中华皮肤科杂志 ›› 2024, Vol. 57 ›› Issue (11): 1022-1028.doi: 10.35541/cjd.20230581

• 论著 • 上一篇    下一篇

皮肤活检标本中海分枝杆菌定量PCR检测方法的建立和价值评估

袁召君1，2    孙乐乐2    孙远航2    张雍2    曹园园2    桑旭2    李紫阁2    王猛2    程艳茹2    李艳艳2    潘晴2    暴芳芳2    刘红2    张福仁2    

  1. 1滨州医学院，烟台  264003；2山东第一医科大学附属皮肤病医院（山东省皮肤病医院） 山东省皮肤病性病防治研究所，济南  250022
  • 收稿日期:2023-10-10 修回日期:2023-12-29 发布日期:2024-10-31
  • 通讯作者: 刘红;张福仁 E-mail:hongyue2519@hotmail.com; zhangfuren@hotmail.com
  • 基金资助:
    国家自然科学基金（82003369）；山东第一医科大学学术推广计划（2019LJ002）

Establishment and evaluation of a quantitative PCR-based assay for the detection of Mycobacterium marinum in skin biopsy specimens

Yuan Zhaojun1,2, Sun Lele2, Sun Yuanhang2, Zhang Yong2, Cao Yuanyuan2, Sang Xu2, Li Zige2, Wang Meng2, Cheng Yanru2, Li Yanyan2, Pan Qing2, Bao Fangfang2, Liu Hong2, Zhang Furen2   

  1. 1Binzhou Medical University, Yantai 264003, Shandong, China; 2Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250022, China
  • Received:2023-10-10 Revised:2023-12-29 Published:2024-10-31
  • Contact: Liu Hong; Zhang Furen E-mail:hongyue2519@hotmail.com; zhangfuren@hotmail.com
  • Supported by:
    National Natural Science Foundation of China （82003369）; Academic Promotion Programme of Shandong First Medical University （2019LJ002）

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摘要： 【摘要】 目的 建立海分枝杆菌皮肤感染定量PCR（qPCR）快速检测方法并分析其临床检测效能。方法 提取海分枝杆菌菌落DNA进行梯度稀释（10-1至10-8），采用既往文献报道的12对海分枝杆菌检测DNA引物探针及本研究设计的6对引物和探针分别进行qPCR扩增，筛选出最灵敏的引物和探针。收集山东第一医科大学附属皮肤病医院2021年确诊的72例海分枝杆菌感染患者（实验组）和68例其他分枝杆菌感染患者（对照组）皮损组织，取实验组和对照组皮损组织分别进行qPCR扩增、γ干扰素释放试验 （IGRA）、抗酸染色和组织培养，评估检测效能。结果 本研究设计的海分枝杆菌增强感染位点2（Mel2）引物和探针的灵敏度最高，其检测限为0.86拷贝/μl（CT值 = 37）；Mel2引物和探针在其他分枝杆菌包括麻风分枝杆菌、葡萄球菌等的检测中qPCR扩增均阴性。实验组72例患者中，qPCR阳性44例（敏感性61.1%，95% CI：49.6% ~ 71.5%），培养阳性47例（敏感性65.2%，95% CI：53.8% ~ 75.3%），对照组68例qPCR、培养均为阴性，特异性均为100%；在65例患者中，31例IGRA阳性（敏感性47.7%，95% CI：36.0% ~ 59.6%），25例对照中16例IGRA阴性（特异性64.0%，95% CI：44.5% ~ 79.8%）；58例患者中，37例抗酸染色阳性（敏感性63.8%，95% CI：50.9% ~ 74.9%），66例对照组中，52例阴性（特异性78.8%，95% CI：67.5% ~ 86.9%）；qPCR与海分枝杆菌培养联合检测的敏感性为93%，对照组均为阴性，特异性100%。结论 本研究建立了一种高敏感性的海分枝杆菌qPCR检测方法，其联合培养方法可进一步提高海分枝杆菌检测的敏感性。

关键词: 分枝杆菌属, 海分枝杆菌, 分枝杆菌感染, 皮肤, 实时荧光定量PCR, 培养, 快速抗酸染色, γ干扰素释放试验

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

引用本文

袁召君, 孙乐乐 孙远航 张雍 曹园园 桑旭 李紫阁 王猛 程艳茹 李艳艳 潘晴 暴芳芳 刘红 张福仁 . 皮肤活检标本中海分枝杆菌定量PCR检测方法的建立和价值评估[J]. 中华皮肤科杂志, 2024,57(11):1022-1028. doi:10.35541/cjd.20230581

Yuan Zhaojun, Sun Lele, Sun Yuanhang, Zhang Yong, Cao Yuanyuan, Sang Xu, Li Zige, Wang Meng, Cheng Yanru, Li Yanyan, Pan Qing, Bao Fangfang, Liu Hong, Zhang Furen. Establishment and evaluation of a quantitative PCR-based assay for the detection of Mycobacterium marinum in skin biopsy specimens[J]. Chinese Journal of Dermatology, 2024, 57(11): 1022-1028.doi:10.35541/cjd.20230581