title: Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay.
abstract: A new high-throughput GenomeLab Gene Expression Profiler (GeXP) analyser-based multiplex PCR assay was developed for the detection of eight reproductive and respiratory pathogens in swine. The reproductive and respiratory pathogens include North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), classical swine fever virus (CSFV), porcine circovirus 2 (PCV-2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). Nine pairs of specific chimeric primers were designed and used to initiate PCRs, and one pair of universal primers was used for subsequent PCR cycles. The specificity of the GeXP assay was examined using positive controls for each virus. The sensitivity was evaluated using serial ten-fold dilutions of in vitro-transcribed RNA from all of the RNA viruses and plasmids from DNA viruses. The GeXP assay was further evaluated using 114 clinical specimens and was compared with real-time PCR/single RT-PCR methods. The specificity of the GeXP assay for each pathogen was examined using single cDNA/DNA template. Specific amplification peaks of the reproductive and respiratory pathogens were observed on the GeXP analyser. The minimum copies per reaction detected for each virus by the GeXP assay were as follows: 1000 copies/μl for PRV; 100 copies/μl for CSFV, JEV, PCV-2 and PPV; and 10 copies/μl for SIV-H1, SIV-H3 and PRRSV-NA. Analysis of 114 clinical samples using the GeXP assay demonstrated that the GeXP assay had comparable detection to real-time PCR/single RT-PCR. This study demonstrated that the GeXP assay is a new method with high sensitivity and specificity for the identification of these swine reproductive and respiratory pathogens. The GeXP assay may be adopted for molecular epidemiological surveys of these reproductive and respiratory pathogens in swine populations.
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China.
authors.forename: Minxiu
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China. Electronic address: xiezhixun@126.com.
authors.forename: Zhixun
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China.
authors.forename: Liji
authors.initials: L
authors.lastname: Xie
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China.
authors.forename: Xianwen
authors.initials: X
authors.lastname: Deng
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China.
authors.forename: Zhiqin
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China.
authors.forename: Sisi
authors.initials: S
authors.lastname: Luo
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China.
authors.forename: Jiabo
authors.initials: J
authors.lastname: Liu
authors.affiliation: Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal and New Technology, Nanning 530001, China.
authors.forename: Yaoshan
authors.initials: Y
authors.lastname: Pang
authors.affiliation: Department of Pathobiology and Veterinary Science, University of Connecticut, 61 North Eagleville Road, Storrs, CT 06269-3089, USA.
authors.forename: Mazhar I
authors.initials: MI
authors.lastname: Khan
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009656:North America; D060737:Reproductive Tract Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology*; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology
chemical_list: D017931:DNA Primers
keywords: GenomeLab Gene Expression Profiler (GeXP); Multiplex PCR; Swine reproductive and respiratory pathogens