16SrDNA结合显色法芯片技术鉴定分枝杆菌的方法学研究  

Methodology research on identification of Mycobacteria through 16SrDNA combining gene chips

作　　者：赵源[1,2] 景奉香[3] 刘志辉[4] 王洁[1] 郑瑞娟[1] 陆俊梅[1] 赵辉[3] 尹俊[2] 胡忠义[1] 

机构地区：[1]上海市肺科医院上海市结核重点实验室,上海200433 [2]内蒙古农业大学生物工程学院 [3]中国科学院微系统与信息技术研究所 [4]广州市胸科医院

出　　处：《中国病原生物学杂志》2008年第6期404-407,F0004,共5页Journal of Pathogen Biology

基　　金：上海市科委重点专项基金资助项目(No.06DZ22320)

摘　　要：目的建立16SrDNA结合显色法芯片技术(玻璃芯片转尼龙膜显色法)鉴定分枝杆菌的方法。方法利用GenBank中细菌16SrDNA保守区序列,设计两对分枝杆菌属特异性通用引物,采用双重PCR法同时扩增16SrDNA的2个不同片段;根据已知16种分枝杆菌的16SrDNA序列,设计合成对应的特异性寡核苷酸探针。先用通用引物PCR扩增所有标准菌株DNA,PCR产物分别与16种分枝杆菌探针的玻璃芯片杂交,通过尼龙膜显色进行分析。结果应用显色法芯片技术分析16种分枝杆菌标准菌株和5种非分枝杆菌菌株,对其双重PCR、杂交、洗涤条件进行优化。确定双重PCR最佳退火温度为52℃,在该温度下除5株非分枝杆菌外均产生2条DNA片段,大小分别为272～280bp和183～192bp;杂交温度32℃、2×SSC洗涤5min、0.2×SSC洗涤1min,16种分枝杆菌的杂交效果最好,特异性达100%。结论用显色芯片法检测16SrDNA,可准确鉴别16种分枝杆菌。Objective To develop 16SrDNA combining gene chips（glass chips turn to nylon membrane coloration methodology） for identifying Mycobacteria. Methods According to conservative gene sequence in 16SrDNA, two pairs of consensus primers for Mycobacteria genus specificity and simultaneously amplifying two different gene sections in 16SrDNA through double PCR technique were designed, and correspondent distinctive oligonucleotide probes were designed. Employ consensus primers to PCR amplification DNA of all Mycobacteria reference strains, PCR products hybridismed with 16 species of Mycobacteria probes in glass chips and make analysis through nylon membrane coloration methodology. Results Analyze 16 species of Mycobacteria reference strains and 5 species of non-Mycobacteria reference strains through gene chips and improve double PCR, hybridisation and washing conditions. The best double PCR renaturation temperature is 52 ℃, under which temperature except the 5 species of non-Mycobacteria others produce two DNA sections-one is 272-280 bp and the other is 183-192 bp. With 32 ℃ hybridisation temperature, 5 min 2×SSC washing and 1 min 0.2 SSC washing as conditions the hybridisation effect is best and 100% specificity is achieved. Conclusion 16 SrDNA combining gene chips enables correct Mycobacteria identification, which technique with high specificity is expected to be an effective clinical identification means.

关 键 词：16SrDNA 显色芯片法 分枝杆菌 

分 类 号：R378.911[医药卫生—病原生物学]