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.

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.

H1-SIV、H3-SIV、NA-PRRSV和EU-PRRSV多重PCR检测方法的建立及应用

猪繁殖与呼吸综合征病毒(PRRSV)感染肺泡巨噬细胞,严重损害免疫系统,导致与其他病毒共感染现象,其中猪流感病毒(SIV)也是病原之一。为了快速检测和区分PRRSV基因型与SIV亚型,本研究针对H1、H3亚型SIV的HA基因和美洲型、欧洲型PRRSV的N、M基因的保守序列,分别设计了4对特异性扩增引物,在引物浓度、退火温度等反应条件的系统优化下,分别对其特异性和敏感性进行验证,建立了能够快速检测以上4种病原的多重PCR方法,并对155份临床样品进行了检测。结果显示,该检测方法特异性好、灵敏度高,对H1-SIV、H3-SIV、NA-PRRSV和EU-PRRSV的最低检出量均低至9.86×10^(0) copies/μL。以单一PCR方法对所有临床样品进行检测后发现,检测结果与多重PCR检测结果一致,证明这种多重PCR方法可以用于这4种病毒的快速检测与分型鉴定。

柑橘3种病毒类病原多重RT-PCR检测技术的建立及应用

【目的】建立柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)、柑橘衰退病毒(citrus tristeza virus,CTV)和啤酒花矮化类病毒(hop stunt viroid,HSVd)的多重RT-PCR检测体系。【方法】设计多重RT-PCR引物,分析其特异性,确定其最佳浓度比、最适退火温度及灵敏度,在此基础上对福建地区的柑橘样品进行检测。【结果】确定了CYVCV-F/R、CTV-F/R和HSVd-F/R等3对引物的最佳浓度比例为1∶1∶2,最适退火温度为52.9℃,灵敏度结果显示该体系可检测模板稀释到10^(-2)的阳性样品。应用该体系对采自福建部分地区的157份柑橘样品进行检测,结果发现,CYVCV、CTV和HSVd的检出率分别为47.1%、56.7%和22.9%。【结论】成功建立了柑橘CYVCV、CTV和HSVd病原的多重RT-PCR检测方法,为该类病害的检测提供准确、快速的检测方法。

抗逆大豆IND-ØØ41Ø-5转化体特异性定性PCR检测方法的建立及其标准化

抗逆大豆IND-ØØ41Ø-5已经批准获得我国进口用作加工原料的安全证书,含有外源HaHB4基因和bar标记基因。本研究根据抗逆大豆IND-ØØ41Ø-5转化体5′端和3′端插入位点旁侧序列信息,设计19对引物,结合罗萨里奥农业生物技术学院公司的1对引物,针对20对引物组合利用定性PCR技术进行引物特异性筛选,结果显示5′端的14号引物特异性良好,优化后获得最佳反应体系和反应程序;经测试,该引物特异性好、稳定性高,检出限达0.1%,扩增片段大小248 bp。经8家有资质实验室对本方法的特异性、检出限、重复性和再现性进行验证,各项参数均达到国家标准要求。本研究成功建立了抗逆大豆IND-ØØ41Ø-5转化体定性PCR检测方法,为IND-ØØ41Ø-5转化体在国内的安全监管提供技术支撑。

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.

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.

Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus,Swine Influenza Virus and Japanese Encephalitis Virus

A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.

Development of Multiplex-PCR for Identification of Pasteurella multocida,Haemophilus parasuis and Actinbacillus pleuropneumoniae

[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Detection and Molecular Characterization of Enteroviruses in Korean Surface Water by Using Integrated Cell Culture Multiplex RT-PCR

Objective To identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture（ICC）multiplex reverse transcription-polymerase chain reaction （RT-PCR） was simultaneously designed to detect coxsackieviruses （CV）, polioviruses （PV）, and reoviruses （RV）. ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay （TCVA）-positive sample-inoculated cell cultures. Results CV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not detected in any sample. Molecular phylogenetic analysis of the VP1 gene sequences revealed that CV types B2 and B4 predominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner. Conclusion CV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.

Rapid Detection Co-infections of Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus by One-step Multiplex RT-PCR

Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.

