一种直接检测结核病痰标本的PCR技术的建立与评价

目的建立并评价聚合酶链式反应(polymerase chain reaction,PCR)在结核病痰标本检测中的应用价值。方法根据结核分枝杆菌复合体IS6110序列设计引物INS1和INS2,并建立PCR反应体系和反应条件。运用PCR方法分别检测标准菌株、结核分枝杆菌PCR检测标准品和拟诊结核病患者痰标本,采用痰涂片和细菌培养为对照。结果比较的统计学分析采用卡方检验。结果PCR方法对结核分枝杆菌、牛分枝杆菌、卡介苗标准株的最小检出浓度分别达到102,103,103个细菌/毫升,能够特异地检出结核分枝杆菌复合体。在PCR检测的574例拟诊病例中,PCR检测阳性病例241例,42%;痰涂片和细菌培养的阳性率分别为19.69%和26.31%,PCR检测阳性率高于传统细菌学检验方法,经χ2检验,差异具有显著统计学意义(χ2=103.67,P<0.01)。以痰培养结果为标准,计算PCR检测方法的敏感度为67.53%。结论PCR检测方法与传统的细菌学检测方法相比可以提高阳性标本检出率,而且具有快速、特异、简便的特点,有望成为结核病大规模筛查和临床快速检测方法。

Establishment and evaluation of a polymerase Chain Reaction technique for detection Mycobacterium tuberculosis in sputum samples from suspect patients with tuberculosis

To establish the polymerase chain reaction (PCR) for detection of sputum samples of TB, and evaluate its perspective in clinical application. Designed a pair of primers (INS1 & INS2) according to the insertion sequence 6110 (IS6110) of Mycobacterium tuberculosis complex (MtbC), and establish PCR reaction condition. The MtbC standard strains, standard Mycobacterium strains and clinical sputum samples of suspected patients with TB were detected by the PCR method. The results were compared to those by smear slide microscopy and culture with chi-square test of statistics analysis. It was found that the minimum detection quantity to M. tuberculosis, M. bovis, BCG was 102,103,103 bacteria/mL respectively, and the MtbC could be detected specifically. Among 574 tested cases, 241 (42%) were positive by PCR. The positive rate of smear slide and culture were 19.69% and 26.31%, respcetively. The positive rate of PCR was much higher than that of bacteriological methods including smear and culture. There was significant difference by means of chi-square statistics analysis (χ2=103.67,P<0.01). Using the results of culture as gold standard, the sensitivity(Se)of PCR was 67.53%. It is evident that PCR used for the direction of MtbC appears to be a method for large scale screening and clinical detection of MtbC.