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

ABSTRACT

BACKGROUND: Trichomonas vaginalis is a protozoan parasite, widely recognized as the most prevalent non-viral sexually transmitted infection (STI) globally. This infection is linked to various complications, including pelvic inflammatory disease, adverse pregnancy outcomes, and an increased risk of acquiring HIV. Current molecular detection methods for T. vaginalis are often costly and technically challenging. METHODS: We developed a novel detection method for T. vaginalis using a multi-enzyme isothermal rapid amplification-clustered regularly interspaced short palindromic repeats (MIRA-CRISPR)/Cas13a-lateral flow device (LFD). This assay targets the repeated DNA sequence (GenBank L23861.1) of T. vaginalis and is performed at a constant temperature of 37 °C for approximately 1 hour. RESULTS: The detection limit of genomic DNA (gDNA) using our protocol was 1 × 10-4 ng/µl. Specificity was confirmed by the absence of cross-reaction with gDNA from various other microorganisms such as Staphylococcus aureus, Lactobacillus taiwanensis, Escherichia coli, Monilia albicans, Giardia lamblia, or Toxoplasma gondii. Among 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 polymerase chain reaction (PCR), 40% (12/30) by MIRA-CRISPR/Cas13a-LFD, and 40% (12/30) by the culture method. Compared with the culture method, the gold standard for diagnosing trichomoniasis, wet mount microscopy showed a sensitivity of 83.3% and moderate diagnostic agreement (kappa value = 0.87). Both nested PCR and MIRA-CRISPR/Cas13a-LFD exhibited 100% sensitivity and excellent diagnostic agreement (kappa value = 1). CONCLUSIONS: The MIRA-CRISPR/Cas13a-LFD method is a convenient, rapid, stable, and accurate diagnostic tool for detecting T. vaginalis. This method has the potential to enhance the diagnosis and management of vaginitis, offering a significant improvement over existing diagnostic techniques.