PCR-膜芯片检测石蜡包埋组织中结核杆菌耐药基因突变

目的探讨PCR-膜芯片技术检测石蜡包埋组织中结核杆菌耐药基因突变的可行性及其在临床病理中的应用价值。方法采集100例石蜡包埋的肺或淋巴结组织标本(临床诊断为结核患者80例,非结核患者20例),TB膜芯片检测标本中的结核杆菌IS6110基因,阳性者利用12对特异性引物进行多重PCR,扩增产物与含有59个探针的耐药-TB膜芯片反向点杂交检测结核杆菌耐药基因突变并测序验证。结果 TB膜芯片检测石蜡包埋组织标本中的结核杆菌IS6110基因,以临床诊断为标准时,灵敏度为52.5%(42/80),特异度为100%(20/20);以抗酸染色结果为标准时,灵敏度为90.5%(19/21),特异度为61.0%(36/59)。耐药-TB膜芯片检测石蜡包埋组织结核杆菌耐药基因突变,在42例结核杆菌IS6110基因阳性的标本中,检出INH基因突变2例,RFP或SM基因突变各1例,INH、RFP和EMB基因同时突变1例,与测序结果基本符合,提示上述5例可能为耐药结核病。结论应用PCR-膜芯片技术可提高石蜡包埋组织中结核杆菌基因的检出率,耐药-TB膜芯片检测结果可为耐药结核病的病理学诊断提供有价值的参考。

PCR-membrane chip for detection of drug-resistant gene mutations of mycobacterium tuberculosis in paraffin-embedded tissues

Purpose To study the feasibility and application of detecting the drug-resistant gene mutation of mycobacterium tuberculosis(MTB) in paraffin-embedded tissues by PCR-membrane chip.Methods A total of 100 paraffin-embedded lung or lymph node tissue specimens were collected(80 cases of TB,20 cases of non-TB).TB membrane chip was applied to detect the MTB IS6110 gene in the specimens.The positves were detected by multiplex PCR with 12 pairs of specific primers and MTB drug-resistant genes mutation were detected by drug resistant-TB reverse dot blot membrane chip which contains 59 probes hybrided with PCR products,and the result was sequenced.Results MTB-IS6110 gene in paraffin-embedded tissue specimens were detected by TB membrane chip: the sensitivity was 52.5%(42/80) and the specificity was 100%(20/20) when clinical diagnosis as the standard,the sensitivity was 90.5%(19/21) and the specificity was 61.0%(36/59) when acid-fast staining results as the standard.MTB drug-resistant gene mutation in paraffin-embedded tissue specimens was detected by drug resistant-TB membrane chip.In 42 cases of IS6110-positive,there were 2 cases of INH,1 case of RFP,1 case of SM gene mutation and there was 1 case of simultaneousINH,RFP and EMB gene mutation.The mutation sites were consistent with the DNA sequencing.Conclusion PCR-membrane chip technology can improve the detection rate in paraffin-embedded tissue of MTB genes,and the results of drug resistant-TB membrane chip can provide valuable reference for the pathological diagnosis of drug-resistant tuberculosis.