Polymerase chain reaction detection of Listeria monocytogenes using oligonucleotide primers targeting actA gene

Abstract

A polymerase chain reaction (PCR) assay targeting the gene encoding actA was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated pork, water and milk. 827-bp PCR product was detected in all 51 L. monocytogenes strains belonging to four different sero-groups (1/2a, 1/2b, 1/2c, and 4b). In contrast, the PCR product was not detected both in all other Listeria spp., including Listeria innocua, Listeria ivanovill, Listeria seelingeri, Listeria welshimeri, or Listeria grayi and in non-Listeria bacteria, indicating that the primer set we use was highly specific for L. monocytogenes. The detection limit of the PCR assay for pure cell cultures was 105 cfu per ml pure cell culture. However, the assay could detect as few as 101 cfu of L. monocytogenes in 25 g of pork and 25 ml milk and water following 16 h of enrichment in Listeria Enrichment broth (LEB) at 30 °C. Only a large number of dead L. monocytogenes cells can cause false positivity, as determined using model pork, milk and water samples artificially contaminated with decimal dilutions of dead L. monocytogenes. The total assay time including enrichment was approximately 18 h. These results suggest that the PCR assay based on amplifying actA gene can used to rapidly detect L. monocytogenes on pork, milk, water and possibly other types of food products.