摘要
目的建立一种快速准确的方法对引起腹泻的常见肠道致病菌做初步筛查,并对早期治疗和最后诊断起指导作用。方法培养大肠埃希氏菌、蜡状芽孢杆菌、铜绿假单胞菌、金黄色葡萄球菌、单核增生性李斯特菌、产气荚膜梭菌、粪肠球菌、志贺菌、伤寒沙门菌、小肠结肠炎耶尔森菌、空肠弯曲菌、副溶血弧菌、艰难梭菌、普通变形杆菌等的标准菌株。根据生物信息学技术设计以16S rRNA序列为靶基因设计各细菌的特异型探针和引物。待测细菌经Cy5标记后的引物以不对称PCR方法扩增后,与固定探针的芯片进行杂交。然后用荧光扫描仪扫描,判定细菌的种类。结果我们建立的针对16S rRNA序列的基因芯片检测系统能够成功地将14属的细菌鉴定到属。在纯培养物的最低检测浓度为103CFU/mL。结论我们所建立的基因芯片系统具有高通量和高准确性,可以作为临床鉴定细菌的一个重要的工具。
Objective To establish and evaluate a quick and accurate DNA microarray method to detect intestinal pathogens directly from human diarrheal stool samples as an alternative to traditional culture methods. Methods Clostridium difficile, Campylobacter jejuni, Clostridium perfringens, Vibrio parahaemolyticus, Proteus spp, Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Enterococcus faecalis, Shigella spp, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus and E. coli were cultured by the culture method. Primers and 21 oligonucleotide probes, based on the sequences of bacterial 16S rRNA gene were arrayed on microarray slides. Hybridization between probes and amplicons was performed. Results Our data showed that The probes of the assay were successful in discriminating 14 genera or species of intestinal pathogens. The limit of detection was approximately 103 CFU/mL for one species of pathogen. Conclusion DNA microarrays with its high efficiency and accuracy could be used as an alternative to culture method.
出处
《胃肠病学和肝病学杂志》
CAS
2008年第10期809-812,共4页
Chinese Journal of Gastroenterology and Hepatology
