To analysis the drug resistance and gene mutation in Mycobasterium tuberculosis , and to investigate the clinical application value of DNA sequence analysis detecting of Mycobasterium tuberculosis isolates streptomyci-resistance. First, traditional drug susceptibility test (DST ) was used to analysis streptomycin-resistance for 49 strains Mycobasterium tuberculosis Then, analysis assay for detection of mutation in two drug resistance associated genes of 49 strains Mycobasterium tuberculosis clinical isolates (rpsl, rrs) by DNA sequencing, and screening for mutations. According to the mutations of genes, the design of primers of DNA Sequence analysis for detection of streptomycin resistance. DST results showed that 16 strains were Streptomycin-resistant strains, 33 strains were streptomycin-sensitive strains. Sequencing results showed that there was only a mutations of rpsl, for the 43rd codon mutation of AAG, mutation forms for AAG - AGG, amino acid from lysine (Lys) into arginine (Arg).There were five site mutations of rrs gene, 513 A to G, 523, 599, 612 of lack of A, 598A to G, respectively. The 513th base mutations existed only in the streptomycin resistant strains, the remaining four mutations in resistant strains and sensitive strains existed. The preliminary think the 43rd codon Rpsl gene mutation and rrs 513th of base mutations associated with streptomycin resistance . Culture-based phenotypic drug susceptibility testing and DNA sequencing to analyze the level of rpsl gene, rrs gene mutation, the results showed 1 strain existed 513th base mutations of rrs genes, 13 strains existed 43th codon mutations of rpsl gene in 16 strains of streptomycin resistant strains. With culture-based phenotypic drug susceptibility testing as the gold standard, the DNA sequencing detected rpsl gene 43th codes or rrs gene 513th base mutations for strains resistant, DNA sequencing results showed that the sensitivity and specificity was 87.5% and 96.97%. It can be concluded that DNA sequencing analysis technology has good sensitivity and specificity to detect streptomycin resistance of Mycobasterium tuberculosis clinical isolates, which is short time-consuming and in early diagnosis of streptomycin drug-resistant TB.%为检测结核分枝杆菌链霉素(streptomycin,SM)耐药性,分析耐药基因突变,探讨DNA测序分析技术在结核分枝杆菌链霉素耐药性检测中的应用价值.采用方法:1、用传统的比例法药敏试验对49株结核分枝杆菌临床分离株进行链霉素耐药性检测.2、用DNA测序分析技术对49株结核分枝杆菌临床分离株进行链霉素耐药相关基因Rpsl和rrs的突变位点分析,筛查耐药相关突变位点.3、探讨DNA测序分析技术对结核分枝杆菌临床分离株进行链霉素耐药性检测的效率.结果:1、传统比例法药敏试验结果显示,49株实验菌株中16株为链霉素耐药株,33株为链霉素敏感株.2、Rpsl基因DNA测序.结果显示:Rpsl基因只存在一个突变位点,为第43位密码子AAG的突变,突变形式为AAG→AGG,氨基酸由赖氨酸(Lys)变为精氨酸(Arg).rrs基因DNA测序结果发现:rrs基因共存在5个突变位点,分别为第513位碱基A突变为G、第523位碱基A缺失、第598位碱基A突变为G、第599位碱基A缺失、第612位碱基A缺失,其中第513位碱基突变只存在于链霉素耐药株中,其余4个突变位点在耐药株和敏感株中均存在.即初步认为Rpsl基因第43位密码子的突变及rrs基因第513位碱基的突变与结核分枝杆菌链霉素耐药性有关.3、分析Rpsl基因和rrs基因突变与结核分枝杆菌链霉素耐药性之间的关系,结果显示:16株链霉素耐药株中13株存在Rpsl基因第43位密码子的突变,1株存在rrs基因第513位碱基的突变.以传统的比例法药敏结果为参照,以DNA测序分析Rpsl基因或rrs基因发现Ppsl基因发生第43位密码子或rrs基因第513位碱基突变为判断菌株耐药的标准,DNA测序分析技术对结核分枝杆菌链霉素耐药性检测的敏感性为87.5%,特异性为96.97%.由此可知,初步认为Rpsl基因第43位密码子的突变及rrs基因第513位碱基的突变与结核分枝杆菌链霉素耐药性有关.用DNA测序分析结核分枝杆菌Rpsl基因和rrs基因链霉素耐药相关突变,判断结核杆菌链霉素耐药性具有较好的灵敏度和特异性,耗时短,在链霉素耐药结核病的快速诊断方面具有一定的应用价值.
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