Establishment Of Triplex Real-time PCR Method For Detection Of Foodborne Pathogens

With the improvement of people's life level,more and more attention was paid to food safety.Every year,the bacterial pathogens,which come from food, compromise people's life seriously.Salmonella strains,Listeria Moncytogenes and enterohemorrhagic Escherichia coli O157:H7 are among the most important foodborne bacterial pathogens.It need to establishment a correct,rapidly method to determind these bactetia.Therefore,we established a few real-time Polymerase chain reaction(real-time PCR) methods to detect the meatborne pathogens,such as Salmonella,Listeria Moncytogenes and E.coli O157:H7.Target genes of different pathogens were determined according the genes previously reported.The primers and the probes were designed according to the published DNA sequences of the Salmonella ttrRSBCA,the L.Moncytogenes hlyA and the E.coli O157:tt7 rfbE locuses.We developed 3 simplex real-time PCR assays, from which a triplex real-time PCR assay was further developed for the detection of the meatborne pathogens.Sensitivity of the tube was improved by optimization. Specificity of each simplex real-time PCR assay was validated by testing various bacteria including positive isolates and negative isolates.The sensitivity of each simplex PCR assay was determined by detecting serially diluted purified DNA and DNA extracted from bacterial culture by boiling.The sensitivity of the triplex real-time PCR assay was determined by detecting serially diluted DNA extracted from bacterial culture by boiling.Each simplex real-time PCR showed good specificity. There were no false-negative in the assays.The sensitivity of each simplex PCR assay was 100 fg genome DNA for Salmonella,100 fg for L.monocytogenes and 10 fg for E.coli O157:H7 DNA per tube.The standard curve with threshold cycle(C_t) value against the mass of DNA molecules added showed good linear correlation(R~2＞0.98) at the range of 100 fg～100 ng for Salmonella,100 fg～100 ng for Listeria monocytogenes,100 fg～100 ng for E.coli O157:H7.In each simplex PCR assay, as few as 235 CFU of Salmonella,13.15 CFU of Escherichia coli O157:H7 and 607.5 CFU of Listeria monocytogenes per tube were detected.The standard curve with C_t value against lg(CFU/mL) showed good linear correlation(R~2＞0.97) at the range of 10~3 CFU/mL～10~8 CFU/mL for Salmonella,10~5 CFU/mL～10~8 CFU/mL for L.monocytogenes and 10~2 CFU/mL～10~8 CFU/mL for E.coli O157:H7. Comparing with the simplex real-time PCR assay,the sensitivity of the triplex PCR assay detecting the Salmonella and the Listeria monocytogenes were not changed, except for the E.coli O157:H7(131.5 CFU/tube).The standard curve with Ct value against lg(CFU/mL) showed good linear correlation(R~2＞0.98) at the range of 10~3 CFU/mL～10~8 CFU/mL for Salmonella,10~5 CFU/mL～10~8 CFU/mL for L. monocytogenes and 10~3 CFU/mL～10~8 CFU/mL for E.coli O157:H7.The triplex real-time PCR provides a rapid,specific,and sensitive method for identifying Salmonella,L.monocytogenes and E.coli O157:H7.In the 539 samples,21 were Salmonella positive,7 were L.monocytogenes positive,13 were E.coli O157:H7 posive.By identification,21 Salmonella positive,5 were L.monocytogenes positive, and there were no E.coli O157:H7 positive.The established triplex real-time PCR assay is quick,convenient,accurate,high efficient and can be applied to detect the foodborne pathogens in meat and clinical samples.And the triplex PCR method is valuable for exploitation and application. Results of this study can also be considered as groundwork for national or regional standard methods.