Hebei Academy of Science and Technology for Inspection and Quarantine, Shijiazhuang 050051, China; Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China.
Hebei Animal Disease Control Center, Shijiazhuang 050050, China.
J Virol Methods. 2018 Jun;256:85-88. doi: 10.1016/j.jviromet.2018.03.005. Epub 2018 Mar 14.
A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. The primers and exo probe were designed to be specific for a portion of spike (S) gene conserved in TGEV, but absent in the closely related porcine respiratory coronavirus (PRCV). The amplification was performed at 40 °C for 20 min. The assay could only detect the TGEV, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed TGEV RNA as template, the limit of detection of the developed RT-RPA was 100 copies per reaction. The assay performance was evaluated by testing 76 clinical samples by RT-RPA and a real-time RT-PCR. Fourteen samples were TGEV RNA positive in RT-RPA (18.4%, 14/76), which were also positive in the real-time RT-PCR. The diagnostic agreement between the two assays was 100% (76/76). The R value of RT-RPA and real-time RT-PCR was 0.959 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of TGEV in resource-limited diagnostic laboratories and on-site facilities.
本研究开发了一种快速且特异的实时逆转录重组酶聚合酶扩增检测法（RT-RPA），用于检测传染性胃肠炎病毒（TGEV）。该引物和外切探针设计针对 TGEV 中保守的刺突（S）基因的一部分，而在密切相关的猪呼吸道冠状病毒（PRCV）中不存在。扩增在 40°C 下进行 20 分钟。该检测法只能检测到 TGEV，与测试的其他病原体没有交叉反应。使用体外转录的 TGEV RNA 作为模板，开发的 RT-RPA 的检测限为每个反应 100 个拷贝。通过 RT-RPA 和实时 RT-PCR 检测 76 份临床样本评估了该检测法的性能。在 RT-RPA 中，有 14 份样本（18.4%，14/76）为 TGEV RNA 阳性，这些样本在实时 RT-PCR 中也为阳性。两种检测法的诊断一致性为 100%（76/76）。通过线性回归分析，RT-RPA 和实时 RT-PCR 的 R 值为 0.959。开发的 RT-RPA 检测法为资源有限的诊断实验室和现场设施提供了一种快速、简便、可靠的检测 TGEV 的有用替代工具。
