变性高效液相色谱法检测结核分枝杆菌利福平耐药基因突变的初步探索

Application of denaturing high perfomance liquid chromatography in detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis

目的探讨变性高效液相色谱（DHPLC）技术在MTB的利福平耐药菌株rpoB基因突变检测中的应用价值。方法PCR扩增MTB的rpoB基因利福平耐药决定区，扩增产物与野生型DNA链形成异源双链，在65．4℃柱温下进行DHPLC分析。对DHPLC不同峰型菌株的rpoB基因片段进行测序，评价DHPLC技术的敏感度和特异度。结果共分析46株MTB菌株，其中42株对利福平耐药，4株对利福平敏感。经DHPLC分析，产生15种不同图谱。测序结果表明，各图谱菌株突变特征不同。除D108和D24菌株的DHPLC峰形相同而突变特征不同外，其他菌株均为DHPLC峰型与基因多态性相一致，即DHPLC峰型相同则基因多态性相同，DHPLC峰型不同则基因多态性不同。结论DHPLC具有简便快捷、高通量和自动化的特点，敏感度和特异度高，可用于快速检测耐药MTB的基因突变。

Objective To explore the value of denaturing high performance liquid chromatography （DHPLC） in detection of rpoB mutations in rifampin-resistant M. tuberculosis, in order to establish a convenient and rapid approach to screening rpoB gene mutations in M. tuberculosis. Methods Rifampin resistance-determining region of rpoB gene in M. tuberculosis was amplified by PER and further analyzed by DHPLC to screen mutations at optimized denaturation temperature （65.4 ℃ ）, which utilized heteroduplex formation between wild-type and mutated DNA strands to identify mutations. The PCR products from strains with different chromatographic profiles were sequenced further to evaluate the sensitivity and specificity. Results There were 46 M. tuberculosis strains including 42 rifampin resistance strains and 4 rifampin sensitive strains. From these strains, 15 different chromatographic profiles were produced by DHPLC. Combined with the results of gene sequencing, it was shown that strains with different chromatographic profiles had distinct mutations. Except D108 and D24 which had same chromatographic profiles but different genetic polymorphisms, all other strains showed consistent ehromatographic profiles with genetic polymovphisms, i.e. if they had identical chromatographic profiles, their genetic polymorphism were the same; but if they had different chromatographic profiles, their genetic polymovphism were also different. Conclusion DHPLC is a simple, efficient, high-throughput and automatic method with high sensitivity and specificity, which may be useful in the rapid detection of rpoB gene mutation in M. tuberculosis.