婴幼儿腹泻粪便标本菌群的16S rDNA PCR-DGGE分析

目的探讨DGGE技术对常见肠道病原菌的区分能力及腹泻病人粪便标本中的菌群组成和变化情况。方法采用细菌的16S rDNAV3区通用引物,以常见肠道病原菌的染色体和腹泻病人粪便标本中的总DNA为模板,PCR扩增后进行DGGE分析及优势条带的序列分析。结果6份粪便标本的DGGE图谱均表现为高度多态性,不同标本之间优势带型存在很大的差异。序列分析显示每份粪便标本中的DGGE优势带型由不同的细菌组成,而且其中非可培养细菌占较大比例,恢复期病人的粪便标本中双歧杆菌和/或乳杆菌成为优势菌型。结论PCR-DGGE能够有效区分不同种属的常见肠道病原菌,技术可为常规的病原菌分离培养方法提供补充,用于腹泻粪便标本的菌群分析,为疾病诊断提供线索。

The fecal bacterial community in infantile diarrheal samples explored by PCR-denatured gradient gel electrophoresis technique based on 16S rDNA

The composition and changes of the fecal bacterial community in infantile diarrheal samples were explored by using the PCR-denatured gradient gel electrophoresis （DGGE） assay based on 16S rDNA, in which the universal primer of the bacterial 16S rDNA -V3 region was used as primer and the chromosome of common intestinal pathogen as well as the total DNA in the fecal specimens of the diarrheal patients were employed as templates in the PCR-DGGE assay. After PCR amplification, DGGE analysis and the sequence analysis of the dominant bands were performed. As demonstrated by sequence analysis, the dominant bands in DGGE of the fecal samples were composed of different bacteria with a large proportion of the un-cultured bacteria. The bifidobacterium or lactobacillus appeared to be the dominant organisms in the fecal samples of convalescent diarrheal patients. It is evident that the PCR-DGGE technique can be used in the bacterial community analysis or used as an alterna-tive method for the isolation of intestinal pathogens.