title: Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical porcine epidemic diarrhea virus.
abstract: Since October 2010, porcine diarrhea outbreaks have occurred widely, resulting in major losses in suckling piglets in China. A variant porcine epidemic diarrhea virus (PEDV), characterized by base deletion and insertion in the S gene, compared to classical PEDV CV777, was shown to be responsible for this outbreak. In this study, a multiplex TaqMan probe-based real-time PCR was developed for detecting PEDV and differentiating the variant from classical PEDV, by using two sets of primers and probes based on the S gene of PEDV. The limits of detection of both variant and classical PEDV were 5×10(2) DNA copies. Specificity was determined using eight other viral pathogens of swine. Reproducibility was evaluated using standard dilutions, with coefficients of variation <1.4%. Standard dilutions included in each test allowed quantification of the amount of PEDV. Among 42 intestinal samples from pigs with severe watery diarrhea, 36 variant PEDV and three classical PEDV samples were detected, with viral loads of 10(2)-10(8) copies/μl and 10(3)-10(5) copies/μl, respectively, which suggested that the variant PEDV was prevalent in China. The multiplex TaqMan probe-based real-time PCR should be a useful tool for quantifying viral load, detecting PEDV, and differentiating variant from classical PEDV.
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Pan-deng
authors.initials: PD
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Bai
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: jiangping41845@163.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Jiang
authors.affiliation: Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing 200001, China.
authors.forename: Tai-shan
authors.initials: TS
authors.lastname: Tang
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Tan
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Shi
mesh_terms: D000818:Animals; D002681:China; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D003967:Diarrhea / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D053485:Porcine epidemic diarrhea virus / Q000145:classification* / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014730:Veterinary Medicine / Q000379:methods
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral
keywords: Multiplex real-time PCR; Porcine epidemic diarrhea virus