铜绿假单胞菌OprD2基因环介导等温扩增快速检测技术的研究  

Rapid detections of OprD2 gene in Pseudomoas aeruginosa using loop-mediated isothermal amplification

作　　者：王欢[1] 黄庆[1] 黄君富[1] 夏涵[1] 杨昭[1] 府伟灵[1] 

机构地区：[1]第三军医大学西南医院检验科,重庆400038

出　　处：《中华检验医学杂志》2013年第6期543-547,共5页Chinese Journal of Laboratory Medicine

基　　金：国家“863”重大专项资助项目（2011AA02A121）;国家科技重大专项资助项目（2012zx10004801-003.006）;全军“十二五”重大专项资助项目（AWS11C001）

摘　　要：目的建立肉眼直接判读检测结果的铜绿假单胞菌OprD2耐药基因环介导等温扩增（LAMP）快速检测方法。方法对2011年12月至2012年6月西南医院微生物室收集的47株铜绿假单胞菌标本进行前瞻性研究。利用LAMP针对铜绿假单胞菌OprD2耐药基因设计两对引物，特异性识别OprD2的6个独立区域来快速扩增。通过在LAMP反应体系中加入核酸染料羟基萘酚蓝（HNB），建立可直接用肉眼判读检测结果的LAMP反应体系，并用该方法检测和分析47株铜绿假单胞菌OprD2耐药基因的分布情况及其与抗生素耐受性的相关性。结果设计出针对铜绿假单胞菌OprD2耐药基因可直接肉眼判读结果的恒温扩增检测方法，其检测体系的灵敏度为17．414μg／L，比常规PCR方法（灵敏度：174．14μg／L）高10倍。49％（23／47）的菌株OprD2基因缺失，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）、57％（13／23）、48％（11／23）、48％（11／23）、48％（11／23）和43％（10／23）。与OprD2基因阳性株相比，OprD2基因缺失株的亚胺培南、左氧氟沙星和美罗培南耐药比例明显升高，差异有统计学意义（χ2值分别为9．155、4．846、4．037，P值分别为0．002、0．028、0．045）。结论建立了可直接肉眼判读检测结果的铜绿假单胞菌OprD2耐药基因LAMP快速技术。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。Objective To establish a rapid detection approach by visual interpretation directly for OprD2 resistance gene of Pseudomonas aeruginosa based on the Loop-mediated isothermal amplification （LAMP）, and provide a quick and effective method for clinical monitoring of Pseudomonas aeruginosa strains. Methods Totally 47 strains of Pseudomonas aeruginosa collected from December 2011 to June 2012 in Southwest Hospital of microorganisms were prospectively studied. Four LAMP primers （two inner, two outer） were designed according to the six zones of the OprD2 gene of Pseudomonas aeruginosa. A positive reaction is indicated by the color change after adding an intercalating dye （hydroxy naphthol blue） to the reaction solution. This method was used to detect and analyze the distribution of OprD2 resistance gene in 47 strains of Pseudomonas aeruginosa and its correlation with antibiotic resistance. Results The LAMP assays showed 100% specificity for the OprD2 gene, and the sensitivity （with the lowest detection limits of 17. 414 μg/L） was 10-fold higher than that of conventional PCR assays. The OprD2 gene was negative in 23 strains by both conventional PCR and LAMP. In OprD2 negative strains, the resistance rate of cefotaxime, levofloxacin, aztreonam, piperacillin, imipenem and meropenem was 100% （23/23）, 57% （ 13/23）, 48% （ 11/23 ）, 48% （ 11/23 ）, 48% （ 11/23 ） and 43% （ 10/23 ）. Compared with the OprD2 positive strains,statistical analysis showed that the resistance rate of imipenem, levofloxacin and meropenem in OprD2 negative strains increased significantly （ chisquare value is 9. 155, 4. 846, 4. 037, P value was 0. 002, 0. 028, 0. 045, and so there was significant difference）. Conclusions The established LAMP approach in this study enables rapid, sensitive and specific detection of OprD2 gene in Pseudomonas aeruginosa by visual interpretation. Deficiency of OprD2 gene confers Pseudomonas aeruginosa a basal level of resistance to carbapenems especially to imipenem. The identificat

关 键 词：假单胞菌 铜绿 抗药性 细菌 

分 类 号：R378.991[医药卫生—病原生物学]