本文關(guān)鍵詞： 結(jié)核病 Taqman PCR 結(jié)核分枝桿菌 支氣管肺泡灌洗液　出處：《吉林大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：結(jié)核�。═uberculosis, TB）是一種古老的慢性傳染病，是由結(jié)核桿菌引起的一種人畜共患傳染病。據(jù)世界衛(wèi)生組織在2009年發(fā)布的最新報(bào)告，每年有940萬(wàn)新增病例，1400萬(wàn)現(xiàn)患病例，其中艾滋病毒陰性者有130萬(wàn)死亡，而艾滋病毒抗體陽(yáng)性者有38萬(wàn)死亡。目前我國(guó)活動(dòng)性肺結(jié)核病人約450萬(wàn)，而患病人數(shù)居世界第二位，僅次于印度,嚴(yán)重影響國(guó)計(jì)民生。耐多藥結(jié)核病及廣泛耐藥結(jié)核病的病例呈現(xiàn)上升趨勢(shì)，結(jié)核病在全球已拉起了警報(bào)。在這種情況下，新的有效藥物對(duì)抗復(fù)制或潛在的病菌、更好的疫苗以及新的診斷方法迫切需要改變和克服。 近年，因現(xiàn)有結(jié)核病病原學(xué)診斷工具在檢測(cè)敏感性和特異性方面有限，臨床需要建立快速診斷病原體的方法以指導(dǎo)臨床早期準(zhǔn)確用藥。傳統(tǒng)實(shí)驗(yàn)室診斷方法存在不同方面的缺陷，對(duì)疾病早期診斷及準(zhǔn)確治療造成影響。聚合酶鏈反應(yīng)（Polymerase Chain Reaction PCR）技術(shù)為基礎(chǔ)的檢測(cè)方法逐步應(yīng)用于結(jié)核病病原體的檢測(cè)。目前在傳統(tǒng)PCR技術(shù)基礎(chǔ)上發(fā)展了更先進(jìn)的實(shí)時(shí)熒光PCR技術(shù)，如Taqman探針PCR。Taqman探針PCR是集PCR高敏感性、DNA雜交探針的高特異性和光譜技術(shù)為一體，擴(kuò)增目標(biāo)基因可以實(shí)時(shí)出現(xiàn)，整個(gè)過(guò)程能夠1-2小時(shí)完成。因此，我們旨在運(yùn)用實(shí)時(shí)熒光PCR技術(shù)，建立和評(píng)估結(jié)核桿菌Taqman探針PCR檢測(cè)方法。 對(duì)象與方法：臨床研究對(duì)象依次選擇從2010年9月至2011年1月43例吉林大學(xué)第一醫(yī)院的非結(jié)核病患者和132例長(zhǎng)春市傳染病院的結(jié)核病患者。收集患者痰及支氣管肺泡灌洗液（BALF）于-70℃保存待檢。結(jié)核病的診斷依據(jù)痰結(jié)核菌培養(yǎng)結(jié)果。根據(jù)國(guó)內(nèi)外文獻(xiàn)報(bào)道，選擇結(jié)核桿菌復(fù)合群IS6110插入序列及人型結(jié)核桿菌12.7kb序列，設(shè)計(jì)并合成引物及探針，建立雙重實(shí)時(shí)熒光PCR的檢測(cè)方法，并對(duì)其反應(yīng)的特異性、敏感性等進(jìn)行了實(shí)驗(yàn)驗(yàn)證，建立了結(jié)核桿菌實(shí)時(shí)熒光PCR檢測(cè)方法。 針對(duì)兩種樣本的涂片、組織活檢病理及實(shí)時(shí)熒光PCR3種檢測(cè)方法的陽(yáng)性率及方法差異性進(jìn)行評(píng)估。本研究結(jié)果表明，132份結(jié)核性樣本中，痰涂片、組織病理及雙重實(shí)時(shí)熒光PCR檢測(cè)（痰、支氣管肺泡灌洗液）陽(yáng)性率分別為11.4%、33.3%、50%、66.7%，43例非結(jié)核性樣本中，雙重實(shí)時(shí)熒光PCR檢測(cè)結(jié)果均為陰性。痰涂片、肺泡灌洗液涂片及組織病理分別與雙重實(shí)時(shí)熒光P CR方法比較，數(shù)據(jù)經(jīng)統(tǒng)計(jì)學(xué)處理，P=0.000（P0.05），證實(shí)差異有顯著意義。雙重實(shí)時(shí)熒光PCR分別檢測(cè)痰及支氣管肺泡灌洗液兩種樣本，，數(shù)據(jù)經(jīng)統(tǒng)計(jì)學(xué)處理，P=0.006（P0.05）,證實(shí)差異有顯著意義。結(jié)果證明所建立的實(shí)時(shí)熒光PCR優(yōu)于其余三種檢查方法，且對(duì)支氣管肺泡灌洗液的檢測(cè)優(yōu)于痰，能夠用于結(jié)核桿菌的檢測(cè)。
[Abstract]:Tuberculosis (Tuberculosis, TB) is a chronic infectious disease old, is caused by Mycobacterium tuberculosis is a zoonotic infectious disease. According to the latest report released by the WHO in 2009, with 9 million 400 thousand new cases a year, 14 million of the cases, there were 1 million 300 thousand deaths among HIV negative and HIV positive persons. 380 thousand. The death of active pulmonary tuberculosis patients in China about 4 million 500 thousand, while the number of patients ranked second in the world, second only to India, seriously affected. Beneficial to the people's livelihood of MDR-TB and XDR-TB cases increasing, TB has raised alarm in the world. In this case, a new effective drugs against bacteria copy or potential, better vaccines and diagnostic methods of new urgent need to change and overcome.
