining data from encyclopaedia

    NARCIS (Netherlands)

    Pajić, V.S.; Pavlović-Lažetić, G.M.; Beljanski, M.V.; Brandt, B.W.; Pajić, M.B.

    2013-01-01

    To associate phenotypic characteristics of an organism to molecules encoded by its genome, there is a need for well-structured genotype and phenotype data. We use a novel method for extracting data on phenotype and genotype characteristics of microorganisms from text. As a resource, we use an

  16. Genotype call for chromosomal deletions using read-depth from whole genome sequence variants in cattle

    DEFF Research Database (Denmark)

    Mesbah-Uddin, Md; Guldbrandtsen, Bernt; Lund, Mogens Sandø

    2018-01-01

    We presented a deletion genotyping (copy-number estimation) method that leverages population-scale whole genome sequence variants data from 1K bull genomes project (1KBGP) to build reference panel for imputation. To estimate deletion-genotype likelihood, we extracted read-depth (RD) data of all...

  17. Evaluating imputation algorithms for low-depth genotyping-by-sequencing (GBS) data

    Science.gov (United States)

    Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordabl...

  18. Hepatitis C viral load, genotype 3 and interleukin-28B CC genotype predict mortality in HIV and hepatitis C-coinfected individuals

    DEFF Research Database (Denmark)

    Clausen, Louise Nygaard; Astvad, Karen; Ladelund, Steen

    2012-01-01

    OBJECTIVE: We hypothesized that hepatitis C virus (HCV) load and genotype may influence all-cause mortality in HIV-HCV-coinfected individuals. DESIGN AND METHODS: Observational prospective cohort study. Mortality rates were compared in a time-updated multivariate Poisson regression analysis....... RESULTS: We included 264 consecutive HIV-HCV-coinfected individuals. During 1143 person years at risk (PYR) 118 individuals died [overall mortality rate 10 (95% confidence interval; 8, 12)/100 PYR]. In multivariate analysis, a 1 log increase in HCV viral load was associated with a 30% higher mortality......) CC genotype was associated with 54% higher mortality risk [aMRR: 1.54 (0.89, 3.82] compared to TT genotype. CONCLUSION: High-HCV viral load, HCV genotype 3 and IL28B genotype CC had a significant influence on the risk of all-cause mortality among individuals coinfected with HIV-1. This may have...

  19. The study of correlation between HBV genotype and the response to transcatheter arterial chemoembolization therapy in hepatocellular carcinoma patients

    International Nuclear Information System (INIS)

    Huang Keyao; Yang Weizhu; Jiang Na; Zheng Qubing

    2004-01-01

    Objective: To evaluate the influence of hepatitis B virus(HBV) genotype on response to transcatheter arterial embolization therapy in patients with HBV-related HCC. Methods: Transcatheter arterial chemoem-bolization therapy was conducted in patients with HBV-related HCC and response to embolization therapy were observed according to the tumor necrosis rate, the HCC recurrence rate, the cumulative incidence of survival rate and the change of AFP. The HBV genotype was determined by sequencing directly the polymerase chain reaction products of the HBV S gene. The response of HCC to embolization therapy was compared between patients who were infected with different genotypic HBV. Results: The tumor necrosis rate of genotype C patients was similar to that of genotype B patients (P=0.099). The HCC recurrence rate of genotype B was lower than that of genotype C patients (P=0.036). The cumulative incidence of survival rates of 2 and 3 years were significantly higher in the genotype B patients (P=0.036 and P=0.013). There was no difference between the two genotypes, patients in the change of AFP (P>0.05). Conclusions: HBV genotype B patients seem to have a better response to embolization therapy as compared to genotype C patients. Determination of HBV genotype may be useful in predicting the outcomes of TACE therapy in HBV-related HCC. (authors)

  20. Simultaneous Cocirculation of Both European Varicella-Zoster Virus Genotypes (E1 and E2) in Mexico City▿

    Science.gov (United States)

    Rodríguez-Castillo, Araceli; Vaughan, Gilberto; Ramírez-González, José Ernesto; Escobar-Gutiérrez, Alejandro

    2010-01-01

    Full-length genome analysis of varicella-zoster virus (VZV) has shown that viral strains can be classified into seven different genotypes: European (E), Mosaic (M), and Japanese (J), and the E and M genotypes can be further subclassified into E1, E2, and M1 through 4, respectively. The distribution of the main VZV genotypes in Mexico was described earlier, demonstrating the predominance of E genotype, although other genotypes (M1 and M4) were also identified. However, no information regarding the circulation of either E genotype in the country is available. In the present study, we confirm the presence of both E1 and E2 genotypes in the country and explore the possibility of coinfection as the triggering factor for increased virulence among severe cases. A total of 61 different European VZV isolates collected in the Mexico City metropolitan area from 2005 to 2006 were typed by using a PCR method based on genotype-specific primer amplification. Fifty isolates belonged to the E1 genotype, and the eleven remaining samples were classified as E2 genotypes. No coinfection with both E genotypes was identified among these specimens. We provide here new information on the distribution of VZV genotypes circulating in Mexico City. PMID:20220168

  1. Temperature Switch PCR (TSP: Robust assay design for reliable amplification and genotyping of SNPs

    Directory of Open Access Journals (Sweden)

    Mather Diane E

    2009-12-01

    Full Text Available Abstract Background Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs. Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP, a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. Results We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Conclusion Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.

  2. Two-temperature LATE-PCR endpoint genotyping

    Directory of Open Access Journals (Sweden)

    Reis Arthur H

    2006-12-01

    Full Text Available Abstract Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. Results Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of

  3. Echinococcus granulosus sensu lato genotypes infecting humans--review of current knowledge.

    Science.gov (United States)

    Alvarez Rojas, Cristian A; Romig, Thomas; Lightowlers, Marshall W

    2014-01-01

    Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1-10 and the 'lion strain'), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1-3 (=E. granulosus sensu stricto) and G6-10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1-3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6-7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by

  4. Automated genotyping of dinucleotide repeat markers

    Energy Technology Data Exchange (ETDEWEB)

    Perlin, M.W.; Hoffman, E.P. [Carnegie Mellon Univ., Pittsburgh, PA (United States)]|[Univ. of Pittsburgh, PA (United States)

    1994-09-01

    The dinucleotide repeats (i.e., microsatellites) such as CA-repeats are a highly polymorphic, highly abundant class of PCR-amplifiable markers that have greatly streamlined genetic mapping experimentation. It is expected that over 30,000 such markers (including tri- and tetranucleotide repeats) will be characterized for routine use in the next few years. Since only size determination, and not sequencing, is required to determine alleles, in principle, dinucleotide repeat genotyping is easily performed on electrophoretic gels, and can be automated using DNA sequencers. Unfortunately, PCR stuttering with these markers generates not one band for each allele, but a pattern of bands. Since closely spaced alleles must be disambiguated by human scoring, this poses a key obstacle to full automation. We have developed methods that overcome this obstacle. Our model is that the observed data is generated by arithmetic superposition (i.e., convolution) of multiple allele patterns. By quantitatively measuring the size of each component band, and exploiting the unique stutter pattern associated with each marker, closely spaced alleles can be deconvolved; this unambiguously reconstructs the {open_quotes}true{close_quotes} allele bands, with stutter artifact removed. We used this approach in a system for automated diagnosis of (X-linked) Duchenne muscular dystrophy; four multiplexed CA-repeats within the dystrophin gene were assayed on a DNA sequencer. Our method accurately detected small variations in gel migration that shifted the allele size estimate. In 167 nonmutated alleles, 89% (149/167) showed no size variation, 9% (15/167) showed 1 bp variation, and 2% (3/167) showed 2 bp variation. We are currently developing a library of dinucleotide repeat patterns; together with our deconvolution methods, this library will enable fully automated genotyping of dinucleotide repeats from sizing data.

  5. Decoding noises in HIV computational genotyping.

    Science.gov (United States)

    Jia, MingRui; Shaw, Timothy; Zhang, Xing; Liu, Dong; Shen, Ye; Ezeamama, Amara E; Yang, Chunfu; Zhang, Ming

    2017-11-01

    Lack of a consistent and reliable genotyping system can critically impede HIV genomic research on pathogenesis, fitness, virulence, drug resistance, and genomic-based healthcare and treatment. At present, mis-genotyping, i.e., background noises in molecular genotyping, and its impact on epidemic surveillance is unknown. For the first time, we present a comprehensive assessment of HIV genotyping quality. HIV sequence data were retrieved from worldwide published records, and subjected to a systematic genotyping assessment pipeline. Results showed that mis-genotyped cases occurred at 4.6% globally, with some regional and high-risk population heterogeneities. Results also revealed a consistent mis-genotyping pattern in gp120 in all studied populations except the group of men who have sex with men. Our study also suggests novel virus diversities in the mis-genotyped cases. Finally, this study reemphasizes the importance of implementing a standardized genotyping pipeline to avoid genotyping disparity and to advance our understanding of virus evolution in various epidemiological settings. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Toward fully automated genotyping: Allele assignment, pedigree construction, phase determination, and recombination detection in Duchenne muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Perlin, M.W.; Burks, M.B. [Carnegie Mellon Univ., Pittsburgh, PA (United States); Hoop, R.C.; Hoffman, E.P. [Univ. of Pittsburgh School of Medicine, PA (United States)

    1994-10-01

    Human genetic maps have made quantum leaps in the past few years, because of the characterization of >2,000 CA dinucleotide repeat loci: these PCR-based markers offer extraordinarily high PIC, and within the next year their density is expected to reach intervals of a few centimorgans per marker. These new genetic maps open new avenues for disease gene research, including large-scale genotyping for both simple and complex disease loci. However, the allele patterns of many dinucleotide repeat loci can be complex and difficult to interpret, with genotyping errors a recognized problem. Furthermore, the possibility of genotyping individuals at hundreds or thousands of polymorphic loci requires improvements in data handling and analysis. The automation of genotyping and analysis of computer-derived haplotypes would remove many of the barriers preventing optimal use of dense and informative dinucleotide genetic maps. Toward this end, we have automated the allele identification, genotyping, phase determinations, and inheritance consistency checks generated by four CA repeats within the 2.5-Mbp, 10-cM X-linked dystrophin gene, using fluorescein-labeled multiplexed PCR products analyzed on automated sequencers. The described algorithms can deconvolute and resolve closely spaced alleles, despite interfering stutter noise; set phase in females; propagate the phase through the family; and identify recombination events. We show the implementation of these algorithms for the completely automated interpretation of allele data and risk assessment for five Duchenne/Becker muscular dystrophy families. The described approach can be scaled up to perform genome-based analyses with hundreds or thousands of CA-repeat loci, using multiple fluorophors on automated sequencers. 16 refs., 5 figs., 1 tab.

  7. Genetic variation of european maize genotypes (zea mays l. Detected using ssr markers

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    Martin Vivodík

    2017-01-01

    Full Text Available The SSR molecular markers were used to assess genetic diversity in 40 old European maize genotypes. Ten SSR primers revealed a total of 65 alleles ranging from 4 (UMC1060 to 8 (UMC2002 and UMC1155 alleles per locus with a mean value of 6.50 alleles per locus. The PIC values ranged from 0.713 (UMC1060 to 0.842 (UMC2002 with an average value of 0.810 and the DI value ranged from 0.734 (UMC1060 to 0.848 (UMC2002 with an average value of 0.819. 100% of used SSR markers had PIC and DI values higher than 0.7 that means high polymorphism of chosen markers used for analysis. Probability of identity (PI was low ranged from 0.004 (UMC1072 to 0.022 (UMC1060 with an average of 0.008. A dendrogram was constructed from a genetic distance matrix based on profiles of the 10 maize SSR loci using the unweighted pair-group method with the arithmetic average (UPGMA. According to analysis, the collection of 40 diverse accessions of maize was clustered into four clusters. The first cluster contained nine genotypes of maize, while the second cluster contained the four genotypes of maize. The third cluster contained 5 maize genotypes. Cluster 4 contained five genotypes from Hungary (22.73%, two genotypes from Poland (9.10%, seven genotypes of maize from Union of Soviet Socialist Republics (31.81%, six genotypes from Czechoslovakia (27.27%, one genotype from Slovak Republic (4.55% and one genotype of maize is from Yugoslavia (4.55%. We could not distinguish 4 maize genotypes grouped in cluster 4, (Voroneskaja and Kocovska Skora and 2 Hungarian maize genotypes - Feheres Sarga Filleres and Mindszentpusztai Feher, which are genetically the closest.

  8. Polymorphism of proteins in selected slovak winter wheat genotypes using SDS-PAGE

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    Dana Miháliková

    2016-12-01

    Full Text Available Winter wheat is especially used for bread-making. The specific composition of the grain storage proteins and the representation of individual subunits determines the baking quality of wheat. The aim of this study was to analyze 15 slovak varieties of the winter wheat (Triticum aestivum L. based on protein polymorphism and to predict their technological quality. SDS-PAGE method by ISTA was used to separate glutenin protein subunits. Glutenins were separated into HMW-GS (15.13% and LMW-GS (65.89% on the basis of molecular weight in SDS-PAGE. At the locus Glu-A1 was found allele Null (53% of genotypes and allele 1 (47% of genotypes. The locus Glu-B1 was represented by the HMW-GS subunits 6+8 (33% of genotypes, 7+8 (27% of genotypes, 7+9 (40% of genotypes. At the locus Glu-D1 were detected two subunits, 2+12 (33% of genotypes and 5+10 (67% of genotypes which is correlated with good bread-making properties. The Glu – score was ranged from 4 (genotype Viglanka to 10 (genotypes Viola, Vladarka. According to the representation of individual glutenin subunits in samples, the dendrogram of genetic similarity was constructed. By the prediction of quality the results showed that the best technological quality was significant in the varieties Viola and Vladarka which are suitable for use in food processing.

