针对Asia-1型FMDV的qRT-PCR终点法中和试验的建立及初步研究  

Establishment and preliminary study of a neutralization assay for Asia-1 type of FMDV using an end-point quantitative reverse transcription PCR

作　　者：宋一鸣[1] 杨洋[1] 张志东[1] SONG Yiming YANG Yang ZHANG Zhidong(Innovation Team of viral diseases in Herbivores, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, Chin)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室草食动物病毒病创新团队,兰州730046

出　　处：《黑龙江畜牧兽医》2016年第10期42-47,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：中国农业科学院创新工程项目

摘　　要：为了缩短病毒中和试验(VNT)在口蹄疫病毒(FMDV)疫苗毒株选择以及病原检测中的时间,减少结果判断时人为因素的影响,试验采用qRT-PCR方法改进了传统的VNT,首先建立基于FMDV 3D序列的Taqman探针法qRT-PCR,之后使用FMDV Asia-1/JSL/06毒株感染IBRS-2细胞,提取不同时间收集的细胞病毒混合物的RNA进行qRT-PCR定量检测,获得FMDV Asia-1/JSL/06毒株在IBRS-2细胞中的病毒复制曲线,确定病毒复制的终点时间,即病毒中和试验中加入细胞维持液之后的第20小时,此时使用TRIzol裂解细胞并提取RNA进行qRT-PCR定量检测。以6作为拷贝浓度对数的临界值,拷贝浓度对数大于6的最低稀释度作为终点稀释度,建立了qRTPCR-VNT方法的终点稀释度判断标准,并应用该方法对19份阴性血清以及4份阳性血清进行qRT-PCR-VNT检测。结果表明:该方法获得的阴性、阳性结果与传统VNT检测获得的结果完全相符。说明该方法具有快速、有效、重复性好等优点,具有代替传统VNT的潜能。To shorten the time of viral neutralization test （VNT） in the selection of foot and mouth disease virus （FMDV） vaccine strain and pathogen detection, and reduce the impact of human factors when judging the results, a quantitative reverse transcription PCR （ qRT - PCR） was used to improve the tonventional VNT. A Taqman probe qRT - PCR assay was established based on FMDV 3D sequences, and then the A- sia - 1/JSL/06 strain of FMDV was used to infect IBRS -2 cells. The RNAs of the mixtures of cells and viruses collected at different time were extracted for qRT- PCR quantitative detection. The replication curve of the Asia- 1/JSL/06 strain in IBRS- 2 ceils was obtained, and then the 20th hour after adding cell - maintenance medium in the VNT was recognized as ending time of viral replication. At this point, the cells were lysed by TRIzol reagent and the total RNA was extracted for qRT - PCR quantitative detection. The judgment standard of qRT - PCR - VNT assay was established according to the endpoint approach, taking 6 as a logarithmic critical value of copy number concentration and the lowest dilution of logarithmic copy number concentration of greater than 6 as the end point dilution. 19 FMDV negative sera and 4 FMDV posi- tive sera were detected by the assay. The results showed that the positive and negative results of all sera detected by qRT - PCR - VNT assay were entirely consistent with those detected by conventional VNT. The result indicates that the qRT - PCR - VNT assay has the advantages of being rapid, efficacious with good repeatability, etc. , and also has the potential to replace the conventional VNT.

关 键 词：口蹄疫病毒 反转录荧光定量聚合酶链式反应(qRT-PCR) 病毒复制曲线 病毒中和试验 终点法 

分 类 号：S854.43[农业科学—临床兽医学]