肠道病毒71型实时荧光RT-PCR检测方法的建立及临床评价  

Establishment and Evaluation of Enterovirus 71 Real-time RT-PCR Detection Kit

作　　者：朱坤[1,2] 高秀杰[2] 邓中平[2] 徐伟文[1] 高旭年[2] 李明[1] 

机构地区：[1]南方医科大学生物技术学院,广州510515 [2]中山大学达安基因股份有限公司,广州510080

出　　处：《热带医学杂志》2010年第5期505-508,519,共5页Journal of Tropical Medicine

摘　　要：目的利用实时荧光RT-PCR技术,并通过系统的分析性能和临床性能评价,建立一种早期快速检测肠道病毒71型(EV71)的方法。方法根据EV71基因组中编码衣壳蛋白VP1基因保守区序列设计一对引物和一条荧光探针,利用实时荧光RT-PCR技术建立了检测EV71的方法,并对扩增产物进行分析,同时进行灵敏度、特异性、精密度评价,在此基础上利用不同标本类型共1104例临床样本对本方法和RT-PCR方法进行对比分析。结果本方法可以特异性的检测EV71,而对种属相近的或引起症状相似的其他病毒均无交叉反应。本方法检测灵敏度达到9.22×102PFU/ml,不同浓度样本的Ct值的变异系数在1.4%～2.9%之间。1104例临床样本的研究显示本方法与RT-PCR方法检测结果的总符合率达到96.74%,阳性样本的检出率要高于RT-PCR方法。结论本方法检测EV71具有灵敏度高、特异性强、精密度高、快速简便的特点,并与RT-PCR方法具有很好的符合率,在手足口病的早期快速诊断和疫情监测方面具有很好的应用前景。Objective To establish and evaluate a method for early and rapid detection of enterovirus 71 （EV71） by real-time RT-PCR. Methods PCR primers and Taqman fluorescent probe were designed according to the conserved gene sequence of EV71 capsid protein VP1. The analytical performance such as specificity,sensitivity and precision were evaluated for the established detection system. 1104 clinical specimens including pharynx swab,anal swab,herpes,stool,urine,blood plasma and serum were collected for the evaluation of the clinical performance. A double-blinded and control trial （DB-CT） was carried out,RT-PCR as a control. Results The results showed that the method was specific for EV71 detection. The detection sensitivity was 9.22×102 PFU / ml. The CV ranged from 1.4% to 2.9%. Compared with RT-PCR,the coincidence rate was 96.74%. Conclusion The method for detection of EV71 by real-time RT-PCR was proved to be sensitive,specific and accurate. The results of real-time RT-PCR were consistent with those of RT-PCR. It could be well used for early diagnosis and surveillance of hand,foot and mouth disease.

关 键 词：肠道病毒71型 实时荧光RT-PCR 性能评价 

分 类 号：R512.5[医药卫生—内科学]