FTA滤膜用于PCR检测肉中的沙门氏菌-食品科学.PDF

254 2009, Vol. 30, No. 16 食品科学 ※分析检测 李伟昊，张会彦，刘卫华，李 岩，李英军，张 伟* (河北农业大学食品科技学院，河北保定 071001) ：在采用浮选与溶剂萃取相结合方法的基础上，使用FTA 滤膜从鲜肉中提取沙门氏菌的DNA ，消除PCR 反应的抑制因子。以沙门氏菌编码吸附和侵袭上皮细胞表面蛋白的invA 基因为靶基因，经过PCR 扩增得到376bp 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 方法检测沙门氏菌， 猪肉、牛肉、羊肉、鸡肉匀浆液的检出限均为7 ×101CFU/ml ，可在6h 内完成对肉中沙门氏菌的检测，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GB/T 4789.4 —2003 方 法做比较，本方法的检出率为27.8% ，检出时间为6h ；GB/T 4789.4 —2003 方法检出率为22.2% ，检出时间为5d 。 结果表明：FTA 滤膜用于PCR 检测肉中沙门氏菌检出率高、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。 ：P C R ；F T A 滤膜；沙门氏菌；肉 FTA Filter Paper-based Template Preparation for the PCR Detection of Salmonella in Meat LI Wei-hao ，ZHANG Hui-yan ，LIU Wei-hua ，LI Yan ，LI Ying-jun ，ZHANG Wei* (College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, China) Abstract ：An assay of PCR was developed for the detection of Salmonella in meat. Based on flotation and solvent extraction technology, FTA filter was used to extract Salmonella DNA from fresh meat to eliminate the inhibitory factors of PCR reaction. An invA gene in surface protein of epithelial cells absorbed and invaded by Salmonella was used to amplify a 376 bp DNA fragment, which could be confirmed by DNA sequencing. The detection limits of Salmonella in pork, beef, mutton and chicken homogenates all were 7 ×101 CFU/ml, and the detection could be finished in 6 h, which was shorter than that of the conventional method of combined enrichment and PCR by 12 －24 h. In this study, 72 samples were detected and the results were compared with the GB/T 4789.4—2003 method. The detection rates of Salmonella by this method and the GB/T 4789.4—2003 method were 27.8% and 22.2%, and the detection by the latter method cost 5 days. In general, this