基于MODS和实时荧光PCR微流控芯片同步检测结核表型耐药和基因型耐药的技术研究

The global drug resistance in tuberculosis is getting worse, standard chemotherapy was inadequate for fighting against TB. So the key points in TB control were early diagnosis and individualized chemotherapy.The conventionlal drug susceptibility test based on M.TB culture had lower positive rate and was time consuming.While kinds of techniques detecting gene mutations correlating with drug resistance were fast,accurate and safe. We designed a new Gene Chip for TB drug resistance detecion and found the sensitivity and specificity weren’t 100%. So that we plan to develep a microfluidics based on MODS and Real-time PCR, which could detect phenotypic and genotypic drug resistance of TB on the same platform: Capture of M.TB quickly using iron-based magnetic nanoparticles tagged with antibodies that will bind to the bacteria; Detection of phenotypic resistance of 11 drugs using MODS ,TEM and Q PCR; Detection of genotypic drug resistance using multiple chanel Real time PCR; This would provide an accurate and relible techenique for drug resistance detection in TB as a guide for individualized chemotherapy.

因全球耐药结核疫情恶化，标准化疗方案的可靠性下降，控制结核的关键问题是早期诊断和个体化药物治疗。传统药敏试验阳性率低、耗时长达2-3月；多种结核耐药基因检测技术快速、准确、安全；前期研究发现Gene Chip检测耐药基因与耐药表型并非完全符合，特别是二线药物。本项目建立一种微流控芯片技术，磁性纳米Fe3O4粒子-结核特异性抗体绑定结核杆菌，磁场快速分离，MODS、抗酸染色、TEM和Q PCR测定11种抗结核药物药敏试验；快速扩增耐药基因，多通道实时荧光PCR-Taqman MGB探针技术同步快速检测耐药基因，有望建立一种准确、可靠的结核耐药检测技术，指导结核个体化化疗，为控制结核疫情作出贡献。

研发结核耐药检测微流控芯片，并与表型药敏结果对照，评价微流控芯片检测耐药结核的可行性和准确性。针对脊柱结核临床样本，采用微流控芯片行分枝杆菌菌种鉴定及耐药基因检测，最终，建立一种优势互补的结核耐药检测体系。.共有186名患者被诊断为耐药脊柱结核。采用二代基因芯片对这186例患者的临床样本进行检测，RIF检测的敏感性为92.74%，特异性为97.64%;INH检测的敏感性为91.26%，特异性为97.25%;EMB检测的敏感性为54.17%，特异性为88.26%; SM检测的敏感性为86.73%，特异性为96.50%; FQs检测的敏感性为84.87%，特异性为97.64%; SLID检测的敏感性为80.90%，特异性为98.41%。.微流控芯片检测INH敏感性和特异性分别为：89.34%和97.43%；RIF敏感性和特异性分别为：91.78%和95.90%；EMB敏感性和特异性分别为：76.29%和95.50%；SM敏感性和特异性分别为：87.97%和97.09%；FQS敏感性和特异性分别为：95.12%和96.76%；AMK敏感性和特异性分别为：88.24%和97.79%；KAN敏感性和特异性分别为：84.29%和97.29%；CPM敏感性和特异性分别为：89.80%和96.99%。.采用微流控芯片对136例脊柱结核临床样本行分枝杆菌菌种鉴定，阳性率为87.50%。在培养阳性样本中，微流控芯片结核分枝杆菌菌种鉴定的敏感性为95.45%。对涂片阳性、培养阳性的样本检测，敏感性为95.80%，涂片阴性，培养阳性的样本检测，敏感性为31.32%。在INH耐药检测方面，微流控芯片的敏感性为80.95%,特异性为91.78%，在RIF耐药检测中，微流控芯片检测敏感性为88.24%,特异性为91.53%；SM耐药检测敏感性为81.82%,特异性为92.77%；FQS耐药检测敏感性为88.89%,特异性为97.53%；EMB耐药检测敏感性为71.43%,特异性为96.43%。.耐药脊柱结核对医生来说是一个巨大挑战，有较高的致残率和死亡率。药物治疗仍是最根本的措施。治疗的关键是明确耐药情况，尽早开始有针对性的个体化化疗。分子药敏技术的出现，明显改善患者预后。本课题设计研发的微流控芯片检测耐药脊柱结核样本敏感性、特异性高，操作简单，耗时短，有较好的应用前景。