Rapid detection of Streptococcus agalactiae using an RPA-MbCas12a CRISPR assay targeting novel sodA and groL markers

Streptococcus agalactiae (Group B Streptococcus, GBS) is a major cause of neonatal and invasive infections, necessitating rapid and sensitive diagnostic approaches beyond conventional culture and PCR. Here, we developed an isothermal recombinase polymerase amplification (RPA)-MbCas12a CRISPR assay for fluorescence-based detection of GBS using a recently characterized MbCas12a variant from Moraxella bovoculi. The assay targets two conserved housekeeping genes, sodA and groL, and operates under isothermal conditions at 37 °C with minimal sample processing. Analytical sensitivity reached 1.6 × 10¹ to 2.5 × 10¹copies per reaction, while analytical specificity was confirmed against non-target bacterial species. The assay successfully detected all 14 tested GBS isolates directly from crude heat lysates and generated results within 1 h. By combining rapid isothermal amplification, simplified sample preparation, and MbCas12a-mediated detection, this platform represents a promising approach for point-of-care GBS screening and infection control in resource-limited settings.