中国结核分枝杆菌临床分离菌株利福平耐药基因rpoB突变特征的初步研究

发布时间：2018-02-27 11:53

本文关键词： 结核分枝杆菌利福平耐药 rpoB基因 DNA序列分析反向点杂交　出处：《大连医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 结核分枝杆菌(Mycobacterium tuberculosis,MTB)的感染是因感染性因素导致人类死亡的最显著原因之一.。近年来,随着人口流动增加,人口密度增高,结核分枝杆菌多重耐药菌株及同人体免疫缺陷病毒双重感染的出现,更加重了结核病在发达国家和发展中国家再度肆虐的态势,每年在世界范围内导致大约200万人死亡[1]。随着结核病发病人数的上升,耐多药结核病(Multi-Drug Resistance Tuberculosis,MDR-TB)的发病率也呈上升趋势。利福平(Rifarmpicin,RFP)作为最有效的抗结核药物之一,对其耐药,尤其对RFP耐药与其它药物耐药相关时,通常会导致临床上结核病的复燃。由于异烟肼和利福平是临床最常用的药物,有报道约90%的结核分枝杆菌耐利福平株同时也耐异烟肼,所以单独进行利福平敏感性分析也可能作为耐多药的筛选指标[2-4]。因此,对主要抗结核药物如利福平的耐药性快速检测对于有效治疗结核病和控制耐多药株的流行是必不可少的。为较好建立寡核苷酸探针反向点杂交方法,本研究从西藏、湖南、河南、四川、福建、安徽和陕西七省收集结核分枝杆菌临床分离菌株,首先对其采用生化反应方法进行菌种鉴定并用比例法进行药物敏感性试验。其后用聚合酶链反应(Polymerase Chain Reaction,PCR)扩增rpoB基因“热点突变区”附近区域进行DNA测序分析,了解我国结核分枝杆菌rpoB基因突变情况,明确其突变位点的分布,主要突变位点及其发生率。选取特征性突变位点,设计检测探针,建立快速检测结核分枝杆菌rpoB基因突变的以PCR为基础的寡核苷酸探针反向点杂交方法。采用SPSS 11.0统计软件对实验数据进行分析,探讨该方法能否作为筛查方法用于临床检测结核分枝杆菌对利福平的敏感性,并评价其临床应用价值。首先,采用生化培养基对从结核病人分离的536株分枝杆菌临床分离株进行了菌种鉴定,482株被鉴定为结核分枝杆菌(MTB),占89.93%;19株为牛结核分枝杆菌(M. bovis),3.54%;33株为非结核分枝杆菌(NTM),6.16%;2株为结核分枝杆菌加非结核分枝杆菌的混合感染,0.37%。采用比例法对筛选出来的482株结核分枝杆菌临床分离株进行药物敏感性试验,结果显示对5种抗结核药物全部敏感的菌株为126株,占26.14%;耐药菌株356株,耐药率达75.52%,其中,单耐药菌株88株,18.26%;多耐药菌株268株,55.60%。其次,针对结核分枝杆菌利福平耐药的相关基因rpoB基因设计引物,应用DNA序列分析对结核分枝杆菌标准株H37Rv及300株已经明确耐药背景的临床分离株进行耐药基因突变检测。经DNA序列分析,181株RFP耐药株中,531位点突变占所有突变的53.61%,526位点突变占27.71%,516位点突变占9.64%,511位点突变占6.02%,这四种突变位点的突变频率之和占到所有突变类型的96.99%;多位点联合突变15株,占所有突变的9.04%,且发现1株发生整个氨基酸密码子插入和5株单碱基或整个氨基酸密码子缺失菌株。67株全敏感株中未发现突变,52株RFP相对敏感株中,有2株发生511、526位点突变。在此基础上,利用反向点杂交技术对300株结核分枝杆菌临床分离株进行rpoB基因突变分析显示:119株利福平敏感株中,67株全敏感株均无突变,52株RFP相对敏感株中有2株检测到突变;181利福平耐药株中,165株检测到突变,16株未见突变。与DNA测序结果比较,168株中有164株被判定为耐药株,4株判定为敏感株;132株测序未发现突变的菌株中129株被判定为敏感株,3株被判定为耐药株。其敏感度为97.62%(164/168),特异度为97.73%(129/132),阳性预测值为98.20%(164/167),阴性预测值为96.99%(129/133)。有293株反向点杂交的检测结果与其DNA测序结果相符,二者的符合度可达97.67%(293/300),经×2检验,差异无统计学意义龴P0.05㖞。综上所述,RFP耐药基因rpoB的突变主要发生在531、526、516、511位点,且92.82%的耐药株发生突变,进一步证实了结核分枝杆菌耐利福平与rpoB基因突变有关,同时也提示了还有其他的机制参与了利福平耐药。本研究所采用的以PCR为基础的反向点杂交技术,一次可检测多个位点的突变,与DNA序列分析的符合率可达97.67%,经х2检验,两者结果的差异在统计上无意义。该方法具有快速、灵敏、特异度高、操作简便、价格低廉等特点,可以适用于结核分枝杆菌临床分离株对利福平敏感性的大批量初筛实验,其作为基因芯片的技术基础,具有广阔的临床应用前景。
[Abstract]:Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) infection is caused by infectious factors leading to the death of a human being one of the most significant reasons. In recent years, with the increase of population, population density increased, the emergence of multiple drug-resistant strains of Mycobacterium tuberculosis and human immunodeficiency virus with double infection, more TB in developed countries and again the developing trend, leading to about 2 million deaths in [1]. with the rise in the number of TB each year in the world, multi drug resistant tuberculosis (Multi-Drug Resistance, Tuberculosis, MDR-TB) the incidence rate is rising. Li Fuping (Rifarmpicin, RFP) as one of the most effective anti tuberculosis drugs, the drug resistance, especially related to RFP drug resistance and other drug resistance, usually leads to clinical TB resurgence. Because isoniazid and Li Fuping are the most commonly used clinical medicine Things have reported about 90% of Mycobacterium tuberculosis resistant strains of Li Fuping also resistant to isoniazid alone, so Li Fuping sensitivity analysis may also be used as a screening index for [2-4]. multi drug resistance so the main anti tuberculosis drugs such as Li Fuping's resistance to rapid detection for effective treatment of tuberculosis and control of multidrug-resistant strains is essential.
To better establish oligonucleotide probe reverse dot blot hybridization method, this study from Tibet, Hunan, Henan, Sichuan, Fujian, Anhui and Shaanxi Province, collected seven clinical isolates of Mycobacterium tuberculosis from the first, using biochemical methods of species identification and drug sensitivity test was carried out using the proportion method. By using polymerase chain reaction (Polymerase Chain Reaction PCR), rpoB gene amplification "hotspot region" near the area DNA sequencing analysis, my understanding of the rpoB gene mutation of Mycobacterium tuberculosis, the clear distribution of mutations, mutation sites and the incidence of major mutations. Feature selection, design of detection probe, oligonucleotide probe reverse point to establish the rapid detection of Mycobacterium tuberculosis rpoB gene mutation of PCR based hybrid method. The experimental data were analyzed by SPSS 11 statistical software, explore the method can make The screening method is used to detect the sensitivity of Mycobacterium tuberculosis to rifampin, and to evaluate its clinical value.
