JIA-2021-2226 同时检测CSFV、ASFV及APPV多重荧光PCR方法的建立及初步应用

doi:10.1016/j.jia.2022.08.115

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (2): 559-567.DOI: 10.1016/j.jia.2022.08.115

JIA-2021-2226 同时检测CSFV、ASFV及APPV多重荧光PCR方法的建立及初步应用

收稿日期:2022-01-05 接受日期:2022-02-24 出版日期:2023-02-20 发布日期:2022-02-24

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus and atypical porcine pestivirus

SONG Xiang-peng*, XIA Ying-ju*, XU Lu, ZHAO Jun-jie, WANG Zhen, ZHAO Qi-zu, LIU Ye-bing, ZHANG Qian-yi, WANG Qin

China Institute of Veterinary Drug Control/National Reference Laboratory for Classical Swine Fever, Beijing 100081, P.R.China

Received:2022-01-05 Accepted:2022-02-24 Online:2023-02-20 Published:2022-02-24
About author:SONG Xiang-peng, E-mail: songxp_2022@163.com; XIA Ying-ju, E-mail: vet_xiayj@163.com; Correspondence ZHANG Qian-yi, Tel: +86-10-61255400, E-mail: zhangqy114@126.com; WANG Qin, Tel: +86-10-62103673, E-mail: wq551@vip.sina.com * These authors contributed equally to this study.
Supported by:
This work was supported by the National Natural Science Foundation of China (31872484) to Zhang Qianyi and the Non-profit Key Program of Veterinary Drug Industry from China Institute of Veterinary Drug Control (GY202011) to Xia Yingju.

摘要/Abstract

摘要：

建立一种同时鉴别诊断猪瘟病毒（classical swine fever virus, CSFV)、非洲猪瘟病毒（African swine fever virus, ASF）和猪非典型瘟病毒（atypical porcine pestivirus, APPV）的快速、灵敏、有效的检测方法。依据GenBank中登录的CSFV (5¢ UTR)、ASFV (B646L) 和 APPV (5¢ UTR) 的高度保守基因序列分别设计和优化了多对特异性引物和Taq-man探针，以保守区基因序列分别制备三种阳性质粒，用矩阵法优化单重/多重荧光PCR的反应体系和条件，为避免荧光通道的交叉干扰多重荧光PCR扩增，结合所标记的荧光报告基团做颜色补偿试验，构建标准曲线的扩增图和对应的线性方程，并进行特异性、敏感性、重复性、符合性以及临床样本的检测等试验。三种病毒的标准曲线相关系数均达到0.995以上，具有良好的线性关系；与其它常见猪病无交叉扩增反应，具有很好的特异性；多重荧光PCR的最低检测量均为1 copy/mL，具有较高的敏感性；组内和组间的变异系数均小于1%，具有很好的重复性。该方法与CSF的国标(GB/T 27540-2011), ASF的国标 (GB/T 18648-2020)，APPV的发明专利 (CN108611442A)检测样本盘的22个毒株样本符合率为100%。本研究建立的多重荧光PCR检测方法具有快速、高效、通量高、特异性好、灵敏度高等特点，可以对CSFV、ASFV和APPV病毒进行鉴别检测，为动物疫病的流行病学调查、疫情的检测提供一种新型的检测手段。本研究结合荧光PCR仪不同荧光通道设计CSFV、ASFV和APPV探针荧光信号强度较高且干扰较小的FAM、CY5和HEX报告基团，建立多重荧光PCR检测方法，用于同时鉴别诊断3种主要猪病毒的检测方法，在临床诊断中具有重要的应用价值。

Abstract:

With the implementation of the C-strain vaccine, classical swine fever (CSF) has been under control in China, which is currently in a chronic atypical epidemic situation. African swine fever (ASF) emerged in China in 2018 and spread quickly across the country. It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety. Atypical porcine pestivirus (APPV) was first detected in Guangdong Province, China, in 2016, which mainly harms piglets and has a local epidemic situation in southern China. These three diseases have similar clinical symptoms in pig herds, which cause considerable losses to the pig industry. They are difficult to be distinguished only by clinical diagnosis. Therefore, developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential. In this study, three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV (5´ UTR), African swine fever virus (ASFV) (B646L), and APPV (5´ UTR), followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay. The results showed that the method did not cross-react with other swine pathogens (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine viral diarrhea virus BVDV). The sensitivity results showed that CSFV, ASFV, and APPV could be detected as low as 1 copy mL^–1; the repeatability results showed that the intra-assay and inter-assay coefficient of variation of ASFV, CSFV, and APPV was less than 1%. Twenty-two virus samples were detected by the multiplex real-time PCR, compared with national standard diagnostic and patented method assay for CSF (GB/T 27540–2011), ASF (GB/T 18648–2020), and APPV (CN108611442A), respectively. The sensitivity of this triple real-time PCR for CSFV, ASFV, and APPV was almost the same, and the compliance results were the same (100%). A total of 451 clinical samples were detected, and the results showed that the positive rates of CSFV, ASFV, and APPV were 0.22% (1/451), 1.3% (6/451), and 0% (0/451), respectively. This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV, ASFV, and APPV.

Key words: classical swine fever virus , African swine fever virus , atypical porcine pestivirus , real-time PCR

. JIA-2021-2226 同时检测CSFV、ASFV及APPV多重荧光PCR方法的建立及初步应用[J]. Journal of Integrative Agriculture, 2023, 22(2): 559-567.

SONG Xiang-peng, XIA Ying-ju, XU Lu, ZHAO Jun-jie, WANG Zhen, ZHAO Qi-zu, LIU Ye-bing, ZHANG Qian-yi, WANG Qin. A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus and atypical porcine pestivirus[J]. Journal of Integrative Agriculture, 2023, 22(2): 559-567.

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JIA-2021-2226 同时检测CSFV、ASFV及APPV多重荧光PCR方法的建立及初步应用

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus and atypical porcine pestivirus

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