Identification Of Foodborne Pathogens In Fresh Vegetables By MALDI-TOF MS

Foodborne pathogenic bacteria contamination on fresh vegetables has become a problem of global common concern. In order to monitor the pollution of foodborne pathogens,reduce its harm to human health,guarantee our agricultural products consumption safety. It is necessary to study on the rapid and reliable detection and identification technology. However, the existing methods have several shortcomings such as the complexity of the processing steps, the large workload and the long detection and identification period, which can’t meet the requirements of simple, fast and high throughput detection. In recent years, although Matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS) could provide a new solution for the identification of foodborne pathogens, the application of MALDI-TOF MS in fresh vegetables is still in the initial stage. In this paper,for establishing a suitable, fast,reliable, and high-throughput method for the detection and identification of pathogens on fresh vegetables, the vital pretreatment process and other factors which influence the mass spectrometry were investigated. The results are as follows:1、The standard strains of Escherichia coli, Escherichia coli O157: H7, Staphylococcus aureus, Salmonella,Listeria monocytogenes were used as model bacterium to investigate factors affecting the identification results such as the selection of matrix solution, cultivation conditions, solvent treatment method and the sample deposition method. Ultimately, the BHI medium being culturing for 24 hours, bacterial protein being extracted with 70% formic acid mixed in the same amount of acetonitrile, the sample solution and matrix solution(1 μL + 1 μL)being added on the target panel respectively were selected. Also, the matrix saturated solution of the α-Cyano-4-hydroxycinnamic acid(CHCA) in the mixed solvent of water, acetonitrile and trifluoroacetic acid(volume ratio is 19:20:1) were used. Different concentrations of bacterial liquid were identified after pretreatment, the results shows that the detection limits of the method is 106~109 cfu/mL; Stability testing shows the average score of the same strain was higher than 95%; The variation coefficient was less than 5%. This meet the requirement of the general detection need after enrichment culture, and the stability and the repeatability were well.2、The 16 S rRNA gene sequence analysis for 93 wild strains and 7 standard strains was used as a reference to evaluate the credibility of MALDI-TOF MS method. The MALDI-TOF MS method showed good stability for rapid detection of those strains and high consistency with the 16 S rRNA gene sequence analysis(83% consistency at the species level and 94% consistency at the genus level). For the 56 strains of foodborne pathogen isolates, MALDI-TOF MS can achieve the consistency of 100% at the genus level, 96.4% at the species level with the 16 S rRNA sequencing result.3、Using traditional culture and isolation with MS MALDI-TOF method, the common foodborne pathogens and other potential pathogenic bacteria were detected in 200 pieces of fresh fruits and vegetables. 82 strains of common foodborne pathogens, including Escherichia coli, Staphylococcus aureus, Salmonella, increasing Listeria monocytogenes, Bacillus cereus were isolated from the 69 samples. And the other potential pathogenic bacteria which can lead to food poisoning were also isolated such as Bacillus mirabilis, Pseudomonas aeruginosa, Enterococcus faecalis and so on. It is indicated that the microbial contamination of fresh vegetables in China deserves our important attention.