  9. GMFilter and SXTestPlate: software tools for improving the SNPlex™ genotyping system

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    Schreiber Stefan

    2009-03-01

    Full Text Available Abstract Background Genotyping of single-nucleotide polymorphisms (SNPs is a fundamental technology in modern genetics. The SNPlex™ mid-throughput genotyping system (Applied Biosystems, Foster City, CA, USA enables the multiplexed genotyping of up to 48 SNPs simultaneously in a single DNA sample. The high level of automation and the large amount of data produced in a high-throughput laboratory require advanced software tools for quality control and workflow management. Results We have developed two programs, which address two main aspects of quality control in a SNPlex™ genotyping environment: GMFilter improves the analysis of SNPlex™ plates by removing wells with a low overall signal intensity. It enables scientists to automatically process the raw data in a standardized way before analyzing a plate with the proprietary GeneMapper software from Applied Biosystems. SXTestPlate examines the genotype concordance of a SNPlex™ test plate, which was typed with a control SNP set. This program allows for regular quality control checks of a SNPlex™ genotyping platform. It is compatible to other genotyping methods as well. Conclusion GMFilter and SXTestPlate provide a valuable tool set for laboratories engaged in genotyping based on the SNPlex™ system. The programs enhance the analysis of SNPlex™ plates with the GeneMapper software and enable scientists to evaluate the performance of their genotyping platform.

  10. Identification of Coxiella burnetii genotypes in Croatia using multi-locus VNTR analysis.

    Science.gov (United States)

    Račić, Ivana; Spičić, Silvio; Galov, Ana; Duvnjak, Sanja; Zdelar-Tuk, Maja; Vujnović, Anja; Habrun, Boris; Cvetnić, Zeljko

    2014-10-10

    Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Hepatitis C virus genotypes among multiply transfused hemoglobinopathy patients from Northern Iraq

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    Adil A Othman

    2014-01-01

    Full Text Available Background and Aim: Owing to the scarcity of data on hepatitis C virus (HCV genotypes in Iraq and due to their epidemiological as well as therapy implications, this study was initiated aiming at determining these genotypes in Northern Iraq. Materials and Methods: A total of 70 HCV antibody positive multi transfused patients with hemoglobinopathies, who had detectable HCV ribonucleic acid, were recruited for genotyping using genotype-specific nested polymerase chain reaction. Results: The most frequent genotype detected was genotype 4 (52.9% followed by 3a (17.1%, 1b (12.9% and 1a (1.4%, while mixed genotypes (4 with either 3a or 1b were detected in 7.1%. Conclusion: The predominance of genotype 4 is similar to other studies from surrounding Eastern Mediterranean Arab countries and to the only earlier study from central Iraq, however the significant high proportion of 3a and scarcity of 1a, are in contrast to the latter study and may be explainable by the differing population interactions in this part of Iraq. This study complements previous studies from Eastern Mediterranean region and demonstrates relative heterogeneity of HCV genotype distribution within Iraq and should trigger further studies in other parts of the country.

  12. Allelopathic interference of alfalfa (Medicago sativa L.) genotypes to annual ryegrass (Lolium rigidum).

    Science.gov (United States)

    Zubair, Hasan Muhammad; Pratley, James E; Sandral, G A; Humphries, A

    2017-07-01

    Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.

  13. [The HVR genotypes and their relationship with the resistance of methicillin-resistant staphylococci].

    Science.gov (United States)

    Liao, F; Fan, X; Lü, X; Feng, P

    2001-06-01

    To investigate the HVR-PCR genotype of methicillin-resistant Staphylococci in local hospitals and compare it with the antibiograms, with aview to selecting effective antibacterial agents, moreover, to discuss preliminarily its role in molecular epidemiology. The minimal inhibitory concentrations(MICs) of 86 MRSA, 10 MRSE(Mc'S. epidemidis), 5 MSSE(Mc'S. epidemidis), 8 MRSH(Mc'S. haemolyticus) and 5 MSSH(Mc'S. haemolyticus) clinical isolates collected from 4 local hospitals were tested by serial two-fold agar dilution method; their DNA were extracted by moved basic lytic method, whose polymerase chain reaction(PCR) products amplified, based on the size of mec-associated hypervariable region(HVR) were analyzed by PAG vertical and agarose gel electrophoresis. MRSA, MRSE and MRSH were grouped into 4, 3 and 2 HVR genotypes respectively according to the size of the PCR products. The PCR products amplified from 9 of 10 MRSE isolates were the same as the products amplified from MRSA isolates. MRSA strains in this study were mainly HVR genotypes A and D, which accounted for 52.32% and 39.53%; Genotypes B and C were the most multi-drug resistant, but genotype D was multi-sensitive. The I genotype of MRSE was multi-drug resistant, but its genotype III was multi-drug sensitive. The genotype a of MRSH was more resistant than genotype b. These results suggest that HVR-PCR genotype method is an easy and fast method for epidemiological investigation of nosocomial infections caused by MRSA, and it is helpful for clinical selection of antibacterial agents. This method can compare the mec determinants of MRSA and Mc'CNSt isolates and hence to search for the origin of the mec determinant.

  14. Mistaken identity of an open reading frame proposed for PCR-based identification of Mycoplasma bovis and the effect of polymorphisms and insertions on assay performance

    Science.gov (United States)

    Mycoplasma bovis is an important cause of disease in cattle and bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories routinely use PCR to replace or complement conventional isolation and identification methods. A frequently used target of such assays is th...

  15. Pervasive Genotypic Mosaicism in Founder Mice Derived from Genome Editing through Pronuclear Injection.

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    Daniel Oliver

    Full Text Available Genome editing technologies, especially the Cas9/CRISPR system, have revolutionized biomedical research over the past several years. Generation of novel alleles has been simplified to unprecedented levels, allowing for rapid expansion of available genetic tool kits for researchers. However, the issue of genotypic mosaicism has become evident, making stringent analyses of the penetrance of genome-edited alleles essential. Here, we report that founder mice, derived from pronuclear injection of ZFNs or a mix of guidance RNAs and Cas9 mRNAs, display consistent genotypic mosaicism for both deletion and insertion alleles. To identify founders with greater possibility of transmitting the mutant allele through the germline, we developed an effective germline genotyping method. The awareness of the inherent genotypic mosaicism issue with genome editing will allow for a more efficient implementation of the technologies, and the germline genotyping method will save valuable time and resources.

  16. AMMI analysis to evaluate the adaptability and phenotypic stability of sugarcane genotypes

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    Luís Cláudio Inácio da Silveira

    2013-02-01

    Full Text Available Sugarcane (Saccharum sp. is one of the most important crops in Brazil. The high demand for sugarcane-derived products has stimulated the expansion of sugarcane cultivation in recent years, exploring different environments. The adaptability and the phenotypic stability of sugarcane genotypes in the Minas Gerais state, Brazil, were evaluated based on the additive main effects and multiplicative interaction (AMMI method. We evaluated 15 genotypes (13 clones and two checks: RB867515 and RB72454 in nine environments. The average of two cuttings for the variable tons of pol per hectare (TPH measure was used to discriminate genotypes. Besides the check RB867515 (20.44 t ha-1, the genotype RB987935 showed a high average TPH (20.71 t ha-1, general adaptability and phenotypic stability, and should be suitable for cultivation in the target region. The AMMI method allowed for easy visual identification of superior genotypes for each set of environments.

  17. Applications of blood group genotyping

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    Mariza A. Mota

    2006-03-01

    Full Text Available Introduction: The determination of blood group polymorphism atthe genomic level facilitates the resolution of clinical problemsthat cannot be addressed by hemagglutination. They are useful to(a determine antigen types for which currently available antibodiesare weakly reactive; (b type patients who have been recentlytransfused; (c identify fetuses at risk for hemolytic disease of thenewborn; and (d to increase the reliability of repositories of antigennegative RBCs for transfusion. Objectives: This review assessedthe current applications of blood group genotyping in transfusionmedicine and hemolytic disease of the newborn. Search strategy:Blood group genotyping studies and reviews were searched ingeneral database (MEDLINE and references were reviewed.Selection criteria: All published data and reviews were eligible forinclusion provided they reported results for molecular basis ofblood group antigens, DNA analysis for blood group polymorphisms,determination of fetal group status and applications of blood groupgenotyping in blood transfusion. Data collection: All data werecollected based on studies and reviews of blood grouppolymorphisms and their clinical applications.

  18. An Efficient, Simple, and Noninvasive Procedure for Genotyping Aquatic and Nonaquatic Laboratory Animals.

    Science.gov (United States)

    Okada, Morihiro; Miller, Thomas C; Roediger, Julia; Shi, Yun-Bo; Schech, Joseph Mat

    2017-09-01

    Various animal models are indispensible in biomedical research. Increasing awareness and regulations have prompted the adaptation of more humane approaches in the use of laboratory animals. With the development of easier and faster methodologies to generate genetically altered animals, convenient and humane methods to genotype these animals are important for research involving such animals. Here, we report skin swabbing as a simple and noninvasive method for extracting genomic DNA from mice and frogs for genotyping. We show that this method is highly reliable and suitable for both immature and adult animals. Our approach allows a simpler and more humane approach for genotyping vertebrate animals.

  19. Grain yield stability of early maize genotypes

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    Chitra Bahadur Kunwar

    2016-12-01

    Full Text Available The objective of this study was to estimate grain yield stability of early maize genotypes. Five early maize genotypes namely Pool-17, Arun1EV, Arun-4, Arun-2 and Farmer’s variety were evaluated using Randomized Complete Block Design along with three replications at four different locations namely Rampur, Rajahar, Pakhribas and Kabre districts of Nepal during summer seasons of three consecutive years from 2010 to 2012 under farmer’s fields. Genotype and genotype × environment (GGE biplot was used to identify superior genotype for grain yield and stability pattern. The genotypes Arun-1 EV and Arun-4 were better adapted for Kabre and Pakhribas where as pool-17 for Rajahar environments. The overall findings showed that Arun-1EV was more stable followed by Arun-2 therefore these two varieties can be recommended to farmers for cultivation in both environments.

  20. PCR-based assays versus direct sequencing for evaluating the effect of KRAS status on anti-EGFR treatment response in colorectal cancer patients: a systematic review and meta-analysis.

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    Lianfeng Shan

    Full Text Available The survival rate of colorectal cancer (CRC patients carrying wild-type KRAS is significantly increased by combining anti-EGFR monoclonal antibody (mAb with standard chemotherapy. However, conflicting data exist in both the wild-type KRAS and mutant KRAS groups, which strongly challenge CRC anti-EGFR treatment. Here we conducted a meta-analysis in an effort to provide more reliable information regarding anti-EGFR treatment in CRC patients.We searched full reports of randomized clinical trials using Medline, the American Society of Clinical Oncology (ASCO, and the European Society for Medical Oncology (ESMO. Two investigators independently screened the published literature according to our inclusive and exclusive criteria and the relative data were extracted. We used Review Manager 5.2 software to analyze the data.The addition of anti-EGFR mAb to standard chemotherapy significantly improved both progression-free survival (PFS and median overall survival (mOS in the wild-type KRAS group; hazard ratios (HRs for PFS and mOS were 0.70 [95% confidence interval (CI, 0.58-0.84] and 0.83 [95% CI, 0.75-0.91], respectively. In sub-analyses of the wild-type KRAS group, when PCR-based assays are employed, PFS and mOS notably increase: the HRs were 0.74 [95% CI, 0.62-0.88] and 0.87 [95% CI, 0.78-0.96], respectively. In sub-analyses of the mutant KRAS group, neither PCR-based assays nor direct sequencing enhance PFS or mOS.Our data suggest that PCR-based assays with high sensitivity and specificity allow accurate identification of patients with wild-type KRAS and thus increase PFS and mOS. Furthermore, such assays liberate patients with mutant KRAS from unnecessary drug side effects, and provide them an opportunity to receive appropriate treatment. Thus, establishing a precise standard reference test will substantially optimize CRC-targeted therapies.

  1. Screening of salinity tolerant jute (corchorus capsularis and c. olitorius) genotypes via phenotypic and phsiology-assisted procedures

    International Nuclear Information System (INIS)

    Hongyu, M.A.; Wang, Z.; Wang, X.

    2011-01-01

    To obtain salt tolerant genotypes, salt tolerance of 10 jute genotypes of different origins was evaluated by relative salt harm rate at germination stage and by index of salt harm at seedling stage, respectively. The results indicated that salt tolerance of germination stage of jute was consistent with that of seedling stage, with a markedly significant (P < 0.01) correlation of 0.8432 (n =10). Two high salt tolerant genotypes (Huang No.1 and 9511) and two salt sensitive genotypes (Mengyuan and 07-21) were screened out by these methods. Further activity analysis of POD, SOD and CAT and determination of MDA content at seedling stage validated that genotypes Huang No.1 and 9511 were more salt tolerant than genotypes Mengyuan and 07-21. Our results indicated that the combination of relative salt harm rate at germination stage and index of salt harm at seedling stage can be used to evaluate salt tolerance of jute genotypes. (author)

  2. Heterogeneous recombination among Hepatitis B virus genotypes.

    Science.gov (United States)

    Castelhano, Nadine; Araujo, Natalia M; Arenas, Miguel

    2017-10-01

    The rapid evolution of Hepatitis B virus (HBV) through both evolutionary forces, mutation and recombination, allows this virus to generate a large variety of adapted variants at both intra and inter-host levels. It can, for instance, generate drug resistance or the diverse viral genotypes that currently exist in the HBV epidemics. Concerning the latter, it is known that recombination played a major role in the emergence and genetic diversification of novel genotypes. In this regard, the quantification of viral recombination in each genotype can provide relevant information to devise expectations about the evolutionary trends of the epidemic. Here we measured the amount of this evolutionary force by estimating global and local recombination rates in >4700 HBV complete genome sequences corresponding to nine (A to I) HBV genotypes. Counterintuitively, we found that genotype E presents extremely high levels of recombination, followed by genotypes B and C. On the other hand, genotype G presents the lowest level, where recombination is almost negligible. We discuss these findings in the light of known characteristics of these genotypes. Additionally, we present a phylogenetic network to depict the evolutionary history of the studied HBV genotypes. This network clearly classified all genotypes into specific groups and indicated that diverse pairs of genotypes are derived from a common ancestor (i.e., C-I, D-E and, F-H) although still the origin of this virus presented large uncertainty. Altogether we conclude that the amount of observed recombination is heterogeneous among HBV genotypes and that this heterogeneity can influence on the future expansion of the epidemic. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Improving the precision of genotype selection in wheat performance trials

    Directory of Open Access Journals (Sweden)

    Giovani Benin

    2013-12-01

    Full Text Available The aim of this study was to verify whether using the Papadakis method improves model assumptions and experimental accuracy in field trials used to determine grain yield for wheat lineages indifferent Value for Cultivation and Use (VCU regions. Grain yield data from 572 field trials at 31 locations in the VCU Regions 1, 2, 3 and 4 in 2007-2011 were used. Each trial was run with and without the use of the Papadakis method. The Papadakis method improved the indices of experimental precision measures and reduced the number of experimental repetitions required to predict grain yield performance among the wheat genotypes. There were differences among the wheat adaptation regions in terms of the efficiency of the Papadakis method, the adjustment coefficient of the genotype averages and the increases in the selective accuracy of grain yield.

