4种食源性致病菌PCRSSCP检测技术研究.docVIP

4种食源性致病菌PCRSSCP检测技术研究 　　摘要:采用细菌的16S rDNA保守区引物作为通用引物,对常见食源性致泻大肠埃希氏菌(Diarrheogenic Escherichia coli)、沙门氏菌(Salmonellae)、金黄色葡萄球菌(Staphylococcus aureus)和蜡样芽胞杆菌(Bacillus cereus)进行PCR扩增,产物及其Xmn I酶切后产物进行单链构象多态性分析。试验结果显示,4种食源性致病菌的PCR产物经Xmn I酶切后进行单链构象多态性分析可以相互区别。因此,PCR-SSCP技术可以方便地用于检测食源性大肠杆菌、沙门氏菌、金黄色葡萄球菌和蜡样芽胞杆菌。 　　关键词:食源性致病菌;聚合酶链式反应;单链构象多态性;检测 　　中图分类号: Q503 文献标识码: A 文章编号:1006-6500(2009)01-0001-0013-03 　　 　　Study on Detection of Four Kinds of Food-borne Pathogenic Bacteria by PCR-SSCP 　　WANG Yong,ZHAO Xin,LAN Qing-kuo,ZHU Zhu,CHENG Yi 　　(Central Lab,Tianjin Academy of Agricultural Sciences,Tianjin 300384,China) 　　Abstract:In order to establish a detection method for identification of four kinds of food-borne pathogenic bacteria including Diarrheogenic Escherichia coli, Salmonellae, Staphylococcus aureus and Bacillus cereus by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), the bacterial 16S rDNA primers conservative region as a universal primers, PCR product and Xmn I digested products of E.coli, Salmonella, Staphylococcus aureus and bacillus cereus were analyzed by single strand conformation polymorphism. The results showed that four kinds of food-borne pathogens could be distinguished between each other, after digesting the PCR products by Xmn I. So, PCR-SSCP technique can be used to facilitate the detection of food-borne E. coli, Salmonella, Staphylococcus aureus and Bacillus cereus. 　　Key words: food-borne pathogenic bacteria;PCR;SSCP;detection 　　 　　食品安全是直接关系到消费者生命、健康和社会稳定的热点问题。食品法典委员会(CAC)将微生物性健康危害列为食源性危害的三大原因之一,我国近年微生物性食物中毒比例高达67%[1]。因此,食源性致病菌的检测与鉴定已成为食品行业不可或缺的关键环节。传统的细菌学检验与鉴定需要进行增菌、分离培养以及生化反应等繁琐步骤,既费时又费力,但对于有些细菌仍无法给出正确的鉴定[2]。随着分子生物学技术的发展,PCR-RFLP、PCR-RAPD、PCR-SSCP、荧光原位杂交技术(FISH)、基因芯片(Microarry)、流式细胞技术(FCM)等技术被广泛用于微生物种群鉴定及临床诊断,上述问题迎刃而解。 　　聚合酶链式反应-单链构象多态性(PCR-SSCP)技术是建立在PCR基础上的单链构象多态性分析技术。SSCP技术在原理上甚至可以检测出基因片段中单个碱基的差异[3-4]。16S rDNA是细菌相对保守的基因,经常用于细菌分类。本试验旨在探讨以16S rDNA保守区段为PCR扩增对象,运用SSCP技术对致泻大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌以及蜡样芽胞杆菌进行分析,以期