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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 establish a rapid method for the detection of L2 cephalosporin β-lactamase. Methods Five specific primers designed for the L2 cephalosporin β-lactamase gene by loop-mediated isothermal amplification of DNA (LAMP) were designed and used to specifically recognize the specific Bla L2 gene Detection of Carbapenems-resistant Stenotrophomonas maltophilia in seven separate areas. The results of real-time turbidimeter monitoring showed that the LAMP reaction was completed within 50 min under the constant temperature of 65 ℃. The visualization of calcein showed that the positive result was green, which was significantly different from the negative result. Results The minimum detectable gene concentration of LAMP was 2.58 pg / μl, which was 100 times more sensitive than PCR and had good specificity. Conclusion The LAMP method has the characteristics of simple process, simple experimental apparatus, distinguishable macroscopic findings, high sensitivity and strong specificity. It is negative for the results of non-L2 cephalosporin β-lactamase gene and is suitable for clinical application of L2 cephalosporin β-lactamase gene for rapid detection.