Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4

• Published in: Applied Microbiology and Biotechnology 2014, Vol.98 (9), p.4179
• Main Authors: Hu, Zhe, Zhu, Chao, Chang, Hao, Guo, Wei, Liu, Diqiu, Xiang, Wenhua, Wang, Xiaojun
• Format: Report
• Language: English
• Subjects: Analysis; Care and treatment; Diagnosis; Equine herpesvirus diseases; Polymerase chain reaction; Risk factors
• ISSN: 0175-7598
• DOI: 10.1007/s00253-014-5626-6

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.sub.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.