本文選題：分枝桿菌 + 結(jié)核　； 參考：《華東師范大學》2006年碩士論文

【摘要】：結(jié)核病(Tuberculosis，TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis)引起的一種慢性傳染性疾病，是現(xiàn)在世界上最普遍和最嚴重的人類傳染病之一，也是全世界由單一致病菌致死最多的疾病之一。由于耐藥性結(jié)核桿菌的出現(xiàn)與傳播、艾滋病的流行以及人口的頻繁流動等原因，結(jié)核病疫情呈現(xiàn)全球性的回升。吡嗪酰胺(Pyrazinamide，PZA)是治療結(jié)核病最有效的一線藥物之一，快速準確的PZA耐藥性檢測技術(shù)對臨床治療及控制結(jié)核病具有重要的意義。吡嗪酰胺耐藥的精確的分子機理還未被闡明，對吡嗪酰胺耐藥相關(guān)基因pncA的突變進行研究有很大的理論及應用參考意義。 【目的】 本研究旨在建立起結(jié)核分枝桿菌吡嗪酰胺耐藥性的噬菌體生物擴增(Phage Amplified Biologically Assay，PhaB)快速檢測方法，并探討和評價所建方法的實際應用價值；同時對結(jié)核分枝桿菌PZA耐藥菌株的PZA耐藥相關(guān)基因pncA的突變進行研究，對PZA耐藥機制進行初步探索，為以后進行PZA耐藥機制研究提供相關(guān)實驗基礎。 【方法】 應用PhaB法，對液體培養(yǎng)基的pH值、PZA藥物濃度、藥物作用時間進行一系列實驗，通過正交法篩選和優(yōu)化檢測條件，并對不同的噬菌體侵染環(huán)境的恢復方法進行比較來確定最佳恢復方法；應用所建方法對109株結(jié)核分枝桿菌臨床分離株進行PZA耐藥性的測定，并將檢測結(jié)果與常規(guī)藥敏檢測方法結(jié)果進行比較，對兩種方法檢測結(jié)果不相符的菌株進行最低抑菌濃度(minimum inhibitory concentration，MIC)測定，以探討和評價所建方法的應用價值；將結(jié)核分枝桿菌PZA耐藥株的耐藥相關(guān)基因pncA進行PCR擴增后對全基因進行測序，對pncA基因突變進行研究。 【結(jié)果】 PhaB法檢測PZA耐藥性的最佳條件為液體培養(yǎng)基pH值為5.5、PZA藥物濃度200ug／ml、37℃作用48小時，確定離心洗滌為噬菌體侵染環(huán)境的恢復方法。對109株結(jié)核分枝桿菌臨床分離株進行PZA耐藥性測定，常規(guī)絕對濃度藥敏法檢測結(jié)果為敏感35株，耐藥74株；PhaB法檢測結(jié)果為敏感34株，耐藥75株，與絕對濃度法檢測結(jié)果的符合率為91.7％，對兩種檢測方法不相符的9株菌株進行MIC測定，結(jié)果6株(3株敏感，3株耐藥)與PhaB法相符合，如以絕對濃度法測定PZA耐藥性結(jié)果為考核標準，，則PhaB法檢測PZA耐藥性的敏感性為94.6％(70／74)、特異性為85.7％(30／35)、陽性預測值為93.3％(70／75)、陰性預測值為88.2％(30／34)、準確性為91.7％(100／109)。與常規(guī)藥敏方法耗時長達6-8周相比，PhaB法檢測PZA耐藥性僅需2-3天，但與常規(guī)藥敏方法符合率高且實驗結(jié)果重復性好。對47株P(guān)ZA耐藥菌株進行pncA基因測序，結(jié)果19株的pncA基因存在各種不同形式的突變，突變率達40.43％，在所有突變形式中，除已經(jīng)見報道的突變形式外，我們還發(fā)現(xiàn)了22個新的突變位點，這22種新的突變位點形成12種新的突變形式。 【結(jié)論】 PhaB法檢測結(jié)核分枝桿菌PZA耐藥性具有快速、操作簡便、與常規(guī)藥敏方法符合率較高等特點，且不需要特殊儀器設備，所用試劑也是一般常
[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.

【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R378.911

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