摘要
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。
We established a multiplex direct PCR for rapid detection of E.coli,Salmonella,Staphylococcus aureus,Listeriaand Yersinia enterocolitica bacteria.Multiplex direct PCR primers were designed according to gene sequences of phoA（E.coli）,invA（Salmonella）,nuc（S.aureus）,hly（Listeria）,and ail（Y.enterocolitica）.After the multiplex direct PCR were established,the specificity and sensitivity of primers were detected.Then,multiplex direct PCR was applied to examine 60 swine product samples,the detection specificity,accuracy and positive predictive value were calculated compared with the gold standard culture method.Results showed that multiplex direct PCR primers could be used for specific detection of E.coli,Salmonella,S.aureus,Listeriaand Y.enterocolitica,with the minimal detectable limit of 10,1,100,1and 1CFU,respectively.For the examination of 60 swine product samples using multiplex direct PCR,15 were positive for E.coli,6positive for Salmonella,21 positive for S.aureus,20 positive for Listeria,and 35 positive for Y.enterocolitica,with all positive detection rates higher than that of culture.The total detection sensitivity was 100%,accuracy was 94%,and positive predictive value was 81.44%.Multiplex direct PCR could be used for specific and sensitive detection of common food-borne pathogens,and the testing time was shorten to be 3hours because of saving time for template extraction. Multiplex direct PCR might serve the detection of food-borne pathogens in food safety risk monitoring much better.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2017年第8期710-715,共6页
Chinese Journal of Zoonoses
基金
农业部"国家农产品质量安全风险评估重大专项"(No.GJFP2016007)资助
留学人员科技活动项目择优资助~~
关键词
多重直接PCR
猪肉
食源性致病菌
multiplex direct PCR
food-borne pathogens
rapid
sensitive
