使用环介导等温扩增技术快速检测金黄葡萄球菌的初步研究

目的 探索建立一种快速的、简单的检测金黄葡萄球菌的环介导等温基因扩增方法(100p-mediated isothermal amplification,LAMP).方法 针对金黄葡萄球菌clfA基因保守序列,根据LAMP方法的原理,设计LAMP检测反应体系和程序,对方法的特异性、灵敏度、重复性及初始拷贝数的对数值与反应时间(荧光信号值为1×10~4时对应的时间)之间的线性关系进行评估.结果 使用LAMP方法可以在0.5 h内获得检测结果,LAMP检测技术的灵敏度高出经典PCR技术10倍以上,与其他相关致病菌无交叉反应,平均试验间变异系数小于5%,反应时间与模板浓度有良好的线性关系(r~2=0.9501),LAMP法检测临床样本中金黄葡萄球菌的灵敏度为100%,特异度为94.4%,符合性为96.6%.结论 该方法快速、灵敏、特异、操作简单、设备要求低的特点适合基层医疗卫生机构和现场查验机构的广泛应用.

Research and development of genetic diagnostic method of Staphyloccocus aureus based on loop-mediated isothermal amplification

Objective To develop a method of loop-mediated isothermal amplification(LAMP) to Staphyloccocus aureus rapidly, specifically, sensitively and simply suited for the primary health agency. Methods According to conserved nucleotide of Staphyloccocus aureus and principle of LAMP, we designed a set of LAMP primers and set up an LAMP reaction system. We evaluated the specificity, sensitivity and re-peatability of the method. In addition,we evaluated the linearity between initial template copies 1g value and reaction time (the time when the fluorescent value is 1×10~4). Results The optimal assay showed that it was no cross-reaction with other closely related members of pathogens, and was 10 times more sensitive than PCR. The coefficient of variance between tests was less than 5% ,and the kinetics curves showed a good line-arity between initial template copies lg value and reaction time(r~2=0. 9501). The detection activity could be finished within 1 h with the sensitivity of LAMP was 100% and the specificity was 94.4%, and the accuracy was 96.6%. Conclusion These findings demonstrated that the LAMP had the potential clinical application for detection and differentiation of Staphyloccocus aureus in the public health agency for its sensitive, specific and simple feature.