伪狂犬病病毒RT-qPCR检测方法的建立与应用  

Establishment and Application of an RT-qPCR Detection Method for Pseudorabies Virus

作　　者：李家奇 周汛 汪涛 吴文明 LI Jiaqi;ZHOU Xun;WANG Tao;WU Wenming(School of Environmental and Chemical Engineering,Wuyi University,Jiangmen 529020,China;School of Petrochemical Engineering,Liaoning Petrochemical University,Fushun 113001,China;Institute of Biological and Medical Engineering,Guangdong Academy of Sciences,Guangzhou 510316,China)

机构地区：[1]五邑大学环境与化学工程学院,广东江门529020 [2]辽宁石油化工大学石油化工学院,辽宁抚顺113001 [3]广东省科学院生物与医学工程研究所,广东广州510316

出　　处：《五邑大学学报（自然科学版）》2024年第4期1-8,共8页Journal of Wuyi University(Natural Science Edition)

基　　金：国家科技计划人才项目(0523129001);江门市科技项目(江科[2023]72号,江科[2023]111号);五邑大学创新创业项目(2022CX20)。

摘　　要：为建立伪狂犬病病毒(PRV)高灵敏度、超快速双重实时荧光定量PCR检测方法,根据PRV经典毒株gD和gE基因的相对保守序列设计了多组引物探针,经试验筛选出2组最优特异性引物探针,通过正交试验对反应体系中引物和探针的浓度进行优化,通过特异性、灵敏度和重复性试验评测所建立检测方法的性能.结果表明,建立的检测方法具有多重优点:高灵敏度,gD和gE基因最低检测灵敏度分别为1.06 copies/μL和1.68 copies/μL;超快速,检测时间可缩短至1 h内;特异性好,阴性对照组中常见猪患病毒均无交叉反应,PRV野毒株出现了双gD和gE基因扩增曲线,缺失gE基因的疫苗株仅出现gD基因扩增曲线;重复性强,3次重复性批内与批间试验的变异系数均低于3%.综上所述,本研究建立的双重RT-qPCR检测方法可用于PRV野毒株和疫苗株感染的快速诊断鉴别,为猪伪狂犬病(PR)的诊断及流行病学监测提供了一种快速准确的检测方法.To establish a highly sensitive and ultra-fast dual real-time fluorescence quantitative PCR(RT-qPCR)method for detecting Pseudorabies Virus(PRV),multiple sets of primer probes were designed based on the relatively conserved sequences of the gD and gE genes of classical PRV strains.Through experimentation,two sets of primers and probes with optimal specificity were selected.The concentrations of primers and probes within the reaction system were optimized through orthogonal experiments.The performance of the developed detection method was evaluated through tests for specificity,sensitivity,and repeatability.The results indicate that the established detection method has high sensitivity,with the lowest detection sensitivities for the gD and gE genes,being 1.06 copies/μL and 1.68 copies/μL,respectively.This method has a fast detection rate and can produce results within 1 h.This detection method has excellent specificity,with no cross-reactivity observed among common swine viruses in the negative control group.The PRV wild strains displaying amplification curves for both gD and gE genes,while the vaccine strains lacking the gE gene exhibited only the gD gene amplification curve.This detection method has strong repeatability,with coefficients of variation for intra-batch and inter-batch tests all below 3%.In conclusion,the dual RT-qPCR assay established in this study can be utilized for the rapid diagnosis and differentiation of PRV infections between wild and vaccine strains,providing a rapid and accurate method for the diagnosis and epidemiological monitoring of Porcine Pseudorabies(PR).

关 键 词：双重RT-qPCR 猪伪狂犬病 伪狂犬病病毒 疫苗株 野毒株 

分 类 号：R183.3[医药卫生—流行病学]