title: Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses.
abstract: Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Xin-Gang
authors.initials: XG
authors.lastname: Xu
authors.forename: Guang-Da
authors.initials: GD
authors.lastname: Chen
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.forename: Li
authors.initials: L
authors.lastname: Ding
authors.forename: Zhao-Cai
authors.initials: ZC
authors.lastname: Li
authors.forename: Ching-Dong
authors.initials: CD
authors.lastname: Chang
authors.forename: Chi-Young
authors.initials: CY
authors.lastname: Wang
authors.forename: De-Wen
authors.initials: DW
authors.lastname: Tong
authors.forename: Hung-Jen
authors.initials: HJ
authors.lastname: Liu
mesh_terms: D000818:Animals; D060085:Coinfection / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004266:DNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004267:DNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012327:RNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology*; D014730:Veterinary Medicine / Q000379:methods; D014773:Virology / Q000379:methods
chemical_list: D017931:DNA Primers