In recent years, due to the existing tuberculosis pathogen diagnostic tools Co. in the detection sensitivity and specificity, establishment of a rapid method for diagnosis of pathogens of clinical need to guide clinical medication. Early accurate diagnosis method of traditional laboratory defects in different aspects, the impact on the accurate and early diagnosis and treatment of disease. The polymerase chain reaction (Polymerase Chain Reaction PCR) detection method based technology has been gradually applied to tuberculosis pathogen. At present on the basis of traditional PCR development of a real-time fluorescent PCR technology is more advanced, such as the Taqman probe PCR.Taqman probe PCR is the set of PCR Gao Min sensitivity, high specificity and spectroscopy of DNA hybridization probe as a whole, the amplified target gene can appear in real time, the whole process can 1-2 hours to complete. Therefore, we aim to use real-time PCR technology, establishment and evaluation of Mycobacterium tuberculosis Taqman PCR probe. Measurement method.
Subjects and methods: the clinical study selected from September 2010 to January 2011 43 cases of No.1 Hospital of Jilin University of non tuberculosis patients and 132 cases of Changchun infectious hospital patients with tuberculosis were collected. Sputum and bronchoalveolar lavage fluid (BALF) at -70 DEG C to be detected. The preserved diagnosis of tuberculosis disease according to sputum culture. According to the literatures at home and abroad and the choice of Mycobacterium tuberculosis complex and IS6110 insertion sequence of Mycobacterium tuberculosis 12.7kb sequence, the primers and probes were designed, establish a detection method of dual real-time PCR, and the specificity of the reaction, the sensitivity experiment method was established for detection of Mycobacterium tuberculosis by real-time PCR.
For the two kinds of samples were assessed with the positive rate of smear and biopsy method and real-time fluorescent PCR3 method for detecting the differences. The research results showed that the samples from 132 cases of tuberculosis, sputum smear, biopsy and double fluorescent real-time PCR detection (sputum, bronchoalveolar lavage fluid) positive rates were 11.4%. 33.3%, 50%, 66.7%, 43 cases of non tuberculous samples, dual real-time PCR test results were negative. Sputum smear, bronchoalveolar lavage fluid smear and histopathology were compared with dual real-time P CR method, data by statistical processing, P=0.000 (P0.05), significant differences were detected in sputum and confirmed. Bronchoalveolar lavage fluid samples of two kinds of dual real-time PCR data by statistical processing, P=0.006 (P0.05), there was significant difference. Real time PCR is better than that of the results proved that the established three examination methods, and the gas branch The detection of pulmonary alveolar lavage fluid is superior to phlegm and can be used for the detection of Mycobacterium tuberculosis.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R378

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