欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2025, Vol. 41 ›› Issue (30): 156-164.doi: 10.11924/j.issn.1000-6850.casb2025-0088

• 畜牧·动物医学·蚕·蜂 • 上一篇    

非洲猪瘟病毒微滴式数字PCR检测方法的建立与优化

王永浩1,2(), 刘鹏翔3, 宋帅1, 陈玉婷1, 温肖会1, 向国庆1, 陈美霞1, 李文超2, 罗胜军1()   

  1. 1 广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站， 广州 510640
    2 安徽科技学院动物科学学院/动物营养调控与健康安徽省重点实验室， 安徽凤阳 233100
    3 佛山大学动物科技学院， 广东佛山 528231
  • 收稿日期:2025-02-12 修回日期:2025-07-28 出版日期:2025-10-25 发布日期:2025-11-04
  • 通讯作者:
    罗胜军，男，1974年出生，安徽望江人，研究员，硕士，主要从事动物疫病防控与监测研究。通信地址：510640广东省农业科学院动物卫生研究所，E-mail：。
  • 作者简介:

    王永浩，男，1997年出生，山东诸城人，硕士，研究方向：动物疫病与防控。通信地址：510640 广东省农业科学院动物卫生研究所，E-mail：。

  • 基金资助:
    广东省科技计划项目“广东省畜禽疫病防治研究重点实验室”(2023B1212060040)

Establishment and Optimization of Droplet Digital PCR Method for African Swine Fever Virus

WANG Yonghao1,2(), LIU Pengxiang3, SONG Shuai1, CHEN Yuting1, WEN Xiaohui1, XIANG Guoqing1, CHEN Meixia1, LI Wenchao2, LUO Shengjun1()   

  1. 1 Institute of Animal Health, Guangdong Academy of Agricultural Sciences/ Guangdong Province Key Laboratory of Livestock Disease Prevention/ Guangdong Provincial Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Technology, Ministry of Agriculture and Rural Affairs, Guangzhou 510640
    2 Anhui Provincial Key Laboratory of Animal Nutrition Regulation and Health, College of Animal Science, Anhui Science and Technology University, Fengyang, Anhui 233100
    3 College of Animal Science and Technology, Foshan University, Foshan, Guangdong 528231
  • Received:2025-02-12 Revised:2025-07-28 Published:2025-10-25 Online:2025-11-04

RichHTML

1

PDF (PC)

3

摘要：

本研究旨在建立一种检测非洲猪瘟病毒(ASFV)的微滴式数字PCR(droplet digital PCR，ddPCR)方法。通过合成ASFV特异性引物和探针，优化退火温度、引物和探针浓度等反应条件，对方法的特异性、灵敏度和重复性进行综合评价，并根据优化后的条件进行初步临床应用。结果显示：建立的ddPCR方法最佳退火温度为61.4℃；最佳引物和探针终浓度分别为700 nmol/L和150 nmol/L；特异性试验显示该方法仅特异性扩增ASFV，与其他常见疫病病毒无交叉反应；最低检测限为1.37 copies/μL，比荧光定量PCR敏感性高10倍，浓度梯度线性分析显示良好线性关系(R2=0.9898)；重复性试验中变异系数均低于10%。利用该方法检测80份不同来源的疑似ASFV临床样本，结果显示均为阴性，与荧光定量聚合酶链式反应(qPCR)方法检测结果一致。综上，本研究建立的ddPCR方法敏感性高、特异性强、重复性好，可用于ASFV临床样本的精确检测分析。

关键词: 微滴式数字PCR(ddPCR), 非洲猪瘟, 定量检测, 拷贝数

Abstract:

The objective of this study is to establish a droplet digital PCR (ddPCR) method for the detection of African swine fever virus. In this study, the specific primers and probes of African swine fever virus were synthesized, and the annealing temperature, primer and probe concentration and reaction conditions were optimized. The specificity, sensitivity and repeatability of the method were comprehensively evaluated, and the preliminary clinical application was carried out according to the optimized conditions. The optimal annealing temperature of the established ddPCR method was 61.4℃. The optimal final primer and probe concentrations were 700 nmol/L and 150 nmol/L, respectively; the results of this method showed that the specific amplification of ASFV was positive, and the amplification of other common epidemic viruses was negative without amplification, and there was no cross-reactivity. The lowest limit of detection of this ddPCR method was 1.37 copies/μL, which was 10-fold more sensitive than that of real-time PCR, the fit of the concentration gradient linear analysis was R2=0.9898, and the coefficient of variation of the repeatability test was less than 10%. The ddPCR method established by the application was used to detect 80 suspected ASFV clinical samples from different sources, and the results were negative, which was consistent with the results of real-time polymerase chain reaction (qPCR). The ddPCR method established in this study has high sensitivity, strong specificity and good reproducibility, and can be used for accurate quantitative analysis of ASFV laboratory detection.

Key words: droplet digital PCR (ddPCR), African swine fever, quantitative detection, number of copies