Development of Reverse Transcriptase Recombinase Polymerase Amplification Assay for the Detection of Rabies Virus

Background: Rabies is a fatal viral encephalitis caused by the rabies virus (RABV), a member of the Lyssavirus genus, Rhabdoviridae family. Despite vaccine availability, rabies continues to cause over 60,000 deaths annually, largely due to delayed diagnosis and lack of rapid, field-suitable diagnostic tools. Methods: The partial glycoprotein (G) gene of RABV was cloned into the pET-32a (+) vector, and specific primers were designed to target conserved regions of the G gene. The Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA) assay was optimized for temperature (35°C–42°C) and time (15–20 min) using the TwistAmp® Basic Kit. Amplification products were visualized by agarose gel electrophoresis, and assay sensitivity was determined using tenfold serial dilutions of the cloned plasmid DNA. Results: The optimized RT-RPA assay efficiently amplified a 300 bp target fragment at 39°C within 20 min. The assay demonstrated high sensitivity, detecting plasmid dilutions up to 10 4 -fold, and exhibited 100% specificity with no cross-reactivity. Conclusion: The developed RT-RPA assay provides a rapid, sensitive, and specific molecular diagnostic tool for detecting RABV. Its minimal equipment requirement and short turnaround time make it suitable for field deployment, particularly in resource-limited settings. This assay can significantly contribute to early diagnosis, surveillance, and control of rabies, thereby improving public health outcomes.