PEDV、TGEV双重<i>Taq</i>Man实时荧光定量PCR方法的建立及病原学调查

陈秋勇; 陈渊泉; 陈如敬; 车勇良; 吴学敏; 刘玉涛; 周伦江; 王隆柏

doi:10.19303/j.issn.1008-0384.2022.011.002

PEDV、TGEV双重TaqMan实时荧光定量PCR方法的建立及病原学调查

Duplex TaqMan qPCR for Detecting Porcine Epidemic Diarrhea and Transmissible Gastroenteritis Viruses and Epidemic Study in Fujian

摘要

摘要:
目的实现猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒（TGEV）的快速鉴别诊断，并调查分析福建省2019–2021年PEDV和TGEV流行情况。
方法根据PEDV的N 基因和TGEV的S 基因序列分别设计特异性引物和标记FAM、VIC荧光报告基团的探针，建立、优化双重荧光定量PCR反应条件和体系，并检测其敏感性、特异性和重复性。应用该方法对福建省2019–2021年收集的297份疑似腹泻病料进行病原检测。
结果建立的方法对PEDV和TGEV的最低检测限均为10拷贝·μL⁻¹，比普通PCR检测灵敏度提高100倍；对其他常见猪病原不发生非特异性反应；批内、批间变异系数均小于1%。福建省297份临床样本检测结果显示，单PEDV感染率为88.89%，单TGEV感染率为1.01%，PEDV和TGEV混合感染率为3.37%。
结论本研究建立的方法具有敏感性高、特异性强、稳定性好等特点。福建省内2019–2021年以PEDV感染为主，TGEV感染率低，存在PEDV、TGEV混合感染的情况。

Abstract:
Objective A TaqMan probe-based duplex real-time PCR for rapid detection of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) was developed. A study was conducted using the methodology to analyze the related 2019–2021 epidemic occurred in Fujian.
Method Specific primers and probes labeled with FAM and VIC were designed to amplify the N gene of PEDV and the S gene of TGEV, respectively. A reaction system for the assay was established, optimized, and tested for sensitivity, specificity, and repeatability. The assay was used for the viral detection on 297 suspected clinic specimens collected from 2019 to 2021 for an epidemiology study.
Result The newly developed duplex qPCR methodology showed a sensitivity of 10 copies·μL⁻¹ on PEDV and TGEV, which was 100 times higher than that of regular PCR. There were no cross reactions with other common viruses. The inter- and intra-assays had variations on Ct values below 1%. On the 297 specimens, the infection rate of PEDV was 88.89%, that of TGEV 1.01%, and that of both PEDV and TGEV 3.37%.
Conclusion The established duplex qPCR had high sensitivity, specificity, repeatability, and reproducibility for detecting PEDV and TGEV. The 2019–2021 epidemic involving the viruses appeared to be mostly PEDV with low incidents of mixed TGEV and PEDV/TGEV infection.

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