RPA-CRISPR Cas13a-Based Point-of-Care Testing established for rapid detection of Methicillin-Resistant Staphylococcus aureus (MRSA) resistance genes.

Abstract

Purpose: This study aimed to develop a streamlined and efficient method for detecting single nucleotide polymorphism (SNP), specifically the S643N mutation in the mecA gene of Staphylococcus aureus (S. aureus). Detecting this mutation is crucial for guiding appropriate antibiotic therapy, avoiding antibiotic misuse, and enhancing infection control measures, especially in the cases of methicillin-resistant S. aureus (MRSA) infections.

Methods: We employed the RPA-CRISPR Cas13a method to detect the S643N mutation. This approach integrated recombinase polymerase amplification (RPA) with CRISPR Cas13a-mediated detection into a single-step procedure, significantly reducing detection time compared to the conventional two-step process.

Results: The established RPA-CRISPR Cas13a-Based Point-of-Care Testing method achieved high sensitivity, detecting as few as 10 copies per reaction and successfully differentiating between wild-type and mutant mecA genes. The one-step procedure streamlined the workflow and reduced detection time to less than 30 min, while delivering results that were consistent with the conventional two-step method.

Conclusion: The one-step RPA-CRISPR Cas13a method significantly facilitates the rapid and accurate detection of single nucleotide mutations, such as the S643N mutation in the mecA gene. This advancement holds substantial clinical value by guiding precise antibiotic therapy and improving the management of S. aureus infections.