| Foodborne pathogen is a major reason for food poisoning. As a kind of new and most important type of foodborne pathogens, Listeria monocytogenes has aroused wide concern in recent years. L. monocytogenes widely found in nature not only led to many human diseases, the mortality rate was 30%-40%, but also could infect animals, and it resulted in serious threat to people's health and caused significant economic losses. In the last two decades, foodborne diseases around the world caused by the L. monocytogenes have been increasing. Epidemiological survey showed that the disease is mainly due to human consumption of food contaminated by L. monocytogenes. Therefore, it is urgent to establish a rapid and accurate method for detection and identification L. monocytogenes in food products. Pyrolysis technology originated from the fifties of last century. High resolution pyrolysis gas chromatography mass spectrometry (HRPGC/MS) is a method to decompose the sample into small fragments and characteristic pyrolyates under the condition of high temperature, and the complex pyrolysates are separated by high-resolution capillary column and the structures of the components are confirmed by mass spectrometer detector.This study was based on the HRPGC/MS technology and the combination of chemometrics software, to establish the fingerprint database of L. monocytogenes and other microorganisms. The homology of L. monocytogenes was also identified in the whole-cell level. Rapid detection of L. monocytogenes in agricultural products was studied here.(1) The L. monocytogenes type strain CMCC54004 was used to evaluate the influence of different HRPGC/MS factors (the type of the capillary column, pyrolysis temperature, pyrolysis time and GC temperature program) on the result of the chromatograms for the pyrolysates. Based on the single factor condition, the optimal operating conditions of HRPGC/MS system were as follows:capillary column type for GC analysis was DB-WAXTER; pyrolysis temperature was 650?; pyrolysis time was 5 s; a three-step temperature-program was used in this study. The pyrolyer was a CDS model 5000 pyrolyzer. The result of the repeated experiment showed that HRPGC/MS was with good accuracy and stability. A normative HRPGC/MS technique has been build up. (2) This method was applied to 6 different Listeria species type strains,20 L. monocytogenes isolates and 10 different genuses of pathogens, which were Escherichia coli, Salmonella anatum, Shigella boydii, Enterobacter aerogenes, Proteus mirabilis, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Monilia albican, Bacillus subtilis and Staphylococcus aureus. By HRPGC/MS detection, the fingerprint databases for these pathogens were preliminarily built up. By cluster analyzing, the dendrogram results showed that this method can be used as a rapid and accurate method for identification of bacteria in genus and species levels. Agricultural products (beef, milk and cucumber) which were not contaminated by L. monocytogenes were analyzed as control samples. By comparing the TICs, the peak with two feature ion fragments of m/z 54,98 at the retention time 19.056 min was selected out as the characteristic peak for L. monocytogenes. Comparing with the standard MS library NIST107, it was with high similarity to 2(5)-furanone,5-methyl-. The feature information was used in the following detection.(3) Selected ion monitoring mode (19.056 min, m/z 54,98) was used to detect agricultural samples (beef, milk and cucumber) artificially contaminated by L. monocytogenes. A standard curve reflects relationship between the concentrations of L. monocytogenes in food products with the peak area of the feature peak. The result showed good linear correlation between the concentration of 6.50×109-1.04×1011 cfu/g, and with a correlation coefficient value up to 0.996. It confirmed that this method for detection of L. monocytogenes in food sample is feasible in theory. |
PDF Full Text Request