Surface-enhanced Raman Spectroscopy For Rapid Detection Of Meat-derived Pathogens

Foodborne pathogen contamination is one of the important reasons for frequent food safety incidents,which brings great harm to human health,economic and social development.Meat food is rich in nutrition and is a good place for the growth and reproduction of pathogens,which leads to the serious contamination of pathogens in meat.Therefore,it is of great significance to establish a rapid detection method for pathogens in meat for the prevention of foodborne diseases.The traditional detection methods of pathogens usually can not achieve rapid and non-destructive detection,while Raman spectroscopy technology can achieve rapid,non-destructive and high sensitivity detection.Surface-enhanced Raman scattering(SERS),which uses rough precious metal surface as the substrate,is a special Raman spectroscopy technique.Its signal is usually stronger and more sensitive than ordinary Raman spectrum signals.However,the existing SERS studies usually used the microscopic Raman spectrometer to detect pure cultured pathogens,which could not achieve rapid and convenient detection,and few studies combined with meat samples for the detection of pathogens in meat samples.The portable Raman spectrometer is easy to carry,therefore,it is worthy to use portable Raman spectrometer for rapid and convenient detection of foodborne pathogens.In this study,a portable Raman spectrometer with Ag Nanoparticles(AgNPs)as a nano-enhanced substrate was used to investigate four kinds food-borne pathogens commonly found in meat and meat products,including Salmonella typhimurium,Escherichia coli,Listeria monocytogenes and Staphylococcus aureus(both single and mixed strains in pure cultures and in beef samples)and two non-pathogens,Listeria innocua and Listeria welshimeri for SERS detection.Based on above,the study aimed to classify the Raman spectroscopy data of different bacteria and to determine the limit of detection(LOD)of different pathogens under different cultivation environment(including pure culture and meat borne pathogens in beef samples).The main results of this study were as follows:(1)The detection conditions of the surface-enhanced substrate AgNPs were optimized.When the particle size of AgNPs was 170 nm and the volume ratio of nano-silver sol to bacterial solution was 5:1,the stability and Raman signal of the prepared AgNPs were the best.(2)Combined with SERS detection and multivariate statistical analysis to achieve qualitative detection of different foodborne pathogens.The pretreatment method of Raman spectrum was determined.The results illustrated that the Raman spectral clustering effect of Standard Normal Variate(SNV)processed meat pathogens of the same Gram type,genus and species is better than that of Multiplicative Scatter Correction(MSC)and the first derivative,the Raman spectra of different types of pathogens could be better separated,so the Raman spectra data pretreated by SNV were selected for analysis.Principal Component Analysis(PCA)and Hierarchical Clustering Analysis(HCA)were used to classify 6 bacterial species,resulting successfully realized the classification of different strains at the level of Gram type,genus and species,and distinguished L.monocytogenes from two non-pathogenic Listeria species.Linear discriminant Analysis(LDA)was used to verify the classification models of PCA and HCA,and the overall average classification accuracy rate was 95.65%.The results show that the classification results of this method are accurate.(3)The pure culture of S.typhimurium,E.coli and their mixed strains were identified by SERS.The comparation of SERS spectra of pure cultured Salmonella typhimurium,E.coli and their two kinds of mixed bacteria indicated that PCA classification trend was obvious.Thus,SERS can realize the classification and identification of the mixed bacteria and single bacteria of the two kinds of bacteria through PCA.(4)SERS method was used to detect pathogens in beef with high sensitivity.Four pathogens,namely S.typhimurium,L.monocytogenes,S.aur and E.coli,were returned to beef samples.SERS analysis showed that the recovery rate of four pathogens in beef samples ranged from 90.51%to 110.17%,the average recovery rate was 99.47%,and the classification accuracy of different pathogens reached 93%.Through analyzing and calculating SERS spectra of four kinds of pathogens at different concentrations(10-10~8 CFU/m L)in pure culture and beef samples,the LOD of S.typhimurium,L.monocytogenes,S.aur and E.coli in pure culture and beef samples were 9 CFU/m L and 4 CFU/m L,9 CFU/m L and 19 CFU/m L,11 CFU/m L and 5CFU/m L,16 CFU/m L and 23 CFU/m L,respectively.The sensitivity is significantly higher than the traditional detection method.To sum up,the portable Raman spectrometer used SERS method to detect pathogenic bacteria(pure culture and pathogenic bacteria in beef samples)within 2 h,the detection time is better than that of the traditional detection method in days and the Portable Raman instrument realized the rapid and convenient qualitative and quantitative detection of pathogenic bacteria.