基于顏色判定的環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)快速檢測(cè)銅綠假單胞菌及其OprD2耐藥基因的研究
發(fā)布時(shí)間：2018-11-17 12:34
【摘要】：目的 銅綠假單胞菌(Pseudomonas aeruginosa，PAE)為條件致病菌，分布廣泛，在正常情況下不致病。但當(dāng)人體免疫力降低時(shí)(如：長(zhǎng)期應(yīng)用激素、腫瘤化療、AIDS)或嚴(yán)重創(chuàng)傷(如大面積燒傷、大型手術(shù))或醫(yī)療操作(如氣管切開(kāi)、插管)后，PAE引起的感染性疾病不斷增加。隨著抗生素的廣泛使用，耐藥銅綠假單胞菌的發(fā)生率增多，特別是多重耐藥菌，使臨床上治療該病原菌引起的相關(guān)感染變得越來(lái)越來(lái)困難。然而傳統(tǒng)的細(xì)菌學(xué)檢查方法時(shí)間長(zhǎng)、敏感性低，一直不能滿足臨床診治的需求。因此建立了一種新的、簡(jiǎn)單、快速的現(xiàn)場(chǎng)檢測(cè)方法，對(duì)銅綠假單胞菌及耐藥性的早期診斷和治療具有重大意義。 本研究目的：了解臨床標(biāo)本中分離的銅綠假單胞菌對(duì)16種抗菌藥物的耐藥情況，為臨床合理使用抗生素提供參考。對(duì)多重耐藥銅綠假單胞菌進(jìn)行相關(guān)耐藥基因的研究。建立一種基于顏色判定的，能夠快速準(zhǔn)確地檢測(cè)銅綠假單胞菌及其OprD2耐藥基因的LAMP方法。 方法 對(duì)臨床收集47株耐藥銅綠假單胞菌采用K-B紙片擴(kuò)散法測(cè)定對(duì)慶大霉素等16種抗菌藥物的敏感性。PCR擴(kuò)增檢測(cè)收集的銅綠假單胞菌標(biāo)本中的耐亞胺培南類IMP、VIM、GES、GIM、OXA-10、OprD2耐藥基因。針對(duì)銅綠假單胞菌OprL、OprD2基因設(shè)計(jì)LAMP引物,通過(guò)引物特異性識(shí)別OprL、OprD2的6個(gè)獨(dú)立區(qū)域來(lái)快速檢測(cè)銅綠假單胞菌特有OprL基因和OprD2耐藥基因。如果在反應(yīng)前添加熒光染料羥基萘酚藍(lán)(HNB)或鈣黃綠素(Calcein)，其結(jié)果可以通過(guò)肉眼觀察反應(yīng)前后顏色變化來(lái)判定并經(jīng)瓊脂糖凝膠電泳驗(yàn)證。對(duì)該技術(shù)進(jìn)行特異性、靈敏度分析，，用LAMP擴(kuò)增法、PCR擴(kuò)增法和臨床傳統(tǒng)方法同時(shí)檢測(cè)臨床標(biāo)本。 結(jié)果 1.47株耐藥PAE對(duì)頭孢噻肟耐藥率最高占97.87%，左旋氧氟沙星耐藥率占40.42%，氨曲南耐藥率占38.30%，哌拉西林耐藥率34.04%，美羅培南耐藥率占29.79％，亞胺培南、環(huán)丙沙星、頭孢哌酮的耐藥率占27.66％。 2.47株P(guān)AE中VIM基因陽(yáng)性12株，陽(yáng)性率25.53%，OXA-10基因陽(yáng)性2株，陽(yáng)性率4.26%，OprD2基因缺失23株，缺失率占48.94%，而IMP、GIM、GES基因均陰性。OprD2基因缺失株的頭孢噻肟、左氧氟沙星、氨曲南、哌拉西林、亞胺培南和美羅培南耐藥率分別為100%(23/23)、56.52%(13/23)、47.83%(11/23)、47.83%(11/23)、47.83%(11/23)和43.48%(10/23)。統(tǒng)計(jì)學(xué)分析結(jié)果顯示：與OprD2基因陽(yáng)性株相比，OprD2基因缺失株的亞胺培南、左旋氧氟沙星、美洛培南耐藥比例明顯升高(卡方值為9.155、4.846、4.037，P值為0.002、0.028、0.045，差異有統(tǒng)計(jì)學(xué)意義)。 3.設(shè)計(jì)出針對(duì)銅綠假單胞菌OprL基因、OprD2耐藥基因可直接肉眼判讀結(jié)果的等溫?cái)U(kuò)增檢測(cè)方法。本等溫?cái)U(kuò)增檢測(cè)法只擴(kuò)增銅綠假單胞菌，對(duì)其他菌不擴(kuò)增，顯示出良好的特異性。其最低檢測(cè)限為17.414pg/μl, PCR方法最低檢出限為174.414pg/μl, LAMP方法檢測(cè)靈敏度是PCR方法檢測(cè)靈敏度的10倍,靈敏度高。LAMP法與PCR法對(duì)臨床銅綠假單胞菌OprL基因和OprD2耐藥基因檢出率相當(dāng)。 結(jié)論 1．臨床銅綠假單胞菌耐藥情況嚴(yán)重，耐藥銅綠假單胞菌OprD2基因缺失較為普遍，47株中缺失23株占(48.94%)。OprD2基因缺失導(dǎo)致銅綠假單胞菌以亞胺培南為主的耐藥，因此明確OprD2基因分布狀況有助于臨床抗菌藥物的選用。 2．LAMP方法用于快速檢測(cè)銅綠假單胞菌具有檢測(cè)過(guò)程簡(jiǎn)單、實(shí)驗(yàn)裝置簡(jiǎn)便、反應(yīng)結(jié)果肉眼可辨別、靈敏度高和特異性強(qiáng)等特點(diǎn)，特別適合用于現(xiàn)場(chǎng)和基層檢疫及醫(yī)療單位的快速診斷。本課題成功建立針對(duì)銅綠假單胞菌OprL基因及其OprD2耐藥基因可直接肉眼判讀檢測(cè)結(jié)果的LAMP技術(shù)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R378.991;R440
本文編號(hào)：2337797
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R378.991;R440
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