环介导等温扩增技术检测铜绿假单胞菌方法的建立及其应用

发布时间：2018-11-14 11:30

【摘要】：背景:铜绿假单胞菌作为自然界中常见的细菌,也是医院外科手术常见的感染病原体之一。临床现象表明铜绿假单胞菌在伤口感染时会形成绿色的脓液,从而诱发败血症、呼吸道感染以及尿路感染等。败血症主要发生于人体大面积烧伤以及心脏手术等,患者常伴有休克或者凝血等症状,患者皮肤上会出现坏死性脓包,通常会在患者的血管内有菌栓的形成;呼吸道感染最为常见的为借助机械呼吸而诱发的呼吸机相关性肺炎;截肢患者会因为置入导尿管而诱发尿路感染,长期使用抗生素治疗同样会诱发患者发生假单胞菌的感染。皮肤感染铜绿假单胞菌常见于机体受到损害而又潮湿的部位,例如婴儿的脐带周围、机体的烧伤部位以及潮湿的脚趾间等。在正常人体内发病率较低,当人体受到外界因素影响而导致抵抗力降低时,该菌可诱发严重的或者致死性的感染。目的:利用环介导等温扩增技术建立了铜绿假单胞菌的一种快速检测方法。方法:以铜绿假单胞菌gbca基因为模板,采用电脑在线引物设计的方法,来设计LAMP引物,利用反应体系的颜色变化及琼脂糖凝胶电泳的差异,检测分析铜绿假单菌,采用LAMP和PCR的方法同时检测不同菌株,考察LAMP方法的特异性和灵敏度。结果:本研究针对铜绿假单胞菌中的gbca基因设计LAMP序列,建立了铜绿假单胞菌的LAMP检测方法。与传统的PCR相比,LAMP不需要将DNA从样本中纯化出来,因此可以较为快速的应用临床样本的检测。但不可否认,LAMP技术也有一定的缺陷,例如相对于PCR而言,LAMP技术需要设计4条引物,其设计要求相对较高,同时,一旦有非特异性扩增的出现,会直观的反应在梯度条带上,误导样本的鉴别,而对于LAMP较为灵敏的特点,实验过程中较容易受到污染。为了更为直观的考察建立的LAMP方法对铜绿假单胞菌鉴定的适用情况,本研究还在反应体系中加入HNB染料,通过颜色变化来判定LAMP技术检测铜绿假单胞菌gbca基因,并对该方法的灵敏度以及特异性进行了考察。从实验结果能够看出:(1)颜色变化以及凝胶电泳的结果显示,只有铜绿假单胞菌的gbca基因在LAMP方法下发生了扩增反应,其他对照菌株没有发生LAMP扩增,说明该方法的特异性较好。(2)LAMP扩增结果显示与染料组凝胶电泳的结果一致,可以成功地检测在8.7 pg/ul浓度时铜绿假单胞菌的总DNA,而PCR法只能检测到浓度为87pg/ul的铜绿假单胞菌的总DNA,说明LAMP的检测灵敏度是PCR的10倍左右。同时采用LAMP和PCR检测临床收集的21份铜绿假单胞菌标本,其它10例对照菌也同样平行检测,结果表明LAMP和PCR的检出率是一致的(21/21),两种方法均未出现假阳性事件。结论:LAMP方法在检测铜绿假单胞菌时具有特异性强、灵敏度高等特点,适合于医疗卫生机构的推广。
[Abstract]:Background: Pseudomonas aeruginosa, as a common bacterium in nature, is also one of the common infectious pathogens in hospital surgery. Clinical manifestations show that Pseudomonas aeruginosa can form green pus in wound infection, which can induce sepsis, respiratory tract infection and urinary tract infection. Septicemia mainly occurs in human body extensive burn and heart surgery, patients often have shock or coagulation symptoms, the skin of patients will appear necrotic purulent, usually in the blood vessel of patients with bacterial thrombus formation; Respiratory tract infection is the most common type of ventilator-associated pneumonia induced by mechanical respiration. Amputation patients will cause urinary tract infection because of catheter placement. Long-term use of antibiotics will also induce pseudomonas infection. Pseudomonas aeruginosa is commonly found in damaged and moist parts of the body, such as around the umbilical cord, burns, and between wet toes. The incidence of the disease is low in the normal human body. When the body is affected by external factors and the resistance is reduced, the bacteria can induce severe or fatal infection. Objective: to establish a rapid detection method for Pseudomonas aeruginosa by ring-mediated isothermal amplification. Methods: using the gbca base of Pseudomonas aeruginosa as the template, the LAMP primers were designed by computer on-line primer design. Using the color change of the reaction system and the difference of agarose gel electrophoresis, the Pseudomonas aeruginosa was detected and analyzed. LAMP and PCR were used to detect different strains at the same time, and the specificity and sensitivity of LAMP method were investigated. Results: in this study, the LAMP sequence of gbca gene in Pseudomonas aeruginosa was designed, and the LAMP detection method for Pseudomonas aeruginosa was established. Compared with the traditional PCR, LAMP does not need to purify DNA from the samples, so it can be used to detect clinical samples more quickly. But it is undeniable that LAMP technology also has some defects. For example, compared with PCR, LAMP technology needs to design 4 primers, and the design requirements are relatively high. At the same time, once non-specific amplification occurs, it will react intuitively on the gradient band. The identification of misleading samples is more sensitive to LAMP, and it is easy to be contaminated in the process of experiment. In order to investigate more intuitively the applicability of the established LAMP method to the identification of Pseudomonas aeruginosa, HNB dyes were added to the reaction system to detect the gbca gene of Pseudomonas aeruginosa by LAMP technique. The sensitivity and specificity of the method were investigated. The results showed that: (1) the results of color change and gel electrophoresis showed that only the gbca gene of Pseudomonas aeruginosa was amplified by LAMP method, but no LAMP amplification was found in other control strains. (2) the results of LAMP amplification were consistent with those of dye group gel electrophoresis, and the total DNA, of Pseudomonas aeruginosa could be detected successfully at the concentration of 8.7 pg/ul. The total DNA, of Pseudomonas aeruginosa with concentration of 87pg/ul detected by PCR showed that the sensitivity of LAMP was about 10 times that of PCR. At the same time, 21 samples of Pseudomonas aeruginosa were detected by LAMP and PCR. The results showed that the detection rate of LAMP and PCR was the same (21 / 21). There were no false positive events in both methods. Conclusion: the LAMP method has the characteristics of high specificity and high sensitivity in the detection of Pseudomonas aeruginosa, and is suitable for the popularization of medical and health institutions.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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