一种快速检测非洲猪瘟病毒I177L基因缺失毒株的荧光定量PCR方法

doi:10.11843/j.issn.0366-6964.2023.06.030

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (6): 2521-2527.doi: 10.11843/j.issn.0366-6964.2023.06.030

一种快速检测非洲猪瘟病毒I177L基因缺失毒株的荧光定量PCR方法

邱英武^1,2, 常昊^3,4, 彭杰^1,4, 易和友^1,3, 王秋梅^1,4, 郭彦辰^1,3, 吴倩雯^1,3, 曹雪珍², 林丽苗², 李薇², 周庆丰², 张桂红^1,3,4, 李群辉^2*, 龚浪^1,3,5*

1. 华南农业大学兽医学院非洲猪瘟防控技术研究中心, 广州 510642;
2. 广东省畜禽健康养殖与环境控制企业重点实验室, 新兴 527400;
3. 国家非洲猪瘟区域实验室(广州), 广州 510642;
4. 广东省动物源性人兽共患病预防与控制重点实验室, 广州 510642;
5. 岭南现代农业科学与技术广东省实验室茂名分中心, 茂名 525000

收稿日期:2022-11-14 出版日期:2023-06-23 发布日期:2023-06-16
通讯作者: 龚浪,主要从事猪冠状病毒与非洲猪瘟病毒的分子流行病学及致病机制研究,E-mail:gonglang@scau.edu.cn;李群辉,主要从事畜禽疾病防控技术研究,E-mail492992437@qq.com
作者简介:邱英武(1998-),男,江西南昌人,硕士研究生,主要从事非洲猪瘟病毒流行病学调查研究,E-mail:1685424689@qq.com;Tel:18720909144。
基金资助:
本研究受广州市重点领域研发计划(202206010036);广州市基础与应用基础研究项目(202201010490);茂名实验室科研启动项目(2021TDQD002);财政部和农业农村部:国家现代农业产业技术体系资助(CARS-35)

A Real-time PCR Method for the Rapid Detection of African Swine Fever Virus I177L Gene Deletion Strain

QIU Yingwu^1,2, CHANG Hao^3,4, PENG Jie^1,4, YI Heyou^1,3, WANG Qiumei^1,4, GUO Yanchen^1,3, WU Qianwen^1,3, CAO Xuezhen², LIN Limiao², LI Wei², ZHOU Qingfeng², ZHANG Guihong^1,3,4, LI Qunhui^2*, GONG Lang^1,3,5*

1. Research Center of African Swine Fever Prevention and Control Technology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. Guangdong Provincial Key Laboratory of Livestock and Poultry Health and Environmental Control, Xinxing 527400, China;
3. National African Swine Fever Regional Laboratory(Guangzhou), Guangzhou 510642, China;
4. Guangdong Provincial Key Laboratory for Prevention and Control of Zoonoses, Guangzhou 510642, China;
5. Guangdong Provincial Laboratory of Modern Agricultural Science and Technology, Maoming Branch Center, Maoming 525000, China

Received:2022-11-14 Online:2023-06-23 Published:2023-06-16

摘要/Abstract

摘要： 本研究旨在建立一种特异、灵敏、快速的ASFV-G-ΔI177L实时荧光定量PCR(FQ-PCR)检测方法。针对ASFV I117L基因的保守区域设计特异性引物/探针对,通过优化反应条件和反应程序,从而提高检测方法的灵敏性,并验证检测方法的重复性和特异性。对170份临床样品进行了检测,并与OIE 推荐的检测方法进行对比。结果表明,基于ASFV I117L基因所设计的特异性引物/探针对,能扩增2.2×10¹~2.2×10¹⁰copies·μL^-1的pMD18-I177L标准质粒,建立的标准曲线R²可达0.995 7,最低检测模板浓度为2.2×10¹copies·μL^-1;该方法扩增反应采用一步法,耗时35 min,批内、批间变异系数均＜1.8%;针对ASFV I177L基因特异性扩增,但对其他5种常见猪病毒核酸样品及无酶水未见阳性扩增;用该方法对170份临床样品进行检测,本方法和OIE推荐的检测方法具有良好的一致性。本研究成功建立了一种特异、灵敏、快速ASFV-G-ΔI177L检测方法,为临床非洲猪瘟病毒的监测诊断提供了良好的技术支撑。

关键词: ASF, ASFV, TaqMan FQ-PCR, 分子诊断

Abstract: The aim of this study was to establish a specific, sensitive and rapid real-time fluorescence quantitative PCR (FQ-PCR) method for the detection of ASFV-G-ΔI177L. Specific primers/probes were designed for the conserved region of ASFV I117L gene to improve the sensitivity of the detection method and verify the repeatability and specificity of the detection method by optimizing the reaction conditions and procedures. A total of 170 clinical samples were tested and compared with OIE recommended testing methods. The results showed that the specific primer/probe pair based on ASFV I117L gene could amplify 2.2×10¹-2.2×10¹⁰ copies·μL^-1 pMD18-I177L standard plasmid, and the established standard curve R² reached 0.995 7. The minimum detection template concentration was 2.2×10¹copies·μL^-1. The amplification reaction was performed by one-step method, which took 35 min, and the coefficient of variation within and between batches was less than 1.8%. Specific amplification of ASFV I177L gene was detected, but no positive amplification was observed for nucleic acid samples of other 5 common porcine viruses and enzyme-free water. This method was used to detect 170 clinical samples, and it was in good agreement with the method recommended by OIE. This study successfully established a specific, sensitive and rapid ASFV-G-ΔI177L detection method, which provides a good technical support for the clinical monitoring and diagnosis of African swine fever virus.

Key words: ASF, ASFV, TaqMan FQ-PCR, molecular diagnosis

中图分类号:

S852.659.1

邱英武, 常昊, 彭杰, 易和友, 王秋梅, 郭彦辰, 吴倩雯, 曹雪珍, 林丽苗, 李薇, 周庆丰, 张桂红, 李群辉, 龚浪. 一种快速检测非洲猪瘟病毒I177L基因缺失毒株的荧光定量PCR方法[J]. 畜牧兽医学报, 2023, 54(6): 2521-2527.

QIU Yingwu, CHANG Hao, PENG Jie, YI Heyou, WANG Qiumei, GUO Yanchen, WU Qianwen, CAO Xuezhen, LIN Limiao, LI Wei, ZHOU Qingfeng, ZHANG Guihong, LI Qunhui, GONG Lang. A Real-time PCR Method for the Rapid Detection of African Swine Fever Virus I177L Gene Deletion Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2521-2527.

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一种快速检测非洲猪瘟病毒I177L基因缺失毒株的荧光定量PCR方法

A Real-time PCR Method for the Rapid Detection of African Swine Fever Virus I177L Gene Deletion Strain

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