Establishment Of A Method For Rapid Detection Of Five Foodborne Pathogens By Antimicrobial Peptide Capture Combined With Multiplex PCR

Foodborne pathogen infection is an important factor affecting public health safety.Rapid detection of foodborne pathogenic bacteria in food is of great value to ensure food safety.Due to the complexity of food matrix and low content of pathogens,the traditional detection of foodborne pathogens requires bacteria enrichment,isolation,identification and other steps to complete the detection,which takes a long time,tedious operation and complex steps.It shows some limitations in the actual detection.The effective enrichment of foodborne pathogens in the process of sample pretreatment will greatly improve the sensitivity of later detection results.Antimicrobial peptides(AMPs)are short peptides with broad-spectrum antibacterial activity and can bind to bacterial cell membranes.In this study,taking advantage of the binding capacity of AMPs and cell membrane,antimicrobial peptides were used to capture foodborne pathogens,and combined with multiplex PCR(mPCR)to detect five foodborne pathogens(Staphylococcus aureus,Escherichia coli,Shigella and Pseudomonas aeruginosa)at the same time.Firstly,the specific genes of S.aureus,E.coli,Shigella and P.aeruginosa were screened by bioinformatics,and based on the K.pneumoniae specific gene reported by Tian et al.(2019),five pairs of specific primers were designed by Primer Explorer V5.PCR specificity evaluation showed that five specific genes could be used as target genes for detection of five foodborne pathogens such as S.aureus,E.coli,Shigella,K.pneumoniae and P.aeruginosa.Based on five specific genes,a mPCR method was established to detect S.aureus,E.coli,Shigella,K.pneumoniae,P.aeruginosa synchronously,and the detection limits were 10~2 CFU/m L,10~3 CFU/m L,10~2CFU/m L,10~2 CFU/m L and 10~2 CFU/m L,respectively.Secondly,taking advantage of the binding of antimicrobial peptide Cathelicidin-DM to the surface of bacterial cell membrane,Cathelicidin-DM was designed as a broad-spectrum bacterial capture element.In the first step,the binding of FITC-labeled Cathelicidin-DM to bacteria was observed by laser confocal microscope.The magnetic bead-ELISA method was used to further verify that the antimicrobial peptide Cathelicidin-DM can capture bacteria.The results of the two methods show that the antimicrobial peptide Cathelicidin-DM can be designed as the capture element of foodborne pathogens.In the second step,a PMAxx-q PCR method was established for quantitative analysis of five target bacteria captured by four different antimicrobial peptides SMAP-29,BMAP-27,Cathelicidin-DM and Chicken CATH-2 coupled with BM.Among them,Cathelicidin-DM had the best capture effect on E.coli,K.pneumoniae,P.aeruginosa,Shigella,S.aureus,and the capture rates were 92.69%,85.82%,84.33%,91.59%and 78.87%,respectively.Finally,the antimicrobial peptide Cathelicidin-DM capture of bacteria and mPCR detection were combined to establish a complete detection method.Comparing the detection limit of antimicrobial peptide capture combined with mPCR detection method with ordinary PCR detection method for artificial contaminated bacteria,the detection limit of the established antimicrobial peptide capture combined with mPCR detection method was significantly higher than that of ordinary PCR detection method.The detection limit for E.coli,S.aureus,Shigella,P.aeruginosa,K.pneumoniae is10~1 CFU/m L、10~2 CFU/m L、10~1 CFU/m L、10~1 CFU/m L、10~1 CFU/m L.In summary,the method of antimicrobial peptide capture combined with mPCR for rapid detection of foodborne pathogenic bacteria can quickly,sensitively and accurately detect five kinds of foodborne pathogenic bacteria of E.coli,S.aureus,Shigella,P.aeruginosa,K.pneumonia.