The Development Of Rapid Detection Technique For Several Foodborn Pathogens

In recent years,food safety incidents are very frequent.Food safety problems hasincreasingly become focus of social concern.Foodborn pathogens including Staphylo-coccus aureus, Salmonella,Vibrio haemolyticus, Shigella, Listeria Momocytogenes,Bacillus cereus, Vibrio alginolyticus are primary factors threatening food safety. Atpresent, traditional method including bacterial culture and biochemical identificationwas used for detection of foodborn pathogens. These methods are very complicated,time-consuming and can only detect one kind of bacterium each time. They usuallytakes about5-7days to get the results,and sensitivity and specificity are low. Therefo-re,establishing a rapid and efficient detection of foodborn pathogens method becomemore and more urgent.As the development of molecular biology, the whole genome of many foodbornpathogens strains has been determined. Molecular methods based on PCR arebecoming most commonly used because of its rapidness and high sensitivity.In thisstudy, according to the sequence of specific gene of foodborn pathogens, Loopmediated isothermal amplification, multiplex polymerase chain reaction and DNAmicroarray were applied for rapid detection of foodborn pathogens. including Vibrioparahaemolyticus, Staphylococcus aureus, Listeria monocytogenes, Salmonellaenteritidi and Shigella Flexneri. These methods would be very critical for rapiddetection of foodborn pathogens in suspected case.Loop-mediated isothermal amplification（LAMP）is a novel DNA amplificationtechnology which amplifies DNA with high specificity, efficiency and rapidity underisothermal conditions. In this paper,LAMP technique was applied for rapid detectionof Vibrio parahaemolyticus. According to the sequence of the specific tdh gene ofVibrio parahaemolyticus, a set of primers were designed to amplify the specifial DNAsequences by LAMP method. Moreover, the reaction conditions were optimized. Theresults showed the optimum LAMP assay for the rapid detection of Vibrio parahaemolyticus was performed at60.8℃for45min. No cross reaction was foundwith other common bacteria which showed a good specificity.The LAMP assay had adetection limit of was35.5fg/tube, it was10times lower than that of PCRassay.The LAMP assay could be finished within an hour requiring only a regularlaboratory water bath for reaction. The detection of the strains isolated from scallopsamples also proved the LAMP assay reliability.Multiplex polymerase chain reaction has the same principle with conventionalPCR, a mumber of primers is added to reaction system, achieve more than one targetgenes amplification in the same system at the same time. It overcomes the drawbackthat conventional PCR and LAMP reaction can only amplify one target gene. Amultiplex polymerase chain reaction was developed for simultaneus detection of fivemajor foodborne pathogens, including Vibrio parahaemolyticus,Staphylococcusaureus, Listeria monocytogenes,Salmonella enteritidi and Shigella Flexneri. Fivepairs of specific primers were designed according to the gene tlh of Vibrioparahaemolyticus,the gene nuc of Staphylococcus aureus, the gene hlyA of Listeriamonocytogenes, the gene invA of Salmonella enteritidi, the gene ipaH of ShigellaFlexneri.The reaction conditions were optimized and the specificity and sensitivitywere tested for this method.The results of detecting samples also proved its reliabilityand practicability.A DNA microarray combined with multiplex PCR was developed forsimultaneous detection of five major foodborn pathogens, including Vibrio parahaemolyticus, Staphylococcus aureus, Listeria monocytogenes, Salmonella enteritidi andShigella Flexneri, Bacillus cereus, Vibrio alginolyticus. Five specific genes, thethermolabile hemolysin gene of Vibrio parahaemolyticus, the heat stable nucleasegene of Staphylococcus aureus，the Listeriolysin O gene of Listeria monocytogenes,the invasion protein A gene of Salmonella, the invasion plasmid antigen H gene ofShigella, nheA gene of Bacillus cereus, rpoX gene of Vibrio alginolyticus were chosenas the amplification targets, labeled with a fluorescence dye by multiplex polymerasechain reaction (PCR),and hybridized to the specific virulence gene probes that hadbeen immobilized on a microchip.15bacterial strains were tested in the study, and only7target bacteria were positive.The detection Limit of the DNA microarray assaywas10times than that of PCR. The reaction conditions were optimized and thespecificity and sensitivity were tested for this method.The results of detecting38samples also proved its reliability and practicability.