作　　者： ; ; ; ; ;

机构地区： 河北农业大学食品科技学院

出　　处： 《生物工程学报》 2005年第6期1009-1013,共5页

摘　　要： 利用FTA滤膜,采用PCR技术可直接检测肉及肉制品中的金黄色葡萄球菌,无需增菌,灵敏度高。在采用浮选与溶剂萃取相结合方法的基础上,使用FTA膜可高效地从鲜肉中提取金黄色葡萄球菌的DNA,消除PCR反应的抑制因子。以金黄色葡萄球菌耐热核酸酶基因(nuc)为靶基因,经过PCR扩增得到279bp的产物。经过DNA测序证实该产物为目的扩增产物。使用FTA滤膜处理样品,再通过PCR方法检测金黄色葡萄球菌,猪肉、牛肉、羊肉、鸡肉匀浆液的检出限均为10cfumL,可在6h内完成对肉中金黄色葡萄球菌的检测,比目前普遍采用的先增菌再进行PCR检测的方法缩短了12～24h。实际检测了72份样品,同时与GB4789.1094方法及两种快速检测致病性金黄色葡萄球菌检测方法做比较,PCR方法的检出率为72.2%,检出时间为6h,GB4789.1094方法检出率为70.8%,检出时间为5d,PrfilmRSA方法的检出率为61.1%,检出时间为18h,BairdParkerR.P.F方法的检出率为69.4%,检出时间为18h,结果表明FTA滤膜用于PCR检测肉中金黄色葡萄球菌检出率高,耗时短。使用FTA滤膜法制备模板DNA,为食品中的致病菌快速检测构建了一个技术平台。 In the present study, an assay using Polymerase Chain Reaction （PCR） was developed for the detection of Staphylococcus aureus in meat. Based on flotation and solvent extraction technology, FTA filter was used to extract S. aureus DNA from artifically contaminated meat. Primers targeting the thermostable nuclease gene （nuc） were used to amplify a 279 bp DNA fragment which was confirmed by DNA sequencing. The detection limit of PCR was10 cfu. g^-1 meat of S. aureus. This novel FTA-PCR assay allows for detection of Staphylococcus aureus in meat in 〈 6h, which is 12 - 24h less than that of conventional PCR with enrichment method. Seventy-two samples were analyzed and the detection rate using the standard cultivation method was 70.8%, detection time was 5d. The detection rate of PCR amplification using filters was 73.6%, detection time was 6h. The detection rate of Baird-Parker R. P. F method was 69.4%, detection timewas lgh. The detection rate of petrfilm RSA method was 61.1%, detection time was 18h. Thus PCR amplification using filters provides a faster and more sensitive method of S. aureus detection than the standard cultivation method. At the same time,it provides an universal process for preparing DNA template.

关 键 词： 滤膜 金黄色葡萄球菌 鲜肉

领　　域： [生物学]