Rapid molecular diagnostic method for Gardnerella vaginalis based on CRISPR-Cas12a and recombinase-aided amplification (RAA)

Imbalance of the vaginal microbiota, particularly the overgrowth of Gardnerella vaginalis, is the primary cause of bacterial vaginosis (BV), which poses a significant threat to women's reproductive health. Therefore, early and rapid diagnosis of BV is crucial. Current laboratory diagnostic methods for BV mainly rely on Amsel's clinical criteria, bacterial culture, and PCR techniques. However, these methods have notable limitations: Amsel's criteria are subject to operator subjectivity, culture methods are time-consuming and require specialized expertise, while PCR necessitates expensive instrumentation. These constraints hinder their widespread clinical application. To address this issue, developing a highly accurate and low-cost molecular diagnostic method holds significant clinical value for BV detection. In recent years, recombinase-aided amplification (RAA) and CRISPR-Cas12a gene-editing technologies have achieved groundbreaking progress in nucleic acid detection. This study innovatively integrates RAA isothermal amplification with CRISPR-Cas12a detection to successfully establish a rapid nucleic acid detection platform for Gardnerella vaginalis. Experimental results demonstrate that this platform achieves a detection sensitivity of 10 copies/mL for Gardnerella vaginalis genomic DNA, with no cross-reactivity against other common reproductive tract pathogens. In validation tests using 44 clinical vaginal swab samples, the platform showed a 100.00 % positive agreement rate compared to qPCR. These findings confirm that the CRISPR-Cas12a-based detection platform exhibits excellent specificity, sensitivity, and reliability, serving as an effective tool for monitoring Gardnerella vaginalis colonization levels. This approach provides a novel molecular diagnostic solution for early BV screening and prevention.