应用多重PCR和基因扫描技术对五指山猪13个家系32个微卫星基因座的遗传分析

五指山猪是我国著名的珍稀畜种,体型小、性成熟早、遗传稳定、近交程度高,可用于医学、药学和生物材料等方面。作者利用多重PCR和基因扫描技术对五指山猪13个家系的32个微卫星基因座进行遗传检测。统计各家系的等位基因组成,计算各家系的平均杂合度和多态信息含量(PIC);结果,各家系32基因座的平均等位基因数为13.66个,平均PIC为0.731,平均杂合度为0.559。表明海南五指山猪具有丰富的遗传多样性。这些结果对于海南五指山猪的保种、定向选育、开发利用都具有重要的指导意义。     

Utility of a C-Jun Microsatellite Marker in Determining Gene Dosage for fatty (fa)

The Zucker fatty (fa) mutation provides a genetic model for obesity and non-insulin dependent diabetes mellitus. The molecular pathogenesis of the metabolic phenotype of these animals is not known. Detailed molecular maps of the region surrounding the fa locus on rat chromosome 5 can be used for positional cloning experiments as well as to permit genotyping of animals from appropriate crosses before the confounding metabolic effects of obesity have occurred. We describe the development of a polymerase chain reaction (PCR) assay for a polymorphic simple sequence repeat (SSR) in the promoter region of the protooncogene c-Jun. This assay was used to position cJun 4.5cM proximal to the fa locus in 111 F2 progeny of a 13MBN fa/+ F1 intercross. Concurrent use of the cJun SSR with a previously described assay for a microsatellite in the glucose transporter, Glutl, permits rapid and accurate assessment of genotypes at the fa locus in animals of any age using minimal amounts of DNA. A strategy is described which minimizes the error rate in assigning genotype at the fatty locus for backcross and intercross progeny.     

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5'' amine modified to allow fixation to the membrane. Primers are 5'' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours).The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.Published applications of mPCR/RLB include detection of antibiotic resistance genes^1,2, typing of methicillin-resistant Staphylococcus aureus^3-5 and Salmonella sp⁶, molecular serotyping of Streptococcus pneumoniae^7,8, Streptococcus agalactiae⁹ and enteroviruses^10,11, identification of Mycobacterium sp¹², detection of genital^13-15 and respiratory tract¹⁶ and other¹⁷ pathogens and detection and identification of mollicutes¹⁸. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.     

Multiplex PCR and RFLP approaches for identification of rabbitfish (Siganus) species using mitochondrial gene regions

Molecular assays are described for the identification of six rabbitfish (Siganus) species. A multiplex PCR assay using primers targeting the mitochondrial cytochrome b region simultaneously identifies four species: Siganus canaliculatus, S. fuscescens, S. javus, and S. spinus. Subsequent RFLP assays of multiplex amplicons differentiate between S. virgatus and S. corallinus based on diagnostic fragments from the mitochondrial cytochrome oxidase I region. Assays were validated with known specimens demonstrating accuracy of the molecular identification. Applied to morphologically indistinguishable early developmental stages, these assays can facilitate studies on species-specific spatio-temporal patterns of larval dispersal and population connectivity to aid fishery management.     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究.结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果.基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略.同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法.     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究。结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果。基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略。同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法。     

Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

Multiplex PCR for both identification of Brazilian Biomphalaria species (Gastropoda: Planorbidae) and diagnosis of infection by Schistosoma mansoni (Trematoda: Schistosomatidae)

A simple and single-step technique based on multiplex PCR (multiplex polymerase chain reaction) has been developed for simultaneous identification of Brazilian Biomphalaria species, the intermediate hosts of Schistosoma mansoni, and their diagnosis of infection by the trematode. We used species-specific primers directed both to the internal transcribed spacer 2 (ITS2) of ribosomal DNA from 3 of the S. mansoni host species and to the mitochondrial DNA (mtDNA) from the trematode. Those primers were used simultaneously in a single multiplex-PCR reaction, and template DNA was obtained from S. mansoni-infected and noninfected snails. The results were visualized in silver stained polyacrylamide gels, revealing the presence of specific bands. The methodology has shown to be efficient, fast, and reproducible for Biomphalaria species identification and diagnosis of snails infected by S. mansoni during prepatent periods.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

