皮肤感染性肉芽肿常见病原菌微孔板反向杂交检测法的研究  

Development of a microplate-reverse hybridization method for the detection and identification of common pathogens in lesions of cutaneous infectious granuloma

作　　者：闫桢桢[1] 姜海琴[1] 孙建方[1] 沈永年[1] 崔盘根[1] 刘伟军[1] 王洪生[1] 刘维达[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所,南京210042

出　　处：《中华皮肤科杂志》2014年第9期633-636,共4页Chinese Journal of Dermatology

基　　金：卫生部部属（管）医院临床学科重点项目（2010-2012-125）

摘　　要：目的 探讨微孔板反向分子杂交技术与PCR-ELISA相结合的＂拟芯片＂法检测和鉴定皮肤感染性肉芽肿常见病原菌的方法.方法 选取、设计真菌和分枝杆菌两对通用引物,及10种皮肤感染性肉芽肿常见的病原菌：结核分枝杆菌、麻风分枝杆菌、海鱼分枝杆菌、偶遇分枝杆菌、脓肿分枝杆菌、申克孢子丝菌、卡氏枝孢霉、裴氏着色真菌、F.Monophora、白念珠菌的特异性探针;采用标准株验证通用引物、特异性探针并检测＂拟芯片＂法的敏感性、特异性;对19株临床分离株进行检测和鉴定.结果 两对通用引物均可扩增出对应的菌种基因序列,PCR产物经测序分析与预期相符;＂拟芯片＂法检测阴性标本吸光度为0.041±0.02,公式计算得临界值为0.101,在此基础上测得其敏感性为1×10～1×102个菌细胞/ml.各病原菌与相应的探针杂交,吸光度均＞ 0.101,为阳性;而与非对应的探针杂交,则吸光度＜0.101,为阴性,特异性高;对临床分离株的菌种鉴定准确率高.结论 ＂拟芯片＂法可应用于皮肤感染性肉芽肿常见病原菌的实验室快速检测和鉴定.Objective To develop a microplate-reverse hybridization method combined with PCR-enzymelinked immunosorbent assay （＆quot;simulant chip＆quot;） for the detection and identification of common species of mycobacteria and fungi in lesions of cutaneous infectious granuloma.Methods Two universal primers for mycobacteria and fungi,as well as specific probes for 10 common pathogens of cutaneous infectious granuloma （including Mycobacterium tuberculosis,Mycobacterium leprae,Mycobacterium marinum,Mycobacterium abscessus,Mycobacterium fortuitum,Sporotrichum schencki,Cladosporium carrionii,Fonseceea pedrosoi,Fonseceea monophora and Candida albicans）,were designed.To test the performance of the universe primers,PCR was performed with them to amplify corresponding fragments from the standard strains of the 10 pathogens followed by sequencing.A ＆quot;simulant chip＆quot; method was developed with the specific probes,and used to detect 10 standard and 19 clinical strains of these pathogens in simulated tissue specimens.The sensitivity and specificity of this method were determined.Results The corresponding fragments were amplified from all the standard strains by PCR with the two pairs of universal primers,and these PCR products were confirmed to match with the expected genes.As the ＆quot;simulant chip＆quot; method showed,the absorbance value was 0.041 ± 0.02 for negative specimens,and the cut-off point was determined as 0.101 for this method.At this cut-off point,the detection limit of the ＆quot;simulant chip＆quot; method was 1 ×101 to 1 × 102 cells/ml for these pathogens.No cross reaction was observed for these specific probes between these 10 pathogens.The genotyping results for these clinical isolates were consistent between the ＆quot;simulant chip＆quot; method and DNA sequencing.Conclusion The established ＆quot;simulant chip＆quot; method is a rapid method for the detection and identification of common pathogens from cutaneous infectious granuloma.

关 键 词：肉芽肿 分枝杆菌感染 真菌感染 寡核苷酸反向杂交技术 

分 类 号：R446.5[医药卫生—诊断学]