关键词: 荧光定量RT-PCR, TaqMan探针, 登革病毒

Abstract: Objective To establish a TaqMan-based real-time PCR assay for detection of dengue 1,2 serotype virus,and the constructed method was primarily used to clinical samples test for dengue. Methods The gene sequences of dengue virus downloaded from the GenBank was aligned using the biologic software and the specific primers and probes were designed in the conserved region of the NS5 for 1 serotype and C gene for serotype. The primers,probes and the reactive condition were optimized to improve the sensitivity and specificity of the assay. The clinical specimens collected from the patients infected with dengue fever were detected by this assay. Results The specificity of the assay was high and there were no cross reactions with Japanese encephalitis virus(JEV),haemorrhagic fever with renal syndrome(HFRS) and other serotype dengue virus. The sensitivity of the assay was 0.1 TCID₅₀ and the viral RNA was directly detected with the clinical specimens by this assay. It took all about three hours to extract viral RNA and the real-time PCR. Conclusion This TaqMan-based real-time PCR assay was a quick,sensitive and specific method for molecular diagnosis of dengue 1,2 serotype virus.