Establishment of TaqMan probe-based fluorescence quantitative PCR for Dengue virus type 1 and its clinical application

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.