中文摘要：

目的探讨建立一种基于毛细管电泳-单链构象多态性（CE-SSCP）技术的血液感染病原菌快速检测方法。方法选取常见血液感染病原菌,查询其基因组序列,在其保守区域设计通用引物进行PCR扩增,然后进行CE-SSCP分析,测定不同病原菌所对应的出峰时间及不同浓度的PCR产物所对应的峰面积;用标准菌株建立各菌的CE-SSCP特异图谱,用临床菌株进行验证;以大肠埃希菌为研究对象,挑取浓度为10～108 CFU/ml的菌液,研究该方法的最低检出浓度。结果所设计引物能够成功扩增所研究的6种病原菌,电泳中未见明显引物二聚体和非特性条带;CE-SSCP分析显示,6种菌的出峰时间各不相同,金黄色葡萄球菌为4082、大肠埃希菌为3999、铜绿假单胞菌为3958、表皮葡萄球菌为4063、肺炎克雷伯菌为3980、粪肠球菌为4166、肺炎链球菌为4055;不同浓度的PCR产物对应不同大小的峰面积,浓度〉10ng/L是两者呈良好的线性关系;CE-SSCP法最低可检测18CFU/ml的菌液。结论利用CE-SSCP技术检测血液感染病原菌快速、灵敏,而且不受抗菌药物影响,尤其适合对难培养菌的检测,可以与传统方法互补。

英文摘要：

OBJECTIVE To establish a method for detection and identification of pathogens in blood samples by single strand conformation polymorphism（SSCP） based on capillary electrophoresis（CE）.METHODS The most common bacterial species isolated from blood cultures in southwest area of China were studied on genomic DNA sequences.A pair of universe primers was selected for the amplification of conserved sequences from those pathogens and PCR products were resolved by CE-SSCP.Different bacteria corresponding to time point,as well as various concentration of PCR products corresponding to peak areas,was determined.The CE-SSCP patterns from clinical strains were compared to those from standard strains.The Escherichia coli cells were diluted different concentrations ranging from 10 CFU/ml to 108 CFU/ml to analyze for the minimum extent.RESULTS The target sequences were amplified successfully with the self-designed primers and no dimers and unspecific amplification fragments were found.Species-specific SSCP patterns were determined for 6 bacterial species,which were E.coli 3999,Staphylococcus aureus 4082,Pseudomonas aeruginosa 3958,S.epidermidis 4063,Klebsiella pneumoniae 3980,S.pneumoniae 4055 and Enterococcus faecalis 4166,respectively;different DNA concentrations corresponded to different peak areas and they were linearly correlated when DNA concentration was higher than 10 ng/μl.The minimum extent was approximately 18 CFU/ml.CONCLUSION It is rapid and sensitive to detect pathogens,especially fastidious micro-organisms,in blood samples by CE-SSCP that complement culture-based assays in microbiology laboratories.