基因芯片法对苏州市结核临床分离株RFP耐药性快速检测价值的研究

目的 通过基因芯片法检测结核分离株RFP的耐药性,为在临床诊断中利用该法快速鉴别苏州市结核RFP耐药菌株的可靠性奠定基础.方法 收集痰液标本45例,利用罗氏固体培养法（金标准）对结核分枝杆菌进行传统药敏试验;利用晶芯结核分枝杆菌耐药检测试剂盒及相关仪器通过核酸提取、PCR扩增、芯片杂交和芯片扫描,对结核分枝杆菌RFP耐药相关基因rpoB的rpoB-RRDR 511,513,516,526,531和533位点进行检测,将基因芯片检测获得的RFP药敏判读结果与金标准法获得的结果进行比较分析;将17例经基因芯片检测过的菌株用PCR法扩增包含rpoB-RRDR的基因片段,经纯化后进行DNA直接测序,将基因芯片对rpoB-RRDR 526,531位点的检测结果与DNA测序获得的结果进行比较分析.结果 与金标准法相比,基因芯片法对菌株RFP药敏检测的准确率为93.3%（42/45）;与DNA测序法相比,基因芯片法对rpoB-RRDR 526,531位点突变型检测的准确率为82.4%（14/17）.结论 rpoB-RRDR 526,531位点突变是导致苏州市结核病RFP耐药的主要因素,通过基因芯片法对rpoB基因相关位点的检测可为临床对RFP耐药结核鉴定提供一种快速、准确和批量性的检测手段,具有极大的应用价值.

Study of the applicability of rifampicin-resistance detecting rapidly with DNA microassay chip for Mycobacterium tuberculosis isolated from clinical tuberculosis patients in Suzhou City of China Authors.

Objective To evaluate the detection applicability of DNA microassay chip for rifampicin （RFP） -resistant Mycobacte- rium tuberculosis （ MTB ） from clinical tuberculosis （TB） patients in Snzhou City. Methods The sputum samples were gathered from TB patients in the Affiliated Hospital of Infectious Diseases of Soochow University. The conventional Lwenstein-Jensen culture medium was a- dopted to test drug sensitivity. Based on examining the RFP-resistance associated gene mutant point rpoB-RRDR 511,513,516,526,531 and 533, DNA microassay chip was adopted to detect RFP resistance. Thirdly, the rpoB-RRDR gene fragment from 17 samples tested by DNA chip was sequencing after amplification through PCR and purification. The results for RFP-sensitivity and point mutation in rpoB- RRDR fragment examined by DNA chip were respectively compared with traditional drug susceptibility test and DNA sequencing. Results Compared with the traditional drug susceptibility testing and DNA sequencing, the rate of accuracy for DNA chip evaluating RFP resist- ance and identifying point mutation in rpoB-RRDR fragment was respectively 93.3% （42/45） and 82.4% （ 14/17 ）. Conclusion In Suzhou city, rpoB-RRDR 526,531 mutation mainly contributes to RFP-resistance for Mycobacterium tuberculosis and can be rapidly detec- ted by using DNA microassay chip.