Study On Rapid Detection Technology For Foodborne Pathogens On Fresh-cut Fruits And Vegetables

Fresh-cut fruits and vegetables have the characteristic of fresh,nutritious,convenient and safe as a new type of fresh agricultural products.Consumers'demands for fresh-cut fruits and vegetables are continuing to grow rapidly and occupying an important position in people's daily life.Artificial mechanical wounding lead to damage of tissues and occurrence of a series of physiological and biochemical reactions such as increasing respiratory rate,stimulating ethylene,accelerating the enzymatic browning,producing some secondary metabolites and so on.More importantly,it is vulnerable to contaminated by foodborne pathogen due to outflow of nutrients after cutting,causing the outbreak of food-borne diseases and endanger consumer health and life safety.At present,the national methods of detecting foodborne pathogenic microorganisms have some disadvantages such as more procedures,time-consuming and detection time is 5-7 d.However,the shelf and preservation life of fresh-cut fruits and vegetables is only 3-5 d.Obviously,the current method of detection didn't assure the microorganisms safety of the product.Therefore,the establishment of efficient,rapid detection and monitor technology is imperative for foodborne pathogen on fresh-cut fruits and vegetables.It has important theory and realistic significance.Fresh-cut fruits and vegetables were chosen in this study.Listeria monocytogenes,Staphylococcus aureus,Escherichia coli 0157:H7,Salmonella typhimurium and Vibrio parahaemolyticus causing disease were selected as the target bacteria,to establish rapid detection technology and provide the technology supporting on detection of foodborne pathogens contaminating fresh agricultural products during processing,logistics,storage,sale.1.Growth potential of foodborne pathogen on fresh-cut fruits was studied.In this study,the effects of different storage temperatures(5??13 ? and 25 ?)on the growth of foodborne pathogen on six types of fresh-cut fruits were analyzed.It indicated that inappropriate storage temperatures(13 ? and 25 ?)caused significant growth of Listeria monocytogenes,Staphylococcus aureus,Escherichia coli 0157:H7 and Salmonella typhimurium on fresh-cut fruits.While foodborne pathogen didn't grow but survival at the low temperature storage(5?).Staphylococcus aureus can only grow and survive on fresh-cut pineapple,while other target bacteria can't grow on fresh-cut pineapple.It showed that foodborne pathogen has reproductive capacity on fresh-cut fruits and potential risks for health.Temperature is the important factor which affect growth of foodborne pathogen and guarantee safety of fresh-cut fruits and vegetables.2.The establishment of multiplex PCR assay for Listeria monocytogenes,Staphylococcus aureus,Escherichia coli 0157:H7 and Salmonella typhimurium on fresh-cut fruits and vegetables.Four pairs of specific primers were designed and screened.The detection limite of multiplex PCR was 3.5×106cfu/mL(L.monocytogenes),2.4×105 cfu/mL(S.aureus),4.8×105 cfu/mL(E.coli 0157:H7)and 1.6×105 cfu/mL(S.typhimurium).The different inoculation concentrations(100,103,106 cfu/mL)were determined using multiplex PCR,detection limit is 100 cfu/mL after 9 h enrichment.It is effective to apply in fresh-cut lettuce,fresh-cut cucumber,fresh-cut papaya,fresh-cut cantaloupe.Therefore,the detection limit of multiplex PCR is 100 cfu/g for pathogen microorganism inoculated on fresh-cut fruits and vegetables.This method could be save labor,reagent and time compared with traditional culture method.The detection time is changed from 5-7 d to 9-12 h.This method has practical significance for the detection of batch samples from professional testing organizations or analysis test centers.3.The mutiplex real-time quantitive PCR rapid assay of four foodborne pathogens on fresh-cut fruits and vegetables was developed.In this study,based on the shortcomings of inability to distinguish dead and living bacteria with molecular biology for nucleic acid detection,combining propidium monoazide(PMA)with mutiplex real-time quantitive PCR can modify the DNA of dead cells in incomplete cell membranes,inhibit DNA amplification and then exclude the interference of false positive.According to the target genes of L.monocytogenes,S.aureus,E.coli 0157:H7 and S.typhimurium,HEX,Cy5,FAM and ROX were selected to label the four probes.After orthogonal experiment optimization,a rapid detection technique for simultaneous detection of four target bacterias was established.The detection limits of the four target bacteria were 5.4×104 cfu/mL,1.8×104 cfu/mL,7.3×104 cfu/mL and 4.2×104 cfu/mL.A double filtration device was developed to filter the residues of fruits and vegetables and to collect foodborme pathogen based on rapid detection.The device was composed of 15 ?m nylon membrane and 0.22 ?m mixed filter membrane.Combining the double filtration device with the PMA-MqPCR,four kinds of foodborne pathogens on fresh-cut fruits and vegetables can be detected quantitatively without culturing.The detection limit was approximately 104 cfu/g,detection time was 3-4h.4.A rapid detection assay of in situ synthesized gene chip for foodborne pathogen was established.The target gene of Listeria monocytogenes,Staphylococcus aureus,Escherichia coli 0157:H7,Salmonella typhimurium and Vibrio parahaemolyticus were designed and screened in the way of tiling type probe.By comparing the specific sequences of the five target bacteria in the NCBI database,the target gene was obtained and the hybridization system was optimized.Result indicated that 141 specific probes were obtained by screening 3227 hybridization probe,that is,26 L.monocytogenes probes,24 S.aureus probes,25 E.coli 0157:H7 probes,20 S.typhimurium probes,46 V.parahaemolyticus probes.No amplification signal was obtained among target bacteria detection.The application of in situ synthesized gene chip to detect the foodborne pathogen on fresh-cut fruits and vegetables.This assay showed the characteristic of strong amplification signal and high accuracy.The detection limit was approximately 103 cfu/g without culturing,detection time was 24 h.The obtained primers and probes have universality and originality and lay a foundation for DNA microarray and visualization chip.The foodborne pathogen detection technology established in this study can effectively detect and monitor foodborne pathogen on fresh-cut fruits and vegetables during processing,cleaning,fresh-cutting,packaging,storage,logistics and sale in the whole chain,then improve quality and safety control technology of fresh-cut fruits and vegetables.