title: Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.
abstract: BACKGROUND
  The porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1.
  
  
  RESULTS
  A pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 10(0) ~ 10(9) copies/μL. This method doesn't detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5%. The serum virus titers of P1 positive in this study were measured by this protocol to be 10(3) to 10(7) copies/mL.
  
  
  CONCLUSIONS
  The established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. kwh2003@yeah.net.|Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Nanjing, 210014, China. kwh2003@yeah.net.
authors.forename: Kong-wang
authors.initials: KW
authors.lastname: He
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Nanjing, 210014, China. wlbwh@hotmail.com.
authors.forename: Li-bin
authors.initials: LB
authors.lastname: Wen
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Nanjing, 210014, China. wys@126.com.
authors.forename: Yong-shan
authors.initials: YS
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. lucp@njau.edu.cn.
authors.forename: Cheng-ping
authors.initials: CP
authors.lastname: Lu
mesh_terms: D000818:Animals; D002681:China / Q000453:epidemiology; D017925:Circovirus / Q000145:classification / Q000302:isolation & purification*; D016014:Linear Models; D053570:Porcine Postweaning Multisystemic Wasting Syndrome / Q000453:epidemiology / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine