(食品质量与安全研讨会)he study of multiplex PCR-based rapid detection technique for foodborne pathogen on fresh-cut fruits and vegetables

1、The study of multiplex PCR-based rapid detection technique for foodborne pathogen on fresh-cut fruits and vegetables Speaker：Ke FengDalian Nationality University 1Introduction2Methods3Results and discussion4ConclusionContentIntroductionList of Selected Multistate Foodborne Outbreak Investigations du

2、ring 9 YearsYearsListeria monocytogenesEscherichia coli O157:H7Staphylococcusaureus2014Dairy ProductsGround beef2013Cheeses SaladsNoodle, Chicken 2012CheeseSpinachDairy Products2011CantaloupeRomaine Lettuce, Hazelnuts2010Cheese2009 Beef, DoughFood2008Beef 2007 Pizza, Beef Pattiesvermicelliroll2006Fr

3、esh SpinachClinical manifestationsNausea and VomitingAbdominal painDiarrheaGastroenteritisDeathProcessing of fresh-cut fruits and vegetablesSales increasing of Fresh-cut productGlobal market volume of Fresh Fruit and VegetableCanned 6% Whole fresh 85%Other 3.5% Frozen 3% Fresh-cut 2.5% The market fo

4、r fresh-cut produce has witnessed dramatic growth in recent years.Processing of fresh-cut fruits and vegetablesIn fresh-cut fruit processing, typical operations such as peeling and cutting can promote microbial adhesion to the tissue, increasing the surface contact and the release of cellular conten

5、t rich in minerals, sugars, vitamins, and other nutrients, ideal substrates for foodborne pathogen growth.Food poisoning cases caused fresh-cut fruit would be an unnegligible problem. Metabolomicselectronic resistancemicrocalorimetryATP-BioluminescenceMolecular biologyDNA probePCRGene microarrayLAMP

6、ImmunologyELISAIMSimmunofluorescencelatexagglutinationBiosensorelectrochemicalsensoroptical Chemical SensorspiezoelectricsensorPhage identificationreporter bacteriophagefluorescencelabelingDetection techniqueDetection products3M Colonycounting mini-VIDASBAX System Q7Colloidal gold test stripMethodsL

7、. monocytogenesE. coli O157:H7S. aureusScreening and design of primerPCR and Condition optimization Multiplex PCR and Condition optimization Multiplex - PCRPMA Enrichment on fresh-cut fruits and vegetablesL. monocytogenesE. coli O157:H7S. aureuspineapplepapayapitayamangohoneydew meloncantaloupewater

8、meloncucumbersensitivity detectionColony countingTechnology roadmapFreshcutDual-filtration inoculated on The virulence factor genesListeria monocytogenesprfA-plcA-hlyA-mpl-actA-plcB; inlA, inlBStaphyloccocus aureusnuc, SmaIE. coli O157:H7wzy , slts, eae, hlyThe different kinds of pathogenic bacteria

9、 can be distinguished with their virulence factors.Heat-resisting nucleic acid enzymeHemolysinO-antigen polymerasePCR detection techniquePMA (propidium monoazide) is a high affinity photoreactive DNA binding dye.It preferentially binds to double stranded DNA with high affinity. Dual-filtration syste

10、mFiltration membranevortexDNA ExtractionChromogenic mediaFruits residueMicroporous filtration membraneResult and discussionMultiplex PCRmPCR react condition 10PCR buffer 2.5L MgCl2 3 mM dNTPs 0.25 mMPrimer (L. monocytlgenes) 0.08 MPrimer (E. coli O157:H7 ) 0.32 MPrimer (S. aureus ) 0.2 M DNA (L. mon

11、ocytlgenes )1.32 ng/L DNA (E. coli O157:H7)1.25ng/LDNA (S. aureus )1.40ng/LrTaq 1 U ddH2O up to 25 LReaction condition95 3min,94 40s,54.2 30s,72 40s,72 10 min.Fig.1 M: 1000bp DNA marker. Lane 1. L. monocytogenes at 285bp, E. coli O157:H7 at 193bp, S. aureus at 159bp. Lane 2-4. S. aureus at 159bp, E.

12、 coli O157:H7 at 193bp and L. monocytogenes at 285bp. Lane 5-6 is negative control. Fig. 1. Multiplex PCR applied to L. monocytogenes, E. coli O157:H7, S. aureus, respectively. 159bp193bp285bp32 cyclesThe specificity of internal primerM: 1000bp DNA marker. Lanes 1. L. monocytogenes (285bp), E. coli

13、O157:H7 (193bp), S. aureus (159bp). Lanes 2. S. aureus (159bp), E. coli O157:H7 (193bp). Lanes 3 L. monocytogenes (285bp), S. aureus (159bp). Lanes 4 S. aureus (159bp). E. coli O157:H7 (193bp). Lanes 5 negative control.Fig. 2. The internal verification and specificity of primer for the L. monocytoge

14、nes, E. coli O157:H7, S. aureus.Fig.2 At lane 1, under optimized multiplex PCR conditions, three pathogen DNA mixtures produced three bands. From Lanes 2 to 4 showed two bands which contained two random pathogens. Bacteria strains (No.)SourcemPCR resulthlynucwzyListeria monocytogenes ATCC 19111+-Lis

15、teria monocytogenes ATCC 19112+-Listeria monocytogenes ATCC 19115+-Listeria monocytogenes ATCC 15313+-Listeria monocytogenes GIM 1.229+-Staphylococcusaureus ATCC 6538-+-EscherichiacoliO157:H7NCTC 12900-+EscherichiacoliO157:H7CICC 21530-+Listeria ivanovii ATCC 19119-Listeria grayi ATCC 25401-Listeria

