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

 West Valley Mosquito and Vector Control District, 1295 E. Locust St., Ontario, CA 91761, USA

Correspondence Address:
Tianyun Su,
West Valley Mosquito and Vector Control District 1295 E. Locust St. Ontario, CA 91761
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.316275

Background & objectives: 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. Methods: 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. Results: 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: 3.96×100 for CHIKV, 3.80×101 for DENV1, 3.20×101 for DENV2, 8.00×10-1 for DENV3, 1.58×100 for DENV4, and 6.20×100 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 1.32×100 for CHIKV, 3.79×100 for DENV1-4 and 2.06×100 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 3.96×10-1, DENV1-4 to 1.14×100 and ZIKV to 3.20×100. In full combination assays, CHIKV was detected to 1.32×10-1, DENV1-4 to 3.79×101 and ZIKV to 1.07×100. Interpretation & conclusion: 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 adoptable protocol for use among vector control and other entities. The established procedures were applied to testing Ae. aegypti and Ae. albopictus collected from the field for CHIKV, DENVs and ZIKV.

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