新型湘云金鳙遗传多样性的RAPD及微卫星分析

湘云金鳙是本实验室经过近十年的工作选育出来的,该种鱼通体金红色,肉质鲜嫩,生长速度快,是一种很有价值的养殖鱼种和实验材料.随机选取12尾湘云金鳙(RBC)和12尾来自湘江流域的普通鳙鱼BC(作为对照)进行RAPD和微卫星分析.在优化RAPD检测条件基础上,从230个随机引物中筛选出来的45个扩增较好且多态性强的引物对这...     

A Novel Simple Method for Determining CYP2D6 Gene Copy Number and Identifying Allele(s) with Duplication/Multiplication

Background

Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.

Methods

We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV) genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.

Results

The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.

Conclusions

Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.     

Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.     

Microsatellite variation within and among recently fragmented populations of the golden lion tamarin (Leontopithecus rosalia)

Four variable microsatellite loci were used toexamine the genetic diversity and differentiation of golden lion tamarins (Leontopithecus rosalia) in four populations recently isolated by habitat fragmentation. Using Rst estimates of genetic differentiation, a considerable genetic divergence was detected among these populations, with an averagedifferentiation of 31%. Significant differences in allele number among these populations were found. However, the heterozygosity among these populations was not statistically different. These results suggestthat loss in allele diversity was faster than loss in heterozygosity. Conservation implications, particularly for golden lion tamarins, are then discussed. Loss of allelic diversity might be as serious a concern to endangered species as heterozygosity or inbreeding.     

Microsatellite DNA markers for American shad (Alosa sapidissima) and cross‐species amplification within the family Clupeidae

Sixteen microsatellite loci were identified and characterized for American shad (Alosa sapidissima). The number of alleles per locus observed ranged from eight to 32 and averaged 15.4 alleles. Average observed heterozygosity was 81.1%. The markers were screened using four other species from the family Clupeidae. Amplification success among Alosa species was 79.2% with 81.6% polymorphism among those markers that amplified successfully. Amplification success was poor in Dorosoma (31.3%). Due to allelic diversity and estimates of heterozygosity, these markers can be useful in A. sapidissima for population level analyses, parentage assignment and broodstock management.     

Development and Linkage Analysis of 104 New Microsatellite Markers for Bay Scallop (Argopecten irradians)

For genetic analysis and linkage mapping of bay scallop (Argopecten irradians), a set of 120 novel simple sequence repeat markers were developed from microsatellite-enriched libraries and expressed sequence tags. An inter-subspecies hybrid bay scallop family (CC5) of 46 progeny was analyzed as the reference population to confirm polymorphism and test the segregation patterns of these loci. A total of 104 microsatellite markers were polymorphic in the reference family, among which 36 in female, 28 in male, and 40 in both parents, respectively. Linkage analysis allowed mapping these markers to 15 linkage groups, which is close to the haploid chromosome number of bay scallop (n = 16). Analysis of the 40 markers segregating in both parents showed a higher recombination rate in the female parent, with the average of female-to-male recombination ratio of 1.09:1 between linked pairs of markers. When null alleles were considered, there were 17 loci showing segregation distortion at the 5% significance level using the chi-square test. The microsatellite markers developed in this study provide a useful resource for future linkage mapping and quantitative loci analysis in A. irradians.     

食源性致病菌多重PCR快速检测方法建立与应用

利用PCR技术,建立多组多重食源性致病菌PCR快速检测方法。设计受试菌特异性引物,反应体系中加入多对引物和多种DNA模板,采用正交试验优化PCR反应条件,进行特异性引物的PCR扩增。建立了多组多重食源性致病菌PCR快速检测方法,方法中所检测受试菌株和模拟样品均出现特异性扩增条带,结果与实际相符。所建立多组多重PCR快速检测体系符合设计要求,可以应用于食源性突发公共卫生事件的应急检测和日常样品检测工作。     

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria,Cylindrospermopsis and Microcystis

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.     

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes)

We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution. Received September 8, 1998; accepted November 23, 1998.