摘要
为建立食品中Escherichia coli O157:H7的微滴数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)快速定量检测方法,针对E.coli O157:H7的特异性单拷贝基因hlyA设计引物探针,并进行特异性、灵敏度和重复性实验,同时通过人工污染三文鱼样品的检测,比较平板计数法、real-time PCR和ddPCR方法的测定值效果。结果表明,所建立的E.coli O157:H7 ddPCR检测方法具有良好的特异性、灵敏性和重复性。细菌纯培养液中定量限为105 CFU/mL,检出限为25 CFU/mL,人工污染三文鱼样品中检出限为110 CFU/g。对不同人工污染水平的三文鱼样品,ddPCR与平板计数的测定值结果无显著性差异(P>0.05),比real-time PCR方法的测定值结果更加稳定、准确。因此,本研究建立的ddPCR方法能够更加快速、准确、灵敏、特异地绝对定量检测食品中E.coli O157:H7。
A droplet digital polymerase chain reaction(ddPCR)method was developed for the rapid and quantitative detection of Escherichia coli O157:H7 in foods.A pair of primers and a probe were designed specific for the single copy gene of hlyA in E.coli O157:H7.The specificity,sensitivity and repeatability of this method were evaluated.At the same time,the plate counting method,real-time PCR and the ddPCR method were compared through the detection of artificially contaminated salmon samples using them.The results indicated that the ddPCR method had excellent specificity,sensitivity and repeatability in E.coli O157:H7 detection.The limit of detection(LOD)and limit of quantification(LOQ)for pure bacterial culture were 105 and 25 CFU/mL,respectively.The sensitivity of this developed method was 110 CFU/g in artificially contaminated salmon samples.At different levels of artificial contamination in salmon samples,there was no significant difference(P>0.05)between the results of ddPCR and plate counting,which were more stable and accurate than the results of real-time PCR.Therefore,the established ddPCR method can detect E.coli O157:H7 in food samples more rapidly,accurately,sensitively and specifically.
作者
魏咏新
马丹
李丹
徐蕾蕊
魏海燕
张西萌
刘莉
曾静
WEI Yongxin;MA Dan;LI Dan;XU Leirui;WEI Haiyan;ZHANG Ximeng;LIU Li;ZENG Jing(Beijing Customs Testing Center,Beijing 100026,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2020年第16期259-265,共7页
Food Science
基金
“十三五”国家重点研发计划重点专项(2017YFC1601602)
国家质量监督检验检疫总局科研项目(2017IK176)。
