环介导等温扩增检测肺炎链球菌方法的建立及临床应用

目的 建立环介导等温扩增(LAMP)快速检测肺炎链球菌的方法.方法 根据美国国家生物信息中心(GenBank)上提交的肺炎链球菌R6株的lytA基因序列(登录号AF2)08540)设计特异LAMP引物,以盐酸胍一苯甲醇法提取阳性血培养瓶中细菌DNA,然后以LAMP技术扩增lytA基因,反应条件为63℃45 min,采用肉眼观察浊度和琼脂糖电泳的方法来观察结果.结果 在196份阳性血培养瓶中,采用LAMP法检测到肺炎链球菌16株,与传统方法比较,其检测肺炎链球菌的灵敏度和特异度均达到100%,且检测时间缩短为1 h左右.模拟标本的检测结果显示该方法的最低检测限为102CFU/ml.结论 该方法灵敏度高,特异性强,操作方便快速,适合从临床标本中检测肺炎链球菌.

Loop-mediated isothermal amplification for detecting Streptococcus pneumoniae and its clinical appfication

Objective To establish loop-mediated isothermal amplification(LAMP)method for detecting Streptococcus pneumoniae in clinical samples.Methods Four Streptococcus pneumoniae-specific LAMP primers were designed according to the published sequence of strain R6(GenBank accession number AE008540).Genomie DNA in positive blood cultures was extracted by nanidine hydrochloride and benzene-methanol.Then lytA was amplified by LAMP at 63℃ for 45 minutes.We observed the turbidity in thereaction tube.For further confirmation.The amplified products were also detected using electrophoresis in 2% agarose gels,followed by ethidium bromide staining.Resuits 16 strains of Streptococcus pneumoniae were detected in 196 positive blood cuhure bottles by LAMP.Compared with traditional method.It8 sensitivity and specificity were both 100%and the detection could be finished in an hour.The assay had a minimum detection limit of 102 CFU/m1.Conclusions This IJAMP-based assay is simple,rapid.Sensitive and specific.It can be used to detect trept OCOCCUS pneumoniae in clinical samples.