鉴别检测裂谷热病毒NSs缺失型疫苗株与野生株模拟物的双重荧光定量RT-PCR方法的建立  

Development of a duplex real-time qRT-PCR method for differential detection of mimics of wild NSs gene deleted rift valley fever virus and mimics of wild strains NSs gene deleted rift valley fever virus strains

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作  者:杨蕾蕾[1] 毛立[1] 李基棕[1] 江杰元[1] 刘茂军[1] 张纹纹[1] 孙敏 李文良[1] YANG Lei-lei;MAO Li;LI Ji-zong;JIANG Jie-yuan;LIU Mao-jun;ZHANG Wen-wen;SUN Min;LI Wen-liang(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture,Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology,Nanjing 210014,China)

机构地区:[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室/江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地,江苏南京210014

出  处:《中国预防兽医学报》2020年第11期1128-1133,共6页Chinese Journal of Preventive Veterinary Medicine

基  金:“十三五”国家重点研发计划(2016YFD0500908)。

摘  要:为了建立一种能够鉴别检测裂谷热病毒野毒株与NSs缺失型疫苗株的荧光定量RT-PCR方法,本研究通过对裂谷热病毒基因组序列进行比较分析,选择其S节段NSs基因及L节段L基因的无二级结构且保守的区段,分别设计多对引物和探针,通过试验验证并优化反应条件,筛选出最佳引物和探针的组合,建立了可以快速检测裂谷热病毒并区分NSs基因缺失疫苗株和野毒株的双重荧光定量RT-PCR方法。利用该方法检测羊临床常见病原,结果显示该方法仅对裂谷热质粒标准品样品检测为阳性,不与羊常见的其他病原出现阳性反应,特异性强;将裂谷热质粒标准品10倍倍比稀释后利用本研究建立的方法进行检测,结果显示,该方法对NSs和L基因重组质粒标准品的检测下限分别为10拷贝/μL和100拷贝/μL,敏感度高;重复性实验结果显示,该方法组内和组间变异系数均小于2%,重复性好。将NSs缺失型和非缺失型质粒分别转染细胞,提取核酸,利用本方法进行检测,结果显示该方法可有效区分NSs基因缺失型和非缺失型核酸样品。本研究建立的双重荧光定量RT-PCR方法为鉴别野生病毒株和NSs缺失型疫苗株奠定了技术基础。To develop a real-time quantitative RT-PCR(qRT-PCR)method for rapid differential detecting wild virus strains and NSs deleted vaccine strains of rift valley fever virus(RVFV),the non-secondary and highly conservative segments of NSs gene in S-segment and L gene in L-segment were selected by comparing and analyzing the genomic sequence of RVFV.A variety of primers and probes combinations targeting NSs and L genes were designed,respectively.The optimal primers and probes combinations were screened out through experiments and the reaction conditions were optimized.A duplex real-time quantitative RT-PCR(qRT-PCR)method was developed for rapid detection of RVFV infection and identification of wild strains and vaccine strains with gene deletion under standardized conditions.Experiments showed that this method had high specificity,and no cross-reactionvity with other common caprine pathogens,as well as the high sensitivity with the limit detection of 10 copies/μL and 100 copies/μL for the NSs gene and L gene,respectively.And the repeatablility was good with a coefficient of variation less than 2%.Using this method,the nucleic acid can be distinguished from the NSs-deleted and non-deleted plasmids transfected cell samples.This method is usful for laiding foundation for the further development of detection technology in identifying wild-type strains and NSs deleted vaccine strains.

关 键 词:裂谷热病毒 双重荧光定量RT-PCR 鉴别 NSs基因 L基因 模拟物 

分 类 号:S852.65[农业科学—基础兽医学]

 

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