title: Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4.
abstract: The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15 pg/μl for EHV-1 and 5 copies/μl or 2.5 fg/μl for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, 427 Maduan Street, Harbin, 150001, People's Republic of China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Hu
authors.forename: Chao
authors.initials: C
authors.lastname: Zhu
authors.forename: Hao
authors.initials: H
authors.lastname: Chang
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
authors.forename: Diqiu
authors.initials: D
authors.lastname: Liu
authors.forename: Wenhua
authors.initials: W
authors.lastname: Xiang
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Wang
mesh_terms: D000818:Animals; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D018397:Equidae; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004861:Herpesvirus 1, Equid / Q000302:isolation & purification*; D023141:Herpesvirus 4, Equid / Q000302:isolation & purification*; D006736:Horses; D025202:Molecular Diagnostic Techniques / Q000379:methods; D009297:Nasal Mucosa / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D044366:Transition Temperature; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D004279:DNA, Viral