针对猪瘟病毒E2蛋白的实时荧光定量PCR方法的建立及应用  

DEVELOPMENT AND APPLICATION OF REAL-TIME FLURESCENCE QUANTITATIVE PCR FOR DETECTION OF CLASSICAL SWINE FEVER VIRUS E2 PROTEIN

作　　者：高飞[1,2] 姜一峰 李国新[1,2] 张玉娇 李丽薇[1] 虞凌雪 周艳君 郑海红[1] 童光志[1,2] GAO Fei;JIANG Yi-feng;LI Guo-xin;ZHANG Yu-jiao;LI Li-wei;YU Ling-xue;ZHOU Yan-jun;ZHENG Hai-hong;TONG Guang-zhi(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2018年第5期23-28,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2016YFE0112500);国家自然科学基金(31670158);中央级公益性科研院所基础科研业务费专项资金项目(2016JB03);973计划(2014CB542701);国家科技支撑计划项目(2015BAD12B01);欧盟Horizon 2020项目SAPHIR(633184)

摘　　要：根据猪瘟病毒(Classical swine fever virus,CSFV)E2基因的保守序列,设计合成一套特异性引物和TaqMan探针,通过对反应体系和反应条件进行优化后,检测其特异性和灵敏性。结果显示,本研究建立的实时荧光定量PCR在101～108范围内线性相关系数为0.997,能够检测到相当于10 copies/μL的病毒核酸,比常规PCR检测方法敏感性强;采用建立的实时荧光定量PCR方法检验猪繁殖与呼吸综合征病毒、猪伪狂犬病毒和猪圆环病毒2型呈现阴性结果,特异性强;变异系数为0.09%～0.7%,小于1%,具有良好的重复性。本研究建立的猪瘟病毒实时荧光定量PCR方法为临床上猪瘟病毒的诊断和定量研究提供了重要的检测工具。A real-time fluorescence quantitative PCR(qPCR)assay was innovated and developed for Classical swine fever virus(CSFV)for convenient,effective and rapid detection of clinical cases.A primer pairs and a probe based on the CSFV E2 gene were designed and its sensitivity and specificity were detected.The assay showed good specificity and linear relationship at a range of 101~108 with the detection limit at 10 copies/μL of viral genome.The real-time qPCR developed here was highly specific to CSFV as it did not react with Porcine reproductive and respiratory syndrome virus(PRRSV),Porcine circovirus type 2(PCV2)and Pseudorabies virus(PRV).The distributions with a coefficient of variation to be 0.09%~0.7%were less than 1%,indicating that the assay had good reproducibility.In this study,the real-time RT-qPCR assay provided a very important tool for CSFV investigation and quantitative research.

关 键 词：猪瘟病毒 荧光定量PCR E2蛋白 

分 类 号：S852.651[农业科学—基础兽医学]