摘要
目的:应用滚环扩增(rolling circle amplification,RCA)技术以DNA芯片为载体建立一种对结核分枝杆菌耐药基因单碱基突变的快速检测方法。方法:根据结核分枝杆菌利福平耐药rpoB基因序列,设计针对该基因常见突变位点的锁式探针,及固定于基因芯片上的捕获探针。针对临床结核分枝杆菌样本的基因组DNA,PCR扩增含有rpoB基因常见突变位点的特异性DNA片段。将与突变型特异性互补结合并环化的锁式探针与芯片上固定的捕获探针进行杂交,并运用滚环扩增技术,将含有生物素标记的dUTP掺入扩增产物,最后通过与亲和素标记的纳米金反应,并银染增强显色。同时与临床样本的测序结果比较。结果:通过优化反应条件,能特异性的检测出结核分枝杆菌耐利福平rpoB基因的单碱基突变,通过对临床样本的检测,结果与测序结果一致。结论:该方法结合了DNA芯片和滚环扩增技术,能够快速有效的检测出耐药结核的单碱基突变,具有高特异性、高灵敏度。
Objective：To establish a method for rapid detection of single base mutations in resistant Mycobacterium tuberculosis by use of rolling circle amplification combined with the DNA chip technology. Methods According to rpoB gene sequence of Mycobacterium tuberculosis rifampicin resistant, padlock probes of the gene mutation types and the capture probe fixed on the gene chip were designed. Amplification of PCR containing rpoB gene mutation types of specific DNA fragments. Padlock probes via connection and cycclization, hybridization with the capture probe fixed on the gene chip. Using rolling circle ampfificatlon doped biotin labeled dUTP into the amplification product, finally through with streptavidin labeled gold nanoparticles reaction, and silver enhancement staining, compared with sequencing. Reults By optimizing the reaction conditions, specific detection of Mycobacterium tuberculosis is rifampicin resistant rpoB gene single nucleotide mutation, the detection of clinical samples, the results are consistent with the sequencing results. Conclusion The method combines DNA chip and single - base mutations rolling circle amplification technology, to be able to quickly and efficiently detect dru^- resistant TB. with high snecificitv, him sensitivitv.
出处
《激光杂志》
CAS
CSCD
北大核心
2013年第2期91-93,共3页
Laser Journal
基金
国家自然科学基金资助项目(编号:81171667)
关键词
滚环扩增
DNA芯片
结核耐药
RPOB基因
rolling circle amplification
DNA chip
Mycobacterium tuberculosis resistance
rpoB gene
