Sexually transmitted infections (STIs) remain a significant global public health challenge. The rapid and precise identification of these pathogens is crucial for effective diagnosis and treatment. Current polymerase chain reaction (PCR)-based assays, despite their widespread use, present several limitations in clinical settings, including high costs and technical complexity. To address these challenges, we developed a portable and automated microfluidic nucleic acid detection device for multiplex detection of three sexually transmitted pathogens via the fluorescence quantitative PCR (qPCR) assay. This integrated platform combines automated sample processing, multiplex qPCR amplification, and fluorescence detection within a single device. The entire closed-loop testing procedure, from automated nucleic acid extraction to result generation, requires only 40 min. The system demonstrates detection limits of 360 copies/ml for Neisseria gonorrhoeae (NG), 140 copies/ml for Ureaplasma urealyticum (UU), and 290 copies/ml for Chlamydia trachomatis (CT). Clinical validation revealed exceptional performance in multiplex detection of infected specimens, showing complete concordance (100 %) with standard diagnostic results while maintaining high specificity. These findings highlight the instrument's clinical potential and establish a new paradigm for automated, multiplex pathogen detection.

Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.