Study on Distribution of Four Pseudomonas Species in Living Environment Using Multiplex PCR

Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.

奶牛乳房炎5种致病菌多重PCR检测方法的建立与应用

为建立一种能快速检测奶牛乳房炎主要病原菌(大肠埃希氏菌、无乳链球菌、肺炎克雷伯氏菌、金黄色葡萄球菌和肠炎沙门氏菌)的多重PCR检测方法,根据大肠埃希氏菌phoA基因、无乳链球菌ef-tu基因、肺炎克雷伯氏菌Khe基因、金黄色葡萄球菌Nuc基因和肠炎沙门氏菌invA基因设计特异性引物,优化PCR反应体系与反应程序并进行特异性试验,同时分别构建含病原菌基因的pMD-19T重组质粒进行灵敏性试验。结果显示,多重PCR反应的最适引物工作浓度为0.8μmol/L,最适退火温度为59.6℃,最佳延伸时间为40 s,最合适的循环数为30次;优化后的多重PCR反应体系可扩增出5种目标病原菌的特异性条带;5种病原菌PCR检测灵敏度可达到5 copies/μL。进一步利用该多重PCR反应体系对采集的108份乳房炎乳样进行检测,检测结果显示,108份样本的阳性检出率为77.8%,大肠埃希氏菌、无乳链球菌、金黄色葡萄球菌、肺炎克雷伯氏菌和肠炎沙门氏菌的检出率分别为23.1%、8.3%、34.3%、8.3%、5.6%。结果表明,建立了一种具有良好特异性和灵敏性的多重PCR检测方法,在临床生产实践中可用于快速鉴定奶牛乳房炎的致病菌,为奶牛乳房炎的诊断、防控与流行病学调查提供了方法基础。

Establishment and Application of a Multiplex PCR System for the Detection of Blast Resistance Genes Pi-ta and Pi-b in Rice

Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.

Establishment of a Multiplex PCR System for Detecting Transgenic Ingredients from Citrus

[Objective] This study aimed to establish a multiplex PCR system for de- tecting transgenic ingredients from Citrus. [Method] Based on the pBI121 plasmid sequences published in GenBank and actin gene sequence of Citrus, the primers specific to CaMV35S promoter, NOS promoter, NOS terminator and actin gene were designed, to establish a multiple PCR system which could detect four types of sequences. In addition, orthogonal tests were performed to determine the optimal concentrations of all the components in PCR reaction system, as well as the optimal PCR cycle parameters. [Result] The optimal PCR reaction system should contain 2.5μl of 10xPCR buffer, 2.0μl of MgCI2 （25 mmol/L）, 2.0 μl of dNTP mixture （2.5 mmol/L of each dNTP）, 1.0 μl of actin gene primers （10μmol/L）, 1.0μl of 35S promoter primers （10 μmol/L）, 1.5 μl of NOS promoter primers （10 μmol/L） and 0.5 μl of NOS terminator primers （10μmol/L）, 0.1 μg of template DNA, 1.25 U of Taq DNA polymerase; ddH20 was added to the total reaction system of 25μl. The PCR reaction program consisted of pre-denaturing at 94℃ for 5 min; 31 cycles of denaturing at 94℃ for 30 s, annealing at 64.1℃ for 45 s and extension at 72℃ for 50 s; final extension at 72℃ for 10 min. The reaction system optimized with the orthogonal tests could detect as less as 0.1% transgenic component in the tested samples. [Conclusion] The MPCR detection system established in this study can meet the requirements in theory for detecting the genetically modified ingredients in Citrus or the deep-processed products.

Establishment of Multiplex PCR for Three Virus of Potato

[Objective]The aim was to establish the multiplex PCR method for three virus of potato：PVA（potato virus A）,TMV（Tobacco mosaic virus）and PVY（potato virus Y）.[Method]According to the PVA,TMV and PVY sequences available in GenBank,pairs of primer were designed for establishing a multiplex PCR method,and constructing recombinant plasmid of target genes by PCR amplified of three viruses as reference standard simple to be used in sensitivity test;PVX（Potato virus X）,PVM（Potato virus M）,PVS（Potato virus S）,PVV（Potato virus V）and CMV（Cucumber mosaic virus）were used to carry out the specificity test and detection of 11 samples which were suspected of virus infected.[Result]The detection limit for PVA,TMV and PVY was 14,14 and 14 copies/ml,respectively.No cross-reactivity was observed with other viruses.Seven of 11 samples were infected by three viruses.[Conclusion]The multiplex PCR for PVA,TMV,PVY three viruses of potato was established successfully,which had provided basis for the detection technology of potato virus.

