皮肤活检组织中分枝杆菌荧光实时PCR鉴定方法的建立  

Establishment of a real-time fluorescence-based PCR method for identification of mycobacteria in skin biopsies

作　　者：刘晓[1] 薛若宁 李若瑜[1] 宋营改[1] Liu Xiao;Xue Ruoning;Li Ruoyu;Song Yinggai(Department of Dermatology and Venereology,Peking University First Hospital,Research Center for Medical Mycology,Peking University,National Clinical Research Center for Skin and Immune Diseases,Beijing Key Laboratory of Molecular Diagnosis of Dermatoses,Beijing 100034,China)

机构地区：[1]北京大学第一医院皮肤性病科,北京大学真菌和真菌病研究中心,国家皮肤与免疫疾病临床医学研究中心,皮肤病分子诊断北京市重点实验室,北京100034

出　　处：《中华皮肤科杂志》2024年第11期1016-1021,共6页Chinese Journal of Dermatology

基　　金：中央高水平医院临床科研业务费资助(北京大学第一医院科研“希望之星”专项)(22cz020301-4803013);军队课题(20SWAQK12)。

摘　　要：目的建立快速鉴定皮肤活检组织中分枝杆菌的荧光实时PCR方法并进行验证。方法自行设计针对分枝杆菌属和14种常见分枝杆菌的引物探针,建立分枝杆菌的荧光实时PCR检测体系和方法。利用12株分枝杆菌标准菌株对该方法进行体外验证,并对2019年至2021年北京大学第一医院皮肤性病科收集的119例临床诊断皮肤分枝杆菌感染患者的组织样本进行检测。结果体外检测显示该方法对12种分枝杆菌标准株均有良好的检测敏感性和特异性。在119例临床诊断分枝杆菌皮肤感染患者活检标本中,培养阳性率仅为21.0%(25/119);荧光实时PCR检测分枝杆菌阳性率为76.5%(91/119),其中,44.5%(53/119)鉴定至菌种水平,以海分枝杆菌为主;31.9%(38/119)仅鉴定至分枝杆菌属水平。结论建立了可在患者皮肤组织样本中高效检测分枝杆菌的荧光实时PCR方法,但仍有漏诊可能,还需结合其他分子诊断方法提高敏感性和特异性。Objective To establish a real-time fluorescence-based PCR method for the rapid identification of mycobacteria in skin biopsies.Methods Primers and probes targeting the genus Mycobacterium and 14 common Mycobacterium species were self-designed,and a real-time fluorescence-based PCR detection system/method was established.Twelve standard strains of Mycobacterium were used for in vitro validation.Tissue samples were collected from 119 patients clinically diagnosed with cutaneous mycobacterial infections at the Department of Dermatology and Venereology,Peking University First Hospital from 2019 to 2021,and were tested with the established real-time fluorescence-based PCR method.Results In vitro testing demonstrated that the method had good sensitivity and specificity for all the 12 standard strains of Mycobacterium.Among the 119 skin biopsy samples from patients clinically diagnosed with cutaneous mycobacterial infections,the culture positivity rate was only 21.0%(25/119);the real-time fluorescence-based PCR yielded a positivity rate of 76.5%(91/119)for the detection of mycobacteria,and 44.5%(53/119)were identified to the species level with Mycobacterium marinum as the predominant species;31.9%(38/119)were identified only to the genus level.Conclusion A real-time fluorescence-based PCR method has been established for the efficient detection of mycobacteria in skin tissue samples;however,there remains a possibility of missed diagnoses,other molecular diagnostic techniques are required to be combined to improve the sensitivity and specificity.

关 键 词：分枝杆菌感染 皮肤活组织检查 荧光实时PCR 分枝杆菌属 培养技术 

分 类 号：R446.5[医药卫生—诊断学] R529.4[医药卫生—临床医学]