16、 seeligeri ATCC 35967-Listeria welshimeri ATCC 35897-Listeria innocua ATCC 33090-Salmenella typhimurium ATCC 14028-Salmonellaparatyphi Type BCMCC 50094-Salmonellatyphi CMCC 50071-Micrococcus luteus CMCC 28001-Proteus mirabilis CMCC 49005-Bacilluscereus CMCC 63301-Escherichiacoli CMCC 44102Escherichi

17、acoli ATCC 8739 -Enteroinvasive E.coliCICC 10661-Enterotoxigenic E. coliCICC 10414-Enteropathogenic E. coliCICC 10372-Vibrio parahemolyticusCICC 21617-Table. 1.The specificity of Multiplex PCRThe result demonstrated L. monocytogenes, S. aureus and E. coli O157:H7 were amplified effectively. No targe

18、t pathogen produced negative result.Sensitivity test of the mPCRFig. 3. The sensitivity of multiplex PCR assay using 10-fold serially diluted the population of L. monocytogenes, E. coli O157:H7 and S. aureus, from 108 cfu/ml to 10 cfu/ml. Lane 1-Lane 8 showed amplicon result for L. monocytogenes, E.

19、 coli O157:H7 and S. aureus (from 108 cfu/ml to 10 cfu/ml). Lane 9 and Lane 10 is negative controlFig.3 The result showed that the sensitivity of the multiplex PCR for mixed genomic DNA was 103 cfu/ml. while for L. monocytogenes was 103 cfu/ml. E. coli O157:H7 was 10 cfu/ml. S. aureus was 102 cfu/ml

20、. Fig. 4. M: 1000bp DNA marker. Lane 1-4 was genome DNA extracted from dead L. monocytogenes, E. coli O157:H7 and S. aureus with PMA treatment (5g/ml, 10g/ml, 20g/ml and 40g/ml). Lane 5-8 was genome DNA extracted from viable L. monocytogenes, E. coli O157:H7 and S. aureus with PMA treatment (5g/ml,

21、10g/ml, 20g/ml and 40g/ml).The detection of viable bacteriaFig. 4. The multiplex PCR with PMA treatmentFig.4 Growth potential of pathogen on fresh-cut fruitsGrowth potential of pathogen on the fresh-cut fruits at 25. ABCTable. 2. The colony count of L. monocytogenes, E. coli O157:H7 and S. aureus in

22、oculated on fresh-cut fruit based dual filtration membrane The filtration of pathogenFiltration (log cfu/ml)Polypropylene membrane (log cfu/ml)Nylon membrane (log cfu/mll)10M20M40M15M40M60MRe-filtration0.220.450.220.450.220.450.220.450.220.450.220.45L. monocytogenes8.907.207.087.457.207.817.638.588.

23、188.608.488.638.11E. coli O157:H78.707.577.187.717.497.857.628.157.978.237.938.348.18S. aureus8.387.697.387.787.547.987.738.047.948.088.048.237.99Different types of filtration were chosen to enhance detection limitation and save detection time. Fig. 8. The LOD of multiplex PCR assay using 10-fold se

24、rially diluted the population of L. monocytogenes, E. coli O157:H7 and S. aureus on fresh-cut fruits. M: 1000bp DNA marker. Lane 1- Lane 8 showed amplicon result for L. monocytogenes, E. coli O157:H7 and S. aureus (from 108 cfu/ml to 10 cfu/ml). Lane 9 and Lane 10 was negative control.The detection

25、limitation of pathogen on fresh-cut fruit Fig.5 The result showed detection limitation for three pathogen was 104 cfu/ml, while for L. monocytogenes was 104 cfu/ml, for E. coli O157:H7 was 102 cfu/ml, S. aureus was 103 cfu/ml. Conclusion1. The newly designed filtration-base PMA-mPCR assay in this pa

26、per was sensitive, specific and rapid for simultaneous detection of viable L. monocytogenes, S. aureus and E. coli O157:H7 on fresh-cut fruit. 2. Combination of the PMA-mPCR assay with filtration membrane could eliminate the effect of inhibitor from food sample effectively.3. This study showed the m

27、ethod was considered to be more successful in detecting L. monocytogenes, S. aureus and E.coli O157:H7.Conclusions已发表论文：1. 冯 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平安控制中的应用，食品工业 科技， 2021，3510：49-52，562. 冯 可，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. 2021，354：392-3963. 萨仁高娃，胡文忠，姜爱丽，马杰，冯可，产单核细胞增生

28、性李斯特氏菌致病机制的研究进展，食品工业科技， 2021，341：372-3764. 萨仁高娃，胡文忠，高春红，姜爱丽，冯可，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，2021，3417：173-176待发表论文：5. 关棣锴，胡文忠，朴永哲，冯可，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，胡文忠，朴永哲，冯可，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. Feng Ke, Hu Wenzhong, Jiang Aili, Fate of Sta

29、phylococcus aureus and Listeria monocytogenes on the fresh-cut melons fruits as impacted by storage temperature and time，International Journal of Food Microbiology, to be Submitted8. Feng Ke, Hu Wenzhong, Jiang Aili, Growth potential of Salmonella spp. and E. coli O157:H7 in seven types of fresh cut

30、 fruits stored at different temperature conditions，Food Microbiology, to be Submitted9. Feng Ke, Hu Wenzhong, Xu yongping, Growth potential of Listeria monocytogenes and Staphylococcus aureus on fresh- cut tropical fruits (pitaya, mango, payaya and pineapple). Journal of Food Protection, to be Submitted10. Feng Ke, Hu Wenzhong, Xu yongping, Modeling the growth of Listeria monocytogenes in fresh-cut cucumber at different temperatures. Food Control, to be Submitted11. 冯可，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，经