摘要
旨在建立一种定量检测猪流行性腹泻病毒(PEDV)和传染性胃肠炎病毒(TGEV)的双重微滴数字PCR (dd PCR)检测方法。根据PEDV M基因(登录号:KX852340.1)和TGEV O RF 1b基因(登录号:DQ811785.1)保守序列分别设计特异性检测引物和探针,通过优化反应体系和扩增条件,建立了一种同时定量检测PEDV和TGEV的双重ddPCR检测方法。为探究该方法的检测性能,分别开展了特异性、灵敏性、稳定性,以及临床样品检测符合性等试验。结果显示:当引物浓度分别为900 nmol/L、探针浓度分别为250和300 nmol/L、退火温度为59.5℃时,ddPCR的扩增效果最优,阴、阳性微滴分布清晰,且阳性微滴分布最集中;该方法对PEDV和TGEV均具有良好的特异性,与猪场常见的猪丁型冠状病毒、猪瘟病毒、猪圆环病毒2型、猪A群轮状病毒和伪狂犬病毒等病原均无交叉反应;对标准质粒pMD-M128和pMD-T116线性模板的检测极限分别为1.8和1.4 copies/反应;组内和组间重复变异系数为3.01%~15.05%;此外,国标(GB/T 36871—2018)和qPCR检出的阳性样本均包含于ddPCR方法中,PEDV检出率40.83%(49/120),TGEV检出率4.17%(5/120),其中1份呈混合阳性。结果表明,建立的双重ddPCR特异、灵敏、稳定,可同时用于PEDV和TGEV的定量检测和鉴别诊断,为监测早期感染及标定核酸标准品提供了一种候选方法。
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 optimizing the reaction system and amplification conditions,a dual ddPCR detection method that enables simultaneous quantitative detection of both viruses were implemented.To evaluate the method’s performance,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 temperature 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 demonstrated 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 successfully 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.
作者
陈千林
杨睿
徐登峰
牟豪
周杰
杨柳
郭庆勇
付利芝
CHEN Qianlin;YANG Rui;XU Dengfeng;MU Hao;ZHOU Jie;YANG Liu;GUO Qingyong;FU Lizhi(Disease Prevention and Control Research Institute,National Center of Technology Innovation for Pigs,Chongqing 402460,China;Veterinary and Veterinary Medicine Research Institute,Chongqing Academy of Animal Sciences,Chongqing 402460,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)
出处
《中国兽医科学》
北大核心
2025年第6期770-779,共10页
Chinese Veterinary Science
基金
重庆英才计划“包干制”项目(cstc2022ycj-hbgzxm0183)
国家生猪技术创新中心先导科技项目(NCTIP-XD/B11)
重庆市财政资金项目(25509C)。
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
porcine epidemic diarrhea virus
transmissible gastroenteritis virus
digital PCR
detection method
