Yamazaki T, Nakamura R M
Department of Bacteriology, National Institute of Health, Tokyo, Japan.
Kekkaku. 1993 Nov;68(11):687-93.
PCR products amplified with the primers YNP-1 and YNP-2 and template DNA from various mycobacterial species showed differences in molecular sizes. By sequencing the PCR products, we found that the DNA fragment from M. tuberculosis and that of M. intracellulare have different DNA sequences. The former was 164 bp, composed of 27 A, 54 C, 57 G, and 26 T, while the latter was 109 bp, composed of 22 A, 37 C, 34 G, and 16 T. We compared these sequences and selected a nucleotide sequence unique to M. intracellulare and used as primer YNP-7. Antisense primer, YNP-8, was constructed from the complementary sequence of YNP-2 which is located about 270 b downstream of YNP-7. Results of PCR using the primers YNP-7 and YNP-8 and template DNA of various mycobacteria showed that positive results were only with the template DNA from M. intracellulare. Bacterial DNA from those other than mycobacteria but appear in sputum were tested in PCR with the primers YNP-7 and YNP-8. None of them showed positive results. Specificity of the PCR products was determined with a specific probe MIP which was constructed using a sequence between YNP-7 and YNP-8 to confirm the specificity of PCR by Southern hybridization. Only the products from M. intracellulare hybridized with the probe MIP, indicating the specificity of YNP-7 and YNP-8 to M. intracellulare.(ABSTRACT TRUNCATED AT 250 WORDS)
用引物YNP - 1和YNP - 2以及来自各种分枝杆菌物种的模板DNA扩增的PCR产物显示出分子大小的差异。通过对PCR产物进行测序，我们发现结核分枝杆菌的DNA片段与胞内分枝杆菌的DNA片段具有不同的DNA序列。前者为164 bp，由27个A、54个C、57个G和26个T组成，而后者为109 bp，由22个A、37个C、34个G和16个T组成。我们比较了这些序列，选择了胞内分枝杆菌特有的核苷酸序列并用作引物YNP - 7。反义引物YNP - 8由位于YNP - 7下游约270 bp处的YNP - 2的互补序列构建而成。使用引物YNP - 7和YNP - 8以及各种分枝杆菌的模板DNA进行PCR的结果表明，仅胞内分枝杆菌的模板DNA产生阳性结果。对痰液中出现的非分枝杆菌的细菌DNA用引物YNP - 7和YNP - 8进行PCR检测。它们均未显示阳性结果。用特异性探针MIP确定PCR产物的特异性，该探针是利用YNP - 7和YNP - 8之间的序列构建的，通过Southern杂交来确认PCR的特异性。只有胞内分枝杆菌的产物与探针MIP杂交，表明YNP - 7和YNP - 8对胞内分枝杆菌具有特异性。（摘要截短于250字）
