TaqMan-MGB荧光定量PCR用于结核分枝杆菌异烟肼耐药的研究

发布时间：2018-06-20 10:34

本文选题：结核分枝杆菌 + 异烟肼耐药　；参考：《中国疾病预防控制中心》2010年硕士论文

【摘要】：近年来,结核病耐药问题已成为全球公共卫生关注的焦点,尤其是耐多药和广泛耐药。耐药结核病的发生和流行成为当今结核病防治工作的难题和最大挑战。耐多药结核病和广泛耐药结核病已成为严重威胁我国广大人民群众身体健康的传染病之一。异烟肼是常用的一线抗结核药物,对病人的治疗发挥重要作用,因此对异烟肼耐药性研究和快速诊断很有必要,可以为结核病的防治提供科学依据。本研究的目的是建立TaqMan-MGB荧光定量PCR技术快速检测结核病异烟肼耐药,并初步应用于临床痰标本,为临床上大批量筛查提供基础。本研究选择来自全国12个省市的274株结核分枝杆菌临床分离株,经比例法药敏试验,了解其耐药概况,尤其是异烟肼耐药。对异烟肼耐药相关基因katG、pre-inhA、inhA、 ndh及oxyR-ahpC采用PCR扩增及DNA测序,了解基因突变位点在中国结核分枝杆菌临床菌株中的分布,筛选出对异烟肼耐药贡献率最大的基因突变位点。根据筛选出的基因位点设计荧光PCR探针和引物,建立TaqMan-MGB荧光定量PCR快速检测结核杆菌katG315突变的方法。用130株结核分枝杆菌,16株非结核分枝杆菌和7株非分枝杆菌标准菌株及阳性质粒标准品一起评价该方法的特异性、敏感性、重复性和最低检测下限。此外用该方法初步应用于161份确诊病人的痰液和5份非结核病呼吸疾病患者的痰液。结果统计学分析采用卡方检验。 274株结核分枝杆菌经四种一线药物(SM、RFP、INH、EMB)药敏试验鉴定单耐药56株,耐多药152株,全敏感55株。222株耐药菌株中异烟肼耐药209株,其次利福平耐药159株。209株INH耐药株测序结果发现主要以katG基因、pre-inhA基因单碱基突变为主,分别占64.58%、24.88%,同时筛选出对INH耐药关系最大的katG315G→C突变,突变率达45.93%。荧光定量PCR目的基因的最低检测下限10copies/μl,低于常规PCR最低检测下限的100倍,检测16株非结核分枝杆菌标准株和7株非分枝杆菌标准株均为阴性,特异性100%。批内批间CT值变异系数均小于1%,重复性好。该方法与130株菌株的药敏试验相比符合率68.46%(89/130)、敏感性59%(59/100)、特异性100%(30/30)。与测序法相比,野生型和突变型探针的敏感性和特异性均为100%。荧光定量PCR检测166份痰液的敏感性84.47%,特异性100%。在确诊病人的痰液中阳性检出率84.47%,明显高于痰涂片40.99%(x,2=46.44,P0.01),痰培养40.37%(χ2=47.89,P0.01), IS6110-PCR50.31%(x2=27.39, P0.01),且有统计学意义。其中荧光PCR在菌阳中检出率94.44%,涂阴培阴的痰液中检出率75.28%。检测61份痰培养异烟肼耐药时,荧光PCR与传统药敏试验相比,两种方法的符合率96.72%(59/61),敏感性33.3%(1/3),特异性100%(58/58)。本研究建立TagMan-MGB荧光定量PCR的方法能特异、灵敏、快速检测结核杆菌菌株或临床痰液katG315G→C突变,同时能诊断痰液中的结核分枝杆菌,提高临床疑似病人的阳性检出率。而且该方法简单、操作简便、时间简短至3小时之内,结果可靠,可成为临床诊断和治疗的一种重要辅助手段。
[Abstract]:In recent years, the problem of tuberculosis resistance has become the focus of global public health concern, especially multi drug resistance and widespread resistance. The occurrence and epidemic of drug-resistant tuberculosis has become a difficult problem and the biggest challenge for the prevention and treatment of tuberculosis. One of the infectious diseases.
Isoniazid is a commonly used anti tuberculosis drug which plays an important role in the treatment of patients. Therefore, it is necessary to study and quickly diagnose isoniazid resistance and provide a scientific basis for the prevention and control of tuberculosis. The purpose of this study is to establish a TaqMan-MGB fluorescence quantitative PCR technique for rapid detection of isoniazid resistance in tuberculosis and its preliminary application. Clinical sputum specimens provide the basis for mass screening in clinical practice.
This study selected 274 clinical isolates of Mycobacterium tuberculosis from 12 provinces and cities throughout the country. The drug resistance of isoniazid, especially isoniazid resistance related genes, katG, pre-inhA, inhA, ndh and oxyR-ahpC, was used to understand the drug resistance of isoniazid, and the gene mutation site was detected by PCR amplification and DNA sequence to understand the gene mutation site in Mycobacterium tuberculosis in China. The distribution of clinical strains screened out the gene mutation site with the largest contribution to isoniazid resistance. Based on the selected gene loci, a fluorescent PCR probe and primers were designed to establish a TaqMan-MGB fluorescence quantitative PCR method for rapid detection of katG315 mutation of tuberculosis bacillus. 