title: A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3.
abstract: Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains.
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: 15283521606@163.com.
authors.forename: Qin
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: zdk24@163.com.
authors.forename: Dekang
authors.initials: D
authors.lastname: Zhu
authors.affiliation: China Rural Technology Development Center, Beijing, 100045, PR China. Electronic address: maguangpeng@163.com.
authors.forename: Guangpeng
authors.initials: G
authors.lastname: Ma
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: chenganchun@vip.163.com.
authors.forename: Anchun
authors.initials: A
authors.lastname: Cheng
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: mshwang@163.com.
authors.forename: Mingshu
authors.initials: M
authors.lastname: Wang
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: sophia_cs@163.com.
authors.forename: Shun
authors.initials: S
authors.lastname: Chen
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: cqrc_jry@163.com.
authors.forename: Renyong
authors.initials: R
authors.lastname: Jia
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: liumafengra@163.com.
authors.forename: Mafeng
authors.initials: M
authors.lastname: Liu
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: sunkunfeng1981@163.com.
authors.forename: Kunfeng
authors.initials: K
authors.lastname: Sun
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: yangqiao721521@sina.com.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: yingzi_no1@126.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China; Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan, 611130, PR China. Electronic address: chenxy_24@sina.cn.
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Chen
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004372:Ducks; D005838:Genotype; D004371:Hepatitis Virus, Duck / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006524:Hepatitis, Viral, Animal / Q000175:diagnosis* / Q000821:virology; D010850:Picornaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers
keywords: DHAV-1; DHAV-3; Duck hepatitis A virus; Duplex rRT-PCR