猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2024.01.040

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (1): 413-418.doi: 10.11843/j.issn.0366-6964.2024.01.040

• 研究简报 • 上一篇

猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用

周建浩¹, 王东方², 刘影², 王淑娟², 马震原², 谢彩华², 赵雪丽², 杨海波², 冯桂丹⁴, 康台生³, 胡煜锋¹, 李博文³, 闫若潜^2*

1. 河南农业大学动物医学院, 郑州 450046;
2. 河南省动物疫病预防控制中心/河南省重大动物疫病监测预警及防控重点实验室, 郑州 450008;
3. 河南科技大学动物科技学院, 洛阳 471000;
4. 上海市动物疫病预防控制中心, 上海 201103

收稿日期:2023-04-11 出版日期:2024-01-23 发布日期:2024-01-24
通讯作者: 闫若潜,主要从事动物疫病防控技术研究,E-mail:yrq1688@126.com
作者简介:周建浩(1997-),河南浚县人,硕士生,主要从事动物疫病分子生物学及免疫学研究,E-mail:1924930995@qq.com
基金资助:
河南省重大科技专项课题（221100110600）；河南省重点研发专项（231111111300）；河南省现代农业产业技术体系（HARS-22-12-T）；河南省重点研发与推广专项（232102111053）

Establishment and Preliminary Application of a Quantitative Droplet Digital PCR Assay for Porcine Epidemic Diarrhea Virus

ZHOU Jianhao¹, WANG Dongfang², LIU Ying², WANG Shujuan², MA Zhenyuan², XIE Caihua², ZHAO Xueli², YANG Haibo², FENG Guidan⁴, KANG Taisheng³, HU Yufeng¹, LI Bowen³, YAN Ruoqian^2*

1. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China;
2. Henan Centre for Animal Diseases Control and Prevention/Henan Provincial Key Laboratory of Monitoring, Early Warning and Prevention and Control of Major Animal Diseases, Zhengzhou 450008, China;
3. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471000, China;
4. Shanghai Animal Disease Prevention and Control Center, Shanghai 201103, China

Received:2023-04-11 Online:2024-01-23 Published:2024-01-24

摘要/Abstract

摘要： 旨在建立一种敏感性高、特异性强、重复性好的猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）微滴式数字PCR（droplet digital PCR， ddPCR）定量检测方法。本研究根据GenBank登录的PEDV（MK862249.1）M基因序列保守区设计合成特异性引物对和探针；通过对反应体系和反应条件的优化，成功建立了可用于检测PEDV的荧光定量PCR（fluorescent quantitative real-time PCR，FQ-PCR）方法，将自行建立的FQ-PCR方法的引物对/探针用来确定ddPCR 方法；对ddPCR方法进行敏感性、特异性、重复性和初步应用试验。结果显示：自行建立的FQ-PCR方法在敏感性、重复性等方面均优于行标FQ-PCR方法；根据自行设计的FQ-PCR方法建立的ddPCR方法最低检测极限为0.15 copies·μL^-1，敏感性高于行业标准（SN/T 1699—2017）FQ-PCR方法（1.0×10¹ copies·μL^-1）；模板浓度为1.0×10⁰~1.0×10⁴ copies·μL^-1时，具有良好的线性关系（R²>0.99）；批内、批间变异系数（CV%）为1.52%~7.40%；对猪丁型冠状病毒、猪伪狂犬病毒等11种对照病毒核酸检测结果均为阴性；分别用建立的ddPCR方法、行业标准FQ-PCR方法对150份临床样品进行PEDV核酸检测，ddPCR方法对行业标准FQ-PCR方法检测阳性样品的检测符合率为100%。本研究成功建立的PEDV ddPCR检测方法可用于临床上PEDV感染的早期检测和定量检测，为PEDV核酸标准物质的研制提供了定量手段。

关键词: 猪流行性腹泻病毒, 微滴式, 数字PCR, 方法, 建立

Abstract: The aim of this study was to develop a highly sensitive, specific and reproducible method for the quantitative detection of porcine epidemic diarrhea virus (PEDV) by droplet digital PCR (ddPCR). In this study, we designed and synthesized specific primer pairs and probes based on the conserved region of the M gene sequence of PEDV (MK862249.1) registered in GenBank, and successfully established a fluorescent quantitative real-time PCR (FQ-PCR) for the detection of PEDV by optimizing the reaction system and reaction conditions. By optimizing the reaction system and reaction conditions, a fluorescent quantitative real-time PCR (FQ-PCR) method for PEDV detection was successfully established, and the primer pairs/probes of the self-established FQ-PCR method were used to determine the ddPCR method; sensitivity, specificity, reproducibility and preliminary application tests of the ddPCR method were performed. The results showed that the self-established FQ-PCR method outperformed the industrial standard FQ-PCR method (SN/T 1699—2017) in terms of sensitivity and reproducibility; the lowest detection limit of the ddPCR method based on the self-designed FQ-PCR method was 0.15 copies·μL^-1, which was more sensitive than the industrial standard FQ-PCR (1.0×10¹ copies·μL^-1); The linearity was good (R²>0.99) at template concentrations of 1.0×10⁰ to 1.0×10⁴ copies·μL^-1. The intra- and inter-batch coefficients of variation (CV%) ranged from 1.52% to 7.40%. The results were negative for 11 control viruses including porcine delta coronavirus and porcine pseudorabies virus. The PEDV nucleic acid test was performed on 150 clinical samples by the established ddPCR and FQ-PCR methods, and the coincidence rate of the ddPCR method with the FQ-PCR method in detecting the positive samples was 100%.The PEDV ddPCR assay successfully established in this study can be used for early detection and quantitative detection of PEDV infection in clinical settings, and provides a quantitative means for the development of PEDV nucleic acid standards.

Key words: porcine epidemic diarrhea virus, droplet, digital PCR, method, establishment

中图分类号:

S852.659.6

周建浩, 王东方, 刘影, 王淑娟, 马震原, 谢彩华, 赵雪丽, 杨海波, 冯桂丹, 康台生, 胡煜锋, 李博文, 闫若潜. 猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用[J]. 畜牧兽医学报, 2024, 55(1): 413-418.

ZHOU Jianhao, WANG Dongfang, LIU Ying, WANG Shujuan, MA Zhenyuan, XIE Caihua, ZHAO Xueli, YANG Haibo, FENG Guidan, KANG Taisheng, HU Yufeng, LI Bowen, YAN Ruoqian. Establishment and Preliminary Application of a Quantitative Droplet Digital PCR Assay for Porcine Epidemic Diarrhea Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 413-418.

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猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用

Establishment and Preliminary Application of a Quantitative Droplet Digital PCR Assay for Porcine Epidemic Diarrhea Virus

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