Research And Appilication Of Multiplex PCR Tcchnology With A Common Primer In The Detection Of Foodborne Pathogens

Aims:Developing a common primer-multiplex PCR assay which can detect eleven foodborne pathogens of Vibrio parahaemolyticus,Vibrio cholerae,Enterohaemorrhagic Escherichia coli O157:H7,Yersinia enterocolitica,Shigella flexneri,Clostridium botulinum type A,Staphylococcus aureus,Salmonella,Clostridium perfringens Alpha toxin,Listeria monocytogenes,Bacillus cereus.Methods:The common primers were first designed by using Primer 5.0 software and the specificity of the designed common primers against bacterial genomic DNA were preliminary verified by online BLAST in NCBI database.Then,eleven foodborne pathogenic genomic DNA were used as templates to further screen common primers with good specificity through temperature gradient PCR experiments.The plasmid template containing sequences of common primers was constructed to detect the sensitivity of each screened common primer.(2)The specific primers for the eleven pathogenic bacteria were ligated with the common primer to form chimeric primers,and the specificity of them was detected by multi-template PCR experiments respectively.The CP-MPCR system was then established and optimized.The specificity and sensitivity of the CP-MPCR system were further tested.(3)The specificity of the optimized CP-MPCR system was further verified with 100 pathogenic isolates collected from the Suzhou CDC.Sixty anal swab samples collected from Suzhou CDC and 16 enrichment cultured solutions of food samples collected from the Suzhou Food Inspection and Testing Center were tested using the CP-MPCR system to verify the practical applicability of the CP-MPCR system.Results:(1)Three common primers Ua,Ub and Uc were successfully designed and screened,none of them generate any amplification with eleven foodborne pathogens by temperature gradient PCR.The sensitivity of Ua,Ub and Uc reached 105,103 and 104 copies/?L respectively.(2)We designed and experimentally screened out specific chimeric primers with high specificity based on the specific genes of 11 foodborne pathogens,and successfully constructed a CP-MPCR system capable of detecting multiple foodborne pathogens.The ability of the CP-MPCR system to analyze multiple templates was further verified through multi-template experiments.The sensitivity of the CP-MPCR detection system was then detected by using cultured bacteria preparations and has been confirmed with a sensitivity of 103-104 CFU/mL,among them,the sensitivity of the CP-MPCR system for Vibrio cholerae and Shigella flexneri can even achieve 102 CFU/mL.The sensitivity of CP-MPCR detection system to genomic DNA of 11 pathogens was also determined and the result was about 0.05 ng/?L.(3)100 pathogenic bacteria isolates verified that the CP-MPCR system constructed in this study has a good specificity,only the target strain produced the target band and the non-target strains did not produce any bands.Among sixty anal swab samples and sixteen enrichment cultured solutions of food samples,the positive samples detected by the CP-MPCR system were verified by sequencing and were also verified by patent primers or standard primers of the People's Republic of China Entry-Exit Inspection and Quarantine Industry,and the results confirmed that they were consistent with the detection results of the CP-MPCR systemConclusion:The CP-MPCR system constructed in this study can realize non-directional screening of 11 common foodborne pathogens in China.The establishment of this method has certain significance for preventing the spread of pathogenic microorganisms and reducing the incidence of foodborne diseases.