Implementation of RT-RAA and CRISPR/Cas13a for an NiV Point-of-Care Test: A Promising Tool for Disease Control

Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription-recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks.