The Establishment And Application Of Listeria Monocytogenes Rapid Detection Method In Food

Listeria monocytogenes is a foodborne pathogen in both human beings and animals, and it may pollute meat, egg products, dairy products, fruits and vegetables, etc.The main detection approach of L.monocytogenes in China is traditional culturing method. To achieve the aim of more rapid and accurate detection, immunology and molecular biology detection technology is developing rapidly.To realize the rapid and accurate detection, this research mainly based on the national food safety testing standards GB 4789.30-2010, combining with a series of rapid detection technology, such as the chromogenic medium, protein test, CPA isothermal amplification method, the real-time fluorescence PCR method and automatic microbial analysis system, etc.Choosing L.monocytogenes and four typical ready-to-use food (meat, seafood, dairy products, fruit and vegetable), four nucleic acid extraction methods (sedimentation method, magnetic beads method, spin columns method, and pyrolysis method) which have been commonly used at home and abroad was compared in the research. Results showed that spin columns method was the preferred method with features of simplicity, stability, high sensitivity and specificity. In addition, the experimental personnel request was not high, and it was easy to master.Using the standard strains and artificially contaminated ready-to-use food matrix (salted duck and salad), the sensitivity and specificity of the above three rapid detection methods were compared. The results showed that the operation of the protein detection method was simple, but the sensitivity was low; CPA isothermal amplification method overcomed the low sensitivity deficiency of the protein detection method, and didn’t need large-scale testing instruments, so it was suitable for the primary laboratory; The real-time fluorescence PCR method has been widely applied in the laboratory with good conditions, because of its’high sensitivity and specificity. And the real-time fluorescence PCR method avoided the electrophoresis process of the conventional PCR, reduced the risk of cross contamination and shortened the test period.It’s a very difficult problem to distinguish L.monocytogenes dead/living bacterium, so eliminating the influence of dead bacteria nucleic acid on test results seems extremely important. Results showed that PMA was better to eliminate the interference of dead bacteria in the pure cultures, and it brought improvement of the accuracy of detection. But the effect was not ideal under the interference of matrix. It was easy to be affected by other double-stranded nucleic acid, causing loss of large amount of PMA.Based on the above study, combining the nucleic acid extraction technology with a series of fast detection technology, the rapid detection method of L. monocytogenes in food was established, and validated and optimizated through the detection of a large number of actual samples. Operation steps were as follows:Firstly, the national standards GB 4789.30-2010 for actual samples of sampling and bacteria culture method were used, then real-time fluorescent PCR method was applied for rapid screening of sample enrichment liquid. The positive samples were streaked to cultivate, combined with chromogenic medium separation and purification of colonies. Finally, the colony was identified through the automatic microbial analysis system. Results indicated that the method was rapid, accurate, saving manpower and it could greatly improve the work efficiency.