CAJ | 학술논문

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.
목적 탐토미공판반향분자잡교기술여PCR-ELISA상결합적“의심편”법검측화감정피부감염성육아종상견병원균적방법.방법 선취、설계진균화분지간균량대통용인물,급10충피부감염성육아종상견적병원균:결핵분지간균、마풍분지간균、해어분지간균、우우분지간균、농종분지간균、신극포자사균、잡씨지포매、배씨착색진균、F.Monophora、백념주균적특이성탐침;채용표준주험증통용인물、특이성탐침병검측“의심편”법적민감성、특이성;대19주림상분리주진행검측화감정.결과 량대통용인물균가확증출대응적균충기인서렬,PCR산물경측서분석여예기상부;“의심편”법검측음성표본흡광도위0.041±0.02,공식계산득림계치위0.101,재차기출상측득기민감성위1×10～1×102개균세포/ml.각병원균여상응적탐침잡교,흡광도균＞ 0.101,위양성;이여비대응적탐침잡교,칙흡광도＜0.101,위음성,특이성고;대림상분리주적균충감정준학솔고.결론 “의심편”법가응용우피부감염성육아종상견병원균적실험실쾌속검측화감정.
Objective To develop a microplate-reverse hybridization method combined with PCR-enzymelinked immunosorbent assay ("simulant chip") 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 "simulant chip" 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 "simulant chip" 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 "simulant chip" 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 "simulant chip" method and DNA sequencing.Conclusion The established "simulant chip" method is a rapid method for the detection and identification of common pathogens from cutaneous infectious granuloma.