环介导恒温扩增法对嗜麦芽寡养单胞菌L2型头孢菌素β内酰胺酶基因的快速检测  

Quick detection of L2 type cephalosporinase beta lactamase gene in Stenotrophomonas maltophilia by loop-mediated isothermal amplification

作　　者：崔茜[1,2,3] 刘威[2] 杨展[2] 陆思静[1] 袁静[2] 李艳[3] 

机构地区：[1]辽宁医学院附属第一医院呼吸科,辽宁锦州121001 [2]军事医学科学院疾病预防控制所,北京100071 [3]军事医学科学院附属医院呼吸科,北京100071

出　　处：《军事医学》2013年第12期943-946,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81071399;81271887);首都医学发展研究基金资助项目(2009-3071)

摘　　要：目的建立快速检测L2型头孢菌素β内酰胺酶的方法。方法利用DNA环介导恒温核酸扩增法(loopmediated isothermal amplification of DNA,LAMP)针对L2型头孢菌素β内酰胺酶基因设计的5条特异性引物,通过引物特异性识别特定Bla L2基因上的7个独立区域实现耐碳青霉烯类嗜麦芽寡养单胞菌的检测。实时浊度仪监测反应结果表明,LAMP反应在65℃恒温条件下50 min内完成;钙黄绿素可视化检测表明阳性结果呈绿色,与阴性结果差异显著。结果 LAMP法最低检出基因的浓度为2.58 pg/μl,其灵敏度为PCR法的100倍,且具有良好的特异性。结论 LAMP法具有过程简单、实验装置简便、结果肉眼可辨别、灵敏度高、特异性强等特点,对非L2型头孢菌素β内酰胺酶基因的结果呈阴性,适用于临床L2型头孢菌素β内酰胺酶基因的快速检测。Objective To develop a quick method for L2 type cephalosporinase beta lactamase detection. Methods A loop-mediated isothermal amplification （LAMP） assay was developed and validated for specific detection of L2 type cepha- losporinase beta lactamase gene by designing 5 pairs of primers that distinguished 7 specoal Blar2 sequences in Stenotroph- omonas maltophilia which is carbapenem resistant. Real time turbidity meter showed that two methods, including monitoring turbidity and adding calcein before reaction, were used to determine the negative and positive results. Results The target DNA was amplified and visualized by the two detection methods within 50 rain at an isothermal temperature of 65℃. LAMP with a detection limit of 2.58 pg/μl was 100 times as sensitive as PCR. Non-L2 type cephalosporinase beta lactamase genes were selected for specificity and the results of the amplification were negative, suggesting that the primers designed had a good specificity. Conclusion LAMP requires a simple process and experimental device, producing discernible reaction results, high sensitivity and specificity. It is suitable for rapid detection of L2 cepbalosporinase beta lactamase gene.

关 键 词：L2型头孢菌素β-内酰胺酶基因 环介导恒温扩增技术(LAMP) 快速检测 嗜麦芽寡养单胞菌 

分 类 号：R446.5[医药卫生—诊断学] R378.99[医药卫生—临床医学]