Development of an RT-LAMP-CRISPR/Cas12a-based nucleic acid detection platform for rapid diagnosis of porcine reproductive and respiratory syndrome virus type 2

Porcine reproductive and respiratory syndrome virus (PRRSV) significantly affects swine production, leading to reproductive failure and respiratory disorders. Current detection methods are limited by slow turnaround times, insufficient sensitivity and specificity, the need for skilled personnel, and costly equipment, which hinder their application in rapid on-site diagnosis and early disease prevention and control. In this study, a novel detection platform was developed by integrating the target specificity of RT-LAMP primers with the dual recognition capability of CRISPR/Cas12a. Optimal crRNA primers targeting conserved sequences in the PRRSV-2 structural protein M gene were designed and screened. Reaction conditions were subsequently optimized, and a cloning vector containing the target gene was constructed. Sensitivity evaluation using PCR, LAMP-CRISPR/Cas12a, and qPCR revealed detection limits of 9.17 × 103 copies/μL, 9.17 × 10-1 copies/μL, and 9.17 × 102 copies/μL for the target plasmid DNA, respectively. Notably, the LAMP-CRISPR/Cas12a assay exhibited the highest sensitivity, with a detection capability 10⁴-fold and 10³-fold greater than that of PCR-CRISPR/Cas12a and qPCR, respectively. Specificity analysis demonstrated that only HP-PRRSV generated a positive signal among the four tested porcine viruses, including PEDV G2b, PRV HNJY, and PPV SD-1. No fluorescent signals were detected for the other viral strains, indicating the absence of cross-reactivity. Fifty porcine serum samples were analyzed to compare the diagnostic performance of the RT-LAMP-CRISPR/Cas12a assay with that of qPCR, and the results showed complete agreement between the two methods. The established RT-LAMP-CRISPR/Cas12a detection system provides a rapid and reliable approach for PRRSV detection and may facilitate improved surveillance and control of PRRSV infections.