Development of the CRISPR-Cas12a-Based Biosensing System for Rapid, Ultrasensitive, and Highly Specific Detection of <i>Streptococcus pyogenes</i>

<i>Streptococcus pyogenes</i> (group A streptococcus, GAS) is the leading bacterial cause of acute pharyngitis in children and adolescents. Rapid and reliable diagnosis of GAS pharyngitis is essential for guiding a timely antibiotic treatment. Here, we developed a rapid, highly sensitive, and specific test platform for the detection of GAS, designated GAS-MCDA-CRISPR. In this diagnostic platform, the multiple cross displacement amplification (MCDA) technique was utilized to preamplify the specific <i>speB</i> gene of GAS. Subsequently, the CRISPR-Cas12a-based biosensing system was employed to decode the MCDA products. MCDA primers, a guide RNA (gRNA), and a quenched fluorescent single-stranded DNA (ssDNA) reporter were designed to target the <i>speB</i> gene of GAS. The GAS-MCDA-CRISPR assay demonstrated the ability to detect GAS genomic DNA at a concentration as low as 45 fg per microliter while exhibiting no cross-reactivity with other non-GAS pathogens. Moreover, 56 clinical samples were correctly detected by the GAS-MCDA-CRISPR assay. These data highlighted that the GAS-MCDA-CRISPR assay is a reliable diagnostic tool for the reliable and quick diagnosis of GAS infection.