论文部分内容阅读
目的建立检测大肠杆菌、沙门氏菌、金黄色葡萄球菌、李斯特菌和小肠耶尔森氏菌5种致病菌的多重直接PCR方法。方法根据大肠杆菌phoA、沙门氏菌invA、金黄色葡萄球菌nuc、李斯特菌hly、小肠耶尔森氏菌ail的基因序列,设计多重直接PCR引物,建立并优化多重直接PCR反应条件,检测引物的扩增特异性和灵敏性,并将所建立的方法应用于实际采集猪肉样本的检测,与金标培养法相比较,计算多重直接PCR检测法的敏感性、准确性以及阳性预测值。结果所设计的多重直接PCR引物对5种菌都有特异扩增,最低检出限量大肠杆菌为10CFU,金黄色葡萄球菌敏感性为100CFU,沙门氏菌、李斯特菌、小肠耶尔森氏菌敏感性可达1CFU。60份猪肉样本中检出大肠杆菌15份、沙门氏菌6份、金黄色葡萄球菌21份、李斯特菌20份、小肠耶尔森氏菌35份,阳性检出率均高于金标培养法,总体检测敏感性为100%,准确性为94%,阳性预测值为81.44%。结论多重直接PCR法实现了同时对各食源性致病菌敏感特异的检测,并且省去了提取模板的步骤,将检测时间缩短至3h左右,便于食品安全风险监测中常见食源性致病菌的通量检测。
Objective To establish a multiplex PCR method for detecting five pathogenic bacteria, Escherichia coli, Salmonella, Staphylococcus aureus, Listeria monocytogenes and Yersinia enterocolitica. Methods Based on the sequence of phoA, Salmonella invA, Staphylococcus aureus nuc, Listeria monocytogenes and Yersinia enterocolitica ail, multiple direct PCR primers were designed to optimize and establish multiplex PCR reaction conditions to detect the amplification of primers Increase specificity and sensitivity, and apply the established method to the actual sampling of pork samples. Compared with gold standard culture method, the sensitivity, accuracy and positive predictive value of multiple direct PCR method were calculated. Results Multiple direct PCR primers were designed to amplify five kinds of bacteria. The minimum detection limit was 10 CFU of Escherichia coli, 100 CFU of S. aureus, Salmonella, Listeria, Yersinia enterocolitica sensitivity Up to 1CFU. Fifteen samples of Escherichia coli, six samples of Salmonella, 21 strains of Staphylococcus aureus, 20 strains of Listeria and 35 strains of Yersinia enterocolitis were detected in 60 samples of pork, the positive detection rate was higher than gold standard culture method, The overall detection sensitivity was 100%, the accuracy was 94%, and the positive predictive value was 81.44%. Conclusions Multiplex PCR method can detect both specific and sensitive foodborne pathogens at the same time, and omit the step of extracting template, which can shorten the detection time to about 3 hours, which is convenient for the common foodborne pathogens in food safety risk monitoring Fungus flux testing.