Tuberculosis (TB) is endemic in China with high prevalence of multiple-drug resistant tuberculous bacilli (MDR-TB).The incidence of new cases of TB reaches 1,300 thousand annually.Among them,5.7 percent are MDR-TB.Staining for acid-fast bacilli in sputum and clinicoradiological examination have been the main diagnostic tools for TB,particularly pulmonary TB.However,the positive rate of sputum Ziehl-Neelsen stain for sputum is disappointedly low,merely 28％ in newly-diagnosed TB.Moreover,the radiological manifestations of the patients suspected of TB are often non-specific.All these facts call for a simple,accurate and rapid diagnostic method to overcome this bottleneck,which hinders the success of satisfactory TB control in China.Employing both hemi-nested RT-PCR and beacon technology with fluorescent probes,the MTB/RIF diagnostic assay specifically amplifies,thus helps detect the rpoB gene,which is unique to Mycobacterium tuberculosis and also a biomolecular-marker of rifampin resistance.As a semi-quantitative analysis,the quantity of Mycobacterium tuberculosis in samples is reflected by the threshold of PCR cycles during MTB/RIF assay.With Mycobacterium tuberculosis culture as the standard reference,for sputum samples from patients suspected of suffering from pulmonary TB,overall diagnostic sensitivity of MTB/RIF assay is 73.1％-90.0％with a specificity of 99.0％-99.5％.For detection of rpoB gene mutations responsible for rifampin-resistance,the sensitivity is 97.2％ and specificity is 98.3％.Following sample loading,the system can automatically complete the diagnostic process and report the results within 2 hours.Targeting the rpoB gene specifically,there is no cross-reaction with non-tuberculosis mycobacteria or other common respiratory pathogens.In addition to sputum samples,the system can be used to detect Mycobacterium tuberculosis in various body fluids (including pleural effusion,urine,cerebrospinal fluid and even bronchoalveolar lavage fluid) and lung tissue biopsy samples.As for sensitivity,the assay is comparable to Mycobacterium tuberculosis culture (for the latter,mean interval between loading and result-reporting is 16 or 30 days (depending upon the culture medium used),and it is 100 times longer than Ziehl-Neelsen stains.Compared with conventional laboratory diagnostic approaches,this assay is much simpler and biohazardous aerosol-free.%我国是结核病及耐药结核高发地区,年新发结核病例130万,新发结核病例耐药率为5.7％.痰涂片抗酸染色和临床放射影像学是结核病尤其是肺结核的主要诊断手段.但痰涂片阳性率偏低,新发肺结核病例涂阳率仅28％,而且结核病的影像学表现缺乏特异性.缺乏简单、准确、快速的诊断手段一直是我国实现结核病防控目标的瓶颈.结核分枝杆菌/利福平耐药(MTB/RIF)检测诊断系统运用半槽式(hemi-nested)RT-PCR及荧光探针报告技术(beacon technology),特异性扩增并检测结核分枝杆菌特有并与利福平耐药密切相关的rpoB基因片段.以PCR循环阈值反映标本中的结核分枝杆菌载量,进行半定量分析.该系统以结核杆菌培养作为标准对照,对疑诊肺结核患者痰标本的诊断总体敏感性为73.1％～90.0％,特异性99.0％～99.5％;对利福平耐药基因(MDR-TB的分子生物学标记物)诊断的敏感性为97.2％、特异性为98.3％.上样后系统可在2h内自动完成诊断及结果打印程序.由于系统特异性扩增rpoB基因片段,因此与非结核分枝杆菌及呼吸系统常见病原体无交叉反应.除外痰标本,该系统还能用于多种体液(胸腔积液、尿、脑脊液以及支气管肺泡灌洗液等)和肺组织匀浆中结核分枝杆菌的检测.该系统的诊断敏感性较抗酸染色高l00倍,与结核分枝杆菌培养相近,但后者从加样至报告的平均时间为16d(平板培养基)或30d(液体培养基).与传统结核诊断实验室诊断技术相比,本系统还具有操作简便、不产生传染性生物气溶胶等优点.
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