title: Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus.
abstract: BACKGROUND
  Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. To date, conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. However, these assays are time consuming, inconvenient, and cannot detect the virus quantitatively. In this study, a rapid and sensitive SYBR Green real-time RT-PCR assay was developed to detect avian HEV RNA quantitatively in serum, liver, spleen, and fecal samples from chickens.
  
  
  RESULTS
  Based on the sequence of the most conserved HEV gene, ORF3, the primers for the assay were designed, and the standard plasmid was constructed. The detection limit of the assay was shown to be 10 copies/μl of standard plasmid/reaction, with a corresponding cycle-threshold value of 29.3. The standard curve exhibited a dynamic linear range across at least 7 log units of DNA copy number. The specificity and reproducibility of this assay was high, showing that the assay detected avian HEV RNA specifically and with little variability. Compared to conventional RT-PCR, the current assay is more sensitive for detecting avian HEV in serum, liver, spleen, and fecal samples from chickens.
  
  
  CONCLUSIONS
  A rapid, specific, and reproducible SYBR Green real-time RT-PCR assay was developed for the diagnosis of avian HEV infection in chickens. This assay can accurately detect avian HEV RNA in serum, liver, spleen, and fecal samples with more sensitivity than conventional RT-PCR.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. qinzhao_2004@sina.com.
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. 602267802@qq.com.
authors.forename: Sha
authors.initials: S
authors.lastname: Xie
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. yanisun_2005@sina.com.
authors.forename: Yani
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. 414165606@qq.com.
authors.forename: Yiyang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. jimingao2006@163.com.
authors.forename: Jiming
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. lihui.xia@163.com.
authors.forename: Huiya
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. wang_xin_jie@126.com.
authors.forename: Xinjie
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. farazvet@yahoo.com.
authors.forename: Shahid Faraz
authors.initials: SF
authors.lastname: Syed
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. lby358891054@126.com.
authors.forename: Baoyuan
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. 917110906@qq.com.
authors.forename: Lizhen
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan, 450002, China. Zhanggaiping2003@163.com.
authors.forename: Gaiping
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. zhouem@nwsuaf.edu.cn.
authors.forename: En-Min
authors.initials: EM
authors.lastname: Zhou
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002645:Chickens; D003959:Diamines; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D006524:Hepatitis, Viral, Animal / Q000175:diagnosis / Q000821:virology*; D052678:Hepevirus / Q000302:isolation & purification*; D009930:Organic Chemicals; D011201:Poultry Diseases / Q000175:diagnosis / Q000821:virology*; D011804:Quinolines; D012327:RNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D003959:Diamines; C400973:ORF3 protein, Hepatitis E virus; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; D014764:Viral Proteins; C098022:SYBR Green I