  4. Tipificación capsular mediante PCR de aislamientos de Haemophilus influenzae no tipificables por aglutinación PCR-based capsular typing of Haemophilus influenzae isolates non-typeable by agglutination

    Directory of Open Access Journals (Sweden)

    G. Weltman

    2005-12-01

    Full Text Available Haemophilus influenzae es reconocido como un agente patógeno responsable de infecciones localizadas y sistémicas. Se han descrito 6 tipos de polisacáridos capsulares antigénicamente distintos (a, b, c, d, e, y f que se pueden identificar por aglutinación en lámina con antisueros específicos. También existen cepas no capsuladas (NC fenotípicamente no tipificables (NT. La introducción de la vacuna conjugada produjo una marcada disminución de las enfermedades invasivas causadas por H. influenzae tipo b. En este contexto, la tipificación capsular mediante PCR es el método más apropiado para distinguir las cepas no capsuladas de las mutantes b deficientes en cápsula (b- y detectar la presencia de cepas pertenecientes a otros serotipos que no puedan ser tipificables por aglutinación. Se determinó el genotipo capsular a 38 aislamientos de Haemophilus influenzae no tipificables por aglutinación, derivados al servicio de Bacteriología Clínica del INEI-ANLIS "Dr. Carlos G. Malbrán" en el período 2002-2004. El 78,9% de los aislamientos provenían de hemocultivos y la mayor parte de ellos estaban asociados a foco respiratorio. El 100% de los aislamientos fueron identificados como H. influenzae no capsulados mediante la técnica de PCR.Haemophilus influenzae is recognized as a pathogenic agent responsible of localized and systemic infections. Six antigenically different capsular polysaccharide types have been described (a, b, c, d, e, and f which can be identified by slide agglutination with specific antisera. Besides there are non capsulated strains that cannot be typed by slide agglutination. The introduction of the conjugated vaccine produced an important reduction of invasive diseases caused by H. influenzae type b. Capsular typing by PCR is the most appropriated method for distinguishing non capsulated strains from capsule deficient type b mutants (b- and for detecting strains of other serotypes that cannot be detected by slide

  5. Hepatitis C virus genotypes in Myanmar.

    Science.gov (United States)

    Win, Nan Nwe; Kanda, Tatsuo; Nakamoto, Shingo; Yokosuka, Osamu; Shirasawa, Hiroshi

    2016-07-21

    Myanmar is adjacent to India, Bangladesh, Thailand, Laos and China. In Myanmar, the prevalence of hepatitis C virus (HCV) infection is 2%, and HCV infection accounts for 25% of hepatocellular carcinoma. In this study, we reviewed the prevalence of HCV genotypes in Myanmar. HCV genotypes 1, 3 and 6 were observed in volunteer blood donors in and around the Myanmar city of Yangon. Although there are several reports of HCV genotype 6 and its variants in Myanmar, the distribution of the HCV genotypes has not been well documented in areas other than Yangon. Previous studies showed that treatment with peginterferon and a weight-based dose of ribavirin for 24 or 48 wk could lead to an 80%-100% sustained virological response (SVR) rates in Myanmar. Current interferon-free treatments could lead to higher SVR rates (90%-95%) in patients infected with almost all HCV genotypes other than HCV genotype 3. In an era of heavy reliance on direct-acting antivirals against HCV, there is an increasing need to measure HCV genotypes, and this need will also increase specifically in Myanmar. Current available information of HCV genotypes were mostly from Yangon and other countries than Myanmar. The prevalence of HCV genotypes in Myanmar should be determined.

  6. Hepatitis C Virus: Virology and Genotypes

    KAUST Repository

    Abdelaziz, Ahmed

    2017-12-01

    Hepatitis C virus (HCV) is a major causative agent of chronic liver disease worldwide. HCV is characterized by genetic heterogeneity, with at least six genotypes identified. The geographic distribution of genotypes has shown variations in different parts of the world over the past decade because of variations in population structure, immigration, and routes of transmission. Genotype differences are of epidemiologic interest and help the study of viral transmission dynamics to trace the source of HCV infection in a given population. HCV genotypes are also of considerable clinical importance because they affect response to antiviral therapy and represent a challenging obstacle for vaccine development.

  7. Detection of genetically modified rice: Collaborative validation study of a PCR based detection of genetically modified rice Oryza sativa commercially available in Saudi Arabia

    OpenAIRE

    Ibrahim, Mohamed; Alaraidh, Ibrahim; Amid, Azura; Farouk, Abd-El Aziem; Bazaid, Salih; Greiner, Ralf; Alghunaim, Abdullah

    2011-01-01

    A collaborative trial study has been conducted for validation of an extraction method and a subsequent PCR for  the detection of transgenic rice sold in Saudi Arabia. The tests were carried out in Saudi Arabia using Real-Time PCR and the positive samples were validated in another lab in Malaysia using PCR and agarose gel visualization.  The samples were tested for the existence of the NOS Terminator. A total of 150 samples were tested out of which three samples tested positi...

  8. Lactococcus garvieae endocarditis in a native valve identified by MALDI-TOF MS and PCR-based 16s rRNA in Spain: A case report

    Directory of Open Access Journals (Sweden)

    V. Heras Cañas

    2015-05-01

    Full Text Available Lactococcus garvieae is a Gram-positive, catalase negative coccus arranged in pairs or short chains, well-known as a fish pathogen. We report a case of Infective Endocarditis (IE by L. garvieae in a native valve from a 68-year-old male with unknown history of contact with raw fish and an extensive history of heart disease. This case highlights the reliability of MALDI-TOF MS compared to conventional methods in the identification of rare microorganisms like this.

  9. Approaches to genotyping individual miracidia of Schistosoma japonicum.

    Science.gov (United States)

    Xiao, Ning; Remais, Justin V; Brindley, Paul J; Qiu, Dong-Chuan; Carlton, Elizabeth J; Li, Rong-Zhi; Lei, Yang; Blair, David

    2013-12-01

    Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.

  10. Resolving ambiguity in the phylogenetic relationship of genotypes A, B, and C of hepatitis B virus

    Science.gov (United States)

    2013-01-01

    Background Hepatitis B virus (HBV) is an important infectious agent that causes widespread concern because billions of people are infected by at least 8 different HBV genotypes worldwide. However, reconstruction of the phylogenetic relationship between HBV genotypes is difficult. Specifically, the phylogenetic relationships among genotypes A, B, and C are not clear from previous studies because of the confounding effects of genotype recombination. In order to clarify the evolutionary relationships, a rigorous approach is required that can effectively explore genetic sequences with recombination. Result In the present study, phylogenetic relationship of the HBV genotypes was reconstructed using a consensus phylogeny of phylogenetic trees of HBV genome segments. Reliability of the reconstructed phylogeny was extensively evaluated in agreements of local phylogenies of genome segments. The reconstructed phylogenetic tree revealed that HBV genotypes B and C had a closer phylogenetic relationship than genotypes A and B or A and C. Evaluations showed the consensus method was capable to reconstruct reliable phylogenetic relationship in the presence of recombinants. Conclusion The consensus method implemented in this study provides an alternative approach for reconstructing reliable phylogenetic relationships for viruses with possible genetic recombination. Our approach revealed the phylogenetic relationships of genotypes A, B, and C of HBV. PMID:23758960

  11. LinkImputeR: user-guided genotype calling and imputation for non-model organisms.

    Science.gov (United States)

    Money, Daniel; Migicovsky, Zoë; Gardner, Kyle; Myles, Sean

    2017-07-10

    Genomic studies such as genome-wide association and genomic selection require genome-wide genotype data. All existing technologies used to create these data result in missing genotypes, which are often then inferred using genotype imputation software. However, existing imputation methods most often make use only of genotypes that are successfully inferred after having passed a certain read depth threshold. Because of this, any read information for genotypes that did not pass the threshold, and were thus set to missing, is ignored. Most genomic studies also choose read depth thresholds and quality filters without investigating their effects on the size and quality of the resulting genotype data. Moreover, almost all genotype imputation methods require ordered markers and are therefore of limited utility in non-model organisms. Here we introduce LinkImputeR, a software program that exploits the read count information that is normally ignored, and makes use of all available DNA sequence information for the purposes of genotype calling and imputation. It is specifically designed for non-model organisms since it requires neither ordered markers nor a reference panel of genotypes. Using next-generation DNA sequence (NGS) data from apple, cannabis and grape, we quantify the effect of varying read count and missingness thresholds on the quantity and quality of genotypes generated from LinkImputeR. We demonstrate that LinkImputeR can increase the number of genotype calls by more than an order of magnitude, can improve genotyping accuracy by several percent and can thus improve the power of downstream analyses. Moreover, we show that the effects of quality and read depth filters can differ substantially between data sets and should therefore be investigated on a per-study basis. By exploiting DNA sequence data that is normally ignored during genotype calling and imputation, LinkImputeR can significantly improve both the quantity and quality of genotype data generated from

  12. Typing of O26 enterohaemorrhagic and enteropathogenic Escherichia coli isolated from humans and cattle with IS621 multiplex PCR-based fingerprinting.

    Science.gov (United States)

    Mainil, J G; Bardiau, M; Ooka, T; Ogura, Y; Murase, K; Etoh, Y; Ichihara, S; Horikawa, K; Buvens, G; Piérard, D; Itoh, T; Hayashi, T

    2011-09-01

    This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS621. Two multiplex PCRs, targeting either the left (5') or right (3') IS/chromosome junction of 12 IS621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty-eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy-one per cent of the 45 EHEC and EPEC with identical IS621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had identical IS621 fingerprints and PFGE profiles. The IS621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. The IS621 printing method represents a rapid (24 h) first-line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology. No claim to Belgian Government works.

  13. Development of a PCR-based marker utilizing a deletion mutation in the dihydroflavonol 4-reductase (DFR) gene responsible for the lack of anthocyanin production in yellow onions (Allium cepa).

    Science.gov (United States)

    Kim, Sunggil; Yoo, Kil Sun; Pike, Leonard M

    2005-02-01

    Bulb color in onions (Allium cepa) is an important trait, but the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that inactivation of the dihydroflavonol 4-reductase (DFR) gene at the transcriptional level resulted in a lack of anthocyanin production in yellow onions. The objectives of the present study were the identification of the critical mutations in the DFR gene (DFR-A) and the development of a PCR-based marker for allelic selection. We report the isolation of two additional DFR homologs (DFR-B and DFR-C). No unique sequences were identified in either DFR homolog, even in the untranslated region (UTR). Both genes shared more than 95% nucleotide sequence identity with the DFR-A gene. To obtain a unique sequence from each gene, we isolated the promoter regions. Sequences of the DFR-A and DFR-B promoters differed completely from one another, except for an approximately 100-bp sequence adjacent to the 5'UTR. It was possible to specifically amplify only the DFR-A gene using primers designed to anneal to the unique promoter region. The sequences of yellow and red DFR-A alleles were the same except for a single base-pair change in the promoter and an approximately 800-bp deletion within the 3' region of the yellow DFR-A allele. This deletion was used to develop a co-dominant PCR-based marker that segregated perfectly with color phenotypes in the F2 population. These results indicate that a deletion mutation in the yellow DFR-A gene results in the lack of anthocyanin production in yellow onions.

  14. Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

    Science.gov (United States)

    Wilkes, Rebecca Penrose; Kania, Stephen A; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chang, Hsiu-Hui; Ma, Li-Juan; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2015-07-01

    Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats. © 2015 The Author(s).

