鹅星状病毒SYBR Green I荧光定量RT-PCR方法的建立  

Development and application of SYBR Green I fluorescence quantitative RT-PCR assay for detection of goose astrovirus

作　　者：白彩霞[1] 张达 赵靓 杨侃侃[1] 王小朋 李新路 李锦春[1] 李永东[2] 王勇[1] BAI Cai-xia;ZHANG Da;ZHAO Liang;YANG Kan-kan;WANG Xiao-peng;LI Xin-lu;LI Jin-chun;LI Yong-dong;WANG Yong(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Ningbo Municipal Center for Disease Control and Prevention,Ningbo 315010,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]宁波市疾病预防控制中心,浙江宁波315010

出　　处：《江苏农业学报》2020年第3期634-638,共5页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31602063)。

摘　　要：为建立快速检测鹅星状病毒(GAstV)的方法,本研究根据GenBank中GAstV ORF2基因序列设计1对特异性引物,构建重组质粒pMD19-T-GAstV,以其作为标准品建立了GAstV的SYBR Green I荧光定量RT-PCR检测方法。优化其反应条件及体系,进行特异性、敏感性和重复性试验以及临床样本检测。试验结果显示,除GAstV外,禽白血病病毒(ALV)、血清4型禽腺病毒(FAdV-4)、鸡传染性喉气管炎病毒(ILTV)和鸡传染性贫血病病毒(CIAV)等常见禽病病原利用该方法检测均呈阴性,表明其特异性良好;建立的荧光定量RT-PCR检出的最低模板含量为1μl 3.75×101拷贝,敏感性较高;组内和组间重复性试验变异系数均小于1%。利用该方法对来自安徽地区的32份临床样品进行检测,阳性检出率为43.75%,常规PCR方法阳性检出率为18.75%,阳性检出符合率100%,表明该方法可用于临床样品检测。该方法的建立为临床样品中GAstV的快速高效检测提供了技术支持。In order to rapidly detect the goose astrovirus(GAstV),the SYBR Green I fluorescence quantitative RT-PCR method was established.Firstly,a pair of specific primers was designed based on the ORF2 gene sequence of the GAstV in GenBank.Secondly,the recombinant plasmid pMD19-T-GAstV was constructed and used as a standard template to generate the standard curve.The reaction conditions and reaction system were optimized,the specificity,sensitivity and reproducibility of the assay were tested.Moreover,the established assay was used in clinical samples detection.The results showed that the detection results of fowl adenovirus serotype 4(FAdV-4),infectious laryngotracheitis virus(ILTV),avian leukosis virus(ALV),chicken infectious anemia virus(CIAV)and other common avian pathogens were negative except the GAstV.It indicated that the assay had good specificity.The lowest template content detected by fluorescence quantitative RT-PCR assay was 3.75×101 copies per milliliter.The variation coefficients in the repeatability test were less than 1%.Thirty-two clinical samples from Anhui province were tested using this assay.The positive detection rate was 43.75%,the positive rate of conventional PCR was 18.75%,and the coincidence rate was 100%.In conclusion,the established assay provides technical support for the rapid and efficient detection of GAstV in clinical samples.

关 键 词：鹅星状病毒 SYBR Green I荧光定量RT-PCR 检测 

分 类 号：S858.33[农业科学—临床兽医学]