Detection of gene mutations in kanamycin-resistant Mycobacterium tuberculosis and development of the method for rapid diagnosis

在污垢分枝杆菌和数量有限的结核分枝杆菌菌株中，16S rRNA基因(RRS)的改变已被证明与卡那霉素耐药有关。为探讨结核分枝杆菌临床分离株中n-S基因特定区域突变与卡那霉素耐药表型的相关性，对43株卡那霉素耐药株、71株卡那霉素敏感株和4株卡那霉素敏感株进行了检测。用聚合酶链式反应(PCR)扩增出携带RRS基因的300bpDNA片段和RRS基因与23S rRNA(RRL)基因之间的插入序列，并进行直接测序。通过核苷酸序列比较，发现43株卡那霉素耐药临床分离株中有29株(67.4%)在1400、1401和1483位发生了突变，而71株敏感株和4种类型菌株均未发生突变。最频繁的替换，从A到G发生在1400位。一次在1401位发现了从C到T的替换。2株临床分离株携带1401位C到A和1483位G到T的双重突变。此外，我们还发现用限制性内切酶TAI和TSP451可以将这些突变与野生型菌株区分开来。此外，我们还发现，用限制性内切酶BST-UI或Dde-I可以区分卡那霉素抗性野生型。

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the n-s gene and the kanamycin-resistant phenotype in clinically isolated M.tuberculosis swains were correlated, 43 kanamycin-resistant swains (MICs, >=200 mu g/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at position 1400, 1401 and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutations can be distinguished from wild-type strains by digestion with the restriction endonucleases Tai I and Tsp 451. Furthermore, we found that the genotypes of kanamycin-resistant swains can be discriminated from each other by digestion with a restriction endonuclease, Bst UI or Dde I.