Dual readout RT-LAMP for rapid on-site dengue virus detection and serotyping

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Dengue is the fastest-growing mosquito-borne disease, with over 14 million reported cases globally in 2024, rendering dengue a major public health issue of global concern. Fast and reliable serotype-specific diagnosis of dengue is critical for patient risk management and epidemiological surveillance. While real-time PCR currently remains the gold standard for dengue virus detection, it requires thermal cyclers and trained personnel in an established laboratory infrastructure, limiting its use in low-resource settings. Loop-mediated isothermal amplification (LAMP) offers a promising alternative, yet currently available reverse transcription assays for dengue either rely on limited sequences used for primer design, thus failing to adequately capture global genetic diversity, or use multiple primer sets, increasing complexity and costs. We developed four serotype-specific dengue virus (DENV) reverse transcription (RT)-LAMP assays in which primer design was informed by 4,277 whole-genome sequences from different geographical regions. Degenerate base positions were incorporated into primers to achieve broad detection capability while minimizing the number of distinct oligonucleotides needed. The assays achieved analytical sensitivities of 96-734 genome equivalents per reaction after 60 min. Clinical evaluation using 95 PCR-confirmed positive samples from Sri Lanka, including serotypes 1-3, demonstrated detection rates of 91.2% for DENV1, 78.6% for DENV2, and 100% for DENV3, with no cross-reactivity observed between serotypes. Evaluation using DENV4-spiked serum demonstrated reliable detection at clinically relevant viral loads, with complete detection at quantification cycle (Cq) values of ~30. A dual readout design enables both real-time fluorescence detection and equipment-free visual colorimetric detection, rendering the assays suitable for point-of-care applications in dengue-endemic regions worldwide. IMPORTANCE: Dengue virus infections pose a growing threat to global health, with cases doubling annually since 2021, yet reliable diagnostic tools remain inaccessible in many affected regions. Current gold-standard molecular tests require expensive equipment and specialized laboratories, creating a critical gap in dengue-endemic areas where rapid, on-site diagnosis could save lives through early patient management and outbreak control. This study addresses this gap by developing dengue virus serotype-specific detection assays that work at a constant temperature without the need for sophisticated and expensive equipment. By analyzing thousands of dengue virus genome sequences and strategically incorporating degenerate bases, we designed primers that can detect diverse viral strains while maintaining simplicity and low cost. The assays provide results in 40-60 min, depending on the workflow, and can be read visually without instruments, making them practical for use in clinics, field settings, and resource-limited laboratories where dengue burden is highest.