  15. Procedures for identifying S-allele genotypes of Brassica.

    Science.gov (United States)

    Wallace, D H

    1979-11-01

    Procedures are described for efficient selection of: (1) homozygous and heterozygous S-allele genotypes; (2) homozygous inbreds with the strong self- and sib-incompatibility required for effective seed production of single-cross F1 hybrids; (3) heterozygous genotypes with the high self- and sib-incompatibility required for effective seed production of 3- and 4-way hybrids.From reciprocal crosses between two first generation inbred (I1) plants there are three potential results: both crosses are incompatible; one is incompatible and the other compatible; and both are compatible. Incompatibility of both crosses is useful information only when combined with data from other reciprocal crosses. Each compatible cross, depending on whether its reciprocal is incompatible or compatible, dictates alternative reasoning and additional reciprocal crosses for efficiently and simultaneously identifying: (A) the S-allele genotype of all individual I1 plants, and (B) the expressions of dominance or codominance in pollen and stigma (sexual organs) of an S-allele heterozygous genotype. Reciprocal crosses provide the only efficient means of identifying S-allele genotypes and also the sexual-organ x S-allele-interaction types.Fluorescent microscope assay of pollen tube penetration into the style facilitates quantitation within 24-48 hours of incompatibility and compatibility of the reciprocal crosses. A procedure for quantitating the reciprocal difference is described that maximizes informational content of the data about interactions between S alleles in pollen and stigma of the S-allele-heterozygous genotype.Use of the non-inbred Io generation parent as a 'known' heterozygous S-allele genotype in crosses with its first generation selfed (I1) progeny usually reduces at least 7 fold the effort required for achieving objectives 1, 2, and 3, compared to the method of making reciprocal crosses only among I1 plants.Identifying the heterozygous and both homozygous S-allele genotypes during

  16. Sources of Wilhelm Johannsen's genotype theory.

    Science.gov (United States)

    Roll-Hansen, Nils

    2009-01-01

    This paper describes the historical background and early formation of Wilhelm Johannsen's distinction between genotype and phenotype. It is argued that contrary to a widely accepted interpretation (For instance, W. Provine, 1971. The Origins of Theoretical Population Genetics. Chicago: The University of Chicago Press; Mayr, 1973; F. B. Churchill, 1974. Journal of the History of Biology 7: 5-30; E. Mayr, 1982. The Growth of Biological Thought, Cambridge: Harvard University Press; J. Sapp, 2003. Genesis. The Evolution of Biology. New York: Oxford University Press) his concepts referred primarily to properties of individual organisms and not to statistical averages. Johannsen's concept of genotype was derived from the idea of species in the tradition of biological systematics from Linnaeus to de Vries: An individual belonged to a group - species, subspecies, elementary species - by representing a certain underlying type (S. Müller-Wille and V. Orel, 2007. Annals of Science 64: 171-215). Johannsen sharpened this idea theoretically in the light of recent biological discoveries, not least those of cytology. He tested and confirmed it experimentally combining the methods of biometry, as developed by Francis Galton, with the individual selection method and pedigree analysis, as developed for instance by Louis Vilmorin. The term "genotype" was introduced in W. Johannsen's 1909 (Elemente der Exakten Erblichkeitslehre. Jena: Gustav Fischer) treatise, but the idea of a stable underlying biological "type" distinct from observable properties was the core idea of his classical bean selection experiment published 6 years earlier (W. Johannsen, 1903. Ueber Erblichkeit in Populationen und reinen Linien. Eine Beitrag zur Beleuchtung schwebender Selektionsfragen, Jena: Gustav Fischer, pp. 58-59). The individual ontological foundation of population analysis was a self-evident presupposition in Johannsen's studies of heredity in populations from their start in the early 1890s till his

  17. SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

    Directory of Open Access Journals (Sweden)

    Velasco Riccardo

    2008-01-01

    Full Text Available Abstract Background Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP discovery and genotyping in grapevine (Vitis vinifera L.. However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs thus providing a valuable source for high-throughput genotyping methods. Results Herein we report the first application of the SNPlex™ genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA methods were used for preparation of genomic DNA for the SNPlex assay. Conclusion Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA, is a good solution for future applications in well-equipped laboratories.

  18. Selection of common bean genotypes for the Cerrado/Pantanal ecotone via mixed models and multivariate analysis.

    Science.gov (United States)

    Corrêa, A M; Pereira, M I S; de Abreu, H K A; Sharon, T; de Melo, C L P; Ito, M A; Teodoro, P E; Bhering, L L

    2016-10-17

    The common bean, Phaseolus vulgaris, is predominantly grown on small farms and lacks accurate genotype recommendations for specific micro-regions in Brazil. This contributes to a low national average yield. The aim of this study was to use the methods of the harmonic mean of the relative performance of genetic values (HMRPGV) and the centroid, for selecting common bean genotypes with high yield, adaptability, and stability for the Cerrado/Pantanal ecotone region in Brazil. We evaluated 11 common bean genotypes in three trials carried out in the dry season in Aquidauana in 2013, 2014, and 2015. A likelihood ratio test detected a significant interaction between genotype x year, contributing 54% to the total phenotypic variation in grain yield. The three genotypes selected by the joint analysis of genotypic values in all years (Carioca Precoce, BRS Notável, and CNFC 15875) were the same as those recommended by the HMRPGV method. Using the centroid method, genotypes BRS Notável and CNFC 15875 were considered ideal genotypes based on their high stability to unfavorable environments and high responsiveness to environmental improvement. We identified a high association between the methods of adaptability and stability used in this study. However, the use of centroid method provided a more accurate and precise recommendation of the behavior of the evaluated genotypes.

  19. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2016-09-01

    Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

  20. Development of a near-infrared spectroscopy method (NIRS) for fast analysis of total, indolic, aliphatic and individual glucosinolates in new bred open pollinating genotypes of broccoli (Brassica oleracea convar. botrytis var. italica).

    Science.gov (United States)

    Sahamishirazi, Samira; Zikeli, Sabine; Fleck, Michael; Claupein, Wilhelm; Graeff-Hoenninger, Simone

    2017-10-01

    This study describes the development of near-infrared spectroscopy (NIRS) calibration to determine individual and total glucosinolates (GSLs) content of 12 new-bred open-pollinating genotypes of broccoli (Brassica oleracea convar. botrytis var. italica). Six individual GSLs were identified using high-performance-liquid chromatography (HPLC). The NIRS calibration was established based on modified partial least squares regression with reference values of HPLC. The calibration was analyzed using coefficient of determination in prediction (R 2 ) and ratio of preference of determination (RPD). Large variation occurred in the calibrations, R 2 and RPD due to the variability of the samples. Derived calibrations for total-GSLs, aliphatic-GSLs, glucoraphanin and 4-methoxyglucobrassicin were quantitative with a high accuracy (RPD=1.36, 1.65, 1.63, 1.11) while, for indole-GSLs, glucosinigrin, glucoiberin, glucobrassicin and 1-methoxyglucobrassicin were more qualitative (RPD=0.95, 0.62, 0.67, 0.81, 0.56). Overall, the results indicated NIRS has a good potential to determine different GSLs in a large sample pool of broccoli quantitatively and qualitatively. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Accurate genotyping across variant classes and lengths using variant graphs

    DEFF Research Database (Denmark)

    Sibbesen, Jonas Andreas; Maretty, Lasse; Jensen, Jacob Malte

    2018-01-01

    of read k-mers to a graph representation of the reference and variants to efficiently perform unbiased, probabilistic genotyping across the variation spectrum. We demonstrate that BayesTyper generally provides superior variant sensitivity and genotyping accuracy relative to existing methods when used...... collecting a set of candidate variants across discovery methods, individuals and databases, and then realigning the reads to the variants and reference simultaneously. However, this realignment problem has proved computationally difficult. Here, we present a new method (BayesTyper) that uses exact alignment...... to integrate variants across discovery approaches and individuals. Finally, we demonstrate that including a ‘variation-prior’ database containing already known variants significantly improves sensitivity....

  2. Genotype by sex and genotype by age interactions with sedentary behavior: the Portuguese Healthy Family Study.

    Directory of Open Access Journals (Sweden)

    Daniel M V Santos

    Full Text Available Sedentary behavior (SB expression and its underlying causal factors have been progressively studied, as it is a major determinant of decreased health quality. In the present study we applied Genotype x Age (GxAge and Genotype x Sex (GxSex interaction methods to determine if the phenotypic expression of different SB traits is influenced by an interaction between genetic architecture and both age and sex. A total of 1345 subjects, comprising 249 fathers, 327 mothers, 334 sons and 325 daughters, from 339 families of The Portuguese Healthy Family Study were included in the analysis. SB traits were assessed by means of a 3-d physical activity recall, the Baecke and IPAQ questionnaires. GxAge and GxSex interactions were analyzed using SOLAR 4.0 software. Sedentary behaviour heritability estimates were not always statistically significant (p>0.05 and ranged from 3% to 27%. The GxSex and GxAge interaction models were significantly better than the single polygenic models for TV (min/day, EEsed (kcal/day, personal computer (PC usage and physical activty (PA tertiles. The GxAge model is also significantly better than the polygenic model for Sed (min/day. For EEsed, PA tertiles, PC and Sed, the GxAge interaction was significant because the genetic correlation between SB environments was significantly different from 1. Further, PC and Sed variance heterogeneity among distinct ages were observed. The GxSex interaction was significant for EEsed due to genetic variance heterogeneity between genders and for PC due to a genetic correlation less than 1 across both sexes. Our results suggest that SB expression may be influenced by the interactions between genotype with both sex and age. Further, different sedentary behaviors seem to have distinct genetic architectures and are differentially affected by age and sex.

  3. Development of duplex real-time RT-PCR based on Taqman technology for detecting simultaneously the genome of pan-enterovirus and enterovirus 71.

    Science.gov (United States)

    Hwang, Seoyeon; Kang, Byunghak; Hong, Jiyoung; Kim, Ahyoun; Kim, Hyejin; Kim, Kisang; Cheon, Doo-Sung

    2013-07-01

    Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks. Copyright © 2013 Wiley Periodicals, Inc.

  4. Genotyping isolates of the entomopathogenic fungus Beauveria ...

    African Journals Online (AJOL)

    Multi-locus denaturing gradient gel electrophoresis (DGGE) analysis was developed to investigate the genotypes of Beauveria bassiana sensu lato. ... These results demonstrated that multi-locus DGGE is a potentially useful molecular marker for genotyping, identifying and tracking the fates of experimentally released ...

  5. Genetic relationship among Musa genotypes revealed by ...

    African Journals Online (AJOL)

    enoh

    2012-03-29

    Mar 29, 2012 ... A banana germplasm was established containing 44 Musa genotypes collected from various locations in Malaysia. To detect their genetic variation and to rule out duplicates among cultivar, microsatellite markers were used in their analysis. The microsatellite profiles of 44 Musa genotypes of various origins.

  6. Finding the right coverage : The impact of coverage and sequence quality on single nucleotide polymorphism genotyping error rates

    NARCIS (Netherlands)

    Fountain, Emily D.; Pauli, Jonathan N.; Reid, Brendan N.; Palsboll, Per J.; Peery, M. Zachariah

    Restriction-enzyme-based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction-enzyme-based methods remain largely unknown.

  7. Nutrient composition of strawberry genotypes cultivated in a horticulture farm.

    Science.gov (United States)

    Hossain, Ashrafi; Begum, Parveen; Salma Zannat, M; Hafizur Rahman, Md; Ahsan, Monira; Islam, Sheikh Nazrul

    2016-05-15

    This article decribes the nutrient composition of four strawberry genotypes cultivated at the Sher-e-Bangla Agriculture University horticulture farm in Dhaka (Bangladesh). AOAC and standard validated methods were employed to analyse the nutrient composition. Protein, fat and ash contents were found to be vary significantly (LSD<0.05), while the variation in moisture (LSD<1.33), dietary fibre (LSD<0.15) and total sugar (LSD<0.09) were found to be insignificant among the genotypes. Vitamin C content ranged from 26.46 mg to 37.77 mg per 100g edible strawberries (LSD<0.060). Amount of carotenoids were found to be very low being in a range of 0.99-3.30 μg per 100g edible fruit. Analysis of mineral revealed that strawberry genotypes contained a wide array of minerals including Ca, Mg, Na, K, P, Mn, Zn, Cu and Fe; most of which varied significantly (LSD<0.05) among the genotypes. Strawberries could be a potential dietary supplement for vitamin C along with minerals, particularly for the children who do not like local fruits, but love to eat the colourful strawberries. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Distribution of hepatitis C virus genotypes among injecting drug users in Lebanon

    Directory of Open Access Journals (Sweden)

    Shamra Sarah

    2010-05-01

    Full Text Available Abstract Background The aim of this study is to determine the prevalence of anti-HCV among injecting drug users (IDUs in Lebanon, to establish the current prevalence of HCV genotypes in this population and to determine whether demographic characteristics and behavioral variables differ between participants who were HCV-RNA positive and those who were HCV-RNA negative or between the different genotypes. Participants were recruited using respondent-driven sampling method. The blood samples were collected as dried blood spots and then eluted to be tested for HCV, HBV and HIV by ELISA. Anti-HCV positive samples were subjected to RNA extraction followed by qualitative detection and genotyping. Results Among 106 IDUs, 56 (52.8% were anti-HCV-positive. The two groups did not differ in terms of age, marital status, and nationality. As for the behavioral variable, there was a trend of increased risky behaviors among the HCV-RNA positive group as compared to the HCV-RNA negative group but none of the variables reached statistical significance. Half (50% of the 56 anti-HCV-positive were HCV-RNA positive. Genotype 3 was the predominant one (57.1% followed by genotype 1 (21% and genotype 4 (18%. Conclusions The predominance of genotype 3 seems to be the predominant genotype among IDUs in Lebanon, a situation similar to that among IDUs in Western Europe. This study provides a base-line against possible future radical epidemiological variant that might occur in IDUs.

  9. Molecular methods for the detection of human papillomavirus infection: new insights into their role in diagnostics and epidemiological surveillance

    Directory of Open Access Journals (Sweden)

    Andrea Piana

    2009-06-01

    Full Text Available Human papillomaviruses (HPVs comprise more than 180 genotypes. HPV infection is mainly diagnosed by molecular methods. The aim of our study was to review the main molecular methods used to diagnose HPV infection, underscoring their characteristics. Several methods have been developed for molecular diagnosis of Papilloma infection, such as those based on PCR technique. Another commercial non-PCR based diagnostic method is Hybrid Capture test; it is the only commercially available HPV DNA detection test approved by the FDA. Several Authors have suggested that viral load and E6/E7 transcripts could be used as surrogate markers of persistent HPV infection, being more specific predictors of progressive disease than the simple presence of HPV DNA. Validating clinical sensitivity and specificity of each technique and improving the interpretation of the results are essential; consequently, there is a clear need for well characterized international quality control panels to compare the various diagnostic methods. HPV DNA testing could be useful both as a primary screening test, alone or in combination with a Pap smear, for the early detection of cervical cancer precursors, and as triage test to select women with minor cytological abnormalities who will need further follow-up and to predict possible treatment failure in women with diagnosed high-grade intraepithelial lesions who have undergone excisional therapy. In the next future surveillance for HPV infections, based on these molecular methods, could represent an important step for the development of primary and secondary prophylactic interventions, such as new vaccines targeted to genotypes who might replace those previously prevalent.