130 strains of Mycobacterium tuberculosis, 16 non tuberculous mycobacteria and 7 non branching strains were used. The standard strain of bacilli and the positive plasmid standard were used together to evaluate the specificity, sensitivity, repeatability and minimum detection limit of the method. In addition, the method was preliminarily applied to sputum of 161 confirmed patients and 5 patients with non tuberculosis respiratory diseases.
274 strains of Mycobacterium tuberculosis were tested by four front-line drugs (SM, RFP, INH, EMB), 56 strains of single drug resistance, 152 resistant to multidrug resistance, 209 of isoniazid resistant strains of all sensitive.222 strains, followed by sequencing results of INH resistant strains of rifampin resistant 159.209 strains, which were mainly katG gene and single base mutation of pre-inhA gene, accounting for 64, respectively. .58%, 24.88%, and screened the katG315G - C mutation with the largest resistance to INH, with a mutation rate of 45.93%.
The lowest detection limit of the fluorescent quantitative PCR target gene is 10copies/ Mu L, which is 100 times lower than that of the conventional PCR minimum detection limit. 16 standard strains of non Mycobacterium tuberculosis and 7 non Mycobacterium strain standard strains are all negative. The CT value variation coefficient in the specific 100%. batch is less than 1% and the reduplication is good. This method and the drug sensitivity test of the 130 strain strains The specific coincidence rate was 68.46% (89/130), sensitivity 59% (59/100) and specificity 100% (30/30). Compared with sequencing, the sensitivity and specificity of wild and mutant probes were 100%.
The sensitivity of 166 sputum by fluorescence quantitative PCR was 84.47%, and the positive rate of specific 100%. was 84.47% in the sputum of the confirmed patients, which was significantly higher than that of sputum smear 40.99% (x, 2=46.44, P0.01), sputum culture 40.37% (x 2=47.89, P0.01), IS6110-PCR50.31% (x2=27.39, P0.01), and statistically significant. The detection rate of fluorescent PCR in bacteria was 94.44%, smear Yin. When the detection rate of sputum in the sputum was detected by 75.28%., 61 phlegm cultures of isoniazid resistance were detected. Compared with the traditional drug sensitivity test, the coincidence rate of the two methods was 96.72% (59/61), the sensitivity 33.3% (1/3), and the specificity 100% (58/58).
The method of establishing TagMan-MGB fluorescence quantitative PCR in this study can be specific, sensitive and rapid detection of katG315G to C mutation in Mycobacterium tuberculosis or clinical sputum, and can diagnose Mycobacterium tuberculosis in sputum and improve the positive detection rate of clinical suspected patients. Moreover, this method is simple and simple, and the time is short to 3 hours, and the result is reliable. It has become an important auxiliary means for clinical diagnosis and treatment.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.911;R3416

【参考文献】