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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 establish a method for the identification of mycobacteria by 16SrDNA combined with chromogenic assay (glass chip to nylon membrane colorimetry). Methods Two pairs of mycobacterium specific primers were designed according to the sequence of 16SrDNA conserved region in GenBank. Two different 16S rDNA fragments were amplified simultaneously by double PCR. Based on the 16S rDNA sequences of 16 mycobacteria, Corresponding specific oligonucleotide probes. All standard DNA samples were amplified by universal primer PCR. PCR products were hybridized with 16 kinds of Mycobacterium probe glass chips and analyzed by nylon membrane colorimetry. Results Sixteen mycobacteria and five non-mycobacteria strains were analyzed by colorimetric microarray technique to optimize their duplex PCR, hybridization and washing conditions. The optimal annealing temperature of dual PCR was determined to be 52 ° C. Two DNA fragments except for 5 non-mycobacteria were produced at this temperature, ranging in size from 272 bp to 280 bp and from 183 bp to 192 bp, respectively. The hybridization temperature was 32 ° C and 2 x SSC 5min, 0.2 × SSC washing 1min, 16 kinds of mycobacterium hybridization best, specificity up to 100%. Conclusion 16SrDNA can be detected by color chip method, which can accurately identify 16 mycobacteria.