Development of a Panel of Six Duplex Real-Time Reverse Transcription Polymerase Chain Reaction Assays for Detecting 12 Mosquito-Borne Viruses

Mosquito-borne virus family consists of more than 20 viruses. Some of them have similar characteristics of geographical distribution and seasonal activity, and produce similar symptoms, making their discrimination by routine methods difficult. Therefore, the goal of the present study was to develop a panel of six duplex real-time reverse transcription polymerase chain reaction (RT-PCR) assays capable of simultaneous detection and distinction of 12 mosquito-borne viruses. The limit of detection of the duplex assays was determined using 10-fold serial dilutions of standards of 12 mosquito-borne viruses and compared to a conventional RT-PCR method for evaluation of the sensitivity. The assessment of the duplex real-time RT-PCR panel was performed with clinical samples and spiked samples. The duplex assays enabled detection and differentiation between 12 mosquito-borne viruses from positive strains and other negative strains; cross-reactivity was not encountered. Moreover, the limit of detection of the duplex assays was at least one or two log dilutions lower than that of the conventional PCR.