结核杆菌Taqman PCR检测方法的建立及评估

发布时间：2018-02-03 02:15

本文关键词： 结核病 Taqman PCR 结核分枝杆菌支气管肺泡灌洗液　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：结核病（Tuberculosis, TB）是一种古老的慢性传染病，是由结核杆菌引起的一种人畜共患传染病。据世界卫生组织在2009年发布的最新报告，每年有940万新增病例，1400万现患病例，其中艾滋病毒阴性者有130万死亡，而艾滋病毒抗体阳性者有38万死亡。目前我国活动性肺结核病人约450万，而患病人数居世界第二位，仅次于印度,严重影响国计民生。耐多药结核病及广泛耐药结核病的病例呈现上升趋势，结核病在全球已拉起了警报。在这种情况下，新的有效药物对抗复制或潜在的病菌、更好的疫苗以及新的诊断方法迫切需要改变和克服。近年，因现有结核病病原学诊断工具在检测敏感性和特异性方面有限，临床需要建立快速诊断病原体的方法以指导临床早期准确用药。传统实验室诊断方法存在不同方面的缺陷，对疾病早期诊断及准确治疗造成影响。聚合酶链反应（Polymerase Chain Reaction PCR）技术为基础的检测方法逐步应用于结核病病原体的检测。目前在传统PCR技术基础上发展了更先进的实时荧光PCR技术，如Taqman探针PCR。Taqman探针PCR是集PCR高敏感性、DNA杂交探针的高特异性和光谱技术为一体，扩增目标基因可以实时出现，整个过程能够1-2小时完成。因此，我们旨在运用实时荧光PCR技术，建立和评估结核杆菌Taqman探针PCR检测方法。对象与方法：临床研究对象依次选择从2010年9月至2011年1月43例吉林大学第一医院的非结核病患者和132例长春市传染病院的结核病患者。收集患者痰及支气管肺泡灌洗液（BALF）于-70℃保存待检。结核病的诊断依据痰结核菌培养结果。根据国内外文献报道，选择结核杆菌复合群IS6110插入序列及人型结核杆菌12.7kb序列，设计并合成引物及探针，建立双重实时荧光PCR的检测方法，并对其反应的特异性、敏感性等进行了实验验证，建立了结核杆菌实时荧光PCR检测方法。针对两种样本的涂片、组织活检病理及实时荧光PCR3种检测方法的阳性率及方法差异性进行评估。本研究结果表明，132份结核性样本中，痰涂片、组织病理及双重实时荧光PCR检测（痰、支气管肺泡灌洗液）阳性率分别为11.4%、33.3%、50%、66.7%，43例非结核性样本中，双重实时荧光PCR检测结果均为阴性。痰涂片、肺泡灌洗液涂片及组织病理分别与双重实时荧光P CR方法比较，数据经统计学处理，P=0.000（P0.05），证实差异有显著意义。双重实时荧光PCR分别检测痰及支气管肺泡灌洗液两种样本，，数据经统计学处理，P=0.006（P0.05）,证实差异有显著意义。结果证明所建立的实时荧光PCR优于其余三种检查方法，且对支气管肺泡灌洗液的检测优于痰，能够用于结核杆菌的检测。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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