猪伪狂犬病病毒变异毒株与经典毒株实时荧光定量PCR鉴别诊断方法的建立和应用  

Establishment and Application of Real-time Quantitative PCR for Differential Diagnosis of Variant and Classical Strains of Porcine Pseudorabies Virus

作　　者：李媛 张盼 唐慧芬 候凤 师小潇 LI Yuan;ZHANG Pan;TANG Huifen;HOU Feng;SHI Xiaoxiao(Tecon Biology Pharmacy Company Limited,Urumqi 830010,China)

机构地区：[1]天康生物制药有限公司,新疆乌鲁木齐830010

出　　处：《中国兽医杂志》2024年第12期58-63,共6页Chinese Journal of Veterinary Medicine

基　　金：天康生物技术中心项目(2022D01A324)。

摘　　要：为了建立猪伪狂犬病病毒(PRV)变异毒株与经典毒株的快速鉴别诊断方法,本试验根据PRV变异毒株与经典毒株的糖蛋白gC基因设计鉴别引物和探针,加入纳米金优化建立实时荧光定量聚合酶链式反应(qPCR)检测方法,验证其敏感性、特异性和重复性。利用建立的qPCR和测序分析方法同时对新疆4个不同地区采集的猪场临床样品进行检测和分析。优化后的qPCR检测结果显示,重组阳性质粒PUC57-Var标准品在1×10^(9)~1×10^(3)copies/μL范围内线性关系良好,相关系数为0.999,斜率为-3.325,最低检测限为1×10^(3)copies/μL,与其他猪源常见病毒未发生交叉反应,组内和组间重复性试验的变异系数均小于3%;该qPCR方法对临床样品的检测结果与测序分析结果一致。结果表明,本试验建立的qPCR方法可用于PRV变异毒株与经典毒株的鉴别诊断,为PRV的准确诊断和疫苗应用提供科学依据。To develop a rapid diagnostic method for distinguishing between variant and classical strains of pseudorabies virus(PRV)in swine,this study designed specific primers and probes targeting the gC gene of PRV variant and classical strains.A real-time quantitative PCR(qPCR)method was optimized with the addition of gold nanoparticles to enhance detection sensitivity.The sensitivity,specificity,and repeatability of the qPCR method were validated.Clinical samples from pig farms in four different regions of Xinjiang were analyzed using both the developed qPCR method and sequencing.The optimized qPCR assay demonstrated a good linear relationship for the recombinant positive plasmid PUC57-Var standard,with concentrations ranging from 1×10^(9) to 1×10^(3) copies/μL,a correlation coefficient of 0.999,and a slope of-3.325.The detection limit was determined to be 1×10^(3) copies/μL.The assay showed no cross-reaction with other common porcine viruses,and the intra-and inter-assay coefficient of variation was below 3%.The results from the qPCR assay were consistent with those from sequencing analysis for clinical samples.This indicates that the established qPCR method can be used for the differential diagnosis of PRV variant and classical strains,providing a reliable tool for accurate PRV diagnosis and vaccine application.

关 键 词：猪伪狂犬病病毒(PRV) 变异毒株 实时荧光定量聚合酶链式反应(qPCR) 

分 类 号：S852.65[农业科学—基础兽医学]