食品标本处理方法对大肠埃希菌检出限影响
Sample preparation for PCR detection of Enterohaemorrhagic E.coli in food
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摘要:目的 研究快速、敏感、低成本的食品标本处理方法。方法 在牛肉馅中接种不同浓度的大肠埃希菌O157:H7, 经离心、过滤, 去除PCR抑制剂, 浓缩菌株, 用煮沸法和酶/冻融法裂解菌株, 制备模板DNA, 通过PCR检测大肠埃希菌O157:H7志贺毒素基因以评价该处理方法的可行性。结果 在未增菌条件下, PCR最低能检测到10 3CFU/g牛肉馅, 增菌6h, 最低能检测1 CFU/g牛肉馅, 整个检测过程只需要12 h。结论 所采用的标本处理方法是一种灵敏、省时、低成本的方法, 能显著地提高PCR检测食品标本中的大肠埃希菌O157:H7的灵敏度。Abstract:Objective To develop a rapid, sensitive and cost-effective sample preparation for PCR detection of EHEC O157:H7 in ground beef.Methods Ground beef samples were inoculated with varying concentrations of E.coli O157:H7.PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation.DNA was purified and concentrated using enzymatic digestion and successive freeze/thaw cycles and the Shiga toxin gene was amplified using PCR to evaluate the sample preparation method.Results Without prior enrichment of cells in broth media, the detection limit was 10 3 CFU/g beef.When a 6 h enrichment step was incorporated, the detection limit was 1 CFU/g beef.The total time required from beginning to end of the procedure was 12 h.Conclusion The sample preparation method can substantially improved the sensitivity of E.coli O157:H7 by PCR detection.
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