23S rRNA基因在常见病原菌鉴定中的应用

Application of 23S rRNA gene in identification of common pathogens

目的 建立基于 2 3SrRNA基因的一种常见病原菌菌种鉴定的分子生物学方法。 方法 使用地高辛标记的通用引物扩增常见病原菌的 2 3SrRNA基因 ,并在尼龙膜上固定根据扩增产物序列设计的菌种特异性探针 ,根据反向杂交结果判断病原菌种类。选取临床分离获得的菌株验证该方法的有效性。结果 通用引物可特异性扩增细菌 2 3SrRNA。应用该方法可鉴定金黄色葡萄球菌、凝固酶阴性葡萄球菌、粪肠球菌、屎肠球菌、肺炎链球菌、大肠埃希菌、肺炎克雷伯菌、奇异变形杆菌、阴沟肠杆菌、鲍曼不动杆菌、绿脓假单胞菌、嗜麦芽窄食单胞菌和洋葱伯克霍尔德菌。检测临床标本结果显示 ,与常规培养方法的符合率为 99%。结论 该方法可用于常见病原菌的鉴定。

Objective To establish a molecular biological method to identify common pathogen based on 23S rRNA gene Method Using digoxin labeled universal primers to amplify bacterial genome, followed by reverse hybridization of the product to the species specific probes bound to nylon membrane To identify the species based on the spot shown on the membrane Result The primers can only amplify bacterial 23S rRNA Staphylococcus aureus, coagulase negative staphylococcus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Proteus spp, Enterobacter cloacae, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia cepacia could be identified correctly The results of clinical isolates identification were 99% accord with the traditional method Conclusion PCR reverse hybridization can be used to identify common bacterial pathogens