Development of Field-Based Real-Time Reverse Transcription-Polymerase Chain Reaction Assays for Detection of Chikungunya and O'nyong-nyong Viruses in Mosquitoes

Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reac- tion (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and spe- cific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitiv- ity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque- forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.