Study Of Rapid Detection Of Important Pathogenic Bacteria In Dairy And Meat Products By Real-time Recombinase Polymerase Amplification(real-time RPA)

China is the leading nation of consuming dairy products and meat around the world,enhancing the importance of microbial safety in food.Pathogenic bacteria like Yersinia enterocolitica and Escherichia coli O157:H7 in dairy products and meat,which can cause food-borne diseases,always receive great concerns.Conventional ways to detect these pathogens in dairy products and meat require long testing period and complicate operation.Molecular biological technologies,taking PCR as a representative,shorten the detection period but increase the operator requirements,and are not suitable for primary laboratories and on-site detection during food safety supervision.As a new isothermal amplification technique,Recombinase Polymerase Amplification(RPA)assay just need simple instruments to achieve specific nucleotide sequences amplification in 5?20 mins at room temperature.The results could be visualized by means of agarose gel electrophoresis,real-time fluorescence signal or others.In this study,pathogenic Yersinia enterocolitica and Escherichia coli O157:H7 were detected by real-time RPA.Specific primers and exo probes were designed according to the sequences of Yersinia enterocolitica pathogenic ail(Attachment Invasion Locus)gene(NC?008800.1)and Escherichia coli O157:H7 rfb E gene(S83460.1).The comparement of three DNA extraction methods and RPA running programs,and the specificity,sensibility and stability studies as well as artificial contamination studies were carried out in succession.Finally,the real-time RPA assay to detect these two pathogens were established and verified in real samples.The results of the real-time RPA assays are listed below.(1)Among the PSS Magtration~R 12GC PLUS Nucl Acid extractor and its Mag DEA~RDNA200(GC)kit,Quick Gene DNA tissue kit and TIANamp Bacteria DNA kit,the TIANamp kit showed the best effect to extract DNA from sample solution.Furthermore,certain experiments showed that the boiling method could be utilized in real-time RPA.(2)The detection could be completed within 20 mins at 37?with good specificity.The detection limit of pathogenic Yersinia enterocolitica DNA and Escherichia coli O157:H7DNA is 0.1 ng/?L and 0.01 ng/?L separately.(3)In stability study,the assay was performed at a series dilution of target DNA from100 ng/?L to 0.001 ng/?L in octuplicate.In pathogenic Yersinia enterocolitica,the relative standard deviation(RSD)of threshold time and maximum fluorescence values of the eight parallels at 0.1 ng/?L DNA were the lowest and?7.06%.In Escherichia coli O157:H7,the DNA concentration of 0.01 ng/?L and above could be detected steadily.(4)In artificially contaminated powdered milk and beef samples with an initial pathogenic Yersinia enterocolitica concentration of 3 CFU/25g,the pathogen could be detected using the real-time RPA method after 20 hours of incubation.When in milk and chicken samples contaminated artificially with an initial Escherichia coli O157:H7concentration of 4 CFU/25g,the pathogen could be detected after 9 hours of incubation.(5)Of each assay,the detection of 20 different real samples showed that the results of real-time RPA assay were consistent with that of GB 4789.8-2016 and GB4789.36-2016,the national standard of China to detect Yersinia enterocolitica and Escherichia coli O157:H7.In conclusion,the real-time RPA assays established in this study to detect pathogenic Yersinia enterocolitica and Escherichia coli O157:H7 in dairy products and meat can detect them indeed with good specificity and sensibility at room temperature and shorten the detection period substantially.The results are consistent with national standards of China.With these advantages,the real-time RPA assays can be used for conducting quick screening and on-site detection of Yersinia enterocolitica and Escherichia coli O157:H7 in dairy products and meat.The real-time RPA assays are practical for qualitative and quick detection of pathogenic Yersinia enterocolitica and Escherichia coli O157:H7 in dairy products and meat at room temperature.