Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for detection of dengue virus type 2

Objective To develop a TaqMan-based reverse transcription-polymerase chain reaction assay (RT-PCR) for the detection of dengue virus type 2 (DENV-2).Methods The whole genome sequences of DENV-2 were downloaded from the GenBank and aligned with the Bioedit software.Specific primers and probes were designed on the conserved region of DENV-2 based on the alignment results to develop a TaqMan-based RT-PCR assay.Results The detection limit of the assay was 10² copies/μL.Besides the obvious amplification of DENV-2,there were no cross reactions with Murray Valley encephalitis virus,Japanese encephalitis virus,West Nile virus,tick-borne encephalitis virus,other types of dengue virus,yellow fever virus,and Kyasanur Forest disease virus.The results of the stability assay showed that the between-and within-group coefficient of variation were less than 2%.For the 87 mosquito specimens detected with two different methods,56 positive samples were confirmed with fluorescence quantitative RT-PCR,while only 16 positive samples were confirmed with the traditional RT-PCR method,with a significant difference (χ²=37.908,P=0.000).Conclusion The developed TaqMan-based one-step reverse transcription-polymerase chain reaction assay is of strong specificity and high sensitivity for molecular diagnosis of DENV-2.