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<p>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&#xa0;°C for 40&#xa0;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 (<i>κ</i> = 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月17日

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  • Shyam Sundar Nandi,
  • Sonali A. Sawant,
  • Pragya D. Yadav,
  • Anita Shete-Aich,
  • Triparna D. Majumdar,
  • Kalpesh C. Bavkar,
  • Anupam Mukherjee,
  • Jagadish M. Deshpande

摘要

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.