4种食源性致病菌多重PCR检测体系的建立及其在乳品检测中的应用

食源性致病菌是通过食物进入人体引起食物中毒和诱发疾病的有害微生物,严重威胁着人类健康和环境安全,为了预防和控制食源性疾病的发生,有效对策之一是对食品中的病原微生物进行安全检测。本研究采用一种多重聚合酶链式反应法(multiplex polymerase chain reaction,MPCR)快速检测乳品中4种食源性致病菌,结果表明,选取单核细胞增生李斯特氏菌prfA基因、蜡样芽胞杆菌gyrB基因、鼠伤寒沙门氏菌invA基因、大肠埃希氏菌O157∶H7 stx2A基因的保守序列设计4对引物,在优化的PCR反应体系和退火温度58℃下,PCR扩增表现出良好的特异性,分别扩增出274、221、482、108 bp条带,无非特异性扩增,4种病原菌检出限达到10~100 CFU/mL;对17份人工染菌牛奶样品进行检测,检出结果与国标培养法完全一致。该研究结果为快速、高效、准确地检测出乳品中单核细胞增生李斯特氏菌、蜡样芽胞杆菌、鼠伤寒沙门氏菌、大肠埃希氏菌O157∶H7提供了一种实用方法,也为其他食源性致病菌的快速检测提供了思路。

Comparison of Multiplex Fluorescent PCR with Serum Type-specific Antibody Detection in Diagnosis of Genital Herpes

Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Methods: We detected HSV infection in 121 speci-mens collected from patients with genital herpesusing both multiplex fluorescent PCR and serum type-specific antibody detection. HSV viral isolation wasused as the standard control.Results: When compared with the viral isolation, thesensitivity and specificity for multiplex fluorescentPCR were 100% and 88.89%, respectively afterdiscrepant analysis. The sensitivity and specificity fortype-specific antibody detection was 77.68 % and77.78 %, respectively. However, the type-specificantibody detected HSV in two asymptomatic patientswhile the multiplex fluorescent PCR couldn’t detectany HSV DNA from those specimens.Conclusions: Multiplex fluorescent PCR is a verysensitive and specific method for detection and typingof HSV in the lesion of genital herpes, it failed todetect HSV DNA from the asymptomatic patients.Serum type-specific antibody detection was a lesssensitive and specific test but could detect the specificantibody from some asymptomatic patients. Thecombination of these two techniques would allow rapid,sensitive and accurate detection and typing of HSVand help clinical diagnosis and epidemiologic survey-ing of genital herpes.

TaqMan多重实时定量PCR快速检测3种常见食源性病原菌

建立一种可同时快速检测大肠杆菌O157:H7、单增李斯特菌和蜡样芽孢杆菌的多重实时定量PCR(qPCR)方法。依据大肠杆菌O157:H7 tir基因、单增李斯特菌mpl基因和蜡样芽孢杆菌entFM基因的保守序列分别设计特异性引物和TaqMan探针,建立多重qPCR反应体系,进行灵敏度、特异性和稳定性试验,同步检测人工染菌牛奶样品中的病原菌并与国家标准方法作对比。结果表明,建立的多重qPCR方法灵敏度高,最低检出限为12 CFU/mL;特异性强,只对3种目标菌进行PCR扩增;稳定性好,各重复性试验中Ct值的变异系数<1%;所有受污染样品阳性检出率均为100%,与国家标准方法检测结果一致,且检测周期缩短至6 h。本研究建立的TaqMan多重qPCR方法能同时快速、准确地检测乳品中的大肠杆菌O157:H7、单增李斯特菌和蜡样芽孢杆菌,为食品安全提供技术支撑。

Multiplex PCR在猪传染性萎缩性鼻炎中的研究

通过一次实验同时检出传染性萎缩性鼻炎的病原体支气管败血波氏杆菌和毒素源性多杀性巴氏杆菌 .PCR扩增后的DNA产物为 2 37bp ,为检出毒素源性败血波氏杆菌toxAgene而设计的引物位于toxA内部的碱基序列 ,扩增后的DNA产物为 338bp.MultiplexPCR可检出败血波氏杆菌模板DNA至 0 .1ng ,毒素源性多杀性巴氏杆菌模板至 0 .1ng.从表现呼吸症状的 7个养猪场的 1 0 0～ 1 2 0日龄肥育猪采取的鼻腔棉拭子中检出支气管败血波氏菌 46例 (85.2 % ) ,毒素源性多杀性巴氏杆菌 8例 (1 4.8% ) ,同时检出上述两种病原体的为 8例 (1 4.8% ) .