  10. Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

    Science.gov (United States)

    Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

    2015-12-01

    We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Detection and genotyping of human papilloma virus in cervical cancer specimens from Saudi patients.

    Science.gov (United States)

    Al-Badawi, Ismail A; Al-Suwaine, Abdulrahman; Al-Aker, Murad; Asaad, Lina; Alaidan, Alwaleed; Tulbah, Asma; Fe Bohol, Marie; Munkarah, Adnan R

    2011-07-01

    To determine the rates and types of human papilloma virus (HPV) infection in cervical cancer specimens from Saudi patients. One hundred specimens were randomly selected and retrieved from the achieved samples stored in the pathology department accessioned under the diagnosis of cervical cancer and carcinoma in situ between the years 1997 and 2007. Human papilloma virus in the clinical samples was detected using polymerase chain reaction amplification methods. Two primer systems are commonly used: the MY09-MY11 primers and the GP5+-GP6+ that amplify a wide range of HPV genotypes. Human papilloma virus isolates were genotyped using DNA sequencing and reverse line blot hybridization assay to identify the high-risk HPV genotypes. Ninety cases fulfilled the diagnostic criteria and were analyzed. The rate of HPV genotype detection among cervical cancer samples was 95.5%. The most common HPV genotype detected by both methods was HPV-16 (63.4%), followed by HPV-18 (11.1%), HPV-45 (4.5%), HPV-33 (3.3%), and HPV-31, HPV-52, HPV-53, HPV-58, HPV-59, and HPV-66 with 2.2% prevalence rate each. Prevalence of HPV genotypes among patients with cervical cancer in Saudi Arabia is comparable to the international rates. The use of the reverse line blot hybridization assay genotyping method could be useful for classifying oncogenic HPV-positive women. It is relatively inexpensive and reliable and can be performed in routine practice or epidemiological study compared with the available standard commercial kits.

  12. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    OpenAIRE

    Hubbard Alan E; Dorsey Grant; Gupta Vinay; Rosenthal Philip J; Greenhouse Bryan

    2010-01-01

    Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary elec...

  13. Lactase persistence genotypes and malaria susceptibility in Fulani of Mali

    Directory of Open Access Journals (Sweden)

    Dolo Amagana

    2011-01-01

    Full Text Available Abstract Background Fulani are a widely spread African ethnic group characterized by lower susceptibility to Plasmodium falciparum, clinical malaria morbidity and higher rate of lactase persistence compared to sympatric tribes. Lactase non-persistence, often called lactose intolerance, is the normal condition where lactase activity in the intestinal wall declines after weaning. Lactase persistence, common in Europe, and in certain African people with traditions of raising cattle, is caused by polymorphisms in the enhancer region approximately 14 kb upstream of the lactase gene. Methods To evaluate the relationship between malaria and lactase persistence genotypes, a 400 bp region surrounding the main European C/T-13910 polymorphism upstream of the lactase gene was sequenced. DNA samples used in the study originated from 162 Fulani and 79 Dogon individuals from Mali. Results Among 79 Dogon only one heterozygote of the lactase enhancer polymorphism was detected, whereas all others were homozygous for the ancestral C allele. Among the Fulani, the main European polymorphism at locus C/T-13910 was by far the most common polymorphism, with an allele frequency of 37%. Three other single-nucleotide polymorphisms were found with allele frequencies of 3.7%, 1.9% and 0.6% each. The novel DNA polymorphism T/C-13906 was seen in six heterozygous Fulani. Among the Fulani with lactase non-persistence CC genotypes at the C/T-13910 locus, 24% had malaria parasites detectable by microscopy compared to 18% for lactase persistent genotypes (P = 0.29. Pooling the lactase enhancer polymorphisms to a common presumptive genotype gave 28% microscopy positives for non-persistent and 17% for others (P = 0.11. Conclusions Plasmodium falciparum parasitaemia in asymptomatic Fulani is more common in individuals with lactase non-persistence genotypes, but this difference is not statistically significant. The potential immunoprotective properties of dietary cow milk as a reason

  14. Genetic diversity of popcorn genotypes using molecular analysis.

    Science.gov (United States)

    Resh, F S; Scapim, C A; Mangolin, C A; Machado, M F P S; do Amaral, A T; Ramos, H C C; Vivas, M

    2015-08-19

    In this study, we analyzed dominant molecular markers to estimate the genetic divergence of 26 popcorn genotypes and evaluate whether using various dissimilarity coefficients with these dominant markers influences the results of cluster analysis. Fifteen random amplification of polymorphic DNA primers produced 157 amplified fragments, of which 65 were monomorphic and 92 were polymorphic. To calculate the genetic distances among the 26 genotypes, the complements of the Jaccard, Dice, and Rogers and Tanimoto similarity coefficients were used. A matrix of Dij values (dissimilarity matrix) was constructed, from which the genetic distances among genotypes were represented in a more simplified manner as a dendrogram generated using the unweighted pair-group method with arithmetic average. Clusters determined by molecular analysis generally did not group material from the same parental origin together. The largest genetic distance was between varieties 17 (UNB-2) and 18 (PA-091). In the identification of genotypes with the smallest genetic distance, the 3 coefficients showed no agreement. The 3 dissimilarity coefficients showed no major differences among their grouping patterns because agreement in determining the genotypes with large, medium, and small genetic distances was high. The largest genetic distances were observed for the Rogers and Tanimoto dissimilarity coefficient (0.74), followed by the Jaccard coefficient (0.65) and the Dice coefficient (0.48). The 3 coefficients showed similar estimations for the cophenetic correlation coefficient. Correlations among the matrices generated using the 3 coefficients were positive and had high magnitudes, reflecting strong agreement among the results obtained using the 3 evaluated dissimilarity coefficients.

  15. Developmental plasticity: re-conceiving the genotype.

    Science.gov (United States)

    Sultan, Sonia E

    2017-10-06

    In recent decades, the phenotype of an organism (i.e. its traits and behaviour) has been studied as the outcome of a developmental 'programme' coded in its genotype. This deterministic view is implicit in the Modern Synthesis approach to adaptive evolution as a sorting process among genetic variants. Studies of developmental pathways have revealed that genotypes are in fact differently expressed depending on environmental conditions. Accordingly, the genotype can be understood as a repertoire of potential developmental outcomes or norm of reaction. Reconceiving the genotype as an environmental response repertoire rather than a fixed developmental programme leads to three critical evolutionary insights. First, plastic responses to specific conditions often comprise functionally appropriate trait adjustments, resulting in an individual-level, developmental mode of adaptive variation. Second, because genotypes are differently expressed depending on the environment, the genetic diversity available to natural selection is itself environmentally contingent. Finally, environmental influences on development can extend across multiple generations via cytoplasmic and epigenetic factors transmitted to progeny individuals, altering their responses to their own, immediate environmental conditions and, in some cases, leading to inherited but non-genetic adaptations. Together, these insights suggest a more nuanced understanding of the genotype and its evolutionary role, as well as a shift in research focus to investigating the complex developmental interactions among genotypes, environments and previous environments.

  16. A SNP Genotyping Array for Hexaploid Oat

    Directory of Open Access Journals (Sweden)

    Nicholas A. Tinker

    2014-11-01

    Full Text Available Recognizing a need in cultivated hexaploid oat ( L. for a reliable set of reference single nucleotide polymorphisms (SNPs, we have developed a 6000 (6K BeadChip design containing 257 Infinium I and 5486 Infinium II designs corresponding to 5743 SNPs. Of those, 4975 SNPs yielded successful assays after array manufacturing. These SNPs were discovered based on a variety of bioinformatics pipelines in complementary DNA (cDNA and genomic DNA originating from 20 or more diverse oat cultivars. The array was validated in 1100 samples from six recombinant inbred line (RIL mapping populations and sets of diverse oat cultivars and breeding lines, and provided approximately 3500 discernible Mendelian polymorphisms. Here, we present an annotation of these SNPs, including methods of discovery, gene identification and orthology, population-genetic characteristics, and tentative positions on an oat consensus map. We also evaluate a new cluster-based method of calling SNPs. The SNP design sequences are made publicly available, and the full SNP genotyping platform is available for commercial purchase from an independent third party.

  17. GENOTYPE-PHENOTYPE STUDY OF FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS TYPE 3

    OpenAIRE

    Sieni , Elena; Cetica , Valentina; Santoro , Alessandra; Beutel , Karin; Mastrodicasa , Elena; Meeths , Marie; Ciambotti , Benedetta; Brugnolo , Francesca; Zur Stadt , Udo; Pende , Daniela; Moretta , Lorenzo; Griffiths , Gillian M.; Henter , Jan-Inge; Janka , Gritta; Arico , Maurizio

    2011-01-01

    Abstract Background: Mutations of UNC13D are causative for FHL3 (OMIM 608898). We present a genotype-phenotype study of 845 FHL3 patients. Methods: A consortium of 3 countries planned to pool in a common database data on presenting features and mutations from individual patients with biallelic UNC13D mutations. Results: 845 FHL3 patients (median age: 4.1 months) were reported from Florence, Italy (n=54), Hamburg, Germany (n=18), Stockholm, Sweden (n=123). Their ethnic origi...

  18. The association of complex liver disorders with HBV genotypes prevalent in Pakistan

    Directory of Open Access Journals (Sweden)

    Qureshi Huma

    2007-11-01

    Full Text Available Abstract Background Genotyping of HBV is generally used for determining the epidemiological relationship between various virus strains and origin of infection mostly in research studies. The utility of genotyping for clinical applications is only beginning to gain importance. Whether HBV genotyping will constitute part of the clinical evaluation of Hepatitis B patients depends largely on the availability of the relevance of the evidence based information. Since Pakistan has a HBV genotype distribution which has been considered less virulent as investigated by earlier studies from south East Asian countries, a study on correlation between HBV genotypes and risk of progression to further complex hepatic infection was much needed Methods A total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Council's (PMRC out patient clinics. Two hundred and twenty six (77% were males, sixty nine (23% were females (M to F ratio 3.3:1. Results Out of 295 patients, 156 (53.2% had Acute(CAH, 71 (24.2% were HBV Carriers, 54 (18.4% had Chronic liver disease (CLD Hepatitis. 14 (4.7% were Cirrhosis and HCC patients. Genotype D was the most prevalent genotype in all categories of HBV patients, Acute (108, Chronic (39, and Carrier (53. Cirrhosis/HCC (7 were HBV/D positive. Genotype A was the second most prevalent with 28 (13% in acute cases, 12 (22.2% in chronics, 14 (19.7% in carriers and 5 (41.7 in Cirrhosis/HCC patients. Mixed genotype (A/D was found in 20 (12.8% of Acute patients, 3 (5.6% of Chronic and 4 (5.6% of carriers, none in case of severe liver conditions. Conclusion Mixed HBV genotypes A, D and A/D combination were present in all categories of patients except that no A/D combination was detected in severe conditions. Genotype D was the dominant genotype. However, genotype A was found to be more strongly associated with severe liver disease. Mixed genotype (A/D did not significantly appear to influence the clinical outcome.

  19. Analysis of HBV genotype distribution and its association with liver cirrhosis in Xinjiang Uygur Autonomous Region, China

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    WANG Xiaozhong

    2014-12-01

    Full Text Available ObjectiveTo investigate the distribution of hepatitis B virus (HBV genotypes among patients in Xinjiang Uygur Autonomous Region, China, and to explore its association with liver cirrhosis. MethodsHBV genotypes of 1018 hepatitis B patients were determined by PCR analysis. The relationship of HBV genotype with clinical outcomes and relevant chronic liver diseases was assessed by contingency chi-square test, Kruskal-Wallis test, and multivariate unconditional logistic regression analysis. ResultsAmong the 828 patients whose HBV genotyping was completed in this study, type C was the major genotype and the percentage was 54.11% (448/828, 25.15% (200/828 had type B, and 16.18% (134/828 had type D. Among the 116 patients with liver cirrhosis, 20.84% had type C, which was significantly more frequent than other genotypes (P<0.00. The multivariate unconditional logistic regression model identified several risk factors for liver cirrhosis, including duration of hepatitis B≥10 years, C genotype, high HBV DNA viral load, and impaired liver function characterized by abnormal alanine aminotransferase test. Among all these factors, genotype C had the highest relevance to liver cirrhosis (OR=2819. ConclusionThe leading genotype of HBV in Xinjiang Uygur Autonomous Region is type C, followed by type B and type D. Genotype C is an independent risk factor for HBV-related liver cirrhosis.

  20. Genotype X/C recombinant (putative genotype I) of hepatitis B virus is rare in Hanoi, Vietnam--genotypes B4 and C1 predominate.

    Science.gov (United States)

    Phung, Thi Bich Thuy; Alestig, Erik; Nguyen, Thanh Liem; Hannoun, Charles; Lindh, Magnus

    2010-08-01

    There are eight known genotypes of hepatitis B virus, A-H, and several subgenotypes, with rather well-defined geographic distributions. HBV genotypes were evaluated in 153 serum samples from Hanoi, Vietnam. Of the 87 samples that could be genotyped, genotype B was found in 67 (77%) and genotype C in 19 (22%). All genotype C strains were of subgenotype C1, and the majority of genotype B strains were B4, while a few were B2. The genotype X/C recombinant strain, identified previously in Swedish patients of indigenous Vietnamese origin, was found in one sample. This variant, proposed to be classified as genotype I, has been found recently also by others in Vietnam and Laos. The current study indicates that the genotype X/C recombinant may represent approximately 1% of the HBV strains circulating in Vietnam. (c) 2010 Wiley-Liss, Inc.

