Fang F, Xiang Z, Chen R
Department of Pediatrics, Tongji Hospital, Tongji Medical University, Wuhan 430030.
J Tongji Med Univ. 2000;20(4):324-6. doi: 10.1007/BF02888193.
In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a tubercle bacillus (Tb)-specific primer were designed based on highly conserved regions and Tb species-specific variable region of bacterial 16s rDNA. A 360 bp fragment was detected in all bacteria tested, and a 210 bp fragment was found only in Tb. 19 species of known bacteria including Tb were used for evaluating specificity, universality and sensitivity of the PCR. Candida albicans and human diploid cell served as controls. It was found that both 210 bp and 360 bp fragments were amplified only in Tb, and only 360 bp fragment was detected in other 18 species of general bacteria. Candida albicans and human cells were negative for both 360 bp and 210 bp fragments. The lowest detectable level of the PCR was 10 fg of DNA for Escherichia coli and 100 fg of DNA for Tb. The results indicated that this multiplex PCR system for the simultaneous detection of Tb and other common bacteria had higher specificity and sensitivity, as well as good universality and might be useful to rapidly diagnose bacterial infections and effectively distinguish tuberculosis from other bacterial involvement.
为了快速诊断结核病并将其与其他细菌感染区分开来，开发了一种基于16S rRNA基因（16s rDNA）的多重PCR系统。在该系统中，根据细菌16s rDNA的高度保守区域和结核杆菌（Tb）物种特异性可变区域设计了一对通用引物和一对结核杆菌特异性引物。在所有测试细菌中均检测到一个360 bp的片段，而仅在结核杆菌中发现一个210 bp的片段。使用包括结核杆菌在内的19种已知细菌来评估该PCR的特异性、通用性和敏感性。白色念珠菌和人二倍体细胞用作对照。结果发现，仅在结核杆菌中扩增出210 bp和360 bp的片段，而在其他18种常见细菌中仅检测到360 bp的片段。白色念珠菌和人细胞对360 bp和210 bp的片段均呈阴性。该PCR对大肠杆菌的最低可检测DNA水平为10 fg，对结核杆菌为100 fg。结果表明，这种用于同时检测结核杆菌和其他常见细菌的多重PCR系统具有较高的特异性和敏感性，以及良好的通用性，可能有助于快速诊断细菌感染并有效区分结核病与其他细菌感染。
