Establishment of a rapid RAA-CRISPR/Cas12a system targeting the recN gene for on-site detection of Streptococcus suis in livestock and fresh pork meat

Abstract

Streptococcus suis is a major bacterial pathogen in the swine industry, causing meningitis, arthritis, and other diseases in infected pigs. It also poses significant public health risks due to its zoonotic potential, particularly in individuals with skin lesions. Current detection methods, including traditional culture-based techniques and PCR assays, are time-consuming, labor-intensive, and lack sufficient accuracy. To address these limitations, this study aimed to develop a rapid and precise detection method for S. suis. By leveraging whole-genome sequencing (WGS) and multiple sequence alignment, the recN gene was identified as a highly specific molecular target. A novel isothermal detection method, integrating recombinase-aided amplification (RAA) with CRISPR/Cas12a, was subsequently established. This RAA-CRISPR/Cas12a-based system demonstrated superior sensitivity compared to conventional PCR (targeting the gdh gene), achieving detection within 30 min without requiring specialized equipment. This method achieves 2.44 × 10¹ copies/µL and 2.1 × 10¹ CFU sensitivity and 100% specificity within 30 min, outperforming conventional PCR in speed and reliability while eliminating dependency on specialized equipment. Designed for field applications, it offers a cost-effective (US$1/test), user-friendly solution for on-site S. suis detection in swine farms and fresh pork meat, enhancing outbreak control and preventive healthcare in the livestock industry.