流行性腮腺炎病毒荧光定量逆转录-聚合酶链反应快速检测方法的建立  

Development of Rapid Real-Time RTPCR Method for Detection of Mumps Virus

作　　者：冯燕[1] 卢亦愚[1] 严菊英[1] 史雯[1] 周敏[1] 

机构地区：[1]浙江省疾病预防控制中心,杭州310009

出　　处：《中国计划免疫》2006年第5期394-398,共5页Chinese Journal of Vaccines and Immunization

基　　金：浙江省科学技术厅攻关项目

摘　　要：目的 建立以TaqMan—MGB荧光探针为特点的荧光定量逆转录-聚合酶链反应（RT—PCR），用于检测流行性腮腺炎（腮腺炎）病毒（MuV）核酸。方法 针对MuV血凝素（H）基因保守区域设计特异性引物与TaqMan—MGB荧光探针，筛选并优化荧光定量RT—PCR反应体系与反应条件，用以提高方法的特异性、灵敏度与准确性；并通过体外克隆技术建立MuV基因拷贝数定量分析模型。结果 引物与探针的优化浓度分别为880mmol／L和280mmool／L，具有良好的保守性和特异性，与其它呼吸道病毒均无交叉反应；方法检测灵敏度为10拷贝／反应，相当于0．01TCID50，标准曲线线性范围为10^7～10拷贝／反应；从病毒核酸提取至检测完成仅需3h左右，操作简便，重现性好。结论 TaqMan-MGB荧光定量RT-PCR方法特异、敏感、快速，为MuV的早期快速检测提供了一种新的检测技术。Objective A real-time reverse transcription-polymerase chain reaction （RRT-PCR）assay based on TaqMan-MGB probe was developed to rapidly detect mumps virus. Methods The ashy, based on primers and TaqMan-MGB probes were selected from highly conserved regions of the hemagglutinin gene of mumps virus was optimized in reactive system and condition to improve the sensitivity, specificity and accuracy. In addition,colone technology was used to develop a quantitative PCR format with virus copies amount. Results The best concentration of primers and probe is 880 mmol/L and 280 mmol/L respectively,with good conservatism and specificity, none of the negative control sample showed false-positive reaction in duplication. The detection limit of the assay was 10 copies per reaction, equaling 0.01TCID50 （50 % tissue culture infective dose）. A linear standard curve was obtained between 10 and 107 DNA copies/reaction. It took only three hours from viral RNA extraction to complete the detection, and besides, the as,say was simple and good reproducibility. Conclusion This realtime RT-PCR assay provides a good method for quick and early detection of mumps virus.

关 键 词：流行性腮腺炎病毒 荧光定量逆转录-聚合酶链反应 TaqMan—MGB探针 

分 类 号：R373.16[医药卫生—病原生物学]