基于颜色判定的环介导等温扩增技术快速检测铜绿假单胞菌及其OprD2耐药基因的研究

发布时间：2018-11-17 12:34

【摘要】：目的铜绿假单胞菌(Pseudomonas aeruginosa，PAE)为条件致病菌，分布广泛，在正常情况下不致病。但当人体免疫力降低时(如：长期应用激素、肿瘤化疗、AIDS)或严重创伤(如大面积烧伤、大型手术)或医疗操作(如气管切开、插管)后，PAE引起的感染性疾病不断增加。随着抗生素的广泛使用，耐药铜绿假单胞菌的发生率增多，特别是多重耐药菌，使临床上治疗该病原菌引起的相关感染变得越来越来困难。然而传统的细菌学检查方法时间长、敏感性低，一直不能满足临床诊治的需求。因此建立了一种新的、简单、快速的现场检测方法，对铜绿假单胞菌及耐药性的早期诊断和治疗具有重大意义。本研究目的：了解临床标本中分离的铜绿假单胞菌对16种抗菌药物的耐药情况，为临床合理使用抗生素提供参考。对多重耐药铜绿假单胞菌进行相关耐药基因的研究。建立一种基于颜色判定的，能够快速准确地检测铜绿假单胞菌及其OprD2耐药基因的LAMP方法。方法对临床收集47株耐药铜绿假单胞菌采用K-B纸片扩散法测定对庆大霉素等16种抗菌药物的敏感性。PCR扩增检测收集的铜绿假单胞菌标本中的耐亚胺培南类IMP、VIM、GES、GIM、OXA-10、OprD2耐药基因。针对铜绿假单胞菌OprL、OprD2基因设计LAMP引物,通过引物特异性识别OprL、OprD2的6个独立区域来快速检测铜绿假单胞菌特有OprL基因和OprD2耐药基因。如果在反应前添加荧光染料羟基萘酚蓝(HNB)或钙黄绿素(Calcein)，其结果可以通过肉眼观察反应前后颜色变化来判定并经琼脂糖凝胶电泳验证。对该技术进行特异性、灵敏度分析，，用LAMP扩增法、PCR扩增法和临床传统方法同时检测临床标本。结果 1.47株耐药PAE对头孢噻肟耐药率最高占97.87%，左旋氧氟沙星耐药率占40.42%，氨曲南耐药率占38.30%，哌拉西林耐药率34.04%，美罗培南耐药率占29.79％，亚胺培南、环丙沙星、头孢哌酮的耐药率占27.66％。 2.47株PAE中VIM基因阳性12株，阳性率25.53%，OXA-10基因阳性2株，阳性率4.26%，OprD2基因缺失23株，缺失率占48.94%，而IMP、GIM、GES基因均阴性。OprD2基因缺失株的头孢噻肟、左氧氟沙星、氨曲南、哌拉西林、亚胺培南和美罗培南耐药率分别为100%(23/23)、56.52%(13/23)、47.83%(11/23)、47.83%(11/23)、47.83%(11/23)和43.48%(10/23)。统计学分析结果显示：与OprD2基因阳性株相比，OprD2基因缺失株的亚胺培南、左旋氧氟沙星、美洛培南耐药比例明显升高(卡方值为9.155、4.846、4.037，P值为0.002、0.028、0.045，差异有统计学意义)。 3.设计出针对铜绿假单胞菌OprL基因、OprD2耐药基因可直接肉眼判读结果的等温扩增检测方法。本等温扩增检测法只扩增铜绿假单胞菌，对其他菌不扩增，显示出良好的特异性。其最低检测限为17.414pg/μl, PCR方法最低检出限为174.414pg/μl, LAMP方法检测灵敏度是PCR方法检测灵敏度的10倍,灵敏度高。LAMP法与PCR法对临床铜绿假单胞菌OprL基因和OprD2耐药基因检出率相当。结论 1．临床铜绿假单胞菌耐药情况严重，耐药铜绿假单胞菌OprD2基因缺失较为普遍，47株中缺失23株占(48.94%)。OprD2基因缺失导致铜绿假单胞菌以亚胺培南为主的耐药，因此明确OprD2基因分布状况有助于临床抗菌药物的选用。 2．LAMP方法用于快速检测铜绿假单胞菌具有检测过程简单、实验装置简便、反应结果肉眼可辨别、灵敏度高和特异性强等特点，特别适合用于现场和基层检疫及医疗单位的快速诊断。本课题成功建立针对铜绿假单胞菌OprL基因及其OprD2耐药基因可直接肉眼判读检测结果的LAMP技术。
[Abstract]:Purpose Pseudomonas aeruginosa (PAE) is a condition pathogenic bacteria and has a wide distribution. Under normal conditions, it does not When the immunity of the human body is reduced (such as long-term application of hormone, tumor chemotherapy, AIDS) or severe trauma (such as large-area burn, large-scale operation) or medical operation (such as tracheotomy and intubation), the infectious disease caused by PAE is In addition, with the extensive use of antibiotics, the incidence of drug-resistant Pseudomonas aeruginosa is increased, especially the multiple drug-resistant bacteria, so that the clinical treatment of the related infection caused by the pathogenic bacteria becomes more and more However, the traditional bacteriological examination method has long time and low sensitivity, and has been unable to meet the clinical diagnosis and treatment. Therefore, a new, simple and rapid field detection method is established, which is of great significance to the early diagnosis and treatment of P. aeruginosa and drug resistance. Significance. The purpose of this study was to understand the drug resistance of the isolated P. aeruginosa to 16 antibacterial drugs in clinical specimens. Reference. Resistance to multiple drug-resistant Pseudomonas aeruginosa A study on the gene of Pseudomonas aeruginosa and its OprD2-resistant gene can be quickly and accurately detected based on color determination. AM P method. The method was used to collect 47 strains of drug-resistant Pseudomonas aeruginosa, and the method of K-B paper diffusion was used to determine the gentamycin and the