Two-Tube Multiplex PCR for Genotyping Tuberculous and Nontuberculous Mycobacterial Species in Pathology Specimens

Background: The incidence of mycobacterial infection, in particular M. tuberculosis complex (MTC), is increasing in some Western countries, while nontuberculous mycobacteria (NTM) may be recognized more frequently in clinical specimens worldwide. The clinical scenario and available histopathology alone are often insufficient to separate these two categories of mycobacterial disease, whose behavior and treatment differ. In particular, NTM may be clinically unsuspected in pathological specimens and the opportunity for culturing missed. Methods: We developed two multiplex PCR assays, which distinguish MTC from NTM by detecting the IS6110 insert in the first tube and discriminating up to 14 NTM reference strains in the second by targeting the 16S-23S rRNA internal transcribed spacer. Test material included 594 routine clinical specimens with diverse pathology;many were granulomas unrelated to mycobacterial infection. About 75% were formalin-fixed paraffin blocks, the remainder mainly cytologic imprints or aspirates on FTA cards submitted on suspicion of mycobacterial infection either to avoid frozen sectioning (with the attendant risk of aerosolisation) or at the time of fine needle aspiration. Results: The paraffinized material yielded 53 MTC positives and the cytological 21 positives. A subset consisting of 337 specimens was also analyzed for NTM and yielded 51 positives. The frequency of simultaneous NTM infection in tuberculous patients was about 17%. Mycobacterium avium complex represented the dominant NTM species overall, showed a predilection for lung and lymph node, and together with M. haemophilum were the second most frequent NTM just behind M. ulcerans/M. marinum in skin and soft tissue, the category displaying the largest NTM diversity. Conclusions: Cytological and deparaffinized tissue analyzed in a new two-tube multiplex PCR allows for specific discrimination of causative agents in mycobacterial infection. MTC is readily distinguished from NTM for appropriate therapy, and NTM presumptively diagnosed at the species level allows appropriate choices of antimicrobials.

Hepatitis B virus in cerebrospinal fluid of a patient with purulent bacterial meningitis detected by multiplex-PCR:A case report

BACKGROUND Bacterial meningitis(BM)is a common central nervous system inflammatory disease.BM may cause serious complications,and early diagnosis is essential to improve the prognosis of affected patients.CASE SUMMARY A 37-year-old man was hospitalized with purulent meningitis because of worsening headache for 12 h,accompanied by vomiting,fever,and rhinorrhea.Head computed tomography showed a lesion in the left frontal lobe.Infectious disease screening showed positivity for hepatitis B surface antigen,hepatitis B e antigen,and hepatitis B core antigen.Cerebrospinal fluid(CSF)leak was suspected based on clinical history.Streptococcus pneumoniae(S.pneumoniae)was detected in CSF by metagenomic next-generation sequencing(mNGS)technology,confirming the diagnosis of purulent BM.After treatment,multiplex PCR indicated the presence of hepatitis B virus(HBV)DNA and absence of S.pneumoniae DNA in CSF samples.CONCLUSION We report a rare case of HBV in the CSF of a patient with purulent BM.Multiplex PCR is more sensitive than mNGS for detecting HBV DNA.

Detection of Herbicide-tolerant Canola by Multiplex PCR

[Objective]This study aimed to establish a multiplex PCR detection method of herbicide-tolerant canola.[Method] An endogenous reference gene(CruA) and three exogenous genes(T-CaMV 35 S, P-CaMV 35 S and pat) were selected for multiplex PCR. Specific primers were designed based on national standards or related literature. The annealing temperature, ratio of primer concentration and sensitivity of the established multiplex PCR system were optimized. The optimal multiplex PCR system was verified with known samples. [Result] The experimental results showed that the optimal annealing temperature of multiplex PCR was 58 ℃; the optimal ratio of primer concentration(μmol/L) was T-CaMV 35S: CruA: P-CaMV 35S: pat=0.1: 0.2: 0.2: 0.2;the detection sensitivity of the established multiplex PCR method was 0.3 ng. The amplified bands of known samples were completely consistent with the molecular characteristics. [Conclusion] This study provided a rapid, accurate and effective multiplex PCR technique for detection of herbicide-tolerant canola.

