5种食源致病菌16SrRNA基因单链构象多态性分析

Analysis on Single-Strand-Conformation Polymorphism of 16S rRNA Gene of Five Species Food-borne Pathogenic Bacteria

目的 对 5种常见食物中毒致病菌 16SrRNA基因单链构象多态性 (Single -strand -conformationpolymor phism ,SSCP)进行分析 ,探讨该技术在食物中毒致病菌快速鉴别诊断中的可能作用。方法 5种常见食源性致病菌标准株增菌后 ,提取DNA ,通过特异引物对菌株的 16SrRNA序列的可变区进行扩增 ,扩增产物进行SSCP分析。结果 各菌株均分离出两条或以上特异性条带 ,条带间距离及相对位置差异明显 ,并有良好的重复性。该方法的灵敏度可达10～ 15个鼠伤寒沙门菌细胞。结论 5种常见食物中毒致病菌 16SrRNA基因单链构象多态性具有种属特异性 ,PCR-SSCP分析法有助于快速、准确的从基因水平上对食物中毒致病菌进行分析检测。

ObjectiveTo detect five species food-borne pathogenic bacteria Salmonella typhimurium,Staphylococcus aureus,Escherichia coli,Shigellosis Flexneri Bacillus cereus to analyse single-strand-conformation polymorphism(SSCP) of the 16S rRNA gene,and investigate the possible role of this technology at rapid indentification of bacteria.MethodsAfter the number of five bacteria strains of food poisoning was increased,the bacteria DNA was extracted.A pair of special primers for amplification located in 16S rRNA sequence flanking a variable region were designed,and analysis of the amplified product was based on the principle of SSCP.ResultsAll the strains separated two or more special bands,and the distances and relative position of band pattern were different from each other but with good repetition.The sensitivity of this technique obtained for Salmonella typhimurium was 10-15 CFU.ConclusionThe 16S rRNA gene of five pathogenic bacteria strains by SSCP assay were species-specific patterns,this technique was helpful to rapid,accurate,molecular identification of pathogenic bacteria of food poisoning.