Simultaneous enrichment broth (SEB) was developed for the single-enrichment simultaneous screening of six major food poisoning bacteria. After enrichment in SEB for 18 h at 37℃, viable counts of six major food poisoning bacteria (Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Bacillus cereus, and Listeria monocytogenes) were sufficient for 5' nuclease multiplex real-time PCR assay using existing primers and probes. By labeling the probes with three different fluorescent dyes, the assay could be carried out in 2 tubes. The whole process, including enrichment and PCR, was completed within 24 h and the detection limits for the target bacteria from the food sample (boiled chicken) were 36 cfu/25 g for S. aureus, 5.3 cfu/25 g for Salmonella spp, 2.9 cfu/25 g for E. coli O157:H7, 2.0 cfu/25 g for V. parahaemolyticus, 5.5 cfu/25 g for B. cereus, and 6.2 cfu/25 g for L. monocytogenes. The results were comparable to conventional methods that require 4-6 days.

In this study, primer sets of L. monocytogenes virulence gene hlyA were designed for the L. monocytogenes-specific PCR assay. To evaluate the performance of these primer sets, the detection sensitivity and specificity were examined and the most suitable primer set was selected. Subsequently, it was subjected to compare the performance for detection of L. monocytogenes with commercially available kits (TaKaRa detection kit and ABI detection kit) on DNA level. Results of real-time PCR showed that the efficiency of this new primer set was 101.6% while TaKaRa and ABI detection kit were 101.1% and 107.4%, respectively. The detection sensitivity of all three methods was equivalent to less than 1 copy per reaction using purified genomic DNA as template. Detection specificity were 100% when testing L. monocytogenes isolates, and for other Listeria isolates were 15.4%, 76.9% and 100% by the method using hlyA L. monocytogenes detection primer set 7, TaKaRa and ABI detection kit, respectively. In summary, this new PCR detection method is relatively sensitive and more specific to L. monocytogenes under certain conditions, could serve as a rapid detection method and has the potential for detection of L. monocytogenes from contaminated food.

To develop a new method for identification of Listeria monocytogenes genetically similar to clinical isolates, single-nucleotide polymorphism (SNP) typing and multi-locus sequence typing (MLST) of 126isolates of L. monocytogenes from clinical and environmental samples were performed based on sequence analysis of parts of four genes (hlyA, clpC, inlA, and plcA). Based on the sequences of the isolates in this study, SNP typing showed that hlyA, clpC, inlA, and plcA genes were categorized into 9, 14, 17, and 21 types, respectively. MLST showed that the isolates were grouped into 35 types including 12 types of clinical isolates. Out of those, four MLST types were found in food or environmental and clinical isolates. In particular, all clinical isolates with serotype 1/2a were grouped into the same hlyA SNP A5 type. A method using real-time PCR combined with Cycling Probe Technology was developed for rapid identification of SNP type of L. monocytogenes genetically similar to the clinical isolates. By using this method, the 1/2a clinical isolates showing MLST-2 were successfully identified with a specific primer set and a cycling probe designed on the basis of sequence of hlyA. Furthermore, clinical isolates of serotype 4b showing MLST-4 or -35 were successfully identified by a method using cycling probes based on sequences of clpC and inlA.Keywords: cycling probe technology, Listeria monocytogenes, real-time PCR, SNP typing *To whom correspondence should be addressed. Email: tmiyamot@agr.kyushu-u.ac.jp IntroductionListeria species are widely distributed in the environment. As they are found in soil and in mammals, they are often contaminants in various types of food, mainly meats and dairy products . Listeria monocytogenes is a significant food-borne pathogen and causes an infectious disease known as listeriosis. In the food industry, contamination of food by this bacterium may lead to serious problems since it can grow even at low temperatures and high salt concentrations during storage of readyto-eat foods such as unsterilized dairy products and raw vegetables.In Japan, although sporadic cases of listeriosis have been reported, no serious epidemic has occurred. However, L. monocytogenes is often detected in foodstuffs, which may lead to a potential outbreak of listeriosis (Okutani et al., 2004). Listeriosis may result in mortality for pregnant women, infants, immunocompromised individuals, and elderly individuals .For identification and subtyping of L. monocytogenes, several techniques, such as phenotypic typing (serological typing, phage typing, and multilocus enzyme electrophoresis) and molecular typing techniques (ribotyping, restriction enzyme analysis, PCR-based typing, and DNA sequencing) have been used in epidemiology (Gasanov et al., 2005). Furthermore, multilocus sequence typing (MLST) based on DNA sequences has extremely high and accurate discriminatory power (Gasanov et al., 2005). MLST has been applied to subtyping of L. monocytogenes (Cai et al., 2002;Salcedo et al., 2003). However, because it includes...