靶向伪狂犬病病毒UL35基因的SYBR GreenⅠ荧光定量PCR方法的建立  

Establishment of SYBR GreenⅠ-based quantitative PCR targeting pseudorabies virus UL35 gene

作　　者：张心雨 吴红霞[2] 李永锋[2] 孙元[2] 付强 仇华吉 ZHANG Xinyu;WU Hongxia;LI Yongfeng;SUN Yuan;FU Qiang;QIU HuaJi(College of Life Science and Engineering,Foshan University,Foshan,Guangdong 528231,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]佛山科学技术学院生命科学与工程学院,广东佛山528231 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国兽医学报》2024年第11期2334-2340,共7页Chinese Journal of Veterinary Science

基　　金：黑龙江省自然科学基金资助项目(JQ2022C007)

摘　　要：对伪狂犬病病毒(pseudorabies virus,PRV)感染后不同时间点UL35和gB基因的进行定量分析,发现在感染细胞后2.5、5.0以及20.0 h时,两者基因组拷贝数具有显著差异,并且在PRV感染早期可以观察到UL35基因的表达。为了确定UL35基因能否作为诊断PRV感染的靶标,本研究根据PRV不同毒株UL35基因保守序列,设计合成特异性荧光定量PCR引物,扩增长度为54 bp的片段。通过优化反应条件和反应体系,结果显示,建立的PRV SYBR GreenⅠ荧光定量PCR检测方法具有良好的重复性和特异性,其标准曲线与模板浓度呈现良好的线性关系;对猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(classical swine fever virus,CSFV)、非洲猪瘟病毒(African swine fever virus,ASFV)核酸扩增均呈阴性;将所建立的方法与同类方法进行比较,敏感性无差异;组间和组内重复性试验的变异系数小于2%。用本试验所建立的方法对PRV感染小鼠的组织样品进行检测,均检测到一定的病毒载量。结果表明,UL35基因可以作为诊断PRV感染的靶标。Quantitative analysis of UL35 and gB genes at different time points after PRV infection showed that there were significant differences between the two at 2.5,5.0 and 20.0 h after infection,and the expression of UL35 gene could be observed in the early stage of PRV infection.To determine whether the UL35 gene can be used as a target for the diagnosis of PRV infection,a specific primer pair was designed and synthesized according to the conserved sequence of the UL 35gene of different PRV strains,and used to amplify a fragment of 54 bp in length.After optimizing the reaction conditions and system,the standard curve for the established by SYBR GreenⅠrealtime PCR detection method showed that it had good repeatability,specificity and a good linear relationship to the template concentration.No amplifications for porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),and African swine fever virus(ASFV)were detected.Compared with the existing similar methods,the established method showed no difference in sensitivity.The coefficient of variation of inter-and intra-group repeatability for the method was less than 2%.Detection of the samples in mice challenged with PRV revealed a certain amount of viral load in tissue.The results showed that UL35 gene can be used as a target for the diagnosis of PRV infection.

关 键 词：伪狂犬病病毒 UL35基因 SYBR GreenⅠ 定量PCR 

分 类 号：S852.651[农业科学—基础兽医学]