牛传染性鼻气管炎病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立与初步应用  

Development and Preliminary Application of the SYBR Green Ⅰ Real-time Fluorescent Quantitative PCR Method for Detection of Infectious Bovine Rhinotracheitis Virus

作　　者：王倩颖 杨森 刘可欣 王超[2] 孙飞雁 张淑琴[1] WANG Qianying;YANG Sen;LIU Kexin;WANG Chao;SUN Feiyan;ZHANG Shuqin(Institute of Special Economic Animal and Plant Sciences of Chinese Academy of Agricultural Sciences,Changchun 130112,China;Heilongjiang Journal Press of Agricultural Science and Technology,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China)

机构地区：[1]中国农业科学院特产研究所,长春130112 [2]黑龙江省农业科学院,黑龙江农业科技杂志社,哈尔滨150086

出　　处：《中国动物传染病学报》2023年第6期140-145,共6页Chinese Journal of Animal Infectious Diseases

基　　金：吉林省重点研发项目(20220202058NC);国家自然科学基金(31602093);基本科研业务费(1610342018004)。

摘　　要：为了建立牛传染性鼻气管炎病毒(IBRV)的快速定量检测方法,本研究针对IBRV基因组gD序列保守区域设计特异性扩增引物,扩增的目的片段大小为105 bp,将其克隆至pMD18-T载体,构建重组质粒标准品pMD-gD-IBRV,优化反应体系后,建立了快速检测IBRV的SYBR Green I荧光定量PCR方法。该方法特异性较强,与牛病毒性腹泻病毒(BVDV)、牛冠状病毒(BCoV)、牛副流感病毒3型(BPIV3)、牛呼吸道合胞体病毒(BRSV)均无交叉反应;敏感性试验结果显示,该方法敏感性高,对重组质粒标准品的最低检出限达4.8×10^(0)copies/μL,高于常规PCR方法;批内、批间重复性试验的变异系数均小于2%,表明该方法重复性良好;该方法检测的样本阳性率高于常规PCR方法,利用该方法检测牛场送检的106份临床样品,结果显示IBRV的阳性率为36.7%,常规PCR检测方法检测显示阳性率为33.0%,二者符合率为96.2%,表明该方法更适用于临床样品检测。本研究建立的IBRV的SYBR GreenⅠ荧光定量PCR检测方法特异性强、敏感性高、重复性好、快速高效,对于IBRV的检测和流行病学调查具有重要意义。To develop a rapid and quantitative assay for detection of Infectious bovine rhinotracheitis virus(IBRV)in this study,specific amplification primers were designed based on the conserved region of gD sequence of IBRV genome and an amplified target fragment of 105 bp in length was ligated into pMD18-T vector ton construct the recombinant plasmid standard pMD-gD-IBRV.Subsequently,the SYBR Green I real-timefluorescence quantitative PCR method was optimized for its reaction parameters and ready for rapid detection of IBRV.The assay was highly specific and had no cross reaction with Bovine viral diarrhea virus(BVDV),Bovine coronavirus(BCoV),Bovine parainfluenza virus type 3(BPIV3)and bovine respiratory syncytial virus(BRSV).The assay also showed high sensitivity with a minimum detection limit of 4.8×10^(0) copies/μL for the recombinant plasmid standard,which was 100 times more sensitive than that of the conventional PCR method.The coefficients of variation between intra assay and inter assay were less than 2%,indicating that the method was reproducible.Then 106 clinical samples from cattle farms were tested using this method and conventional PCR.The positive rates of IBRV was 36.7%for the SYBR Green I real-timefluorescence quantitative PCR method and 33.0%for the conventional PCR.The coincidence of these two methods was 96.2%,which indicated that the SYBR GreenⅠreal-timefluorescence quantitative PCR method was more suitable for the detection of clinical samples.In summary,the SYBR GreenⅠreal-timefluorescence quantitative PCR assay developed in in this study was specific,highly sensitive,reproducible,fast and efficient,which was of great significance for use in the detection and epidemiological investigation of IBRV.

关 键 词：牛传染性鼻气管炎病毒 检测方法 GD基因 实时荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]