Development and analytical validation of a dual-gene isothermal assay for Nipah virus detection

Nipah virus (NiV) is a highly pathogenic zoonotic virus characterized by fatal encephalitis and severe respiratory illness, with case-fatality rates reaching 75%. Given the lack of licensed vaccines or therapeutics, rapid and accurate detection is paramount for outbreak containment. While real-time reverse transcription polymerase chain reaction (qRT-PCR) remains the gold standard, its requirement for sophisticated laboratory infrastructure limits its utility in resource-constrained field settings. In this study, we developed and validated a rapid, colorimetric dual-target reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the conserved matrix and nucleocapsid genes to enhance diagnostic reliability. Optimized at 62 °C for 40 min, the assay provides a clear visual colorimetric interpretation. A total of 137 samples were evaluated: in vitro-transcribed (IVT) RNA and tissue culture fluid isolate dilutions for limit of detection assessment, 57 non-NiV viral samples for cross-reactivity assessment, and 10 experimentally spiked positive clinical matrices used as proxy clinical specimens together with 50 confirmed NiV-negative clinical swabs for preliminary proxy diagnostic performance evaluation. The dual-target assay demonstrated 100% analytical sensitivity (95% confidence interval or CI, 72.2-100.0) and 100% specificity (95% CI, 92.9-100.0), showing perfect agreement (κ = 1.00) with real-time reverse transcription polymerase chain reaction (qRT-PCR). The composite "matrix or nucleocapsid" interpretation rule supported complete detection in the tested proxy positive clinical-matrix specimens. This simple, cost-effective, and portable molecular tool is well-suited for decentralized diagnostics, supporting early detection and surveillance in NiV-endemic regions.