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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。
The chromosomal DNA of human Mycobacterium tuberulosis TB was used as a template to select the two fragments located at 884 ~ 865 and 568 ~ 588 base pairs in IS6110 as primers, and a specific fragment of 317bp was amplified. It was cloned into the pUC19 vector. Enzyme digestion and DNA sequencing confirmed that the target fragment. The fragment was DIG-labeled, respectively, with 11 kinds of Mycobacterium DNA Southern hybridization, the results prove that only with human-type hybrid Mycobacterium hybridization. Using the PCR detection technique established by the pair of primers, 74 samples of sputum of tuberculosis were detected and compared with the results of clinical bacterial rapid culture, 48 of them were positive by PCR and 11 were found by 26 samples of clinical negative PCR test positive. Hybridization of the sample PCR product with the cloned probe showed the exact agreement between the two. It shows that the PCR detection system has reliable results and its sensitivity is obviously higher than the current clinical methods and can be used as a routine technique for the clinical detection of tuberculosis.