猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用

doi:10.11843/j.issn.0366-6964.2023.10.038

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (10): 4422-4427.doi: 10.11843/j.issn.0366-6964.2023.10.038

猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用

邹宏^1,2, 夏应菊², 李玲², 徐璐², 赵俊杰², 王团结², 张乾义^2*, 宋振辉^1*

1. 西南大学动物医学院, 重庆 402460;
2. 中国兽医药品监察所国家/WOAH猪瘟参考实验室, 北京 100081

收稿日期:2022-10-31 出版日期:2023-10-23 发布日期:2023-10-26
通讯作者: 张乾义,主要从事猪瘟分子病毒学和流行病学研究,E-mail:zhangqy114@126.com;宋振辉,主要从事兽医微生物与免疫学研究,E-mail:szh7678@swu.edu.cn
作者简介:邹宏(1996-),女,四川宜宾人,硕士,主要从事猪瘟病毒感染及免疫机制研究,E-mail:767713928@qq.com;夏应菊(1982-),女,四川成都人,博士,高级兽医师,主要从事猪瘟、非洲猪瘟等重大猪病毒病检验检测技术及免疫机制研究,E-mail:vet_xiayj@163.com。
基金资助:
“十四五”国家重点研发计划项目《兽用生物制品标准物质研制》子课题"牛羊等草食动物疫苗检验用菌毒种库建立"（2022YFD1800601-4）

Establishment and Preliminary Application of a Dual Real-time RT-PCR Assay for CSFV and BVDV

ZOU Hong^1,2, XIA Yingju², LI Ling², XU Lu², ZHAO Junjie², WANG Tuanjie², ZHANG Qianyi^2*, SONG Zhenhui^1*

1. College of Veterinary Medicine, Southwest University, Chongqing 402460, China;
2. National/WOAH Reference Laboratory for Classical Swine Fever, China Institute of Veterinary Drugs Control, Beijing 100081, China

Received:2022-10-31 Online:2023-10-23 Published:2023-10-26

摘要/Abstract

摘要： 本研究旨在建立一种快速、高效、灵敏的猪瘟病毒（CSFV）和牛病毒性腹泻病毒（BVDV）双重鉴别荧光定量PCR检测方法，针对CSFV E2基因和BVDV 5'UTR基因序列的高度保守区域设计特异性引物和探针，经过反应条件及体系的优化，建立双重RT-qPCR方法。结果表明，该方法最低检测值均为5 copies·μL^-1，比普通PCR灵敏约200倍，敏感性高；可准确区分CSFV和BVDV，且与口蹄疫病毒、猪伪狂犬病病毒等猪常见病原均无交叉反应，特异性强；组内和组间重复变异系数均小于2%，重复性良好。对109份临床样品及7份商品化牛血清分别采用构建的双重RT-qPCR检测方法及CSFV RT-qPCR检测方法（GB/T 27540—2011）和BVDV RT-qPCR检测方法（GB/T 18637—2018）进行检测和对比，符合率为100%，同时，检测结果还证实目前商品化牛血清中仍存在BVDV污染，为CSFV、BVDV的鉴别诊断及流行病学的调查提供了技术支持。

关键词: 猪瘟病毒, 牛流行性腹泻病毒, 双重荧光定量PCR

Abstract: The aim of this study was to establish a rapid, efficient and sensitive assay for the dual detection of classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) by real-time PCR. Specific primers and probes were designed according to the highly conserved regions of CSFV E2 gene and BVDV 5'UTR gene sequences, respectively, and optimized the reaction conditions and program to establish a dual RT-qPCR method. The results showed that the limit detection of this method was 5 copies·μL^-1, which was about 200 times more sensitive than the traditional PCR. This method with high specificity could distinguish CSFV and BVDV, and had no cross-reactivity with common pig pathogens such as foot-and-mouth disease virus (FMDV) and pseudorabies virus (PRV). The coefficients of variations of intra- and inter-group data were less than 2%, demonstrating the good repeatability of this assay. Finally, 109 clinical samples and 7 commercial bovine sera were detected to compare the dual RT-qPCR assay with the CSFV RT-qPCR assay (GB/T 27540-2011) and BVDV RT-qPCR assay (GB/T 18637-2018). The coincident rate was 100%, and the results also illustrated that the BVDV contamination was existed in some commercial fetal bovine serums. Together, this assay could provide technical support for the differential diagnosis and epidemiological field investigation of CSFV and BVDV.

Key words: classical swine fever virus, bovine epidemic diarrhoea virus, dual real-time RT-PCR

中图分类号:

S852.659.6

邹宏, 夏应菊, 李玲, 徐璐, 赵俊杰, 王团结, 张乾义, 宋振辉. 猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用[J]. 畜牧兽医学报, 2023, 54(10): 4422-4427.

ZOU Hong, XIA Yingju, LI Ling, XU Lu, ZHAO Junjie, WANG Tuanjie, ZHANG Qianyi, SONG Zhenhui. Establishment and Preliminary Application of a Dual Real-time RT-PCR Assay for CSFV and BVDV[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4422-4427.

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猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用

Establishment and Preliminary Application of a Dual Real-time RT-PCR Assay for CSFV and BVDV

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