TaqMan MGB探针实时聚合酶链反应检测登革病毒

目的建立一种敏感、特异、重复性好的登革病毒(DengueVirus,DV)鉴定方法。方法根据DV3’端非编码区基因保守序列,设计一套特异性引物和TaqManMGB探针。利用1998~2004年收集的26份登革热病人临床血清标本,15例乙脑病人血清,DV4个血清型标准毒株及23株1978~1997年DV地方流行株和相关西尼罗病毒、日本脑炎病毒、麻疹病毒、基孔肯亚病毒等毒株,同时用克隆了1型DV基因组3’端非编码区序列片段的质粒DNA作为阳性对照,检测所建立方法的特异性、敏感性。结果所建立方法的最低检测限约为每反应5个基因拷贝。用该方法检测4个血清型DV标准毒株、23株DV地方流行株和26例分离到DV的阳性血清标本,检出率为100%;用该方法检测15例乙脑病人血清、10例麻疹病人血清和西尼罗病毒、日本脑炎病毒、麻疹病毒、基孔肯亚病毒,结果均为阴性。结论新建的TaqManMGB探针检测DV方法是实验室早期诊断登革热较理想的方法。

Detection of Dengue virus by real-time polymerase chain reaction with TaqMan MGB probe

To develop a sensitive, specific and reproductive method to detect Dengue virus (DV) in clinical specimens, a set of specific primer and TaqMan MGB probe used in the real-time polymerase chain reaction was designed according to the conservative gene of 3' terminal non-coding region of DV, and the specificity and sensitivity of the developed method were to be determined by testing with 26 serum samples of patients with Dengue fever collected from 1998 to 2004, 15 serum samples of patients with Japanese B encephalitis, 4 standard serotype DV strains, 23 local isolates of DV isolated from 1978 to 1997, and the relevant viruses of West Nile virus, Japanese B encephalitis virus, measles virus and Chikungunya virus. The plasmid DNA containing the gene fragment of 3' terminal non-coding region of DV type 1 was used as the positive control. It was found that the lowest detection limit of the developed assay was about 5 genomic copies per reaction, and all the DV and the serum samples of patients with Dengue fever showed positive results, with a corresponding negative results in all the non-DV relative samples. It is evident this newly developed method of assay to detect DV with TaqMan MGB probe may be considered as an ideal method for the early laboratory diagnosis of Dengue fever.