title: Simultaneous typing of seven porcine pathogens by multiplex PCR with a GeXP analyser.
abstract: A novel high-throughput method was developed for simultaneous detection and differentiation of seven porcine pathogens by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyser. The pathogens included in this study were pseudorabies virus (PRV), classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2) and Japanese encephalitis virus (JEV). Seven pairs of chimeric primers, consisting of a pathogen-specific sequence fused to a universal sequence at the 5' end, were used to initiate the PCR, after which a set of universal primers was used for the subsequent cycles of the PCR. Amplification products were separated by capillary electrophoresis and identified using fluorescence spectrophotometry. The specificity of the GeXP assay was examined with single and mixed pathogen cDNA/DNA templates. The specific DNA product amplification peaks of seven pathogens were observed on the GeXP analyser. Negative controls did not produce DNA products. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of the plasmids containing the target sequence. Under optimised conditions this assay achieved a sensitivity of 100-1000 copies/μL for a single virus and 1000 copies/μL when all of the seven pre-mixed viral targets were present. Furthermore, the GeXP-PCR assay was 100% specific when 58 clinical samples were tested in comparison with the conventional PCR method. In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for simultaneously detecting seven pathogens that infect swine.
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China; Zigong Center for Animal Disease Control and Prevention, Zigong 643000, Sichuan, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Hu
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China.
authors.forename: Xingyu
authors.initials: X
authors.lastname: Lin
authors.affiliation: Chongqing Entry-Exit Inspection and Quarantine Bureau of China, Chongqing 400020, China; Chongqing Import and Export Food Safety Engineering Center, Chongqing 400020, China.
authors.forename: Fuping
authors.initials: F
authors.lastname: Nie
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China.
authors.lastname: ZexiaoYang
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China.
authors.forename: Xueping
authors.initials: X
authors.lastname: Yao
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China.
authors.forename: Guili
authors.initials: G
authors.lastname: Li
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China.
authors.forename: Xulong
authors.initials: X
authors.lastname: Wu
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China.
authors.forename: Meishen
authors.initials: M
authors.lastname: Ren
authors.affiliation: Animal Quarantine Laboratory, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611100, Sichuan, China. Electronic address: yaanwangyin@tom.com.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary; D057166:High-Throughput Screening Assays / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014730:Veterinary Medicine / Q000379:methods*; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D014780:Viruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers
keywords: GeXP analyser; Multiplex detection; Reproductive pathogens