Abstract: 【Abstract】 Objective To establish a rapid quantitative PCR （qPCR） technique for Mycobacterium marinum skin infections, and to analyze its clinical diagnostic efficiency. Methods DNA was extracted from Mycobacterium marinum colonies and serially diluted （10-1 to 10-8）. Twelve pairs of previously reported primers and probes, as well as 6 pairs of newly designed primers and probes in this study, were used for qPCR amplification to identify the most sensitive primers and probes for the detection of Mycobacterium marinum. Skin lesion tissues were collected from 72 patients with confirmed Mycobacterium marinum infections （experimental group） and 68 with other mycobacterial infections （control group） at Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology, Shandong First Medical University & Shandong Academy of Medical Sciences in 2021. These skin tissues were subjected to qPCR amplification, interferon-gamma release assay （IGRA）, acid-fast staining, and tissue culture to evaluate the diagnostic efficacy. Results The newly designed primers and probes targeting the mycobacterial enhanced infection locus 2 （Mel2） demonstrated the highest sensitivity, with a detection limit of 0.86 copies/μl （cycle threshold value = 37）; the qPCR amplification with the Mel2 primers/probes did not yield positive results when used for the detection of other mycobacteria （including Mycobacterium leprae and Staphylococcus spp）. Among the 72 patients in the experimental group, 44 were positive for qPCR with a sensitivity of 61.1% （95% CI: 49.6% － 71.5%）, and 47 were positive for culture with a sensitivity of 65.2% （95% CI: 53.8% － 75.3%）; all the 68 controls were negative for both qPCR and culture, with their specificities both being 100%. Among 65 patients subjected to IGRA, 31 were positive with a sensitivity of 47.7% （95% CI: 36.0% － 59.6%）, while 16 out of 25 controls were negative for IGRA with a specificity of 64.0% （95% CI: 44.5% － 79.8%）. Among 58 patients subjected to acid-fast staining, 37 were positive with a sensitivity of 63.8% （95% CI: 50.9% － 74.9%）, and 52 out of 66 controls were negative for acid-fast staining with a specificity of 78.8% （95% CI: 67.5% － 86.9%）. The combination of qPCR and culture resulted in a sensitivity of 93% and a specificity of 100% for the detection of Mycobacterium marinum. Conclusion In this study, a highly sensitive qPCR assay was developed for the detection of Mycobacterium marinum, and its combination with culture could further improve the detection sensitivity.

Key words: Mycobacterium, Mycobacterium marinum, Mycobacterium infections, Skin, Real-time fluorescence-based quantitative PCR, Culture, Acid-fast staining, Interferon-γ release assay