鸡圆圈病毒3型可视化LAMP检测方法的建立及应用  

A Visual LAMP Assay for Detection of Chicken Gyrovirus 3

作　　者：张民秀 谢芝勋 张艳芳 谢志勤 谢丽基 李孟 罗思思 曾婷婷 王粲 ZHANG Minxiu;XIE Zhixun;ZHANG Yanfang;XIE Zhiqin;XIE Liji;LI Meng;LUO Sisi;ZENG Tingting;WANG Can(Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Veterinary Biotechnology/Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs,Nanning 530001,China)

机构地区：[1]广西壮族自治区兽医研究所/广西兽医生物技术重点实验室/农业农村部中国(广西)—东盟跨境动物疫病防控重点实验室,广西南宁530001

出　　处：《河南农业科学》2024年第4期128-136,共9页Journal of Henan Agricultural Sciences

基　　金：广西科技基地与人才专项(桂科AD17195083);“广西八桂学者”专项(2019A50)。

摘　　要：建立鸡圆圈病毒3型(GyV3)可视化环介导等温扩增(LAMP)检测方法,为临床上快速检测鸡GyV3提供一种诊断方法。参考GyV3的VP3基因序列,设计1套LAMP特异性引物,分别通过温度优化、引物浓度优化和反应时间的优化,确定鸡GyV3可视化LAMP诊断方法的扩增温度、引物浓度和反应时间,通过特异性试验、敏感性试验、重复性试验和临床样品检测,验证鸡GyV3可视化LAMP诊断方法的特异性、敏感性、稳定性和可靠性。结果显示,鸡GyV3 LAMP检测方法的反应体系:总体积为20.0μL,包括DNA模板1.0μL,2×Master Mix 10.0μL,内引物GyV3-FIP和GyV3-BIP混合液(工作浓度16.0μmol/L)2.0μL,外引物GyV3-F3和GyV3-B3混合液(工作浓度2.0μmol/L)2.0μL,环引物GyV3-LF和GyV3-LB混合液(工作浓度8.0μmol/L)2.0μL,ddH_(2)O 3μL;鸡GyV3 LAMP检测方法的扩增程序:66℃反应20 min,80℃灭活5 min。鸡GyV3可视化LAMP检测方法能特异性检测GyV3,最低检测浓度为10^(1)拷贝/μL,组内和组间变异系数小于5%;与常规PCR方法检测临床样品的结果比较,两者结果符合率达100%。建立的鸡GyV3可视化LAMP检测方法能特异性检测GyV3,具有特异性好、敏感性高及污染小等优点,且检测结果可通过肉眼观察进行判断,适用于GyV3的临床快速筛查。To establish a visual LAMP detection method for chicken gyrovirus 3(GyV3)and provide a detection method for rapid clinical diagnosis of GyV3 infection,a set of specific primers for loop⁃mediated isothermal amplification(LAMP)was designed according to the VP3 gene of GyV3.The amplification temperature,primer concentration,and reaction time of the visual LAMP diagnostic method were determined by optimization of the temperature,primer concentration,and reaction time.The specificity,sensitivity,stability and reliability of the chicken GyV3 LAMP method were verified through the specificity test,sensitivity test,repeatability test and detection of clinical samples.The reactions were optimized in a final volumes of 20.0μL,including 1.0μL DNA template,10.0μL 2×Master Mix,2μL mixture of inner primers GyV3⁃FIP and GyV3⁃BIP(working concentration 16.0μmol/L),2.0μL mixture of outer primers GyV3⁃F3 and GyV3⁃B3(working concentration 2.0μmol/L),2.0μL mixture of loop primers GyV3⁃LF and GyV3⁃LB(working concentration 8.0μmol/L),3μL ddH_(2)O.The amplification procedures of GyV3 LAMP were determined:reaction at 66℃for 20 min,followed by heating at 80℃for 5 min.The visual LAMP assay for the detection of chicken GyV3 could specifically detect GyV3;the lowest limit of GyV3 detection was 10^(1) copies/μL;the coefficients of variation of intra⁃batch and inter⁃batch assay between groups were both less than 5%;the coincidence rate between the detection results and the conventional PCR detection results was 100%.The visual LAMP assay for the detection of chicken GyV3 can specifically detect GyV3,with the advantages of good specificity,high sensitivity,and low pollution.The detection results can be judged by naked eye observation,which is suitable for rapid clinical screening of GyV3.

关 键 词：鸡圆圈病毒3型(GyV3) 可视化LAMP 特异性引物 诊断方法 敏感性 

分 类 号：S854.44[农业科学—临床兽医学]