数字农科院2.0

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

文献类型： 外文期刊

作者： Hu, Zhe;Zhu, Chao;Chang, Hao;Guo, Wei;Liu, Diqiu;Xiang, Wenhua;Wang, Xiaojun

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关键词： EvaGreen;Duplex real-time PCR;EHV-1;EHV-4

期刊名称： APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

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收录情况： SCI

摘要： 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 ℃ 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 probebased 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.

分类号： Q939.9`Q81

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