RPA-primed droplet-partitioned CRISPR/Cas12a assay for low-copy detection of Candida albicans in vaginal specimens

Candida albicans is a major cause of vulvovaginal candidiasis, and sensitive detection of low-abundance fungal DNA remains challenging in clinical specimens. In this study, we developed a two-stage assay combining rapid recombinase polymerase amplification (RPA) with droplet-partitioned CRISPR/Cas12a fluorescence detection. Target DNA was first amplified by RPA for 10 min and subsequently analyzed in vortex-generated polydisperse droplets without microfluidic chip fabrication. Using predefined NTC-based analytical criteria, the operational LoDs were 10 copies/μL for the bulk RPA-Cas12a assay and 1 copy/μL for the droplet-partitioned format under the tested conditions. No cross-reactivity was observed with the tested non-target organisms, including additional clinically relevant non- albicans Candida species. In a preliminary evaluation of 28 residual vaginal swab specimens, the assay showed complete concordance with a routine composite clinical reference using an exploratory cohort-derived droplet-count cutoff. Because the comparator was not species-specific for C. albicans , these findings represent preliminary concordance rather than definitive diagnostic accuracy. The complete workflow was performed within approximately 1 h using basic laboratory equipment. These findings demonstrate that droplet partitioning can improve the low-copy analytical readout of RPA-Cas12a detection and provide a practical approach for qualitative identification of C. albicans DNA in vaginal specimens.