基于探针熔解曲线分析的结核分枝杆菌不均一耐药的高灵敏检测

Early detection and therapy of drug resistant tuberculosis is the key point for the preventing and controlling of the tuberculosis. The new molecular methods has many advantages over the drug susceptibility testing (DST), such as efficiency and Simplicity, However, these methods still can’t detect 1% drug heteroresistance in Mycobacterium tuberculosis (MTB). Using probe inhabitation strategy, we had setup a highly sensitive method, which can now detect isoniazid heteroresistance in MTB samples with 1% proportion of resistance by simplex PCR. Compare to other molecular methods, our new method is not only more sensitive for heteroresistance, but also more simple and convenient. In this project, we are going to extend this method to a mid-throughput multiplex detection system with high sensitivity for heteroresistance. By only one reaction, we are able to detect rifampin or isoniazid resistance. After completion of the project, we will finally setup a mutation enrichment and detection method under our own intellectual property. Its application in MTB drug resistance detection will greatly benefit the preventing and controlling of the tuberculosis.

耐药结核病的早诊断、早治疗是当前结核病防控的关键所在。新兴的分子耐药检测方法在很大程度上克服了传统药敏试验周期长、操作繁琐的问题，但对不均一耐药样本的检测能力仍无法达到临床要求的1%的水平。此前，申请人建立了一种新的高灵敏的不均一耐药检测方法，采用探针压制野生型模板扩增进而富集突变型模板的策略，结合探针熔解曲线分析技术，在单重PCR体系中实现了异烟肼耐药常见突变位点的高灵敏不均一耐药检测，检出能力达到了1%。与现有的基因检测方法相比，新方法不但对不均一耐药的检测能力大为提高，也更加的简便快速。在本项目中，我们拟将该技术进一步发展为一个具有高灵敏不均一耐药检测能力的多重中通量检测体系，分别在一个反应管中实现对结核分枝杆菌利福平和异烟肼耐药的基因突变检测。该项目的完成，将最终建立一种具有自主知识产权的突变富集检测技术。其在结核分枝杆菌耐药突变检测中的应用将大大改善现有结核耐药检测技术的不足。

耐药结核病的早诊断、早治疗是当前结核病防控的关键所在。新兴的分子耐药检测方法在很大程度上克服了传统药敏试验周期长、操作繁琐的问题，但对不均一耐药样本的检测能力仍无法达到临床要求的1%的水平。在本研究中，申请人建立了一种新的高灵敏的不均一耐药检测方法，采用探针压制野生型模板扩增进而富集突变型模板的策略，结合探针熔解曲线分析技术，在多重PCR体系中实现了对耐多药结核常见突变位点的高灵敏不均一耐药检测，检出能力达到了1%。分析性能评估和临床验证结果显示该方法具有良好的临床应用前景。目前，相应的成果已启动产学研转化，正在进行我国三类医疗器械证书的申报工作。与现有的基因检测方法相比，新方法不但对不均一耐药的检测能力大为提高，也更加的简便快速。该项目的完成，最终建立一种具有自主知识产权的突变富集检测技术。其在结核分枝杆菌耐药突变检测中的应用将大大改善现有结核耐药检测技术的不足。本项目在研究期间共申请国家发明专利1项，发表SCI论文2篇。