  1. RT-PCR for detection of all seven genotypes of Lyssavirus genus.

    Science.gov (United States)

    Vázquez-Morón, S; Avellón, A; Echevarría, J E

    2006-08-01

    The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.

  2. Reducing Bias of Allele Frequency Estimates by Modeling SNP Genotype Data with Informative Missingness

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    Wan-Yu eLin

    2012-06-01

    Full Text Available The presence of missing single-nucleotide polymorphism (SNP genotypes is common in genetic data. For studies with low-density SNPs, the most commonly used approach to deal with genotype missingness is to simply remove the observations with missing genotypes from the analyses. This naïve method is straightforward but is appropriate only when the missingness is random. However, a given assay often has a different capability in genotyping heterozygotes and homozygotes, causing the phenomenon of ‘differential dropout’ in the sense that the missing rates of heterozygotes and homozygotes are different. In practice, differential dropout among genotypes exists in even carefully designed studies, such as the data from the HapMap project and the Wellcome Trust Case Control Consortium. In this study, we propose a statistical method to model the differential dropout among different genotypes. Compared with the naïve method, our method provides more accurate allele frequency estimates when the differential dropout is present. To demonstrate its practical use, we further apply our method to the HapMap data and a scleroderma data set.

  3. Haptoglobin 2-2 Genotype, Patient, and Graft Survival in Renal Transplant Recipients

    DEFF Research Database (Denmark)

    Dupont, Laust; Eide, Ivar Anders; Hartmann, Anders

    2017-01-01

    Background: Cardiovascular disease is the leading cause of death in renal transplant recipients. An association between haptoglobin genotype 2-2 and cardiovascular disease has been found in patients with diabetes mellitus and liver transplant recipients. To date, the role of haptoglobin genotype...... after renal transplantation has not been studied. Methods: In this single-center retrospective cohort study of 1975 adult Norwegian transplant recipients, who underwent transplantation between 1999 and 2011, we estimated the risk of all-cause and cardiovascular mortality and overall and death...... transplant recipients, we could not demonstrate any association between haptoglobin 2-2 genotype and patient or graft survival after renal transplantation....

  4. Hepatitis C Genotypes in Libya: Correlation with Patients’ Characteristics, Level of Viremia, and Degree of Liver Fibrosis

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    Abdel-Naser Elzuoki

    2017-09-01

    Full Text Available Objectives: Our study sought to determine the distribution of hepatitis C virus (HCV-genotypes among patients attending two tertiary care hospitals in Benghazi and Tripoli, Libya, and correlate this with patient’s characteristics, viral load, and degree of fibrosis. Methods: We conducted a retrospective study of 286 HCV-RNA positive Libyan patients referred from different health care facilities in east and west Libya for specific HCV treatment. HCV genotyping was carried out by gene amplification. Liver histology was graded by Metavir score according to the stage of fibrosis. Results: HCV genotypes 1, 2, 3, and 4 were found in 24.1%, 10.8%, 3.4%, and 61.5% of the patients, respectively. Genotype 4 was detected more frequently in patients from east Libya (Benghazi compared to west Libya (Tripoli (75.9% vs. 41.6%, p = 0.245. Genotype 1 was more frequent in patients from west Libya compared to east Libya (34.1% vs. 16.8%, p = 0.657. There was a significant correlation between HCV genotype distribution and viral load. Patients with genotype 4 exhibited a higher degree of liver fibrosis (p < 0.001. Conclusions: HCV genotype 4 is the predominant genotype in Libya followed by genotype 1. However, as we go from the east to the west of the country, genotype 1 increases. Genotype 4 was associated with higher level of viremia and higher stage of liver fibrosis. It is important to note that both genotypes 1 and 4 are associated with a poor response to pegylated interferon and ribavirin combination therapy. The findings emphasize the need to develop improved strategies in Libya for the successful treatment of HCV infection with novel newly available antiviral drugs.

  5. Anal infections with concomitant Chlamydia trachomatis genotypes among men who have sex with men in Amsterdam, the Netherlands

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    van der Loeff Maarten

    2011-03-01

    Full Text Available Abstract Background Lymphogranuloma venereum (LGV proctitis is caused by Chlamydia trachomatis (Ct genotype L and is endemic among men who have sex with men (MSM in western society. Genotype L infections need to be distinguished from non-LGV (genotypes A-K Ct infections since they require prolonged antibiotic treatment. For this purpose, an in-house developed pmpH based LGV polymerase chain reaction (PCR test is used at the Amsterdam STI outpatient clinic. We investigated retrospectively the anal Ct genotype distribution, and the frequency of concomitant genotype infections in MSM infected with LGV and non-LGV Ct infections. To detect concomitant Ct genotype infections, the pmpH LGV PCR and genoTyping Reverse Hybridization Assay (Ct-DT RHA were used. Methods A total of 201 Ct positive rectal swabs from MSM were selected, which were previously diagnosed as either LGV (n = 99 or non-LGV Ct infection (n = 102 according to the algorithm of Ct detection by the commercially available Aptima Combo 2 assay followed by an in-house pmpH LGV PCR. The samples were retested with the commercially available Ct-DT RHA, which differentiates between 14 major genotypes and is able to detect concomitant Ct genotypes. Results Excellent genotyping agreement was observed between the Ct-DT RHA and the pmpH LGV PCR (Kappa = 0.900, 95%CI = 0.845-0.955, McNemar's p = 1.000. A concomitant non-LGV genotype was detected in 6/99 (6.1% LGV samples. No additional LGV infections were observed with the Ct-DT RHA among the non-LGV Ct group. In the non-LGV group genotype G/Ga (34.3% was seen most frequent, followed by genotype D/Da (22.5% and genotype J (13.7%. All LGV infections were caused by genotype L2. Conclusions Concomitant non-LGV genotypes do not lead to missed LGV proctitis diagnosis. The pmpH LGV PCR displayed excellent agreement with the commercially available Ct-DT genotyping RHA test. The genotypes G/Ga, D/Da and J were the most frequent non-LGV Ct strains in MSM.

  6. RAPD-based genotyping of Malassezia pachydermatis from Domestic and wild animals

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    Franciele Cristina Kagueyama

    2016-10-01

    Full Text Available Malassezia pachydermatis (M. pachydermatis is a fungus of importance in human and veterinary medicine. Although a part of the normal microbiota, it can sometimes be present in its pathogenic form, particularly causing otitis and dermatitis in animals. Among human beings, it mainly affects immune compromised patients and newborns, causing simple pustulosis, seborrheic dermatitis, tinea versicolor or fungemia. This study aimed to analyze the genomic polymorphism in M. pachydermatis samples isolated from Canis familiaris (domestic dog, Felis catus (domestic cat, and Myrmecophaga tridactyla (giant anteater. Two hundred and fourteen samples were collected and cultured in Sabouraud agar with chloranphenicol (100mg L-1 and incubated at 37 °C for a period of 7 to 10 days. One hundred and sixty six samples that appeared morphologically comparable to yeast cultures were processed for DNA extraction and PCR was performed for a specific region in the Internal Transcribed Spacer (ITS of M. pachydermatis. Among these, seven (4.21% were negative and 159 (95.79% were positive. Of the 159 positive samples, 102 (64.15% were from animals with clinical signs and 57 (35.85% without clinical signs. Fifty-seven samples were selected at random for RAPD-PCR based genotyping and distributed into four genetic groups. Types I and II were more frequent in animals with clinical signs while type III was frequent in healthy animals. Type IV occurred evenly across animals with or without clinical signs. These results indicate differences in pathogenicity of the fungus based on the genotype.

  7. Hepatitis B virus genotypes circulating in Brazil: molecular characterization of genotype F isolates

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    Virgolino Helaine A

    2007-11-01

    Full Text Available Abstract Background Hepatitis B virus (HBV isolates have been classified in eight genotypes, A to H, which exhibit distinct geographical distributions. Genotypes A, D and F are predominant in Brazil, a country formed by a miscegenated population, where the proportion of individuals from Caucasian, Amerindian and African origins varies by region. Genotype F, which is the most divergent, is considered indigenous to the Americas. A systematic molecular characterization of HBV isolates from different parts of the world would be invaluable in establishing HBV evolutionary origins and dispersion patterns. A large-scale study is needed to map the region-by-region distribution of the HBV genotypes in Brazil. Results Genotyping by PCR-RFLP of 303 HBV isolates from HBsAg-positive blood donors showed that at least two of the three genotypes, A, D, and F, co-circulate in each of the five geographic regions of Brazil. No other genotypes were identified. Overall, genotype A was most prevalent (48.5%, and most of these isolates were classified as subgenotype A1 (138/153; 90.2%. Genotype D was the most common genotype in the South (84.2% and Central (47.6% regions. The prevalence of genotype F was low (13% countrywide. Nucleotide sequencing of the S gene and a phylogenetic analysis of 32 HBV genotype F isolates showed that a great majority (28/32; 87.5% belonged to subgenotype F2, cluster II. The deduced serotype of 31 of 32 F isolates was adw4. The remaining isolate showed a leucine-to-isoleucine substitution at position 127. Conclusion The presence of genotypes A, D and F, and the absence of other genotypes in a large cohort of HBV infected individuals may reflect the ethnic origins of the Brazilian population. The high prevalence of isolates from subgenotype A1 (of African origin indicates that the African influx during the colonial slavery period had a major impact on the circulation of HBV genotype A currently found in Brazil. Although most genotype F

  8. Forensic SNP genotyping with SNaPshot

    DEFF Research Database (Denmark)

    Fondevila, M; Børsting, C; Phillips, C

    2017-01-01

    to routine STR profiling, use of SNaPshot is an important part of the development of SNP sets for a wide range of forensic applications with these markers, from genotyping highly degraded DNA with very short amplicons to the introduction of SNPs to ascertain the ancestry and physical characteristics......This review explores the key factors that influence the optimization, routine use, and profile interpretation of the SNaPshot single-base extension (SBE) system applied to forensic single-nucleotide polymorphism (SNP) genotyping. Despite being a mainly complimentary DNA genotyping technique...... of an unidentified contact trace donor. However, this technology, as resourceful as it is, displays several features that depart from the usual STR genotyping far enough to demand a certain degree of expertise from the forensic analyst before tackling the complex casework on which SNaPshot application provides...

  9. HMSRP Hawaiian Monk Seal Microsatellite Genotypes

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Currently ~2,400 Hawaiian monk seal specimens have been analyzed genetically, providing genotypes at 18 microsatellite loci. These data are organized by individual,...

  10. Global distribution of novel rhinovirus genotype

    DEFF Research Database (Denmark)

    Briese, Thomas; Renwick, Neil; Venter, Marietjie

    2008-01-01

    Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years Udgivelsesdato...

  11. Assessment of antibiotic susceptibilities, genotypic characteristics ...

    African Journals Online (AJOL)

    Jane

    2011-09-28

    Sep 28, 2011 ... Staphylococcus aureus and Salmonella Typhimurium ... This study was designed to evaluate the antibiotic susceptibilities, genotypic characteristics and ..... Distribution of reference and virulence genes among antibiotic-sensitive S. aureus (SAS), .... environmental factors such as temperature, water activity,.

  12. Hepatitis C Virus: Viral Quasispecies and Genotypes

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    Kyoko Tsukiyama-Kohara

    2017-12-01

    Full Text Available Hepatitis C virus (HCV mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs, which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

  13. Hepatitis C Virus: Viral Quasispecies and Genotypes.

    Science.gov (United States)

    Tsukiyama-Kohara, Kyoko; Kohara, Michinori

    2017-12-22

    Hepatitis C virus (HCV) mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs), which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

  14. Early seedling development of Medicago truncatula genotypes ...

    African Journals Online (AJOL)

    adel

    2014-01-08

    Jan 8, 2014 ... heat shock proteins; ABA, abscisic acid. Page 2. Amar et al. 323. Figure 1. Seed vigor of M. truncatula genotypes under different salt stress conditions. Results are means ..... (HSPs) that accumulate during seed late maturation.

  15. Hearing impairment in genotyped Wolfram syndrome patients.

    NARCIS (Netherlands)

    Plantinga, R.F.; Pennings, R.J.E.; Huygen, P.L.M.; Bruno, R.; Eller, P.; Barrett, T.G.; Vialettes, B.; Paquis-Fluklinger, V.; Lombardo, F.; Cremers, C.W.R.J.

    2008-01-01

    OBJECTIVES: Wolfram syndrome is a progressive neurodegenerative syndrome characterized by the features "DIDMOAD" (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). We sought to study the audiometric data of genotyped Wolfram syndrome patients with sensorineural hearing impairment.

  16. First insight into the genotypic diversity of clinical Mycobacterium tuberculosis isolates from Gansu Province, China.

    Directory of Open Access Journals (Sweden)

    Jie Liu

    Full Text Available BACKGROUND: Investigations of Mycobacterium tuberculosis genetic diversity in China have indicated a significant regional distribution. The aim of this study was to characterize the genotypes of clinical M. tuberculosis isolates obtained from Gansu, which has a special geographic location in China. METHODOLOGY/PRINCIPAL FINDINGS: A total of 467 clinical M. tuberculosis strains isolated in Gansu Province were genotyped by 15-locus mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR and spoligotyping. The results showed that 445 isolates belonged to six known spoligotype lineages, whereas 22 isolates were unknown. The Beijing genotype was the most prevalent (87.58%, n = 409, while the shared type 1 was the dominant genotype (80.94%, n = 378. The second most common lineage was the T lineage, with 25 isolates (5.35%, followed by the H lineage with 5 isolates (1.07%, the MANU family (0.64%, 3 isolates, the U family (0.43%, 2 isolates and the CAS lineage with 1 isolate (0.21%. By using the VNTR15China method, we observed 15 groups and 228 genotypes among the 467 isolates. We found no association between the five larger groups (including the Beijing genotype and sex, age, or treatment status, and there was no noticeable difference in the group analysis in different areas. In the present study, seven of the 15 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. CONCLUSIONS/SIGNIFICANCE: The Beijing genotype is the predominant genotype in Gansu province. We confirm that VNTR15China is suitable for typing Beijing strains in China and that it has a better discriminatory power than spoligotyping. Therefore, the use of both methods is the most suitable for genotyping analysis of M. tuberculosis.