First of all, using biochemical medium isolated from 536 strains of Mycobacterium tuberculosis clinical isolates were identified, 482 strains were identified as Mycobacterium tuberculosis (MTB), accounting for 89.93%; 19 strains of Mycobacterium tuberculosis (M. bovis), 3.54%; 33 strains were non tuberculous mycobacteria (NTM), 6.16%; 2 strains of Mycobacterium tuberculosis bacillus mixed non Mycobacterium tuberculosis infection, 0.37%.
For screening out of 482 clinical isolates of Mycobacterium tuberculosis drug sensitivity test was carried out using proportion method, the results showed that for all 5 kinds of anti tuberculosis drug sensitive strains accounted for 26.14% and 126 strains, 356 strains; drug resistance, drug resistance rate was 75.52%, among them, the single drug resistant strains of 88, 18.26%; 268 strains of multi drug resistant strains 55.60%..
Secondly, according to the related gene of Mycobacterium tuberculosis rifampin resistant rpoB gene primers of Mycobacterium tuberculosis standard strain H37Rv and 300 strains have clear background resistance of clinical isolates resistant gene mutation detection using DNA sequence analysis. The result of DNA sequence analysis, 181 strains of RFP resistant strains, 531 site mutations accounted for 53.61% of all mutations 526 mutation, 516 mutation accounted for 27.71%, accounted for 9.64%, accounted for 6.02% of the 511 mutation, four mutation frequency and mutation accounted for 96.99% of all types of mutations; multilocus mutation combined 15 strains accounted for 9.04% of all mutations, and found that 1 strains had the amino acid codon insertion and 5 strains of single the base or the entire amino acid codon deletion mutant.67 strains sensitive strains of mutations were found in 52 strains, RFP relatively sensitive strains, 2 strains had 511526 mutation.
On this basis, the use of reverse dot blot in 300 clinical isolates of Mycobacterium tuberculosis rpoB gene mutation analysis showed that 119 rifampin sensitive strains, 67 strains of sensitive strains had no mutation, 52 strains of RFP relatively sensitive strains in 2 strains detected mutations; 181 rifampin resistant strains, 165 strains were detected the mutation, no mutation was found in 16 strains. Compared with DNA sequencing, 168 strains in 164 strains were determined as resistant strains, 4 strains were determined as sensitive strains; 132 strains sequenced 129 strains were judged to be not found in sensitive strains mutation strains, 3 strains were determined as resistant strains. The sensitivity was 97.62% (164/168), the specificity was 97.73% (129/132), the positive predictive value was 98.20% (164/167), the negative predictive value was 96.99% (129/133). The detection results of 293 strains of reverse dot blot and DNA sequencing results are consistent, up to 97.67% with the two X 2 (293/300), after inspection, the difference was not statistically significant P0.0? 5.?
In summary, the mutation of RFP resistance gene of rpoB mainly occurred in 531526516511 sites, and 92.82% strains of resistance mutations, further confirmed that the rpoB gene mutation and rifampin resistance of Mycobacterium tuberculosis, but also suggests that there are other mechanisms involved in resistance to rifampin. Hybridization technique used in this study is based on PCR and reverse. A mutation detection of multiple loci, and DNA sequence analysis of the coincidence rate was 97.67%, the 2 were test results no difference in statistics. The method is rapid, sensitive, high specificity, simple operation, low price and other characteristics, can be applied to clinical isolates of Mycobacterium tuberculosis in the bulk of rifampicin sensitivity screening test, the gene chip technology, it has broad prospects of clinical application.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

【引证文献】