环介导等温扩增法直接检测阳性血培养瓶中大肠埃希菌

Direct detection of Escherichia coli in positive blood culture bottles by loop-mediated isothermal amplification method

目的应用环介导等温扩增技术(LAMP)建立一种快速敏感的直接检测阳性血培养瓶中大肠埃希菌的方法。方法根据美国国立卫生研究院基因序列数据库(GenBank)上大肠埃希菌的lacZ(登录号:M74750)基因序列,设计4条特异引物(2条内引物、2条外引物),对lacZ基因进行扩增,对扩增反应进行优化。分别验证该方法的特异性及敏感性,并与传统培养法进行比较。结果在300份阳性血培养瓶中,采用LAMP法检测到68株大肠埃希菌,所设计引物对大肠埃希菌扩增的特异性及敏感性均较好,与传统培养法比较,灵敏度和特异性均达100%,但传统培养法的检出时间为3d,而LAMP法的检出时间只需1h。结论LAMP检测大肠埃希菌是快速、低成本、特异性强、灵敏度高的方法,适合临床开展。

Objective To establish a rapid ,sensitive direct method for detecting Escherichia coli （E .coli） in the positive blood culture bottles by using the loop‐mediated isothermal amplification （LAMP） .Methods Based on the lacZ （accession number ：M74750） gene sequence of E .coli in the gene sequence GenBank by the National Institutes of Health （NIH） ,4 specific primers （2 inner primers and outer primers） were designed ,the lacZ gene was amplified and the amplification reaction was optimized .The sen‐sitivity and specificity of this method were verified and its comparison with the traditional culture method was conducted .Results Sixty‐eight strains of E .coli were detected in 300 positive blood culture bottles by adopting the LAMP method .The designed prim‐ers had good sensitivity and good specificity for the amplification of E .coli .The sensitivity and specificity of LAMP all reached 100% compared with the traditional method ,but the traditional method needed 3 d to get the results ,while LAMP only needed 1 h . Conclusion LAMP is extremely rapid for detecting E .coli with low cost ,strong specificity and high sensitivity and is suitable for clinical application .