like. Sensitivity of 16 antimicrobial agents. Iimipenem-resistant species IMP, VIM, GES, GIM, OXA-1 in the samples of P. aeruginosa collected by PCR amplification 0. OprD2-resistant gene. The LAMP primer was designed for the OprL and OprD2 genes of P. aeruginosa, and the specific OprL group of P. aeruginosa was quickly detected by the primer-specific identification of the 6 independent regions of OprL and OprD2. The result can be determined by observing the color change before and after the reaction by the naked eye if the fluorescent dye hydroxyethylphenol blue (HNB) or calcein is added before the reaction. and through agarose gel electrophoresis, the specificity, the sensitivity analysis, the LAMP amplification method, the PCR amplification method and the clinical transmission are carried out on the technology. the system Results 1. The drug-resistant PAE of 1. 47 strains had the highest drug-resistance rate of 97. 87%, the resistance rate of levofloxacin was 40. 42%, the drug-resistant rate of amcinolone was 38. 30%, the drug-resistant rate was 34. 04%, and the drug-resistant rate of melange was 29. 79%. The resistance rate of Ceftrione was 27. 66%. The positive rate of VIM gene in PAE was 25.53%, the positive rate of OXA-10 gene was 25.53%, the positive rate of OXA-10 gene was 4.26%, and that of OprD2 gene was 23, and the deletion rate was 48. 9. The drug resistance of the OrD2 gene was 100% (23/ 23), 56. 52% (13/ 23), 47. 83% (11/ 23), 47. 83% (11/ 23), 47. 83% (11/ 23), 47. 83% (11/ 23), 47. 83% (11/ 23), 47. 83% (11/ 23), 47. 83% (11/ 23), 47. 83% (11/ 23), and 47. 83% (11/ 23), respectively. (11/ 23) and 43. 48% (10/ 23). The statistical analysis showed that the resistance of the OprD2 gene was significantly higher than that of the OprD2 gene (9.155, 4.846, 4.037, P = 0. 002, 0).. 028, 0.045, with statistical significance). 3. Designed for Pseudomonas aeruginosa OprL gene, OprD 2. The isothermal amplification detection method of the two-drug resistance gene can be directly interpreted by naked eyes. The isothermal amplification detection method only amplifies the copper, and the lowest detection limit of the PCR method is 174.414pg/ mul, the detection limit of the PCR method is 174.414pg/ mul, and the LAMP method The sensitivity is 10 times the detection sensitivity of the PCR method, and the sensitivity is high. The LAMP method and the PCR method are used for the clinical P. P. Cytospora The detection rate of OprL gene and OprD2 gene was comparable. Conclusion 1. The drug resistance of P. aeruginosa is serious, and the deletion of the drug-resistant P. aeruginosa OprD2 is more common, and 23 of the 47 strains (48.94%).). The deletion of the OprD2 gene results in Pseudomonas aeruginosa as the main resistance to imipenem. and 2, the LAMP method is used for quickly detecting the pseudomonas aeruginosa has the advantages of simple detection process, simple and convenient experimental device, no visual discrimination of the reaction result and high sensitivity. and has the characteristics of strong specificity and the like, and is particularly suitable for rapid diagnosis of field and grass-root quarantine and medical units.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378.991;R440

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