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。
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.
出处
《中国卫生检验杂志》
CAS
2011年第4期790-793,共4页
Chinese Journal of Health Laboratory Technology
