乙型脑炎病毒的实时荧光定量逆转录PCR检测方法的建立  

Development of one-step RT-qPCR for detection of Japanese encephalitis virus infection

作　　者：孙莉[1] 陈路 王华贵 赵相胜 胡小许 迟洪霞[4] 祁荣[4] 吴伟立[3] 

机构地区：[1]华北石油管理局总医院检验科,河北任丘062552 [2]北京宏微特斯生物科技有限公司,北京101300 [3]中国科学院北京基因组研究所,北京100101 [4]华北石油管理局总医院消化科,河北任丘062552

出　　处：《分子诊断与治疗杂志》2017年第5期341-346,共6页Journal of Molecular Diagnostics and Therapy

摘　　要：目的建立一种可以准确、快速、特异检测乙型脑炎病毒(Japanese encephalitis virus,JEV)的一步法实时荧光定量逆转录PCR(reverse transcription-quantitative real time PCR,RT-q PCR)体系。方法参照NCBI数据库的乙脑病毒全基因组序列,经过一致性比对分析,在乙脑病毒保守的3′非翻译区(3′untranslated region,3′UTR)区段,设计相应的特异性引物以及Taqman-MGB探针,优化检测体系及反应条件,建立一步法RT-q PCR方法。利用登革热、森林脑炎病毒、肠道病毒71型(EV71)、柯萨奇病毒A16型(Cox A16)、狂犬病毒培养物和临床诊断为登革热及手足口病人的血清样本检测其交叉反应。选择15例疑似脑炎症状的病人血清样本,以国家食品药品监督管理总局(CFDA)批准的乙脑病毒检测试剂盒作对照试剂盒,检测其临床适用性。结果所建立的乙脑病毒一步法RT-q PCR检测方法重复性试验Ct值的变异系数CV在0.46%~0.53%之间,最低可检出5 copies/反应样本。检测体系与登革热、森林脑炎病毒等黄病毒及EV71、Cox A16、狂犬病毒培养物等未发现交叉反应;64份临床诊断为登革热和手足口病人的血清样本检测也均为阴性。15份疑似乙脑感染的病人血清样本的检测结果与国家食品药品监督管理总局批准的乙型脑炎病毒检测试剂盒检测结果一致,但本方法操作步骤更少,检测时间更短。结论本实验建立的乙型脑炎病毒一步法实时荧光RT-q PCR检测方法具有较高灵敏度、特异性,可用于乙型脑炎病毒的检测。Objective To establish a sensitive and specific one-step reverse transcription-quantitative real time PCR（RT-q PCR） method for detection of Japanese encephalitis virus（JEV） infection.Methods Based on the whole genome sequence of the encephalitis virus in the NCBI database, the corresponding specific primers and Taqman-MGB Probe were designed in the 3′untranslated region（UTR） of the Japanese encephalitis virus. The detection system and reaction conditions were optimized. The cross-reactivity was measured by using dengue fever virus, forest encephalitis virus, enterovirus 71（EV71）,coxsackie virus A16（Cox A16）, rabies virus cultures and clinical sera samples of clinically diagnosed dengue fever and hand-foot-mouth patients. 15 patients with suspected encephalitis were enrolled in this study. The Japanese encephalitis virus test kit was registered by the China Food and Drug Administration（CFDA） used as a control kit to detect its clinical applicability. Results The repeatability test revealed that coefficient of variation（CV） of Ct value is 0.46%-0.53%. The sensitivity attends to 5 copies/reaction. No cross-reactions were found to dengue, forst encephalitis virus, EV71, Cox A16, rabies virus. The test result of 37 dengue fever and 27 hand-foot-mouth patients were also negative. The results of present developed method to suspended patients are consistent with the CFDA approved kit while with less manual handling steps and shorted operating time. Conclusion The developed one-step RT-q PCR method for detection of encephalitis virus infection with high sensitivity and specificity is suitable to the clinical use.

关 键 词：乙型脑炎病毒 Taqman-MGB 流行性乙型脑炎 RT-QPCR 

分 类 号：R440[医药卫生—诊断学] R512.32[医药卫生—临床医学]