  17. Blood group genotyping: from patient to high-throughput donor screening.

    Science.gov (United States)

    Veldhuisen, B; van der Schoot, C E; de Haas, M

    2009-10-01

    Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

  18. Evaluating Imputation Algorithms for Low-Depth Genotyping-By-Sequencing (GBS Data.

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    Ariel W Chan

    Full Text Available Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS methods, such as Genotyping-By-Sequencing (GBS, offer an inexpensive alternative to array-based genotyping. Although affordable, datasets derived from HTS methods suffer from sequencing error, alignment errors, and missing data, all of which introduce noise and uncertainty to variant discovery and genotype calling. Under such circumstances, meaningful analysis of the data is difficult. Our primary interest lies in the issue of how one can accurately infer or impute missing genotypes in HTS-derived datasets. Many of the existing genotype imputation algorithms and software packages were primarily developed by and optimized for the human genetics community, a field where a complete and accurate reference genome has been constructed and SNP arrays have, in large part, been the common genotyping platform. We set out to answer two questions: 1 can we use existing imputation methods developed by the human genetics community to impute missing genotypes in datasets derived from non-human species and 2 are these methods, which were developed and optimized to impute ascertained variants, amenable for imputation of missing genotypes at HTS-derived variants? We selected Beagle v.4, a widely used algorithm within the human genetics community with reportedly high accuracy, to serve as our imputation contender. We performed a series of cross-validation experiments, using GBS data collected from the species Manihot esculenta by the Next Generation (NEXTGEN Cassava Breeding Project. NEXTGEN currently imputes missing genotypes in their datasets using a LASSO-penalized, linear regression method (denoted 'glmnet'. We selected glmnet to serve as a benchmark imputation method for this reason. We obtained estimates of imputation accuracy by masking a subset of observed genotypes, imputing, and

  19. Evaluating Imputation Algorithms for Low-Depth Genotyping-By-Sequencing (GBS) Data.

    Science.gov (United States)

    Chan, Ariel W; Hamblin, Martha T; Jannink, Jean-Luc

    2016-01-01

    Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordable, datasets derived from HTS methods suffer from sequencing error, alignment errors, and missing data, all of which introduce noise and uncertainty to variant discovery and genotype calling. Under such circumstances, meaningful analysis of the data is difficult. Our primary interest lies in the issue of how one can accurately infer or impute missing genotypes in HTS-derived datasets. Many of the existing genotype imputation algorithms and software packages were primarily developed by and optimized for the human genetics community, a field where a complete and accurate reference genome has been constructed and SNP arrays have, in large part, been the common genotyping platform. We set out to answer two questions: 1) can we use existing imputation methods developed by the human genetics community to impute missing genotypes in datasets derived from non-human species and 2) are these methods, which were developed and optimized to impute ascertained variants, amenable for imputation of missing genotypes at HTS-derived variants? We selected Beagle v.4, a widely used algorithm within the human genetics community with reportedly high accuracy, to serve as our imputation contender. We performed a series of cross-validation experiments, using GBS data collected from the species Manihot esculenta by the Next Generation (NEXTGEN) Cassava Breeding Project. NEXTGEN currently imputes missing genotypes in their datasets using a LASSO-penalized, linear regression method (denoted 'glmnet'). We selected glmnet to serve as a benchmark imputation method for this reason. We obtained estimates of imputation accuracy by masking a subset of observed genotypes, imputing, and calculating the

  20. Rare variant association analysis in case-parents studies by allowing for missing parental genotypes.

    Science.gov (United States)

    Li, Yumei; Xiang, Yang; Xu, Chao; Shen, Hui; Deng, Hongwen

    2018-01-15

    The development of next-generation sequencing technologies has facilitated the identification of rare variants. Family-based design is commonly used to effectively control for population admixture and substructure, which is more prominent for rare variants. Case-parents studies, as typical strategies in family-based design, are widely used in rare variant-disease association analysis. Current methods in case-parents studies are based on complete case-parents data; however, parental genotypes may be missing in case-parents trios, and removing these data may lead to a loss in statistical power. The present study focuses on testing for rare variant-disease association in case-parents study by allowing for missing parental genotypes. In this report, we extended the collapsing method for rare variant association analysis in case-parents studies to allow for missing parental genotypes, and investigated the performance of two methods by using the difference of genotypes between affected offspring and their corresponding "complements" in case-parent trios and TDT framework. Using simulations, we showed that, compared with the methods just only using complete case-parents data, the proposed strategy allowing for missing parental genotypes, or even adding unrelated affected individuals, can greatly improve the statistical power and meanwhile is not affected by population stratification. We conclude that adding case-parents data with missing parental genotypes to complete case-parents data set can greatly improve the power of our strategy for rare variant-disease association.

  1. Empirical Statistical Power for Testing Multilocus Genotypic Effects under Unbalanced Designs Using a Gibbs Sampler

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    Chaeyoung Lee

    2012-11-01

    Full Text Available Epistasis that may explain a large portion of the phenotypic variation for complex economic traits of animals has been ignored in many genetic association studies. A Baysian method was introduced to draw inferences about multilocus genotypic effects based on their marginal posterior distributions by a Gibbs sampler. A simulation study was conducted to provide statistical powers under various unbalanced designs by using this method. Data were simulated by combined designs of number of loci, within genotype variance, and sample size in unbalanced designs with or without null combined genotype cells. Mean empirical statistical power was estimated for testing posterior mean estimate of combined genotype effect. A practical example for obtaining empirical statistical power estimates with a given sample size was provided under unbalanced designs. The empirical statistical powers would be useful for determining an optimal design when interactive associations of multiple loci with complex phenotypes were examined.

  2. Existence of various human parvovirus B19 genotypes in Chinese plasma pools: genotype 1, genotype 3, putative intergenotypic recombinant variants and new genotypes.

    Science.gov (United States)

    Jia, Junting; Ma, Yuyuan; Zhao, Xiong; Huangfu, Chaoji; Zhong, Yadi; Fang, Chi; Fan, Rui; Lv, Maomin; Zhang, Jingang

    2016-09-17

    Human parvovirus B19 (B19V) is a frequent contaminant of blood and plasma-derived medicinal products. Three distinct genotypes of B19V have been identified. The distribution of the three B19V genotypes has been investigated in various regions or countries. However, in China, data on the existence of different B19V genotypes are limited. One hundred and eighteen B19V-DNA positive source plasma pool samples collected from three Chinese blood products manufacturers were analyzed. The subgenomic NS1/VP1u region junction of B19V was amplified by nested PCR. These amplified products were then cloned and subsequently sequenced. For genotyping, their phylogenetic inferences were constructed based on the NS1/VP1-unique region. Then putative recombination events were analyzed and identified. Phylogenetic analysis of 118 B19V sequences attributed 61.86 % to genotype 1a, 10.17 % to genotype 1b, and 17.80 % to genotype 3b. All the genotype 3b sequences obtained in this study grouped as a specific, closely related cluster with B19V strain D91.1. Four 1a/3b recombinants and 5 new atypical B19V variants with no recombination events were identified. There were at least 3 subtypes (1a, 1b and 3b) of B19V circulating in China. Furthermore, putative B19V 1a/3b recombinants and unclassified strains were identified as well. Such recombinant and unclassified strains may contribute to the genetic diversity of B19V and consequently complicate the B19V infection diagnosis and NAT screening. Further studies will be required to elucidate the biological significance of the recombinant and unclassified strains.

  3. Measles Outbreak with Unique Virus Genotyping, Ontario, Canada, 2015.

    Science.gov (United States)

    Thomas, Shari; Hiebert, Joanne; Gubbay, Jonathan B; Gournis, Effie; Sharron, Jennifer; Severini, Alberto; Jiaravuthisan, Manisa; Shane, Amanda; Jaeger, Valerie; Crowcroft, Natasha S; Fediurek, Jill; Sander, Beate; Mazzulli, Tony; Schulz, Helene; Deeks, Shelley L

    2017-07-01

    The province of Ontario continues to experience measles virus transmissions despite the elimination of measles in Canada. We describe an unusual outbreak of measles in Ontario, Canada, in early 2015 that involved cases with a unique strain of virus and no known association among primary case-patients. A total of 18 cases of measles were reported from 4 public health units during the outbreak period (January 25-March 23, 2015); none of these cases occurred in persons who had recently traveled. Despite enhancements to case-patient interview methods and epidemiologic analyses, a source patient was not identified. However, the molecular epidemiologic analysis, which included extended sequencing, strongly suggested that all cases derived from a single importation of measles virus genotype D4. The use of timely genotype sequencing, rigorous epidemiologic investigation, and a better understanding of the gaps in surveillance are needed to maintain Ontario's measles elimination status.

  4. HPV genotype-specific concordance between EuroArray HPV, Anyplex II HPV28 and Linear Array HPV Genotyping test in Australian cervical samples

    Directory of Open Access Journals (Sweden)

    Alyssa M. Cornall

    2017-12-01

    Full Text Available Purpose: To compare human papillomavirus genotype-specific performance of two genotyping assays, Anyplex II HPV28 (Seegene and EuroArray HPV (EuroImmun, with Linear Array HPV (Roche. Methods: DNA extracted from clinican-collected cervical brush specimens in PreservCyt medium (Hologic, from 403 women undergoing management for detected cytological abnormalities, was tested on the three assays. Genotype-specific agreement were assessed by Cohen's kappa statistic and Fisher's z-test of significance between proportions. Results: Agreement between Linear Array and the other 2 assays was substantial to almost perfect (κ = 0.60 − 1.00 for most genotypes, and was almost perfect (κ = 0.81 – 0.98 for almost all high-risk genotypes. Linear Array overall detected most genotypes more frequently, however this was only statistically significant for HPV51 (EuroArray; p = 0.0497, HPV52 (Anyplex II; p = 0.039 and HPV61 (Anyplex II; p=0.047. EuroArray detected signficantly more HPV26 (p = 0.002 and Anyplex II detected more HPV42 (p = 0.035 than Linear Array. Each assay performed differently for HPV68 detection: EuroArray and LA were in moderate to substantial agreement with Anyplex II (κ = 0.46 and 0.62, respectively, but were in poor disagreement with each other (κ = −0.01. Conclusions: EuroArray and Anyplex II had similar sensitivity to Linear Array for most high-risk genotypes, with slightly lower sensitivity for HPV 51 or 52. Keywords: Human papillomavirus, Genotyping, Linear Array, Anyplex II, EuroArray, Cervix

  5. CMV genotyping using different samples in post renal transplant recipients with CMV disease

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    Ramya Barani

    2017-10-01

    Full Text Available CMV is the most common viral infection which occurs in post renal transplant recipients (PTR. There are four different gB genotypes (gB1 to gB4 which exist in CMV. Studies have reported that mixed infection with different genotypes will cause severe clinical manifestations as well as co-infection with other herpesvirus including Epstein-Barr virus (EBV [1]. CMV can cause compartmentalized disease involving different organs with different genotypes. There are reports in immuno compromised individuals with different genotypes [2, 3]. Institutional ethics committee approval was obtained prior to conduct of the study (IEC-NI/08/DEC/07/46. Whole blood, saliva and urine were collected from PTR. DNA were extracted (Qiagen DNA mini kit and CMV quantitative PCR targeting ppUL83 gene was performed with CMV R-gene™ using an ABI 7900 Fast real time PCR (SDS Version: 2.4. PTR who had high viral load (>1000 copies/ml in any three or two samples were included for CMV genotyping PCR targeting gB region (410-bp [2]. DNA sequencing was performed in ABI 3730 GA platform by Sanger method and sequences were analyzed by reference strains. A total of 24 samples were collected from 9 PTR. Among these four PTR had high viral load in all three samples (whole blood, urine & saliva and those with high viral load (n=5 in 2 samples (Whole blood & urine/saliva were screened for CMV genotyping. Majority of the strains belonged to genotype B1 and only one PTR was infected with genotype B2 in three samples. In PTR with genotype B1, gastro intestinal infection (GI was predominantly found in 78% (n=7 followed by graft dysfunction (GDF in 56% (n=5 of the PTR. PTR who detected with genotype B2 was associated with fever, leukopenia (CMV syndrome, GDF and also found with EBV infection. Co-infection with EBV was observed in 44% (n=4; VZV and HSV type 1 was also observed. Genotypes are associated with the severity of the disease and co-infection with other herpes virus infections. In

  6. Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

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    Haque Kashif A

    2005-09-01

    Full Text Available Abstract Background Whole genome amplification (WGA promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA quantity. We evaluated the performance of multiple displacement amplification (MDA WGA using gDNA extracted from lymphoblastoid cell lines (N = 27 with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA was performed using three DNA quantification methods (OD, PicoGreen® and RT-PCR. Two panels of N = 15 STR (using the AmpFlSTR® Identifiler® panel and N = 49 SNP (TaqMan® genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. Results The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. Conclusion The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of

