Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments

Recent outbreaks of listeriosis have emphasized the urgent need for rapid and reliable detection methods for Listeria spp., especially in food. Haemolysin production is a major factor in the pathogenesis of listeriosis and the polymerase chain reaction (PCR) was used to amplify two specific DNA fragments of the alpha- and the beta-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with alpha-haemolysin primers. In the analysis of 50 samples of cooked sausage products, bacterial colonies suspected to be Listeria spp. were isolated by conventional methods from six samples. PCR analysis identified three of six as L. monocytogenes. Subsequent serotyping showed perfect agreement with the PCR results. Since enrichment is the most time consuming step in conventional methods a PCR procedure which allows the direct detection of L. monocytogenes in milk was developed. Pasteurized milk was artificially contaminated with various levels of L. monocytogenes. The detection limit was determined to be 10 bacteria/10 ml milk and direct detection and identification of L. monocytogenes took less than two working days. These results show that this haemolysin gene amplification system is very rapid and reliable and therefore avoids cumbersome and lengthy cultivation steps.