A Stable and Dependable Visual Technique for On-Site Nipah Virus Nucleic Acids Detection.

The Nipah Virus (NiV) is a zoonotic pathogen with the mortality rate of up to 75%, recurring in Asia over the past two decades. Due to increasing the risk of human transmission mediated by various intermediate hosts such as pigs and bats, it is necessary to produce an accurate and reliable point-of-care molecular detection method for NiV field diagnosis. In this study, we designed two pairs of primers targeting the conserved G and P genes and developed a point-of-care nucleic acid detection (POC-NAD) system by integrating one-step RT-PCR, lateral flow immunoassay, and microfluidic technologies. The POC-NAD system shows high specificity and sensitivity, with a Limit of Detection (LoD) of 199.1 copies/rxn. The primers aiming to the conserved sequences enables simultaneous detection of both NiV-M and NiV-B strains. Continuous evaluation of 21 simulated clinical samples demonstrated 100% concordance with RT-PCR results. Lateral flow-based visualization improves the display time and legibility of RT-PCR results. Additionally, microfluidic chips or chambers offer disposable reagent containers and consistent PCR amplification results across various field conditions. Therefore, the diagnostic tool is suitable for real-time nucleic acid testing and NiV surveillance in resource-limited field environments.