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1、The The study study of of multiplex multiplex PCR-PCR-based based rapid rapid detection detection technique technique for for foodborne foodborne pathogen pathogen on on fresh-cut fruits and vegetables fresh-cut fruits and vegetables Speaker：Ke FengDalian Nationality University 1IntroductionIntroduc
2、tion2MethodsMethods3Results and Results and discussiondiscussion4ConclusionConclusionContentContentIntroductionList of Selected Multistate Foodborne Outbreak Investigations during 9 YearsYearsListeriamonocytogenesEscherichiacoliO157:H7Staphylococcusaureus2014DairyProductsGroundbeef2013CheesesSaladsN
3、oodle,Chicken2012CheeseSpinachDairyProducts2011CantaloupeRomaineLettuce,Hazelnuts2010Cheese2009Beef,DoughFood2008Beef2007Pizza,BeefPattiesvermicelliroll2006FreshSpinachClinicalmanifestationsNauseaandVomitingAbdominalpainDiarrheaGastroenteritisDeathProcessingoffresh-cutfruitsandvegetablesSalesincreas
4、ingofFresh-cutproductGlobalmarketvolumeofFreshFruitandVegetableCanned6%Wholefresh85%Other3.5%Frozen3%Fresh-cut2.5%Themarketforfresh-cutproducehaswitnesseddramaticgrowthinrecentyears.Processingoffresh-cutfruitsandvegetablesInfresh-cutfruitprocessing,typicaloperationssuchaspeelingand cutting can promo
5、te microbial adhesion to the tissue,increasingthesurfacecontactandthereleaseofcellularcontentrich in minerals, sugars, vitamins, and other nutrients, idealsubstratesforfoodbornepathogengrowth.Foodpoisoningcasescausedfresh-cutfruitwouldbeanunnegligibleproblem.Metabolomicselectronicresistancemicrocalo
6、rimetryATP-BioluminescenceMolecularbiologyDNAprobePCRGenemicroarrayLAMPImmunologyELISAIMSimmunofluorescencelatexagglutinationBiosensorelectrochemicalsensoropticalChemicalSensorspiezoelectricsensorPhageidentificationreporterbacteriophagefluorescencelabelingDetectiontechniqueDetectionproducts3MColonyc
7、ountingmini-VIDASBAXSystemQ7ColloidalgoldteststripMethodsMethodsL.monocytogenesE.coliO157:H7S.aureusScreeninganddesignofprimerPCRandConditionoptimizationMultiplexPCRandConditionoptimizationMultiplex-PCRPMA Enrichmentonfresh-cutfruitsandvegetablesL.monocytogenesE.coliO157:H7S.aureuspineapplepapayapit
8、ayamangohoneydewmeloncantaloupewatermeloncucumbersensitivitydetectionColonycountingTechnologyroadmapFreshcutDual-filtration inoculatedonThevirulencefactorgenesListeriamonocytogenesprfA-plcA-hlyA-mpl-actA-plcB;inlA,inlBStaphyloccocusaureusnuc,SmaIE.coliO157:H7wzy,slts,eae,hlyThe different kinds of pa
9、thogenic bacteria can bedistinguishedwiththeirvirulencefactors.Heat-resistingnucleicacidenzymeHemolysinO-antigenpolymerasePCRdetectiontechniquePMA(propidiummonoazide)isahighaffinityphotoreactiveDNAbindingdye.ItpreferentiallybindstodoublestrandedDNAwithhighaffinity.Dual-filtrationsystemFiltrationmemb
10、ranevortexDNAExtractionChromogenicmediaFruitsresidueMicroporousfiltrationmembraneResultanddiscussionMultiplexPCRmPCRreactcondition10PCRbuffer2.5LMgCl23mMdNTPs0.25mMPrimer(L.monocytlgenes)0.08MPrimer(E.coliO157:H7)0.32MPrimer(S.aureus)0.2MDNA(L.monocytlgenes)1.32ng/LDNA(E.coliO157:H7)1.25ng/LDNA(S.au
11、reus)1.40ng/LrTaq1UddH2Oupto25LReactioncondition953min,9440s,54.230s,7240s,7210min.Fig.1M:1000bpDNAmarker.Lane1.L. monocytogenes at285bp,E. coliO157:H7at193bp,S. aureus at159bp.Lane2-4.S. aureus at159bp,E. coliO157:H7at193bpandL. monocytogenes at285bp.Lane5-6isnegativecontrol. Fig.1.MultiplexPCRappl
