可视化微孔板芯片检测结核分枝杆菌利福平及异烟肼耐药性研究

Research on the drug resistance to rifampicin and isoniazid in the detection of Mycobacterium tuberculosis by visible microplate chip

目的构建基于可视化微孔板芯片技术的结核分枝杆菌利福平和异烟肼耐药性检测方法。方法将针对结核分枝杆菌利福平和异烟肼耐药性相关基因设计的探针按预设模式固定于芯片表面构建基因芯片,并用其检测结核分枝杆菌的基因突变以判断其耐药性,阳性结果在芯片表面形成裸眼可见的色点。结果可视化微孔板芯片展示了稳定的试验结果,在50株异烟肼耐药株中检出40株(80%),主要为katG基因315位突变;在20株利福平耐药株中检出15株(75%),主要为rpoB基因531及526位突变。芯片鉴定阳性样本经聚合酶链反应(PCR)测序比对,其结果与芯片结果完全相符。结论可视化微孔板芯片用于检测结核分枝杆菌利福平和异烟肼耐药性具有较高的特异性和敏感性,该方法可以快速、准确、方便地应用于结核分枝杆菌耐药性的检测。

Objective To establish a method based on visible microplate chip for the detection of rifampicin and isoniazid resistance to Mycobacterium tuberculosis. Methods Gene chip was constructed by immobilizing default mode probes against genes associated with rifampicin and isoniazid resistance to Mycobacterium tuberculosis on the surface of gene chip. Drug resistance was identified by the prepared gene chip to detect the relative gene mutation, and the positive results, a color dot on the surface of gene chip, can be observed by naked eye. Results The prepared visible microplate chip showed stable detection results. The 40 of 50 （ 80% ） strains were resistant to isoniazid with a high mutation site of 315 codon within katG gene, and 15 of 20 （75%） strains were resistant to rifampicin with a high mutation site of 531 and 526 codons within rpoB gene. The results from the gene chip detection were consistent with the polymerase chain reaction（PCR） sequencing analysis. Conclusions The prepared visible microplate chip with good specificity and sensitivity is a fast, accurate and convenient mothod, and is promising for the detection of rifampicin and isoniazid resistance to Mycobacterium tuberculosis.