羊种布氏杆菌M5-90△26基因缺失疫苗双重实时荧光定量PCR方法的建立

为了寻找一种能够鉴别布氏杆菌M5-90△26疫苗株与其他布氏杆菌菌株的双重实时荧光定量PCR检测方法。针对布氏杆菌菌株的16S rRNA和Bp26基因,各设计一套引物和探针,并优化反应体系和反应程序。以布氏杆菌A19、S2、M5-90和M5-90△26四种疫苗株以及大肠杆菌、金黄色葡萄球菌、李斯特菌、沙门氏菌、链球菌进行双重荧光定量PCR扩增,评价该方法的特异性。克隆构建PCR_16S rRNA_Bp26标准品,通过倍比稀释进行双重荧光定量PCR扩增,评价该方法的灵敏度。使用该方法与虎红平板凝集试验、试管凝集试验进行检测结果对比,评价该方法的可行性。结果显示,本方法特异性好,布氏杆菌A19、S2、M5-90三种疫苗株均同时出现Bp26基因和16S rRNA基因的扩增,布氏杆菌M5-90△26疫苗株只出现16S rRNA基因的扩增,大肠杆菌、金黄色葡萄球菌、李斯特菌、沙门氏菌、链球菌均未出现扩增曲线。该方法建立的16S rRNA基因序列扩增通道和Bp26基因序列扩增通道对标准品的最低检测限均达5 copies/μL。该方法对临床样本的检测结果与虎红平板凝集试验、试管凝集试验检测结果符合率较高,说明建立的羊种布氏杆菌M5-90△26基因缺失疫苗双重实时荧光定量PCR检测方法可用于临床的检测,为鉴别布氏杆菌M5-90△26疫苗株免疫与布氏杆菌野毒株感染提供一种可行的检测方法。

A simple multiplex PCR for assessing prevalence of extended-spectrumβ-lactamases producing Klebsiella pneumoniae in Intensive Care Units of a referral hospital in Shiraz,Iran

Objective:To identify three common genes(bla_(TEM),bla_(SHV)and bla_(CTX-M)responsible for ESBL production in Klebsiella pneumnniae(K.pneumnniae)isolated from Intensive Care Units of Namazi Hospital,Shiraz,Iran.Methods:A total of 60 non-repetitive nosocomial isolates from 60patients were selected during 2009-2010.The phenotypic identification of ESBL production was confirmed by Double Disk Synergy Test(DDST)according to CLSI guidelines.The ESBL's genotype was then analyzed by multiplex PCR of bla_(TEM),bla_(SHV)and bla_(CTX-M)genes and DNA sequencing.Results:The primary susceptibility tests of K.pneumoniae showed that among 10 examined antibiotics,the most resistant and susceptible antibiotics identified in this study were ampicillin and imipenem,respectively.The phenotypic determination of ESBL by DDST showed that 60%(n=36)of isolates produced ESBL.Multiplex PCR of genes among K.pneuunoniae isolates showed that 39%(n=18)of them have TEM,39%in=18)of then have both CTX-M and TEM and 13%(n=8)of them have TEM,SHV,CTX-M.Conclusions:Our findings reveal the high prevalence(60%)of ESBL prcxducing K.pneumnniae from ICL patients along with a new pattern of bla_(TEM)distribution differ from other countries.

A Single-Tube, Functional Marker-Based Multiplex PCR Assay for Simultaneous Detection of Major Bacterial Blight Resistance Genes Xa21, xa13 and xa5 in Rice

In marker-assisted breeding for bacterial blight(BB) resistance in rice, three major resistance genes, viz., Xa21, xa13 and xa5, are routinely deployed either singly or in combinations. As efficient and functional markers are yet to be developed for xa13 and xa5, we have developed simple PCR-based functional markers for both the genes. For xa13, we designed a functional PCR-based marker, xa13-prom targeting the In Del polymorphism in the promoter of candidate gene Os8N3 located on chromosome 8 of rice. With respect to xa5, a multiplex-PCR based functional marker system, named xa5 FM, consisting of two sets of primer pairs targeting the 2-bp functional nucleotide polymorphism in the exon II of the gene TFIIA5(candidate for xa5), has been developed. Both xa13-prom and xa5 FM can differentiate the resistant and susceptible alleles for xa13 and xa5, respectively, in a co-dominant fashion. Using these two functional markers along with the already reported functional PCR-based marker for Xa21(p TA248), we designed a single-tube multiplex PCR based assay for simultaneous detection of all the three major resistance genes and demonstrated the utility of the multiplex marker system in a segregating population.