12、iedtoL. monocytogenes, E. coli O157:H7,S. aureus,respectively. 159bp193bp285bp32cyclesThespecificityofinternalprimerM: 1000bp DNA marker. Lanes 1. L. monocytogenes (285bp), E. coli O157:H7 (193bp), S. aureus (159bp). Lanes 2. S. aureus (159bp), E. coli O157:H7 (193bp). Lanes 3 L. monocytogenes (285b
13、p), S. aureus (159bp). Lanes 4 S. aureus (159bp). E. coli O157:H7 (193bp). Lanes 5 negative control.Fig.2.TheinternalverificationandspecificityofprimerfortheL. monocytogenes,E. coli O157:H7,S. aureus.Fig.2At lane 1, under optimized multiplex PCR conditions, three pathogen DNA mixtures produced three
14、 bands. From Lanes 2 to 4 showed two bands which contained two random pathogens. Bacteriastrains(No.)SourcemPCRresulthlynucwzyListeriamonocytogenesATCC19111+-ListeriamonocytogenesATCC19112+-ListeriamonocytogenesATCC19115+-ListeriamonocytogenesATCC15313+-ListeriamonocytogenesGIM1.229+-Staphylococcusa
15、ureusATCC6538-+-EscherichiacoliO157:H7NCTC12900-+EscherichiacoliO157:H7CICC21530-+ListeriaivanoviiATCC19119-ListeriagrayiATCC25401-ListeriaseeligeriATCC35967-ListeriawelshimeriATCC35897-ListeriainnocuaATCC33090-SalmenellatyphimuriumATCC14028-SalmonellaparatyphiTypeBCMCC50094-SalmonellatyphiCMCC50071
16、-MicrococcusluteusCMCC28001-ProteusmirabilisCMCC49005-BacilluscereusCMCC63301-EscherichiacoliCMCC44102EscherichiacoliATCC8739-EnteroinvasiveE.coliCICC10661-EnterotoxigenicE.coliCICC10414-EnteropathogenicE.coliCICC10372-VibrioparahemolyticusCICC21617-Table.1.ThespecificityofMultiplexPCRTheresultdemon
17、stratedL.monocytogenes,S.aureusandE.coliO157:H7wereamplifiedeffectively.Notargetpathogenproducednegativeresult.Sensitivity test of the mPCRFig.3.ThesensitivityofmultiplexPCRassayusing10-foldseriallydilutedthepopulationofL. monocytogenes,E. coli O157:H7andS. aureus,from108cfu/mlto10cfu/ml.Lane1-Lane8
18、showedampliconresultforL. monocytogenes,E. coli O157:H7andS. aureus (from108cfu/mlto10cfu/ml).Lane9andLane10isnegativecontrolFig.3TheresultshowedthatthesensitivityofthemultiplexPCRformixedgenomic DNA was 103 cfu/ml. while for L. monocytogenes was 103cfu/ml.E.coliO157:H7was10cfu/ml.S.aureuswas102cfu/
19、ml. Fig. 4. M: 1000bp DNA marker. Lane 1-4 was genome DNA extracted from dead L. monocytogenes,E. coli O157:H7andS. aureuswithPMAtreatment(5g/ml,10g/ml,20g/mland 40g/ml). Lane 5-8 was genome DNA extracted from viable L. monocytogenes, E. coli O157:H7andS. aureuswithPMAtreatment(5g/ml,10g/ml,20g/mlan
20、d40g/ml).ThedetectionofviablebacteriaFig.4.ThemultiplexPCRwithPMAtreatmentFig.4Growthpotentialofpathogenonfresh-cutfruitsGrowthpotentialofpathogenonthefresh-cutfruitsat25.ABCTable.2.ThecolonycountofL. monocytogenes,E. coli O157:H7andS. aureusinoculatedonfresh-cutfruitbaseddualfiltrationmembraneThefi
21、ltrationofpathogenFiltration(logcfu/ml)Polypropylenemembrane(logcfu/ml)Nylonmembrane(logcfu/mll)10M20M40M15M40M60MRe-filtration0.220.450.220.450.220.450.220.450.220.450.220.45L. monocytogenes8.907.207.087.457.207.817.638.588.188.608.488.638.11E. coliO157:H78.707.577.187.717.497.857.628.157.978.237.9
