基于双重 SYBR Green I 的实时 PCR 检测方法用于快速检测犬诺如病毒和犬星状病毒。
Duplex SYBR Green I-based real-time PCR assay for the rapid detection of canine kobuvirus and canine astrovirus.
机构信息
Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, PR China.
Anhui Provincial Center for Animal Disease Control and Prevention, Hefei, 230000, PR China.
出版信息
J Virol Methods. 2021 Apr;290:114066. doi: 10.1016/j.jviromet.2021.114066. Epub 2021 Jan 13.
A duplex SYBR Green I-based real-time PCR assay was established for the simultaneous detection of canine kobuvirus (CaKoV) and canine astrovirus (CaAstV). This assay can easily distinguish the two viruses according to their different melting temperatures (Tm) of 80 °C for CaKoV and 86.5 °C for CaAstV; other canine enteroviruses used as controls showed no specific melting peaks. The detection limit of this assay was determined to be 10 copies/μL for both viruses. This method exhibited high repeatability and reproducibility, with a coefficient of variation less than 1.5 %. A total of 48 fecal samples were collected for clinical testing by real-time PCR and confirmed by sequencing. Real-time PCR assay showed a 10.4 % CaKoV-positive rate and a 4.2 % CaAstV-positive rate, and the positive rate of co-infection of the two viruses was 2.1 %, which was consistent with the sequencing results. This assay has many advantages over conventional PCR: it is rapid, sensitive, specific, and reliable for detecting these two viruses in one sample, and it can be used as a tool to detect CaKoV and CaAstV infection or co-infection in clinical settings.
建立了一种用于同时检测犬诺如病毒（CaKoV）和犬星状病毒（CaAstV）的双重 SYBR Green I 实时 PCR 检测方法。该方法可根据 CaKoV 和 CaAstV 的不同熔解温度（Tm），即 80°C 和 86.5°C，轻松区分这两种病毒；用作对照的其他犬肠道病毒则没有特异性的熔解峰。该检测方法对两种病毒的检测限均为 10 拷贝/μL。该方法重复性和重现性高，变异系数小于 1.5%。共收集 48 份粪便样本进行实时 PCR 临床检测，并通过测序进行确认。实时 PCR 检测法显示 CaKoV 的阳性率为 10.4%，CaAstV 的阳性率为 4.2%，两种病毒同时感染的阳性率为 2.1%，与测序结果一致。与传统 PCR 相比，该检测方法具有许多优势：它快速、敏感、特异且可靠，可用于在一个样本中同时检测这两种病毒，可作为临床检测 CaKoV 和 CaAstV 感染或合并感染的工具。
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