Development of a point-of-care diagnostic method for FMDV SAT2 using RT-RAA-CRISPR technology

In recent years, the foot-and-mouth disease virus (FMDV) serotype SAT2 has expanded beyond its traditionally recognized endemic areas on the African continent, leading to continuous dissemination in Middle Eastern countries and presenting a risk of further spread to Asia. In this study, the highly conserved sequence of the FMDV SAT2 genome was initially selected as the target, and five pairs of reverse transcription-recombinase-aided amplification (RT-RAA) primers were designed. By comparing fluorescence signal intensities, the CRISPR RNA (crRNA) with the highest sensitivity and specificity was identified and subsequently integrated with the CRISPR/Cas13a gene-editing system to establish a novel nucleic acid detection method. This method possesses a robust capacity for differential diagnosis and shows no cross-reaction with other serotypes of FMDV. Moreover, this method demonstrated high specificity and no cross-reactivity with the nucleic acid sequences of various common pathogens in porcine and bovine populations. The test results are readily interpretable and can be directly visualized using a fluorescence reader or lateral flow test strips (LFSs). In simulated clinical samples, this method achieved a concordance rate of 100% with the detection results of fluorescence quantitative RT-PCR. This study successfully developed a highly sensitive and specific FMDV SAT2 nucleic acid detection method based on RT-RAA-CRISPR/Cas13a technology. This method is straightforward to perform, does not require costly experimental equipment, and is suitable for rapid onsite detection, offering a convenient and efficient diagnostic tool for the early diagnosis, prevention, and control of FMDV SAT2 epidemics.