22、38.348.18S. aureus8.387.697.387.787.547.987.738.047.948.088.048.237.99Differenttypesoffiltrationwerechosentoenhancedetectionlimitationandsavedetectiontime.Fig.8.TheLODofmultiplexPCRassayusing10-foldseriallydilutedthepopulationofL. monocytogenes,E. coli O157:H7andS. aureus onfresh-cutfruits.M:1000bpD
23、NAmarker.Lane1-Lane8showedampliconresultforL. monocytogenes,E. coli O157:H7andS. aureus (from108cfu/mlto10cfu/ml).Lane9andLane10wasnegativecontrol.Thedetectionlimitationofpathogenonfresh-cutfruitFig.5The result showed detection limitation for three pathogen was 104cfu/ml,whileforL.monocytogeneswas10
24、4cfu/ml,forE.coliO157:H7was102cfu/ml,S.aureuswas103cfu/ml.Conclusion1.Thenewlydesignedfiltration-basePMA-mPCRassayinthispaperwassensitive,specificandrapidforsimultaneousdetectionof viable L. monocytogenes, S. aureus and E. coli O157:H7 onfresh-cutfruit.2. Combination of the PMA-mPCR assay with filtr
25、ationmembranecouldeliminatetheeffectofinhibitorfromfoodsampleeffectively.3. This study showed the method was considered to be moresuccessful in detecting L. monocytogenes, S. aureus and E.coliO157:H7.Conclusions已已发表表论文：文：1.冯可可，胡文忠，姜爱丽，徐永平，萨仁高娃，预测微生物学在鲜切果蔬产品质量安全控制中的应用，食品工业科技，2014，35（10）：49-52，562.冯可可
26、，胡文忠，姜爱丽，徐永平，萨仁高娃.单核细胞增生性李斯特菌分子生物学检测技术的研究进展，食品工业科技.2014，35（4）：392-3963.萨仁高娃，胡文忠，姜爱丽，马杰，冯可可，产单核细胞增生性李斯特氏菌致病机制的研究进展，食品工业科技，2013，34（1）：372-3764.萨仁高娃，胡文忠，高春红，姜爱丽，冯可可，马杰，不同初始浓度的单增李斯特菌在营养肉汤中生长预测模型的建立，食品工业科技，2013，34（17）：173-176待待发表表论文：文：5.关棣锴，胡文忠，朴永哲，冯可可，鲜切甜瓜中单核细胞增多性李斯特菌的荧光定量PCR快速检测方法的研究，食品工业科技，印刷中6.关棣锴，胡文
27、忠，朴永哲，冯可可，TaqMan探针法荧光定量PCR检测单核细胞增多性李斯特菌的研究，食品工业科技，印刷中7.FengKe,HuWenzhong,JiangAili,FateofStaphylococcusaureusandListeriamonocytogenesonthefresh-cutmelonsfruitsasimpactedbystoragetemperatureandtime，InternationalJournalofFoodMicrobiology,tobeSubmitted8.FengKe,HuWenzhong,JiangAili,GrowthpotentialofSalm
28、onellaspp.andE.coliO157:H7inseventypesoffreshcutfruitsstoredatdifferenttemperatureconditions，FoodMicrobiology,tobeSubmitted9.FengKe,HuWenzhong,Xuyongping,GrowthpotentialofListeria monocytogenesandStaphylococcus aureusonfresh-cuttropicalfruits(pitaya,mango,payayaandpineapple).JournalofFoodProtection,
29、tobeSubmitted10.FengKe,HuWenzhong,Xuyongping,ModelingthegrowthofListeria monocytogenesinfresh-cutcucumberatdifferenttemperatures.FoodControl,tobeSubmitted11.冯可可，胡文忠，姜爱丽，植物精油的抑菌活性及其在鲜切果蔬中的应用，食品工业科技12.萨仁高娃，胡文忠，修志龙，姜爱丽，冯可可，经微酸性电解水处理的鲜切皇冠梨上的单增李斯特菌生长模型的建立，食品工业科技13.纪懿芳，胡文忠，姜爱丽，冯可可，董妍，海产品中副溶血弧菌检测方法研究进展，食品工
30、业科技14.董妍，胡文忠，姜爱丽，冯可可，吕晓萌，纪懿芳，食源性大肠杆菌快速检测技术研究进展，食品工业科技Papers会会议发表表论文：文：1.FengKe,HuWenzhong,JiangAili,Sarengawa,FateofListeria monocytogen，Staphylococcus aureus, Salmonella SPPO157:H7 onseventypesoffresh-cutfruits,17thWorldCongressofFoodScienceandTechnology,Canada,2014.8.15-22(Poster)2.冯可可,胡文忠,姜爱丽,萨仁高
31、娃，预测微生物学在鲜切果蔬质量安全控制中的应用，CIFST-中国食品科学技术学会第十届年会暨第七届东西方食品业高层论坛，南京3.冯可可,胡文忠,姜爱丽,萨仁高娃，单核细胞增生性李斯特菌分子生物学检测技术的研究进展，CIFST-中国食品科学技术学会第十届年会暨第七届东西方食品业高层论坛，南京4.冯可可,胡文忠,姜爱丽,萨仁高娃，生物传感器在食源性致病菌检测中的研究进展，CIFST-中国食品科学技术学会第十届年会暨第七届东西方食品业高层论坛，南京5.冯可可，胡文忠，徐永平，姜爱丽，萨仁高娃，张爽，张炜佳，单增李斯特菌和金黄色葡萄球菌在鲜切瓜类水果中的生长潜力，中国中国食品科学技术学会第十一届年会，杭州6.冯可可，胡文忠，徐永平，姜爱丽，萨仁高娃，鲜切黄瓜中单增李斯特菌的预测模型的建立，中国中国食品科学技术学会第十一届年会，杭州7.冯可可，胡文忠，徐永平，姜爱丽，萨仁高娃，谷娜，多重PCR检测三种食源性致病菌的方法建立，中国中国食品科学技术学会第十一届年会，杭州8.冯可可，胡文忠，徐永平，姜爱丽，萨仁高娃，植物精油的抑菌活性及在鲜切果蔬中的应用，中国中国食品科学技术学会第十一届年会，杭州9.冯可可，胡文忠，徐永平，姜爱丽，萨仁高娃，张炜佳，张爽，鼠伤寒沙门氏菌和大肠埃希氏菌O157:H7在七种鲜切水果中的生长潜力，中国中国食品科学技术学会第十一届年会，杭州论文发表Thankyou!
