猪丁型冠状病毒微滴式数字PCR定量检测方法的建立  

Establishment of a droplet digital PCR method for the quantitative detection of porcine deltacoronavirus

作　　者：刘影 周建浩 王东方[1] 谢彩华[1] 王淑娟[1] 马震原[1] 柴茂 杨海波[1] 赵雪丽[1] 王翠[1] 闫若潜[1] LIU Ying;ZHOU Jianhao;WANG Dongfang;XIE Caihua;WANG Shujuan;MA Zhenyuan;CHAI Mao;YANG Haibo;ZHAO Xueli;WANG Cui;YAN Ruoqian(Henan Key Laboratory of Animal Diseases Monitoring&Early Warning&Control and Prevention/Henan Center for Animal Disease Control and Prevention,Zhengzhou 450000,China;College of Animal Medical,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]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,河南郑州450000 [2]河南农业大学动物医学院,河南郑州450046

出　　处：《中国兽医科学》2025年第3期369-374,共6页Chinese Veterinary Science

基　　金：河南省重点研发与推广专项(232102110085,232102110089,232102111053);河南省重点研发专项(231111111300);河南省重大科技专项(221100110600);河南省现代农业产业技术体系项目(HARS-22-12-T)。

摘　　要：为建立一种检测猪丁型冠状病毒的微滴式数字PCR定量方法,选择猪丁型冠状病毒M基因作为靶基因,设计并合成1组引物对和探针,并对反应体系及退火温度进行优化。结果显示:所建立的微滴式数字PCR定量方法检测猪丁型冠状病毒M基因的最低限为1.37 copies/μL,当模板浓度为10^(0)~10^(4)copies/μL范围内与检测值具有良好的线性关系(R^(2)>0.99);检测的批内、批间变异系数为0.63%~4.63%,均低于5%,检测10种猪常见疫病病原(PEDV、PRV、TGEV、SADS-CoV、PoRV、PPV、PRRSV、PCV2、CSFV、FMDV-O、FMDV-A)核酸均为阴性;对130份临床样品进行猪丁型冠状病毒检测,微滴式数字PCR方法敏感性高于行业标准推荐的荧光PCR方法。本研究成功建立了一种特异性强、灵敏度高、重复性好的猪丁型冠状病毒微滴式数字PCR定量检测方法,为猪丁型冠状病毒的早期检测、定量诊断以及疫病净化提供技术保障。To establish a droplet digital PCR method for detecting porcine deltacoronavirus(PDCo V),the M gene of PDCo V was selected as the target gene,and a set of primer pairs and probe were designed and synthesized,and the reaction system and annealing temperature were optimized.The results showed that the minimum detection limit of the established droplet digital PCR quantitative method for the M gene of PDCo V was 1.37 copies/μL,and there was a good linear relationship between the detection value and the dilution when the template concentration in the range of 10^(0)copies/μL—10^(4)copies/μL(R^(2)>0.99).The intra-and inter-batch coefficients of variation were from 0.63% to 4.63%,which was lower than 5%.This method was positive for detecting PDCo V but negative for detecting 10 common porcine disease pathogens(PEDV,PRV,TGEV,SADS-CoV,PoRV,PPV,PRRSV,PCV2,CSFV,FMDV-O,FMDV-A).The droplet digital PCR method was more sensitive than the industry standard recommended fluorescence PCR method in the detection of PDCo V in 130 clinical samples.In this study,a droplet digital PCR quantitative detection method with strong specificity,high sensitivity and good repeatability was successfully established,which provided technical support for early detection,quantitative diagnosis and disease purification of PDCo V.

关 键 词：猪丁型冠状病毒 微滴式数字PCR 荧光定量PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]