A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes

Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations.

Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes.

In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x10⁰ for CHIKV, 3.80x10¹ for DENV1, 3.20x10¹ for DENV2, 8.00x10⁴ for DENV3, 1.58x10⁰ for DENV4, and 6.20x10⁰ for ZIKV When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x10⁰ for CHIKV, 3.79x10⁰ for DENV1-4 and 2.06x10⁰ for ZIKV All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10^-1, DENV1-4 to 1.14x10⁰ and ZIKV to 3.20x10⁰. In full combination assays, CHIKV was detected to 1.32x10⁴, DENV1-4 to 3.79x10¹ and ZIKV to 1.07x10⁰.

This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.