Deployable, field-sustainable, reverse transcription-polymerase chain reaction assays for rapid screening and serotype identification of dengue virus in mosquitoes.
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 spiked with inoculated A. 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 < or =70 genomic equivalents. Sample processing 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-deployed conditions.
McAvin
James C
JC
U.S. Air Force Institute for Operational Health, Brooks City-Base, San Antonio, TX 78235-5237, USA.
Powers
Michael D
MD
Blow
Jamie A
JA
Putnam
John L
JL
Huff
William B
WB
Swaby
James A
JA
D000818:Animals; D009033:Culicidae / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics* / Q000302:isolation & purification; D019223:Densovirinae / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008297:Male; D008403:Mass Screening; D008952:Mobile Health Units; D010865:Pilot Projects; D012313:RNA; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D012703:Serotyping / Q000295:instrumentation / Q000379:methods; D013785:Thailand; D013997:Time Factors
D012313:RNA