Deployable, Field-Sustainable, Reverse Transcription-Polymerase Chain Reaction Assays for Rapid Screening and Serotype Identification of Dengue Virus in Mosquitoes

摘要

Dengue virus universal and serotype 1 to 4 fluorogenic probe hydrolysis, reverse transcription (RT)-polymerase chain reaction (PCR) assays and positive-control RNA template were freeze-dried in a thermally stable, hydrolytic enzyme-resistant format and deployed for testing in a dengue fever- endemic region of Thailand. The study site presented austere testing conditions. Field-collected Aedes aegypti mosquitoes and individual and pooled, field-collected, A. aegypti, A. albopictus, and Culex tritaeniorhynchus mosquitoes were used for RT-PCR assay evaluations. For dengue virus-inoculated A. aegypti mosquitoes and spiked samples, in vitro sensitivity and specificity results for all five assays were concordant with indirect fluorescent antibody assay results. A single pool of field-collected, female A. aegypti mosquitoes was identified as dengue virus positive. Cross-reactivity was not observed across heterologous serotypes, mosquito vectors, or human DNA. The limit of detection was > 7 to &(number sign)8805; 70 genomic equivalents. Sample processing and and analysis required > 2 hours. These results show promise of field-formatted RT- PCR reagents for rapid, sensitive, specific dengue virus screening and serotype identification in mosquitoes under field-deployable conditions.