人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

The Preparation of Mycobacterium tuberculosis Specific Probe and the PCR Diagnosis of Human Tuberculosis

采用人结核分枝杆菌(Mycobacterium tuberulosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段,将其克隆进pUC19载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PCR检测技术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PCR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。

Mycobacterium tuberculosis (TB) is the pathogenic bacteria of human tuberculosis. A specific fragment of IS6110, an insert sequence repeated multiple times in the chromosome of M. tuberculosis was used as template to amplify a 317bp through PCR technique. The 317bp fragment was proved as a specific probe for M. tuberculosis but not for other mycobacterium in the Southern blot. A PCR assay for the rapid diagnosis of pulmonary tuberculosis was developed by using above primers. The PCR results were compared with the clinical bacteria culture for the detection of M. tuberculosis in 74 sputum samples. 48 positive samples were found in the culture and all of those were PCR positive. Another 16 PCR positive sputum were seen in 26 negative cultures. We proved that all the positive PCR products could hybridize with cloned probe through Dot-blotting. The specific DNA probe prepared in this experiment provided the material for the research of epidemiology and molecular biology of M. tuberculosis. The PCR assay set up here offered a sentitive and specific technique for the diagnosis of human tuberculosis.