An extraction-free and one-pot two-temperature CRISPR/Cas12b system for visual detection of Group B <i>Streptococcus</i> by integrating with RPA

This study presents a rapid, convenient, and highly accurate method for Group B <i>Streptococcus</i> (GBS) detection by integrating the CRISPR/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.