利用环介导等温扩增技术针对DeoR家族转录调控因子基因进行表皮葡萄球菌快速鉴定

A Novel Loop-Mediated Isothermal Amplification Assay Targeting DeoR Family Transcriptional Regulator Gene to Rapidly Detect Staphylococcus Epidermidis

表皮葡萄球菌是一种重要的机会致病性微生物,是院内交叉感染的一个重要原因.开发快速有效的检测技术对其防治具有重要意义.利用生物信息学方法进行分析,发现DeoR家族转录调控因子基因可用于表皮葡萄球菌鉴定.针对此基因设计引物,采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)进行检测,在65℃条件下反应进行60 min后,电泳检测可见明显的阶梯状条带.比较了3株表皮葡萄球菌临床分离株和8株其他细菌(金黄色葡萄球菌、溶血葡萄球菌、弗劳氏枸橼酸杆菌、肺炎克雷伯菌、铜绿假单胞菌、粪肠球菌、屎肠球菌和化脓性链球菌),结果显示该方法具有良好的特异性.构建DeoR家族转录调控因子基因质粒载体后考察了检测的灵敏度,结果显示其最低检测限为105拷贝/反应.由此针对DeoR家族转录调控因子基因建立的LAMP检测方法可快速、简便、特异以及敏感的鉴定表皮葡萄球菌.

Staphylococcus epidermidis is an opportunistic bacterium that causes a variety of infections including nosocomial infection. The development of effective techniques for rapid detection of this pathogen is essential for prevention and cure of its infection. In present study,a loop- mediated isothermal amplification（ LAMP） assay targeting DeoR family transcriptional regulator gene to rapidly detect S. epidermidis is developed. This assay is performed in60 min at an optimal temperature of 65 ℃ and visualized as a ladder on a 1% agarosege. Specificity of the assay is evaluated by testing three S. epidermidis clinical isolates and eight non- S. epidermidis bacteria（ Staphylococcus aureus,Staphylococcus haemolyticus,Citrobacter freundii,Klebsiella pneumoniae,Pseudomonas aeruginosa,Enterococcus faecalis,Enterococcus Faecium,and Streptococcus pyogenes）. No ladder pattern is seen with any of the other non-S. epidermidis bacteria. A positive vector which contains targeted region of DeoR family transcriptional regulator gene is constructed and used to evaluate sensitivity of this assay. The sensitivity of LAMP is found to be approximately 10^5 copies per reaction when an incubation period of 60 min is used. In conclusion,LAMP assay targeting the DeoR family transcriptional regulator gene can detect S. epidermidis rapidly and effectively.