Rapid Visual Detection of Mycoplasma Hominis Using an RPA-CRISPR/Cas12a Assay

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Mycoplasma hominis (MH) is an opportunistic pathogen linked to urogenital infections and adverse pregnancy outcomes.
• Current detection methods (culture, qPCR) are slow and require complex equipment, limiting rapid diagnostics.

Purpose of the Study:

• To develop a rapid, sensitive, and specific visual biosensing platform for Mycoplasma hominis detection.
• To integrate recombinase polymerase amplification (RPA), CRISPR/Cas12a, and lateral flow biosensors for MH identification.

Main Methods:

• Developed a visual readout biosensing platform combining RPA, CRISPR/Cas12a, and lateral flow biosensors.
• Targeted the MH-specific 16S rRNA gene for detection.
• Optimized reaction parameters including Cas12a-crRNA ratio and cleavage time.

Main Results:

• Achieved a limit of detection as low as 2 copies/reaction with a total assay time of 60 minutes.
• Demonstrated exceptional specificity, with no cross-reactivity against common urogenital pathogens.
• Validated the assay on 18 clinical samples, showing high specificity.

Conclusions:

• The developed CRISPR-LFA platform offers rapid, sensitive, and specific detection of Mycoplasma hominis.
• This assay has promising potential for clinical applications, especially where rapid testing is needed.
• This study introduces the first contamination-control protocol for an MH-specific CRISPR-LFA assay.