本文選題：結(jié)核分枝桿菌 + 異煙肼耐藥��； 參考：《中國疾病預(yù)防控制中心》2010年碩士論文

【摘要】：近年來,結(jié)核病耐藥問題已成為全球公共衛(wèi)生關(guān)注的焦點(diǎn),尤其是耐多藥和廣泛耐藥。耐藥結(jié)核病的發(fā)生和流行成為當(dāng)今結(jié)核病防治工作的難題和最大挑戰(zhàn)。耐多藥結(jié)核病和廣泛耐藥結(jié)核病已成為嚴(yán)重威脅我國廣大人民群眾身體健康的傳染病之一。 異煙肼是常用的一線抗結(jié)核藥物,對(duì)病人的治療發(fā)揮重要作用,因此對(duì)異煙肼耐藥性研究和快速診斷很有必要,可以為結(jié)核病的防治提供科學(xué)依據(jù)。本研究的目的是建立TaqMan-MGB熒光定量PCR技術(shù)快速檢測(cè)結(jié)核病異煙肼耐藥,并初步應(yīng)用于臨床痰標(biāo)本,為臨床上大批量篩查提供基礎(chǔ)。 本研究選擇來自全國12個(gè)省市的274株結(jié)核分枝桿菌臨床分離株,經(jīng)比例法藥敏試驗(yàn),了解其耐藥概況,尤其是異煙肼耐藥。對(duì)異煙肼耐藥相關(guān)基因katG、pre-inhA、inhA、 ndh及oxyR-ahpC采用PCR擴(kuò)增及DNA測(cè)序,了解基因突變位點(diǎn)在中國結(jié)核分枝桿菌臨床菌株中的分布,篩選出對(duì)異煙肼耐藥貢獻(xiàn)率最大的基因突變位點(diǎn)。根據(jù)篩選出的基因位點(diǎn)設(shè)計(jì)熒光PCR探針和引物,建立TaqMan-MGB熒光定量PCR快速檢測(cè)結(jié)核桿菌katG315突變的方法。用130株結(jié)核分枝桿菌,16株非結(jié)核分枝桿菌和7株非分枝桿菌標(biāo)準(zhǔn)菌株及陽性質(zhì)粒標(biāo)準(zhǔn)品一起評(píng)價(jià)該方法的特異性、敏感性、重復(fù)性和最低檢測(cè)下限。此外用該方法初步應(yīng)用于161份確診病人的痰液和5份非結(jié)核病呼吸疾病患者的痰液。結(jié)果統(tǒng)計(jì)學(xué)分析采用卡方檢驗(yàn)。 274株結(jié)核分枝桿菌經(jīng)四種一線藥物(SM、RFP、INH、EMB)藥敏試驗(yàn)鑒定單耐藥56株,耐多藥152株,全敏感55株。222株耐藥菌株中異煙肼耐藥209株,其次利福平耐藥159株。209株INH耐藥株測(cè)序結(jié)果發(fā)現(xiàn)主要以katG基因、pre-inhA基因單堿基突變?yōu)橹?分別占64.58%、24.88%,同時(shí)篩選出對(duì)INH耐藥關(guān)系最大的katG315G→C突變,突變率達(dá)45.93%。 熒光定量PCR目的基因的最低檢測(cè)下限10copies/μl,低于常規(guī)PCR最低檢測(cè)下限的100倍,檢測(cè)16株非結(jié)核分枝桿菌標(biāo)準(zhǔn)株和7株非分枝桿菌標(biāo)準(zhǔn)株均為陰性,特異性100%。批內(nèi)批間CT值變異系數(shù)均小于1%,重復(fù)性好。該方法與130株菌株的藥敏試驗(yàn)相比符合率68.46%(89/130)、敏感性59%(59/100)、特異性100%(30/30)。與測(cè)序法相比,野生型和突變型探針的敏感性和特異性均為100%。 熒光定量PCR檢測(cè)166份痰液的敏感性84.47%,特異性100%。在確診病人的痰液中陽性檢出率84.47%,明顯高于痰涂片40.99%(x,2=46.44,P0.01),痰培養(yǎng)40.37%(χ2=47.89,P0.01), IS6110-PCR50.31%(x2=27.39, P0.01),且有統(tǒng)計(jì)學(xué)意義。其中熒光PCR在菌陽中檢出率94.44%,涂陰培陰的痰液中檢出率75.28%。檢測(cè)61份痰培養(yǎng)異煙肼耐藥時(shí),熒光PCR與傳統(tǒng)藥敏試驗(yàn)相比,兩種方法的符合率96.72%(59/61),敏感性33.3%(1/3),特異性100%(58/58)。 本研究建立TagMan-MGB熒光定量PCR的方法能特異、靈敏、快速檢測(cè)結(jié)核桿菌菌株或臨床痰液katG315G→C突變,同時(shí)能診斷痰液中的結(jié)核分枝桿菌,提高臨床疑似病人的陽性檢出率。而且該方法簡(jiǎn)單、操作簡(jiǎn)便、時(shí)間簡(jiǎn)短至3小時(shí)之內(nèi),結(jié)果可靠,可成為臨床診斷和治療的一種重要輔助手段。
[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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378.911;R3416

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相關(guān)期刊論文 前3條

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本文編號(hào)：2043960