Field Evaluation of a Deployable RT-PCR Assay System for Real-Time Identification of Dengue Virus

摘要

Dengue fever and the more severe form of the disease, dengue hemorrhagic fever, occurs in tropical and subtropical regions globally through infection by one or more of four viral serotypes, DEN-1, DEN-2, DEN-3 and DEN- 4. Viral transmission to humans is through mosquito vectors, primarily Aedes aegypti. Dengue universal and DEN 1-4 serotype specific fluorogenic, real-time RT-PCR assays and positive control nucleic acid were field-formatted by lyophilization and adapted for use on a field-durable, real-time, fluorimetric thermocycler. Nucleic acid extract isolated from DEN1-4 inoculated Aedes aegypti, field-captured Aedes aegypti and Culex spp, and field-captured Aedes aegypti and Culex spp spiked with total nucleic acid from DEN1-4 inoculated Aedes aegypti were used to construct a test panel (n = 64). In blind testing each of the five assays were 100% concordant in in vitro sensitivity and 100% concordant in specificity with dengue virus inoculated and spiked field- captured mosquitoes. A field-captured Aedes aegypti pool consisting of five females and two males was identified as dengue virus positive by the dengue universal assay; however, serotype identification was inconclusive. This study demonstrates the operational utility of a deployable assay system for rapid, sensitive, and specific screening and serotype identification of dengue virus in mosquito vectors.