中国兽医科学2025，Vol.55Issue(6)：770-779,10.DOI:10.16656/j.issn.1673-4696.2025.0100
猪流行性腹泻病毒和传染性胃肠炎病毒双重微滴数字PCR检测方法的建立与应用
Establishment and application of a dual droplet digital PCR assay for porcine epidemic diarrhea virus and transmissible gastroenteritis virus
摘要
Abstract
This study aims to establish a dual droplet digital PCR(ddPCR)detection method for the quantitative assessment of porcine epidemic diarrhea virus(PEDV)and transmissible gastroenteritis virus(TGEV).Specific primers and probes were designed based on the conserved sequences of the PEDV M gene(accession number:KX852340.1)and the TGEV ORF 1b gene(accession number:DQ811785.1).After opti-mizing the reaction system and amplification conditions,a dual ddPCR detection method that enables si-multaneous quantitative detection of both viruses were implemented.To evaluate the method's perfor-mance,specificity,sensitivity,stability,and compliance with clinical sample detection protocols were assessed.The optimal amplification conditions were achieved with primer concentrations of 900 nmol/L for each,probe concentrations of 250 nmol/L and 300 nmol/L respectively,and an annealing tem-perature of 59.5 ℃.Under these conditions,the ddPCR amplification exhibited clear distribution of negative and positive droplets,with a notable concentration of positive droplets.This method demon-strated high specificity for both PEDV and TGEV,showing no cross-reactivity with common swine pathogens including porcine deltacoronavirus,classical swine fever virus,porcine circovirus 2,porcine rotavirus type A,and pseudorabies virus.The detection limits for the standard plasmids pMD-M128 and pMD-T116 linear templates were found to be 1.8 copies/reaction and 1.4 copies/reaction,respectively,with coefficients of variation ranging from 3.01％to 15.05％within and between groups.Moreover,all positive samples identified by the national standard method(GB/T 36871-2018)and qPCR were success-fully detected by the ddPCR method,achieving detection rate of 40.83％(49/120)for PEDV and 4.17％(5/120)for TGEV.Additionally,one sample was confirmed to be co-positive via this method.In conclusion,the dual ddPCR method established in this study is characterized by its specificity,sensitivity,and stability.It serves as a valuable tool for the quantitative detection and differential diagnosis of PEDV and TGEV,providing robust framework for monitoring early infections and calibrating nucleic acid standards.关键词
猪流行性腹泻病毒/传染性胃肠炎病毒/数字PCR/检测方法Key words
porcine epidemic diarrhea virus/transmissible gastroenteritis virus/digital PCR/detection method分类
农业科技引用本文复制引用
陈千林,杨睿,徐登峰,牟豪,周杰,杨柳,郭庆勇,付利芝..猪流行性腹泻病毒和传染性胃肠炎病毒双重微滴数字PCR检测方法的建立与应用[J].中国兽医科学,2025,55(6):770-779,10.基金项目
重庆英才计划"包干制"项目(cstc2022ycjh-bgzxm0183) （cstc2022ycjh-bgzxm0183）
国家生猪技术创新中心先导科技项目(NCTIP-XD/B11) （NCTIP-XD/B11）
重庆市财政资金项目(25509C) （25509C）
