猪A群轮状病毒荧光定量PCR检测方法的建立及应用  

Establishment and Application of TB Green Real-time Fluorescence Quantitative RT-PCR for Detection of Porcine RVA

作　　者：陈小飞 严楠 张斌[2] 廖明 CHEN Xiao-fei;YAN Nan;ZHANG Bin;LIAO Ming(National and Regional Joint Engineering Laboratory for Medicament for Zoonoses Prevention and Control,Key Laboratory of Zoonoses Ministry of Agriculture,Key Laboratory for Prevention and Control of Zoonotic Diseases of Guangdong Province,College of Veterinary Medicine,South China Agricultural University,Guangzhou,Guangdong,510642,China;College of Life Science and Technology,Southwest University for Nationalities,Chengdu,Sichuan,610041,China)

机构地区：[1]华南农业大学兽医学院/人兽共患病防控制剂国家地方联合工程实验室/广东省动物原性人兽共患病防控重点实验室/农业农村部人兽共患病重点实验室,广东广州510642 [2]西南民族大学生命科学与技术学院,四川成都610041

出　　处：《动物医学进展》2021年第2期41-45,共5页Progress In Veterinary Medicine

摘　　要：根据近5年来GenBank发布的猪A群轮状病毒(RVA)VP6基因序列,针对保守区域设计1对特异性引物,优化反应体系及条件,建立了TB Green实时荧光定量PCR方法。该方法在模板浓度为1.06×10^8 copies/μL~1.06×10^2 copies/μL范围内线性关系良好,相关系数R^2=0.9975,最低检出浓度为106 copies/μL;批内及批间变异系数小于2%。利用该方法对猪RVA亚型G3、G4、G5、G9和G26检测均为阳性,而对猪常见病原(猪瘟病毒、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒、猪呼肠孤病毒MRV、猪捷申病毒、巴氏杆菌、链球菌以及猪等孢球虫)检测均为阴性。对91份临床腹泻样品进行检测,研究建立的猪RVA TB Green实时荧光定量PCR检出率(43.96%,40/91)高于已报道的猪RVA SYBR Green荧光定量PCR(42.86%,39/91)和国家标准RVA的普通RT-PCR(12.09%,11/91),且符合率为100%。建立的RVA荧光定量PCR检测方法为流行病学调查和早期诊断提供了技术支撑。In the study,according to the latest sequences of RVA VP6 gene released by GenBank in the past five years,a pair of specific primers were designed in the conservation region.The TB Green real-time fluorescence quantitative PCR method was established by optimizing the reaction system and conditions.This method has good linearity in the range of template concentration of 1.06×10^8 copies/μL-1.06×10^2 copies/μL,R^2=0.9975,and the lowest detection concentration was 106 copies/μL,and the coefficient of variation within and between batches was less than 2%.The method could detect genotypes G3,G4,G5,G9 and G26 of RVA,but it was negative to detect for CSFV,PRRSV,PEDV,MRV,PTV,PM,SS,and Isospora suis.The rate of detection of TB Green real-time fluorescence quantitative PCR(43.96%,40/91)was higher than that of SYBR Green real-time PCR(42.86%,39/91)and common RT-PCR(12.09%,11/91),the coincidence rate was 100%.In this study,the RVA detection method provided a powerful tool for epidemiological investigation and early diagnosis.

关 键 词：猪轮状病毒 荧光定量PCR VP6基因 

分 类 号：S852.659.4[农业科学—基础兽医学] S858.28[农业科学—兽医学]