靶向伪狂犬病病毒UL35基因的SYBR Green Ⅰ荧光定量PCR方法的建立
Establishment of SYBR Green Ⅰ-based quantitative PCR targeting pseudorabies virus UL35 gene
张心雨 1吴红霞 2李永锋 2孙元 2付强 3仇华吉1
作者信息
- 1. 佛山科学技术学院生命科学与工程学院,广东佛山 528231;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
- 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
- 3. 佛山科学技术学院生命科学与工程学院,广东佛山 528231
- 折叠
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 Green Ⅰ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.
Abstract
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 infec-tion,and the expression of UL35 gene could be observed in the early stage of PRV infection.To de-termine whether the UL 35 gene can be used as a target for the diagnosis of PRV infection,a spe-cific primer pair was designed and synthesized according to the conserved sequence of the UL35 gene 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 Green Ⅰ real-time PCR detection method showed that it had good repeatability,specificity and a good linear rela-tionship to the template concentration.No amplifications for porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),and African swine fever virus(AS-FV)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/Green/Ⅰ/定量PCRKey words
pseudorabies virus/UL35 gene/SYBR Green Ⅰ/real-time PCR引用本文复制引用
出版年
2024
NETL
NSTL
万方数据