【摘要】 目的:针对NDM-1超级耐药菌建立PCR/LAMP快速筛检方法。方法:根据GENBANK登录的NDM-1基因序列分别设计PCR/LAMP引物。分别优化PCR/LAMP反应条件和反应体系,并对已知特性的41株标准株、1株NDM-1阳参克隆株以及275株不同地区上送的地方株进行检测,验证两方法的特异性、敏感性。结果:两方法特异性均好,除阳参克隆株能扩增出阳性对应条带,其它标准株及地方上送株则检测不出相应条带;两方法检测限稍有差异,其中LAMP检测限为31 cfu/反应,PCR检测限为123 cfu/反应;LAMP法从菌株核酸提取至检测完成仅需1.5 h~2 h左右,实现反应及产物检测一步完成;PCR法因扩增产物需凝胶电泳再经紫外观察,故时间相对较长,共需3 h~4 h。结论:本研究针对超级耐药菌NDM-1基因建立的PCR/LAMP方法其特异性、灵敏度均相对较好,可用于今后应对NDM-1超级细菌应急检测技术储备以及目前针对超级细菌的监控防控。更多还原

【Abstract】 Objective:Establishing rapid PCR/LAMP screening detection methods for NDM-1(New Delhi Metallo-beta-Lactamase-1)superbug.Methods: Designing the primers of PCR/LAMP for superbug based on the sequences of NDM-1 from Genbank and optimizing the reaction conditions for PCR/LAMP.Forty one standard strains,one positive clone and two hundred and seventy five strains collected from different regions were tested for the specificity and sensitivity of PCR/LAMP methods.Results: These two methods show good specificity,and no strain was positvie except the positive clone.The detection limits of these two methods were different,with detection limits of 31 cfu/reaction and 123 cfu/reaction for LAMP and PCR,respectively.LAMP can be finished in one step in 1.5 to 2 hours from the DNA extraction to resμlts identification,however,it was time consuming for PCR and need about 3~4 hours for the whole process.Conclusion: The specificity and sensitivity of PCR/LAMP methods established for the detection of superbug NDM-1gene appeared to be pretty good,and these two methods can be acted as one of the emergency detection reserve methods and used in the monitoring survey of superbug now.更多还原