关键词:

肺结核, 利福平耐药, 实时荧光定量核酸扩增检测, rpoB基因, 突变特征

Abstract:

Objective　To analyze the mutation characteristics of rpoB gene in 256 cases of rifampicin-resistant real-time fluorescence quantitative nucleic acid amplification test (Gene Xpert MTB/RIF) positive pulmonary tuberculosis in Xi'an area. Methods　Two hundred and fifty-six MTB strain strains were isolated from outpatients and inpatients with Gene Xpert MTB/RIF-positive pulmonary tuberculosis who visited Shaanxi Provincial Tuberculosis Control Hospital from June 2020 to June 2022. The strains were not collected repeatedly. They were from 174 sputum samples and 82 bronchoalveolar lavage fluid samples. The patients were (45.67±8.36) years old. The PCR products of rpoB gene of 256 rifampicin-resistant strains were analyzed by DNA direct sequencing. The 256 rifampicin-resistant strains were divided into low-, medium-, and high-resistant MTB strains according to the degree of rifampicin resistance, and the mutation sites of the three kinds of strains were compared by χ² test. Three rifampicin-resistant MTB strains were artificially induced, and the PCR products of rpoB gene were analyzed by DNA direct sequencing. Results　The sequencing report showed that 253 of the 256 rifampicin-resistant strains had rpoB locus mutation, and the mutation rate was 98.83% (253/256). There were 10 mutation types including C→T, T→G, C→G, A→T, C→A, A→G, G→A, G→T, T→C, and A→C. There were 14 amino acid codons including serine, leucine, alanine, histidine, tyrosine, glutamic acid, lysine, arginine, aspartic acid, proline, methionine, valine, isoleucine, and glycine, all of which were point mutations. The mutations of rifampicin-resistant strains were mainly concentrated at locus 531 [53.75% (136/253)] and 526 [23.32% (59/253)]. Other sites included locus 513, 516, 533, 515, 513, 532, 522, 511, 519, 518, and 533. Compared with those of low- [37.88% (25/66)] and medium-resistant MTB strains [37.04% (20/54)], the incidence of amino acid mutation at locus 531 of high-resistant MTB strains [66.91% (91/136)] was increased (χ²=22.154,P<0.001). There was no statistically significant difference in the incidence of amino acid mutation at locus 531 in low- and medium-resistant MTB strains (P>0.05). There was no statistically significant difference in the incidence of amino acid mutation at locus 526 among the low-, medium-, and high-resistant MTB strains [22.73% (15/66) vs. 25.93% (14/54) vs. 22.06% (30/136)] (χ²=0.331, P=0.847). Three artificially induced rifampicin-resistant MTB strains all had rpoB gene locus mutation. The mutation of low- and medium-resistant MTB strains were located at locus 526, and the mutation of high-resistant MTB strain was located at locus 531. Conclusions　The mutation rate of rpoB gene in Gene Xpert MTB/RIF-positive pulmonary tuberculosis patients in Xi'an area was high, mainly point mutations, mainly concentrated at locus 531 and 526. The 531 TCG→TTG mutation had the highest mutation frequency among the rpoB gene mutation types. The mutation of low- and medium-resistant MTB strains were located at locus 526, which was associated with high drug resistance.

Key words:

Pulmonary tuberculosis, Rifampicin resistance, Real-time fluorescence quantitative nucleic acid amplification detection, rpoB , gene,  , Mutation characteristics