  7. Evaluation of Drought Tolerance in 16 Genotypes of Safflower(Carthamus tinctoriusL

    Directory of Open Access Journals (Sweden)

    s.M Azimzadeh

    2011-02-01

    Full Text Available Abstract In order to study drought tolerance of 16 genotypes of safflower an experiment was conducted in Research Farm of Shirvan Islamic Azad University during 2005 –2006 growing season. The experiment was performed as randomized complete block design with 4 replications in two separate irrigated and rainfed conditions. The seed rate was 20 seed per square meter, hand planted in each plot. During growing season some agronomic traits including number of grains per heads, grain yield of total head per plant, TKW and grain yield per hectare were recorded. To select drought tolerant genotypes 4 methods including stress tolerance index, stress susceptibility index, cell membrane stability and relative water content were applied. The results showed that two genotypes of LRV-51-51 and CW74 had the highest drought tolerance index and the lowest drought susceptibility index compared with the other genotypes. Grain yield of these two genotypes was 1520 and 1452 kg/ha, respectively which were more than other genotypes. According to these traits the genotypes LRV-51-51 and CW74 are recommended to plant in dry regions with low annual rainfall. Keywords: Safflower, Drought tolerance index, Drought susceptibility index, Yield

  8. Identifying the Genotypes of Hepatitis B Virus (HBV) with DNA Origami Label.

    Science.gov (United States)

    Liu, Ke; Pan, Dun; Wen, Yanqin; Zhang, Honglu; Chao, Jie; Wang, Lihua; Song, Shiping; Fan, Chunhai; Shi, Yongyong

    2018-02-01

    The hepatitis B virus (HBV) genotyping may profoundly affect the accurate diagnosis and antiviral treatment of viral hepatitis. Existing genotyping methods such as serological, immunological, or molecular testing are still suffered from substandard specificity and low sensitivity in laboratory or clinical application. In a previous study, a set of high-efficiency hybridizable DNA origami-based shape ID probes to target the templates through which genetic variation could be determined in an ultrahigh resolution of atomic force microscopy (AFM) nanomechanical imaging are established. Here, as a further confirmatory research to explore the sensitivity and applicability of this assay, differentially predesigned DNA origami shape ID probes are also developed for precisely HBV genotyping. Through the specific identification of visualized DNA origami nanostructure with clinical HBV DNA samples, the genetic variation information of genotypes can be directly identified under AFM. As a proof-of-concept, five genotype B and six genotype C are detected in 11 HBV-infected patients' blood DNA samples of Han Chinese population in the single-blinded test. The AFM image-based DNA origami shape ID genotyping approach shows high specificity and sensitivity, which could be promising for virus infection diagnosis and precision medicine in the future. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Genotypic carriers of the obesity-associated FTO polymorphism exhibit different cardiometabolic profiles after an intervention

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    GREICE G. MORAES

    Full Text Available ABSTRACT Background: Children and adolescents with at-risk genotypes (AA/AT of the rs9939609 polymorphism in FTO, a fat mass and obesity-associated gene, may exhibit different cardiometabolic profile responses than subjects with the TT genotype after an interdisciplinary intervention. Methods: The sample consisted of 36 school children from southern Brazil. We used DNA quantitation and real-time polymerase chain reaction (PCR for polymorphism genotyping. We measured anthropometric parameters (body mass index (BMI, waist circumference, hip circumference, waist-hip ratio, body fat percentage and skinfold sum, biochemical parameters (glucose, lipid profile, ultra-sensitive C-reactive protein, uric acid, alanine aminotransferase, aspartate aminotransferase, insulin and adiponectin and blood pressure. The 4-month intervention consisted of physical education classes, nutritional counseling, and postural and oral health counseling. Results: We observed no significant differences among the groups (AA, AT and TT after the intervention. However, we observed improvements in three parameters (waist circumference, hip circumference and C-reactive protein in the AT/AA genotype group and in two parameters (hip circumference and uric acid in the TT genotype group. Conclusions: After an intervention program, carriers of at-risk genotypes for obesity (AA/AT do not exhibit differences in biochemical parameters, blood pressure and anthropometric parameters compared with carriers of the TT genotype.

  10. Soil nutrient patchiness and genotypes interact on the quantity, quality and decomposition of roots versus shoots of Triticum aestivum.

    NARCIS (Netherlands)

    He, W.M.; Shen, Y.; Cornelissen, J.H.C.

    2012-01-01

    Aims: The purpose of this study was to test the hypotheses that soil nutrient patchiness can differentially benefit the decomposition of root and shoot litters and that this facilitation depends on plant genotypes. Methods: We grew 15 cultivars (i. e. genotypes) of winter wheat (Triticum aestivum

  11. Lack of influence of CYP2D6 genotype on the clearance of (R)-, (S)- and racemic-methadone

    DEFF Research Database (Denmark)

    Coller, J K; Joergensen, C; Foster, D J R

    2007-01-01

    OBJECTIVE: To investigate the influence of CYP2D6 genotype on the oral clearance of (R)-, (S)- and rac-methadone. METHODS: In this retrospective study, CYP2D6 genotypes were identified in 56 methadone maintained subjects. Plasma concentrations of (R)-, (S)- and rac-methadone were determined by st...

  12. Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods.

    Science.gov (United States)

    Nishitani, Yosuke; Sasaki, Eiki; Fujisawa, Tomohiko; Osawa, Ro

    2004-02-01

    A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.

  13. In vitro regeneration of some Iranian alfalfa (Medicago sativa L. genotypes via somatic embryogenesis

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    Majid Shokrpour

    2014-12-01

    Full Text Available An effective in vitro regeneration system is one of the prerequisites for genetic manipulation of alfalfa (Medicago sativa L. varieties and genotypes. In this research, somatic embryogenesis of four alfalfa genotypes, 6-18 (synthetic, 4-14 (Kara Yonje- Karakozlu, 3-27 (Kara Yonje Maraghe and y-6 (Regen-SY, were investigated using leaf and petiole explants. Formation of callus and somatic embryogenesis was significantly influenced by the explant type and interaction of genotype and culture medium. Petiole explants of genotype 4-14 produced the highest yield of callus (0.406 gr fresh weight of callus. Percentage of somatic embryogenesis and the number of embryos per callus in petiole explants of genotype 4-14 was higher than those of other genotypes and explants. In genotype 6-18, the highest percentage of somatic embryogenesis was achieved on MS medium containing 5 mg/L 2,4-D and 2 mg/L kinetin. There was no significant differences between genotypes and explants in terms of embryo conversion to plantlet, and on average, 58% of somatic embryos converted to plantlet on MS medium. The petiole explants of genotype 6-18 did not exhibit somatic embryogenesis response in medium containing low ratio of 2,4-D:Kinetin (5 mg/L 2,4-D and 2 mg/L kinetin. While, these explants showed somatic embryogenesis in higher ratio of 2,4-D:Kinetin (5:1. The plantlet conversion efficiency of somatic embryos produced through this study was relatively higher and therefore, the method presented in this study could be used in alfalfa genetic manipulation and molecular studies.

  14. The Correlation between Different Risk Factors of Hepatitis C and Different Genotypes

    Science.gov (United States)

    Mokhtari, Mozhgan; Basirkazeruni, Hanieh; Rostami, Mojtaba

    2017-01-01

    Background: Hepatitis C infection is one of the health problems in the world. Several known risk factors are responsible in transmission of this infection. We are going to study the prevalence of these risk factors for different genotypes of hepatitis C and if possible, specify probable relations between each risk factor and transmission of each genotype. Materials and Methods: This is a cross-sectional study done on 270 people who had positive anti-hepatitis C virus (HCV) antibody and HCV RNA. Demographic specificity and possible risk factors were collected using a questionnaire, and statistical analysis was done by SPSS software (version 20). Chi-square test used to estimate the prevalence and relation between each qualitative risk factor and HCV genotype transmitted. Analysis of variance was used for studying the prevalence and relation between quantitative risk factors and HCV genotypes. Results: The sample size was 270 persons. Of these, 217 (80.4%) were men and 185 (68.5%) were infected with genotype Type III. Most people were in age range of 31–40 years old 92 (34%). Single people were 126 (46.7%) and 169 (62.6%) were high school and university graduated. Tattooing as a risk factor had a meaningful relation with hepatitis C genotype (P < 0.001). Conclusions: According to the findings, most people in central provinces of Iran with hepatitis C are carrying genotype III, with most prevalent risk factors such as intravenous drug use and unsafe sexual activity. Besides, tattooing had a significant association with hepatitis C genotype, so that in these groups of people, genotype I was more frequent isolated virus. PMID:28503500

  15. GENETIC DIVERGENCE AMONG Passiflora cristalina Vanderpl & Zappi. GENOTYPES BASED ON FLOWER AND FRUIT CHARACTERISTICS

    Directory of Open Access Journals (Sweden)

    GREICIELE FARIAS DA SILVEIRA

    Full Text Available ABSTRACT This study aimed to evaluate the genetic divergence among Passiflora cristalina genotypes and quantify the relative contribution of 30 flower and fruit characteristics, seeking to support the preservation and characterization of genetic resources of the species for preservation and use in future breeding programs. We evaluated 150 fruit and 150 flowers collected in 15 genotypes with naturally occurring in the municipality of Alta Floresta, MT. The characterization of genotypes was performed through 30 morphological characteristics of flowers and fruits, 21 of these for flower and 9 for fruit. Data were evaluated using the principal components and cluster methods obtained by UPGMA method from the similarity matrix (Euclidian mean distance, using the Genes software. By principal component analysis, it has been found that the first three components have absorbed 52.11% of the accumulated variation. The characteristics that most contributed to the discrimination of genotypes were fresh fruit weight, stigma length, length of corona filaments, fruit width, petal width and pulp weight, which are more responsive for the selection of P.cristalina genotypes. Smaller contributions to diversity were obtained from anther width, bract width and fruit length. The smallest contributions for diversity were obtained from the following characteristics: anther width, bract width and fruit length. Through UPGMA clustering method, it was found that there is a large genetic divergence among genotypes analyzed because all genotypes were grouped with over 50% of dissimilarity. This study identified genotypes 4, 5 and 9 as the most divergent and therefore the most suitable for breeding in future breeding programs and genetic conservation of the species.

  16. Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial

    DEFF Research Database (Denmark)

    Woudstra, C.; Skarin, H.; Anniballi, F.

    2013-01-01

    Two real-time PCR arrays based on the GeneDisc® cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated wi...

  17. Cotton genotypes selection through artificial neural networks.

    Science.gov (United States)

    Júnior, E G Silva; Cardoso, D B O; Reis, M C; Nascimento, A F O; Bortolin, D I; Martins, M R; Sousa, L B

    2017-09-27

    Breeding programs currently use statistical analysis to assist in the identification of superior genotypes at various stages of a cultivar's development. Differently from these analyses, the computational intelligence approach has been little explored in genetic improvement of cotton. Thus, this study was carried out with the objective of presenting the use of artificial neural networks as auxiliary tools in the improvement of the cotton to improve fiber quality. To demonstrate the applicability of this approach, this research was carried out using the evaluation data of 40 genotypes. In order to classify the genotypes for fiber quality, the artificial neural networks were trained with replicate data of 20 genotypes of cotton evaluated in the harvests of 2013/14 and 2014/15, regarding fiber length, uniformity of length, fiber strength, micronaire index, elongation, short fiber index, maturity index, reflectance degree, and fiber quality index. This quality index was estimated by means of a weighted average on the determined score (1 to 5) of each characteristic of the HVI evaluated, according to its industry standards. The artificial neural networks presented a high capacity of correct classification of the 20 selected genotypes based on the fiber quality index, so that when using fiber length associated with the short fiber index, fiber maturation, and micronaire index, the artificial neural networks presented better results than using only fiber length and previous associations. It was also observed that to submit data of means of new genotypes to the neural networks trained with data of repetition, provides better results of classification of the genotypes. When observing the results obtained in the present study, it was verified that the artificial neural networks present great potential to be used in the different stages of a genetic improvement program of the cotton, aiming at the improvement of the fiber quality of the future cultivars.

  18. Usefulness of Dried Blood Spots (DBS) to perform hepatitis C virus genotyping in drug users in Senegal.

    Science.gov (United States)

    Ndiaye, O; Gozlan, J; Diop-Ndiaye, H; Sall, A S; Chapelain, S; Leprêtre, A; Maynart, M; Gueye, M; Lo, G; Thiam, M; Ba, I; Lacombe, K; Girard, P M; Mboup, S; Kane, C T

    2017-03-01

    The aim of this pilot study was to analyze the Hepatitis C Virus (HCV) genotypes circulating in Senegal among Drug User (DUs), using Dried Blood Spots (DBS) as RNA source for molecular assays. Heroin and/or cocaine users (n = 506) were recruited in Dakar from April to July 2011, using a Respondent Driven Sampling (RDS) method. DBS preparation consisted of five drops of whole blood from finger applied to a Whatman paper card. HCV infection was screened by the detection of anti-HCV antibodies, using a rapid immune-chromatographic test. HCV RNA was quantified on anti-HCV positive DBS, using the Abbott RealTime HCV® Genotyping was performed on DBS with detectable viral load with Versant® HCV Genotype 2.0 Assay (LiPA) and Abbott RealTime HCV Genotype II assay®. Among the 506 participants, 120 were tested as positive for anti-HCV antibodies and their samples were analyzed for HCV RNA viral load and genotype. Out of the 120 DBS tested, HCV RNA was detected on 25 (20.8%). The median viral load was 15,058 IU/ml (ranging from 710 to 766,740 IU/ml). All positive DBS were suitable for the genotyping assay, that showed a predominance of genotype 1 (21/25) including 16 genotypes 1a and 5 genotypes 1b. HCV genotype 1 prevails in a DU population in Dakar. DBS could be useful for HCV RNA genotyping, but optimal storage conditions should required avoiding RNA impairment. Acknowledging this limitation, DBS could be a great interest for detecting and genotyping HCV viremic patients. J. Med. Virol. 89:484-488, 2017. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  19. Study of correlation between polymorphism of angiotensin II-1 receptor gene A1166 genotype and complications in atrial fibrillation

    International Nuclear Information System (INIS)

    Wang Yueping; Gong Wuxing; Shi Li

    2