RT-qPCR Detection of Nipah Virus (NiV) Targeting the Nucleocapsid (N) Gene v1

The genus Henipavirus (family Paramyxoviridae) includes Hendra virus (HeV) and Nipah virus (NiV), highly pathogenic zoonotic viruses capable of causing severe respiratory and/or encephalitic disease in a broad range of mammalian species. Natural infections have been documented in humans, horses, pigs, dogs, and cats, while experimental infections have been studied in guinea pigs, golden hamsters, and ferrets. Old World fruit bats serve as the natural reservoir hosts, typically exhibiting subclinical infection. Among henipaviruses, NiV is a bat-borne zoonotic pathogen associated with particularly severe disease and high case-fatality rates in humans and animals. Here, we describe a one-step real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the detection and quantification of NiV RNA, targeting the nucleocapsid (N) gene. This assay is a modified version of the method originally described by Feldman et al. (2009), optimized to improve analytical sensitivity and specificity. The RT-qPCR is suitable for detection of NiV RNA in a wide range of biological matrices, including clinical, animal, and environmental samples, when performed in laboratories with appropriate biosafety capacity for handling high-risk pathogens. Viral RNA is extracted from a defined volume of sample using either the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) or the Quick-DNA/RNA Pathogen Miniprep Kit (Zymo Research, Irvine, CA, USA), according to the manufacturers’ instructions. The assay combines reverse transcription and real-time PCR amplification in a single reaction using the SuperScript III Platinum One-Step Quantitative RT-PCR Kit (Invitrogen, Carlsbad, CA, USA) with primers and a modified probe adapted from Feldman et al. (2009). Each 25 μL reaction contains the supplied reagents, primers, probe, and 5 μL of extracted RNA template. Appropriate positive and negative controls are included in each run to ensure assay performance and result validity.