Establishment of TaqMan RT-PCR for detection of Koutango virus

ObjectiveTo establish a real-time quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay for the rapid detection of Koutango virus (KOUTV) in mosquito borne arbovirus surveillance.

MethodsAll gene sequences of KOUTV were downloaded from GenBank for multiple sequence alignment. Specific primers and probes were designed for the highly conserved region of NS5 gene. The specificity of established TaqMan RT-PCR assay was evaluated with other 9 viruses from 5 families, and the stability of the assay was verified by parallel repeated experiments. Moreover, an absolute quantitative analysis model for the NS5 gene copy number of KOUTV was set up with in vitro transcribed RNA standards.

ResultsThe established TaqMan RT-PCR assay has good specificity and sensitivity. No cross reaction with other arbovirus was observed and the sensitivity was 1.0×10² copies/reaction. The coefficients of variation of Ct values in repeated detection of same sample were all less than 1.5%. A total of 6 328 mosquito samples in 112 batches collected in Xinjiang were tested with the established assay and the results showed KOUTV negative in all samples.

ConclusionA high specificity and sensitivity TaqMan RT-PCR assay for KOUTV was successfully established.