结核分枝杆菌吡嗪酰胺耐药性的噬菌体检测技术与耐药基因突变研究

发布时间：2018-05-05 20:18

本文选题：分枝杆菌 + 结核　；参考：《华东师范大学》2006年硕士论文

【摘要】：结核病(Tuberculosis，TB)是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性传染性疾病，是现在世界上最普遍和最严重的人类传染病之一，也是全世界由单一致病菌致死最多的疾病之一。由于耐药性结核杆菌的出现与传播、艾滋病的流行以及人口的频繁流动等原因，结核病疫情呈现全球性的回升。吡嗪酰胺(Pyrazinamide，PZA)是治疗结核病最有效的一线药物之一，快速准确的PZA耐药性检测技术对临床治疗及控制结核病具有重要的意义。吡嗪酰胺耐药的精确的分子机理还未被阐明，对吡嗪酰胺耐药相关基因pncA的突变进行研究有很大的理论及应用参考意义。【目的】本研究旨在建立起结核分枝杆菌吡嗪酰胺耐药性的噬菌体生物扩增(Phage Amplified Biologically Assay，PhaB)快速检测方法，并探讨和评价所建方法的实际应用价值；同时对结核分枝杆菌PZA耐药菌株的PZA耐药相关基因pncA的突变进行研究，对PZA耐药机制进行初步探索，为以后进行PZA耐药机制研究提供相关实验基础。【方法】应用PhaB法，对液体培养基的pH值、PZA药物浓度、药物作用时间进行一系列实验，通过正交法筛选和优化检测条件，并对不同的噬菌体侵染环境的恢复方法进行比较来确定最佳恢复方法；应用所建方法对109株结核分枝杆菌临床分离株进行PZA耐药性的测定，并将检测结果与常规药敏检测方法结果进行比较，对两种方法检测结果不相符的菌株进行最低抑菌浓度(minimum inhibitory concentration，MIC)测定，以探讨和评价所建方法的应用价值；将结核分枝杆菌PZA耐药株的耐药相关基因pncA进行PCR扩增后对全基因进行测序，对pncA基因突变进行研究。【结果】 PhaB法检测PZA耐药性的最佳条件为液体培养基pH值为5.5、PZA药物浓度200ug／ml、37℃作用48小时，确定离心洗涤为噬菌体侵染环境的恢复方法。对109株结核分枝杆菌临床分离株进行PZA耐药性测定，常规绝对浓度药敏法检测结果为敏感35株，耐药74株；PhaB法检测结果为敏感34株，耐药75株，与绝对浓度法检测结果的符合率为91.7％，对两种检测方法不相符的9株菌株进行MIC测定，结果6株(3株敏感，3株耐药)与PhaB法相符合，如以绝对浓度法测定PZA耐药性结果为考核标准，，则PhaB法检测PZA耐药性的敏感性为94.6％(70／74)、特异性为85.7％(30／35)、阳性预测值为93.3％(70／75)、阴性预测值为88.2％(30／34)、准确性为91.7％(100／109)。与常规药敏方法耗时长达6-8周相比，PhaB法检测PZA耐药性仅需2-3天，但与常规药敏方法符合率高且实验结果重复性好。对47株PZA耐药菌株进行pncA基因测序，结果19株的pncA基因存在各种不同形式的突变，突变率达40.43％，在所有突变形式中，除已经见报道的突变形式外，我们还发现了22个新的突变位点，这22种新的突变位点形成12种新的突变形式。【结论】 PhaB法检测结核分枝杆菌PZA耐药性具有快速、操作简便、与常规药敏方法符合率较高等特点，且不需要特殊仪器设备，所用试剂也是一般常
[Abstract]:Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). It is one of the most common and most serious human infectious diseases in the world. It is also one of the most fatal diseases in the world. The epidemic and the frequent flow of population show a global recovery. Pyrazinamide (PZA) is one of the most effective first-line drugs for the treatment of tuberculosis. Rapid and accurate detection of PZA resistance is of great importance to the clinical treatment and control of tuberculosis. The precise molecular mechanism of the drug resistance of the drug is the exact molecular mechanism of the drug resistance. It has not been elucidated yet. It is of great theoretical and practical significance to study the mutation of pncA.
[Objective] to establish a rapid detection method of Phage Amplified Biologically Assay (PhaB) for the drug resistance of Mycobacterium tuberculosis, and to explore and evaluate the practical application value of the method, and the mutation of PZA resistance related gene pncA of Mycobacterium tuberculosis resistant strain of Mycobacterium tuberculosis. The aim of this study is to explore the mechanism of PZA resistance and provide experimental basis for future research on the mechanism of PZA resistance.
[Methods] a series of experiments were carried out on the pH value of liquid medium, the concentration of PZA drug and the time of drug action by using PhaB method. The optimum recovery method was compared by orthogonal method and the methods of different phage infection environment were compared to determine the best recovery method. 109 strains of Mycobacterium tuberculosis were used in clinical practice. The isolates were tested for PZA resistance and compared with the results of conventional drug sensitivity test. The minimum inhibitory concentration (minimum inhibitory concentration, MIC) of the two strains which were not consistent with the test results were measured to explore and evaluate the application value of the established method, and the PZA resistant strains of Mycobacterium tuberculosis were used. The pncA gene was sequenced by PCR amplification, and the pncA gene mutation was studied.
[results] the best conditions for the detection of PZA resistance by PhaB were that the pH value of liquid medium was 5.5, the concentration of PZA was 200ug / ml, and the effect of 37 degrees centigrade was 48 hours. The method of centrifugal washing as the phage infection environment was determined. The drug resistance of 109 Mycobacterium tuberculosis clinical isolates was determined by PZA resistance, and the routine absolute concentration drug sensitivity test results were sensitive. There were 35 strains and 74 resistant strains. The results of PhaB detection were 34 sensitive, 75, 91.7%, and 9 strains which were not consistent with two detection methods. The results of 6 strains (3 sensitive, 3 resistant) were in conformity with the PhaB method, for example, the determination of the result of PZA resistance by absolute concentration method was P, then P The sensitivity of the haB method was 94.6% (70 / 74), the specificity was 85.7% (30 / 35), the positive predictive value was 93.3% (70 / 75), the negative predictive value was 88.2% (30 / 34), and the accuracy was 91.7%. Compared with the conventional drug sensitivity method, the PhaB method was only required to detect the PZA resistance, but it was with the conventional drug sensitivity method. The pncA gene was sequenced in 47 strains of PZA resistant strains, and the results of the 19 strains of pncA gene had various forms of mutation, the mutation rate was 40.43%. In all the mutation forms, we also found 22 new mutation sites except the reported mutation, and the 22 new mutation sites formed 1. 2 new forms of mutation.
[Conclusion] the detection of PZA resistance of Mycobacterium tuberculosis by PhaB is rapid, easy to operate, high in coincidence with conventional drug sensitivity methods, and does not require special instruments and equipment, and the reagents used are generally common.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378.911

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