用于结核分枝杆菌耐药性分子检测的技术
1.线性探针技术（LPA） LPA可同时检测利福平和异烟肼的耐药基因突变，可用于涂阳标本和结核分枝杆菌临床分离株的结核分枝杆菌及其耐药检测。有代表性的LPA试剂盒有AID LPA （AID Diagnostika，Germany），INNO LPA （Innogenetics，Belgium），GenoType MTBDRplus和Geno Type MTBDRsl。GenoType MTBDRplus试剂盒可用于检测结核分枝杆菌及异烟肼、利福平耐药，GenoType MTBDRsl试剂盒可检测结核分枝杆菌对氟喹诺酮类和二线注射类抗结核药物耐药情况。AID试剂盒可检测利福平、异烟肼、链霉素、卷曲霉素、阿米卡星、乙胺丁醇和氟喹诺酮类耐药。Meta分析显示，以表型药敏结果为金标准的LPA，检测利福平耐药的灵敏度和特异度分别为96.7%和98.8%，检测异烟肼耐药的灵敏度和特异度分别为90.2%和99.2%。GenoType MTBDRsl检测氟喹诺酮类、阿米卡星、卷曲霉素、卡那霉素和乙胺丁醇耐药的灵敏度分别为87.4%、82.6%、82.0%、44.4%和67.9%；特异度分别为97.1%、99.5%、97.3%、99.3%和79.9%。
2.Xpert MTB/RIF技术 此技术实现了样本准备、扩增、检测自动化。对MTB特异，敏感性与培养相似，通过检测rpoB基因81 bp的RRDR区域而诊断利福平耐药。Meta分析显示Xpert MTB/RIF在肺结核中检测利福平耐药的灵敏度为95%，特异度为98%，在肺外结核中检测利福平耐药的灵敏度为95%，特异度为98.7%。Xpert XDR可以同时检测异烟肼、氟喹诺酮类和氨基糖苷类耐药，Xie YL等报道Xpert XDR检测异烟肼、氧氟沙星、莫西沙星、卡那霉素和阿米卡星的灵敏度分别是83.3%、88.4%、87.6%莫西沙星（0.5 μg/ml）/96.2%莫西沙星（2.0 μg/ml）、71.4%和70.7%；特异度均高于94.3%，除了莫西沙星（2.0 μg/ml）为84.0%。在耐药高负担地区Xpert XDR在快速诊断XDR—TB中将发挥重要作用。WHO关于Xpert推荐意见（2013）为：① Xpert应当用于MDR和HIV感染高发病率地区的快速筛查；② Xpert用于儿童结核病的早期诊断；③ 推荐Xpert替代涂片、培养等技术用于肺外结核的快速诊断。有文献报道NTM病可能会导致Xpert MTB/RIF假阳性，如脓肿分枝杆菌细菌载量超过106 CFU/ml，可能被误判为检出结核分枝杆菌；rpoB基因突变，海分枝杆菌细菌载量超过104 CFU/ml，可能被误判为检出结核分枝杆菌，rpoB基因无突变。
3.Xpert MTB/RIFultra技术 新一代Xpert MTB/RIF检测技术，可用于超敏结核分枝杆菌和利福平耐药检测。4个分子信标探针识别利福平耐药突变，通过rpoB突变体Tm的偏移，区分耐药突变与利福平敏感。可用于现有相同的GeneXpert系统（包括GeneXpert仪器、计算机、显示器、键盘、条形码阅读器）。该技术试剂盒内的变化包括一个更大的DNA扩增仓室和两个额外的分子靶标来检测结核分枝杆菌，意味着更大量的标本用于DNA的扩增和检测。这些变化解释了该技术增加的敏感性，使其进一步增强了检测低数量细菌的能力。与目前的Xpert MTB/RIF试剂盒相比，在用于检测结核分枝杆菌菌量较少的标本中的结核分枝杆菌时显示出更加优越的性能（灵敏度增加），特别是涂片阴性、培养阳性标本（如来源于合并感染艾滋病病毒患者的标本）、儿科标本和肺外标本（特别是脑脊液），该技术显著检出更多TB脑膜炎。总体而言，该技术比Xpert MTB/RIF技术的敏感性提高5%，但特异性降低3.2%。对涂片阴性—培养阳性和HIV—TB双感患者的痰标本，敏感性提高明显（分别为17%和12%）；对有结核病史人群的特异性降低要高于无结核病史者（—5.4% vs—2.4%），但在结核病低负担的情况下，该技术特异性非常高（99.3%）；对肺外结核和儿童结核，该技术比Xpert MTB/RIF技术的敏感性大幅度提高（脑脊液：95% vs 45%；儿童呼吸道标本：71% vs 47%）。该技术与Xpert MTB/RIF技术比较见下表8—3—2—1。
表8—3—2—1 Xpert MTB/RIF 和新一代Xpert MTB/RIFultra 的比较
4.耐药检测基因芯片 基因芯片是基于特定基因位点突变与耐药性的相关性，通过PCR扩增和核酸杂交技术，检测荧光信号的有无，判断特定位点的突变情况和细菌耐药性的检测方法。结核分枝杆菌耐药检测基因芯片基于rpoB/katG/inhA的基因突变，快速检测分离株或者痰样本中结核分枝杆菌耐药性（利福平、异烟肼）。
5.探针熔解曲线 耐药基因突变，导致DNA双链的结合能力下降，从而导致相应的DNA熔解温度（Tm）下降，Tm下降的幅度与错配的碱基数目、类型和位置有关。野生型基因有特定的Tm，据此可区分和检测出突变型和野生型。四种耐药检测方法比较见下页表8—3—2—2。
表8—3—2—2 四种耐药检测技术的比较
6.Genedrive MTB/RIF 2012年英国某公司研发，机器精巧便携，目标基因：rep13E12和rpoB，检测标本是否含有结核分枝杆菌及利福平耐药性，45 ～75 min完成检测，检测限为（0.25 ～2.5）×105 CFU/ml。Genedrive检测效能与其他方法比较见表8—3—2—3 。
表8—3—2—3 Genedrive 检测效能比较
（王桂荣 钱雪琴）
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