利用寡核苷酸芯片快速检测结核分枝杆菌利福平耐药rpoB基因突变

目的:开发快速检测结核分枝杆菌利福平耐药rpoB寡核苷酸突变的基因芯片。方法:根据结核杆菌rpoB基因序列设计探针和引物并制备基因芯片,从临床痰标本中提取结核菌的基因组DNA,PCR扩增含有rpoB基因突变位点的特异DNA片段,荧光标记后与芯片上的特异性寡核苷酸探针进行杂交,同时与DNA测序法比较。结果:22株临床痰标本有18株可用寡核苷酸芯片检测出来,正确率可达82%;患者痰液中少量DNA足够进行芯片检测,无须细菌培养。结论:利用寡核苷酸芯片检测结核菌对利福平的耐药性具有较高的准确性和敏感性,可以应用于临床耐药性诊断。

Rapid detection of mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis by oligonucleotide microarray

Objective:To develop a new oligonucleotide microarray for rapid detection of rpoB mutation rifampin-resistant Mycobacterium tuberculosis isolated from clinical laboratorial specimen. Methods: According to the sequence of Mycobacterium tuberculosis rpoB gene the oligonucleotide primers and probes were designed. Then oligonucleotide microarray was fabricated. The DNA fragment which contaied hot mutation sites of rpoB gene was amplified by PCR and labeled with cy3 fluorescence. Then it was hybridized with oligonucleotide microarray. DNA of wild type was used as the control. Results: 18 among 22 clinical spuat specimens were examined by oligonucleotide microarray, the accuracy rate was about 82%. Furthermore, a little DNA of M. tuberculosis without being incubated in the sick sputa was sufficient for oligonucleotide microarray detection. Conclusion: Oligonucleotide microarray has high accuracy and sensitivity in detection of rifampin-resistant Mycobacterium tuberculosis and may be applied in clinical diagnosis.