A novel detection method based on MIRA-CRISPR/Cas13a-LFD targeting the repeated DNA sequence of Trichomonas vaginalis

Abstract Trichomonas vaginalis ( T. vaginalis ) is a protozoan parasite that is widely recognized as the most prevalent non-viral sexually transmitted infection (STI) worldwide. This infection is associated with various complications such as pelvic inflammatory disease, adverse pregnancy outcomes, and an increased risk of acquiring HIV. Existing molecular detection methods for T. vaginalis are often expensive or technically challenging. In this study, we developed a novel detection method based on MIRA-CRISPR/Cas13a-LFD, targeting the repeated DNA sequence (Genebank: L23861.1) of T. vaginalis . The MIRA-CRISPR/Cas13a-LFD assay was performed at a constant temperature of 37℃ for approximately 1 hour, and the results were visualized and determined using lateral flow device (LFD). The detection limit of gDNA was 1×10 -4 ng/μl by our developed protocol, and no cross-reaction occurred either with the gDNA of Staphylococcus aureus, Lactobacillus taiwanensis , Escherichia coli , Monilia albicans , Giardia lamblia , and Toxoplasma gondii . Among the 30 clinical samples tested, the positive rates of T. vaginalis detection were 33.33% (10/30) by wet mount microscopy, 40% (12/30) by nested PCR, 40% (12/30) by MIRA-CRISPR/Cas13a-LFD, and 40% (12/30) by culture method. When compared to the culture method, which served as the gold standard for diagnosing trichomoniasis, wet mount microscopy demonstrated a sensitivity of 83.3% and moderate diagnostic agreement (Kappa value = 0.87). In contrast, both nested PCR and MIRA-CRISPR/Cas13a-LFD exhibited 100% sensitivity and excellent diagnostic agreement (Kappa value = 1). These results demonstrate that the MIRA-CRISPR/Cas13a-LFD method is a convenient, rapid, stable, and accurate diagnostic tool for T. vaginalis detection, which can enhance the diagnosis and management of vaginitis.