3种食源性致病菌的实时荧光PCR快速检测

摘要

The genome DNA of Staphylococcus aureus was extracted by four different method and finally chose a method combined steel ball destruction and acetone,protease K removing impurities to get high purity and output DNA. Then the protein gene (egc) of Staphylococcus aureus,a NA gene of Salmonella,and ipaH gene (iapH) of Shigella were used as the gene targets. And the samples were determined by real-time PCR tech-nique. The results showed that the primers were highly specific. No fluorescence signal was determined in other 3 control pathogens (Listeria monocytogenes,Vibrio parahaemolyticus and Bacillus cereus) but target bacteria. The RT-PCR assay could be specific and rapid,and the detection limits were 10-6,10-6,10-7 ng/μL .%DNA提取方法采用先钢珠机械破坏细菌细胞壁，再添加丙酮、蛋白酶K溶解去除杂质，大大提高了食源性致病菌基因组DNA的得率和纯度。继而针对金黄色葡萄球菌egc基因、沙门氏菌NA（not availabale）基因、志贺氏菌ipaH基因为靶基因设计特异性引物对样品建立实时荧光定量PCR检测方法。结果试验设计的引物对3种细菌具有很强特异性，除目标细菌外，对单核细胞增生李斯特氏菌、副溶血性弧菌、蜡样芽孢杆菌3种对照菌均未检测到荧光信号。对金黄色葡萄球菌、沙门氏菌和志贺氏菌的检测灵敏度分别达到10-6、10-6、10-7 ng/μL。