摘要
目的分析西安地区256例利福平耐药实时荧光定量核酸扩增检测技术(Gene Xpert MTB/RIF)阳性肺结核患者rpoB基因突变特征。方法本研究为回顾性分析。256株结核分枝杆菌(MTB)菌株分离自2020年6月至2022年6月于陕西省结核病防治院就诊的Gene Xpert MTB/RIF阳性肺结核门诊及住院患者,菌株无重复收集,来自痰液标本174份、支气管肺泡灌洗液标本82份,患者年龄(45.67±8.36)岁。采用DNA直接测序法对256株利福平耐药MTB菌株rpoB基因的PCR产物进行分析。将256株利福平耐药MTB菌株根据利福平耐药程度分为低、中、高耐药MTB菌株,采用χ^(2)检验比较3种菌株突变位点。人工诱导3株利福平耐药MTB菌株,采用DNA直接测序法对其rpoB基因的PCR产物进行分析。结果测序报告显示,256株利福平耐药MTB菌株中有253株发生rpoB基因位点突变,突变率为98.83%(253/256)。突变类型包括C→T、T→G、C→G、A→T、C→A、A→G、G→A、G→T、T→C、A→C共计10种,涉及丝氨酸、亮氨酸、丙氨酸、组氨酸、酪氨酸、谷氨酸、赖氨酸、精氨酸、天冬氨酸、脯氨酸、蛋氨酸、缬氨酸、异亮氨酸、甘氨酸共14个氨基酸密码子,均为点突变。利福平耐药菌株突变主要集中在531位[53.75%(136/253)]、526位[23.32%(59/253)],其他位点包括513、516、533、515、513、532、522、511、519、518、533。高耐药MTB菌株531位氨基酸突变发生率与低、中耐药MTB菌株比较[66.91%(91/136)比37.88%(25/66)、37.04%(20/54)],差异有统计学意义(χ^(2)=22.154,P<0.001);低、中耐药MTB菌株531位氨基酸突变发生率比较,差异无统计学意义(P>0.05)。低、中、高耐药MTB菌株526位氨基酸突变发生率比较[22.73%(15/66)、25.93%(14/54)、22.06%(30/136)],差异无统计学意义(χ^(2)=0.331,P=0.847)。人工诱导的3株利福平耐药MTB菌株均发生rpoB基因位点突变,低、中耐药MTB菌株突变均位于526位点,高耐药MTB菌株突变位于531位点。结论西安地区Gene Xpert MTB/RIF阳性肺结核患者rpoB基因突变率较高,以点突变为主,主要集中在531位、526位,531位TCG→TTG突变在rpoB基因突变类型中突变频率最高,且与高耐药有关。
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 byχ^(2) 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(χ^(2)=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)](χ^(2)=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.
作者
崔尖
贺志清
Cui Jian;He Zhiqing(Clinical Lab,Shaanxi Provincial Tuberculosis Control Hospital,Xi'an 710110,China;Clinical Lab,Hanzhong Hospital of Traditional Chinese Medicine,Hanzhong 723000,China)
出处
《国际医药卫生导报》
2024年第12期1988-1992,共5页
International Medicine and Health Guidance News
基金
陕西省自然科学基础研究计划(2021JM-051)。
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
Pulmonary tuberculosis
Rifampicin resistance
Real-time fluorescence quantitative nucleic acid amplification detection
rpoB gene
Mutation characteristics