笋用林3种竹笋夜蛾多重PCR鉴定技术的建立与应用

【目的】建立鉴定竹笋夜蛾物种的多重PCR技术,用于浙江省笋用竹的3种竹笋夜蛾幼虫的物种鉴定。【方法】针对COI基因的变异区设计物种特异性多重PCR引物;优化影响多重PCR反应的参数,建立鉴定竹笋夜蛾的多重PCR技术;采用标准DNA模板评价多重PCR技术的敏感性和特异性;利用多重PCR技术对林间采集的竹笋夜蛾幼虫进行物种鉴定。【结果】针对3种竹笋夜蛾COI基因的变异区设计了3条物种特异性多重PCR引物,与通用引物LCO1490配合使用,扩增竹笋基夜蛾、竹笋禾夜蛾和笋秀夜蛾COI基因片段,大小分别为290、390和590 bp。优化后的多重PCR鉴定技术的反应体系为:2×HotStart Taq PCR预混试剂10μL,10μmol·L^(-1)的引物LCO1490、JYE290、HYE390和QTYE590各0.5μL,DNA模板1μL,加ddH2O补足20μL。优化后的多重PCR鉴定技术的反应条件为:94℃预变性5 min;94℃变性30 s,59℃退火30 s,72℃延伸30 s;72℃下延伸10 min,循环数为35。多重PCR鉴定技术对笋秀夜蛾的最低检出限为0.01 ng·μL^(-1),对竹笋禾夜蛾和竹笋基夜蛾的最低检出限低于0.001 ng·μL^(-1)。林间样品鉴定结果表明,所有38份DNA都能扩增出明显的特异性条带,鉴定成功率100%;经测序验证,本研究的技术鉴定的物种与经COI基因序列鉴定的物种一致。【结论】研究建立了浙江省笋用竹的夜蛾的多重PCR鉴定技术,能够快速高效鉴定浙江省笋用林内竹笋夜蛾幼虫的物种,该技术具有鉴定周期短、灵敏度高、特异性强、准确度高等优点。

基于多重PCR靶向测序的烟草1.8K SNP育种液相芯片的开发与应用

本研究根据公开发表的烟草K326基因组和烟草430K SNP固相芯片检测数据,以7份种质两两组合之间每条染色体上20个多态标记为目标,基于多重PCR扩增的精准定位测序分型技术(mGPS,Genotyping by Pinpoint Sequencing of multiplex PCR products)开发出烟草1.8K育种液相芯片(YT1.8K.1)。利用该芯片对上述7份种质两两之间杂交的21个杂交组合进行基因分型检测,每个杂交组合之间的平均差异位点数为650个,能同时满足每个组合定向改良筛选高遗传背景回复率单株的需要。利用该芯片对23个烟草品种进行基因型分型检测和聚类分析,聚类分类结果与品种系谱基本吻合;利用该芯片从367个BC2F1群体中筛选出5个背景回复率高于94.96%的单株,高于理论均值87.5%,表明该育种芯片可应用于烟草种质资源聚类分析、定向改良育种的遗传背景筛选。

Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers

In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.

香蕉枯萎和细菌性软腐病菌的多重PCR检测

香蕉枯萎病菌Fusarium oxysporum f.sp.cubense和细菌性软腐病菌Dickeya zeae的复合侵染为害给香蕉产业发展带来严重挑战,有必要建立相关病害的多重聚合酶链式反应(multiplex polymerase chain reaction, multiplex PCR)检测技术。本文基于尖孢镰刀菌古巴专化型1号生理小种(F.oxysporum f.sp.cubense race 1,FOC1)基因组contig 438区间(35 631-37 693 bp)(GenBank:AMGP01000438.1)和4号生理小种(F.oxysporum f.sp.cubense race 4,FOC4)基因组contig 195区间(4 028-6 126 bp)(GenBank:AMGQ01000195.1)存在160 bp插入序列差异设计特异扩增引物FOC-F/-R,同时以香蕉细菌性软腐病菌D.zeae的促旋酶B亚单位基因(the subunit B of gyrase gene)(GenBank:JQ284039)序列设计特异扩增引物gyrB-F/-R。多重PCR检测结果显示:本技术可在一次PCR扩增反应内同时检测香蕉枯萎病菌1号、4号生理小种和细菌性软腐病菌;多重PCR的灵敏度结果表明:检测香蕉枯萎病菌的DNA浓度最低限为0.1 ng/μL,细菌性软腐病菌的灵敏度为103cfu/mL;检测结果稳定可靠。因此,本研究建立的多重PCR检测方法可有效应用于检测香蕉发病组织中的香蕉枯萎病菌和细菌性软腐病菌,也可用于香蕉种苗和田间土壤带病菌的监测,为香蕉种植保驾护航。

