牛细小病毒2型SYBR GreenⅠ荧光定量PCR检测方法的建立与应用  

Establishment and application of SYBR GreenⅠFluorescent quantitative PCR detection method for bovine parvovirus type 2

作　　者：杨澳飞 刘飞飞 陈慧敏[1,2,3] 余祖华 韩新雨[1,2,3] 贾东鹭[1,2,3] 贾钰航 陈建 魏颖 何雷 马雪连 陈松彪 丁轲[1,2,3] YANG Aofei;LIU Feifei;CHEN Huimin;YU Zuhua;HAN Xinyu;JIA Donglu;JIA Yuhang;CHEN Jan;WEI Ying;HE Lei;MA Xuelian;CHEN Songbiao;DING Ke(College of Animal Science and Technology/Laboratory of Functional Microbiology and Animal Health,Henan University of Science and Technology,Luoyang 471003,China;Luoyang Key Laboratory of Living Carrier Biomaterial and Animal Disease Prevention and Control,Luoyang 471003,China;Key Laboratory of Animal Disease and Public Health,Henan University of Science and Technology,Luoyang 471003,China;Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]河南科技大学动物科技学院功能微生物与畜禽健康实验室,河南洛阳471003 [2]洛阳市活载体生物材料与动物疫病防控重点实验室,河南洛阳471003 [3]河南科技大学动物疫病与公共卫生重点实验室,河南洛阳471003 [4]新疆农业大学,新疆乌鲁木齐830052

出　　处：《中国兽医科学》2025年第3期362-368,共7页Chinese Veterinary Science

基　　金：河南省科技研发计划联合基金项目(232103810029);河南省自然科学基金项目(232300421263)。

摘　　要：本试验旨在建立一种牛细小病毒2型(bovine parvovirus,BPV-2)的SYBR GreenⅠ荧光定量PCR检测方法。首先参考数据库中不同BPV-2序列,设计VP2基因保守区域特异性引物,优化反应体系,初步建立一种BPV-2特异性SYBRGreenⅠ荧光定量PCR(qPCR)方法。结果显示,扩增VP2基因片段长度约156 bp,符合目的基因片段大小,经鉴定所构建的重组质粒正确。用该质粒测得SYBR GreenⅠ荧光定量PCR的最低检测限为3.32×10^(1)copies/μL,而常规PCR方法检测限为3.32×10^(3)copies/μL,表明本研究方法敏感性较高。用该方法检测牛细小病毒Ⅰ型、牛病毒性腹泻病毒、牛轮状病毒、牛冠状病毒、牛疱疹病毒I型时结果均为阴性,表明本方法特异性强。重复性试验结果表明,组内和组间重复变异率均小于1%,表明该方法具有良好的重复性。分别采用本研究所建立的方法和文献中发表的方法对80份腹泻牛粪便进行检测比较。结果显示,两种方法的阳性检出率均为11%(9/80),总符合率达100%。以上结果表明本研究建立基于BPV-2 VP2基因的SYBR GreenⅠ荧光定量PCR检测方法具有良好特异性、灵敏性、重复性,为BPV-2的临床流行病学调查及快速检测提供了新的技术支撑。This study aimed to establish a SYBR GreenⅠfluorescent quantitative PCR method for the detection of bovine parvovirus 2(BPV-2).First,specific primers were designed for the highly conserved region of the VP2 gene of BPV-2 based on different BPV-2 sequences in the database.The reaction system was optimized,and a BPV-2-specific SYBR GreenⅠfluorescent quantitative PCR method was initially established.The results showed that the amplified VP2 gene fragment was approximately 156 bp in length,which was consistent with the size of the target gene.The constructed recombinant plasmid was correctly identified.The lowest de tection limit of SYBR GreenⅠfluorescent quantitative PCR detecting the plasmid was 3.32×10^(1)copies/μL,while the detection limit of conventional PCR was 3.32×10^(3)copies/μL,indicating that the present study method was more sensitive.The results of detection of bovine parvovirus typeⅠ,bovine viral diarrhea virus,bovine rotavirus,bovine coronavirus,and bovine herpesvirus typeⅠwere all negative,indicating the specificity of the method.The results of the repeatability test showed that the variation rate of both intra-group and inter-group were less than 1%,indicating that the method had good repeatability.The method established in this study and the method published in the literature were used to detect 80 samples of diarrhea cow feces.The results showed that the positive detection rates of the two methods were 11%(9/80),and the total coincidence rate was 100%.The results above indicated that the SYBR GreenⅠfluorescent quantitative PCR method based on BPV-2 VP2 gene in this study had good specificity,sensitivity and repeatability,and provided a new technical support for the clinical epidemiological investigation and rapid detection of BPV-2.

关 键 词：牛细小病毒2型 VP2基因 SYBR GreenⅠ荧光定量PCR 建立 应用 

分 类 号：S852.659.2[农业科学—基础兽医学]