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The classical bacteriological method for the detection of Listeria monocytogenes in dairy products, includes an enrich-ment in fluid medium (2 days), a cultivation on selective agar medium (2 days) in order to obtain colonies, followed by a set of confirmation assays which takes typically 8 to 10 days to complete.
The time for confirmation of the species and genus identity can be decreased by the application of a DNA identification. A commercial DNA probe assay (GenProbe, San Diego, CA) was evaluated for the identification of L. monocytogenes colonies, grown on the agarmedium. All results, obtained with the DNA probe assay were confirmed with the classical biochemical and morphological identification method. By this very elegant and non-radioactive method, L. monocytogenes is identified on half a day. The total assay takes 4,5 days. Four days are still necessary for bacterial culturing.
In order to simultaneously identify the colonies, grown on the agar plate, to the genus level (Listeria spp.) and to the species level (L. monocytogenes), a multiplex PCR method was developed. For the identification of Listeria spp., two specific primers were deduced from the 16S rRNA sequence. For the identification of L. monocytogenes, the specific primers were deduced from the invasion associated protein gene sequence. The two amplified DNA fragments, respectively 1003 bp and 593 bp, were separated by agarose gelelectrophoresis. The results, obtained by the multiplex PCR method were in agreement with these obtained by the classical bacteriological method for the 64 dairy samples with presumptive positives.
By means of PCR it is possible to shorten not only the identi-fication procedure but also the bacterial culturing.
Starting from a pure bacterial culture, it is possible to detect 10 L. monocytogenes cells per ml. In order to test the sensitivity of PCR for detecting L. monocytogenes in cheese samples, an artifical contamination procedure was applied after the enrichment incubation of cheese samples. The PCR is stron-gly inhibited by cheese components and not by the ingre-dients of the enrichment broth. A variation of the detection limit from 10 till 106 cfu/ml was noticed, depending on the soft and semi-soft cheeses analyzed.
Therefore, PCR can only be applied for shortening of the bacterial culturing, when a decent sample preparation method is available. It is even possible to avoid bacterial cutluring and to directly detect L. monocytogenes in the food product by PCR.
A method for direct detection of L. monocytogenes in 25 ml of raw milk was developed. The detection limit can be situated between 5 and 10 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers, specific for L. monocytogenes. The results of this method are obtained within one day.
By application of a direct detection method, the question arises if dead L. monocytogenes cells are detectable by PCR. Non culturable L. monocytogenes cells are detected by PCR after treatment with a variety of desinfectants and after different heat conditions. The sensitivity of this PCR detection is strongly dependent on the treatment applied. Dissolving L. monocytogenes cells in pure ethanol for 30 days only reduced the PCR sensitivity 100 times compared to untreated cells. Treatment with 1% HCl and sterilization at 124°C for 15 min prevented PCR detection after 1 hour. Because of the possible detection of dead L. monocytogenes cells by PCR, it is often advisa-ble to opt for a short, overnight enrichment, followed by a simple sample preparation method and a PCR identification. So, it is possible to obtain a reliable results in 24-48 hour detection time.
By molecular typing methods, it is possible to identify a bacterial strain to the subspecies level. This is helpfull to trace a contamination source which is important for epidemiolo-gical studies and for the industry.
The molecular biological typing method RAPD (Randomly Amplified Polymorphic DNA Profile) was applied on 100 L. monocytogenes strains. RAPD is based on PCR with short arbitrary choosen primers. To evaluate the reproducibility of this method, we participate with the international WHO trial. This study showed that especially different thermocyclers and small variations in PCR buffer concentration are the cause of the poor reproducibi-lity obtained.
Molecular typing of Listeria spp. and L. monocytogenes was also possible by repetitive element sequence based PCR. Families of repetitive elements are dispersed throughout the genome of different bacterial species. Two families were used, namely the 35 to 40 bp repetitive extragenic palindromic sequence (REP) and the 124 to 127 bp enterobacterial repetitive intergenic consensus sequence (ERIC).
REP-PCR showed a higher discrimina-tive potential than ERIC-PCR, comparable with RAPD, combining 3-4 primers. The advantage of this method is its simplicity and its high reproducibility.
In this project, a specific PCR identification method was developed for Brucella spp. the specific primers were deduced from the 16S rRNA sequence on the basis of a sequence align-ment between Brucella abortus and Agrobacterium tumefaciens.
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