Abstract:

Objective　To establish a TaqMan probe?based fluorescence quantitative PCR assay for rapid detection of Dengue virus type 1 (DV1) to facilitate the clinical diagnosis. Methods　A set of specific primers and TaqMan probes were designed for the RT?PCR according to the conservative gene sequences at the 5′-terminal non?coding regions of DV1. A total of 40 sera samples were collected from patients with dengue fever, and four serotypes of standard DV strains were used as the control. The specificity of the established TaqMan?based fluorescence quantitative PCR assay was determined using the RNA templates obtained through in vitro transcription in the RT?PCR of the standard strains as a positive control. The sensitivity of the assay was then compared with that of the DV?IgM/IgG?based ELISA by assessing the sera samples. Results　The lowest detection limit of the established method was approximately 10 gene copies per reaction. As to the positive results among the sera samples collected from patients at different stages after onset, the RT?PCR had the highest positive detection rate during the first three days after onset (81.25%), while the ELISA?IgM had the highest positive detection rate from day 4 to day 6 after onset (85.00%). After 7 d, ELISA?IgG had the highest positive detection rate (75.00%). Conclusion　The established RT?PCR assay was a highly sensitive, specific and reproducible approach for rapid detection of DV1, conducive to the early diagnosis of dengue fever.

Key words: Dengue virus type 1, Fluorescence quantitative RT?PCR, TaqMan probe, Detection

摘要：

目的　建立登革热1型病毒（DV1）TaqMan荧光定量PCR快速检测方法及应用于临床诊断。方法　根据DV1 5′端非编码区基因保守序列，设计一套特异性引物和TaqMan探针。用4个血清型DV标准毒株为对照，收集40份DV1临床血清为检测标本。通过对DV1标准毒株RT?PCR后，采用体外转录方式获得RNA模板作为阳性对照，检测所建立TaqMan荧光定量PCR方法的特异性。取DV?IgM/IgG 用ELISA检测患者血清，将TaqMan荧光定量PCR和DV?IgM/IgG ELISA进行敏感性比较。结果　所建立方法的最低检测限约为每反应10个基因拷贝。发病后不同时间采集的登革热患者血清标本检测结果为：发病1～3 d的患者RT?PCR阳性检出率最高（81.25%）；4～6 d ELISA?IgM检出率最高（85.00%）；7 d后ELISA?IgG检出率最高（75.00％）。结论　在登革热的早期诊断中建立的RT?PCR具有较高的敏感性、特异性和重复性，可作为DV1的快速诊断方法。

关键词: 登革热1型病毒, 荧光定量RT?PCR, TaqMan探针, 检测