4种常见食源性病原菌多重PCR检测技术研究

为了解决常规方法检测食源性病原菌存在操作繁琐、周期长的问题,基于4种常见食源性病原菌特异基因序列扩增提出了一种快速、高效的多重PCR(multiplex polymerase chain reaction, MPCR)检测方法。根据单增李斯特菌iap基因、蜡样芽孢杆菌gyrB基因、产志贺毒素大肠埃希氏菌stxⅠ基因、肠炎沙门氏菌invA基因的特异序列分别设计引物后建立MPCR反应体系,测试其特异性、敏感性和可行性,并与国标培养法进行比较。结果表明:MPCR扩增出4个目标基因的特异性条带大小依次为371、221、432、171 bp;在58℃的退火温度下,MPCR扩增表现出良好的特异性,无非特异性扩增,4种病原菌最低检出限达到102CFU/mL;MPCR方法和国标培养法对多种食源性病原菌感染的牛奶样品的检测结果完全一致,检测周期由原来的5~7 d缩减到8~9 h。所建立的MPCR技术作为一种快速、高效的检测方法,可为食品安全生产提供保障。

Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray

Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified （GM） soybean. Seventeen capture probes （PCR products） and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean （GTS40-3-2）, maize （MonS10, Nk603, GA21）, canola （T45, MS1/RF1）, and rice （SCK） in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean （Roundup Ready GTS40-3-2） and canola （MS1/RF1）. Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS 1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Development of Genomic Microsatellite Multiplex PCR Using Dye-Labeled Universal Primer and Its Validation in Pedigree Analysis of Pacific Oyster(Crassostrea gigas)

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction （PCR） and capillary electrophoresis （MPCE） assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catorrholis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebactefium diphthefiae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 252 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE with high specificity and sensitivity. This assay survey of respiratory pathogens. assay is a rapid, reliable, and high-throughput method has great potential in the molecular epidemiological.

Rapid Detection of Haemophilus influenzae and Haemophilus parainfluenzae in Nasopharyngeal Swabs by Multiplex PCR

Objective To establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children. Methods Multiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 165 rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae. Results The sensitivity of the 165 rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden＇s Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 165 rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% （0/666）, 0.15% （1/666）, 1.20% （8/666）, 0.15% （1/666）, 1.20% （8/666）, 1.80% （12/666）, 95.50% （636/666） respectively. Conclusion The multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

Identification and Detection of Actinobacillus pleuropneumoniae in Infected and Subclinically Infected Pigs by Multiplex PCR Based on the Genes ApxIVA and OmlA

PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely related species bacteria. To improve veracity and specificity of PCR, a species-specific multiplex PCR assay was developed to identify and detect A. pleuropneumoniae, based on the 3＇-terminus of the species-specific apxlVA gene and the already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A. pleuropneumoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of ten strains representing eight different species bacteria including species normally found in the respiratory tracts of swine. All of these strains turned out negative in the multiplex PCR. All sequences of products of multiplex PCR randomly sampled were also correct. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR and bacterial isolation were compared to determine their sensitivities by using experimentally infected pigs and clinical disease pigs. The multiplex PCR was more sensitive than bacterial isolation. The multiplex PCR was also evaluated on mixed bacterial cultures from clinical healthy pigs. 26/100 （26%） of the subclinically infected pigs were detected from clinical healthy pigs. The results indicate that the multiplex PCR assay is a sensitive, highly specific, and effective diagnostic tool for identification and detection of A. pleuropneumoniae.

Development of a Multiplex PCR for Diagnosis of Staphylococcus aureus, Escherichia coli and Bacillus cereus from Cows with Endometritis

Staphylococcus aureus, Escherichia coli and Bacillus cereus are the major agents of cow endometritis in dairy cows. A multiplex PCR （SEB-mPCR） was established based on the conserved genes of S. aureus, E. coli and B. cereus, and the detection limits were 103, 102 and 103 CFU mL-1, respectively. SEB-mPCR could not amplify genomic DNA of pathogenic bacteria of other common bovine diseases. A total of 309 vaginal discharge samples from cows with endometritis were tested by SEB-mPCR. Of the samples, 23.95% had the three kinds of bacteria detected, 17.15% had S. aureu and E. coli, 9.39% had E. coli and B. cereus, and 9.71% had S. aureus and B. cereus. The rates of infections with S. aureus, E. coli and B. cereus were 11.35, 16.18 and 9.06%, respectively. Therefore, SEB-mPCR has a potential as a diagnosis tool for endometritis in dairy cows.