The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48–100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48–100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

The DNA and RNA analytical sensitivity of the DENV 1–4 RPA/LFD tests was determined using 10-fold serial dilutions of both plasmid DNA and RNA-transcripts.

Analytical specificity for detection of different dengue serotypes was tested using high concentrations (10⁵ copies/µL) of RNA transcripts from each of the four DENVs

Analytical specificity for detection of different flaviviruses (FV) was tested using RNA extracted from ZIKV, JEV, MVEV, and WNV_KUN cell culture supernatant (5–7 log₁₀ TCID₅₀/ml)

Individual mosquito testing was tested by comparing rapid DENV 1–4 serotyping tests to qRT-PCR

Pooled mosquito testing was also tested by comparing rapid DENV 1–4 serotyping tests to qRT-PCR

The contents of each data file are described in the Readme.txt file.