水疱性口炎病毒免提取核酸荧光定量PCR鉴别检测方法的建立  

Development of A Real-time PCR Assay Without RNA Extraction for Detection and Identification of Vesicular Stomatitis Viruses

作　　者：张彩虹[1] 林彦星[1] 杨俊兴[1] 曹琛福[1] 吕建强[1] 黄超华[1] 祁振强 林庆燕[1] 花群义[1] ZHANG Cai-hong;LIN Yan-xing;YANG Jun-xing;CAO Chen-fu;LV Jian-qiang;HUANG Chao-hua;QI Zhen-qiang;LIN Qing-yan;HUA Qun-yi(Animal&Plants Inspection and Quarantine Technology Center,Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shengzhen,518045,,China;Shenzhen Boostie Biomedicine Co.Ltd,Shengzhen,518101,China))

机构地区：[1]深圳出入境检验检疫局动植物检验检疫技术中心,广东深圳518045 [2]深圳市宝舜泰生物医药股份有限公司,广东深圳518101

出　　处：《动物医学进展》2018年第11期25-29,共5页Progress In Veterinary Medicine

基　　金："十三五"国家重点研发计划课题(2016YFD0500708);国家质检总局科技项目(2017IK270)

摘　　要：为建立可快速鉴别水疱性口炎病毒印第安纳型(VSV-IND)和新泽西型(VSV-NJ)免提核酸的荧光定量PCR检测方法,根据VSV-IND和VSV-NJ两种血清型相对保守的L基因序列,设计2对特异性引物和2条探针,探针用不同的荧光基团进行标记。经过对各反应条件的优化,建立了直接从样品中鉴别VSV-IND和VSV-NJ的荧光定量PCR方法,并对该方法的特异性和敏感性进行分析。结果显示,该直扩荧光定量PCR能准确鉴别VSV-IND和VSV-NJ,两者没有交叉反应现象,对其他几种相似病原的灭活抗原,如口蹄疫病毒(FMDV)、猪水疱病病毒(SVDV)、蓝舌病病毒(BTV)、鹿流行性出血热病毒(EHDV)、小反刍兽疫病毒(PPRV)、牛病毒性腹泻病毒(BVDV)等检测均呈阴性。对10倍系列稀释的水疱性口炎灭活病毒液的检测敏感性可达10-5,与传统的荧光定量PCR的检测敏感性相当。该方法无需提取核酸,可在2h内完成对样品的检测,结果准确、快速、灵敏,为印第安纳型和新泽西型水疱性口炎病毒的特异性鉴别提供了一种切实可行的方法。A real-time PCR assay without RNA extraction was developed for rapid detection and identification of two vesicular stomatitis virus serotypes:VSV-IND and VSV-NJ.Two sets of specific primers and probes were designed based on the L gene sequences of VSV-IND and VSV-NJ respectively.The results showed that:with the optimized concentrations of primers and probes,the direct real-time PCR assay could differentiate VSV-IND and VSV-NJ from other related disease viruses,such as FMDV,SVDV,BTV,EHDV,PPRV,BVDV and CSFV.There was no cross reaction between the two serotypes.The sensitivity of this method was comparable to that of the traditional real-time fluorescent PCR method for the inactivated virus.Compared to the traditional real-time PCR,this method is rapid and the whole test can be completed within 2 hours.Samples can be simply applied to this method without RNA extraction and purification.Due to its high specificity,sensitivity,and repeatability,the real-time PCR assay without RNA extraction provided a novel,rapid,and practical tool for the specific serotype detection of VSV.

关 键 词：水疱性口炎病毒 直扩荧光定量PCR 鉴别检测 

分 类 号：S852.65[农业科学—基础兽医学]