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

在耻垢分枝杆菌和有限数量的结核分枝杆菌菌株中，16 S rRNA基因（rrs）的改变参与了对卡那霉素的耐药性。为了研究临床分离的结核分枝杆菌swain中n-s基因特定区域的突变与卡那霉素耐药表型的相关程度，检测了43个卡那霉素耐药swain（MIC>=200 μ g/ml）、71个卡那霉素敏感菌株和4个模式菌株。通过PCR扩增携带rrs基因和rrs基因与23 S rRNA（rrl）基因片段之间的插入序列的300-bp DNA片段，并进行基于PCR的直接测序。43株卡那霉素耐药株中有29株（67.4%）在1400、1401和1483位发生了碱基替换，而71株敏感株和4株典型株均未发生碱基替换。最常见的替换，从A到G，发生在位置1400。在位置1401处发现一次从C到T的取代。两个临床分离株携带1401位C至A和1483位G至T的双重突变。此外，我们发现，这些突变可以区分与野生型菌株的限制性内切酶Tai I和Tsp 451消化。此外，我们发现卡那霉素抗性swain的基因型可以通过用限制性内切酶Bst UI或Dde I消化来彼此区分。

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.