title: Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus.
abstract: Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV.
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China. Electronic address: zhuxd87@126.com.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China; College of Life Science, Northeast Agricultural University, Harbin 150030, China. Electronic address: wangyufei0204@163.com.
authors.forename: Yufei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China. Electronic address: chenjianfei@126.com.
authors.forename: Jianfei
authors.initials: J
authors.lastname: Chen
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China. Electronic address: zhangxin2410@163.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China. Electronic address: shy2005y@163.com.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Shi
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China. Electronic address: dashi198566@163.com.
authors.forename: Da
authors.initials: D
authors.lastname: Shi
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China; College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, No. 2 Xinyang Road, Sartu District, Daqing 163319, China. Electronic address: arisia@126.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Gao
authors.affiliation: Division of Swine Digestive System Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No.427 Maduan Street, Nangang District, Harbin 150001, China. Electronic address: fl@hvri.ac.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Feng
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003967:Diarrhea / Q000662:veterinary / Q000821:virology; D005243:Feces / Q000821:virology; D052699:Kobuvirus / Q000235:genetics / Q000302:isolation & purification*; D057230:Limit of Detection; D010850:Picornaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral
keywords: Conventional RT-PCR; Porcine kobuvirus (PKV); TaqMan real-time RT-PCR