Abstract:
Background: Dengue viruses are mainly transmitted by the Aedes aegypti mosquito, which grows in breeding containers maintained by rain or human activity. The Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) reaction was found to be more sensitive and can detect 10 copies of RNA template, whereas real-time PCR (qPCR) had detection limits of 100 copies.
Methods: This prospective analytical study was conducted at the Department of Virology, BSMMU (January–December 2017). A total of 2,280 field-collected adult mosquitoes were sorted, and 1,348 female Aedes aegypti were stored at –80°C for serotype testing. Individual mosquito heads were lysed for virus detection. Serotype-specific RT-LAMP primers were designed from the 3′ NCR of all four dengue serotypes. RT-LAMP was performed using a 25 µL reaction mixture and incubated at 63°C for 60 minutes, with results monitored by turbidity, SYBRGreen color change, and agarose gel electrophoresis. Sensitivity and specificity were evaluated using serial dilutions and chikungunya-positive controls. qRT-PCR served as the gold-standard confirmation, using extracted RNA, TaqMan chemistry, and 45 cycles on an Applied Biosystems 7500 system.
Results: A total of 2,280 wild-caught Aedes aegypti mosquitoes were collected from Dhaka, of which 1,348 (59.12%) were females. RT-LAMP screening detected dengue virus in 217 (16.10%) of these females, and qPCR confirmation produced identical results. Among the positive samples, DEN-2 was the dominant serotype, detected in 184 mosquitoes (84.79%), while DEN-1 was identified in 33 mosquitoes (15.21%). No DEN-3 or DEN-4 serotypes were found. Four visual RT-LAMP detection methods were used: amplification curves, gel electrophoresis, color change using HNB dye, and turbidity assessment. Positive samples showed characteristic step-ladder bands, real-time amplification curves, and a blue color change with HNB dye, whereas negative controls exhibited no amplification and retained a purple color. These findings indicate active circulation of dengue virus—predominantly DEN-2—within Dhaka’s mosquito population and demonstrate the reliability of RT-LAMP for rapid field-level serotype detection.
Conclusion: The RT-LAMP is a relatively inexpensive, fast, and simple tool for the accurate detection and serotyping of DENV from wild-caught mosquitoes.
Biography:
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