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248Jpn.

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...

Reverse transcription-PCR targeting the VP0 gene of human parechoviruses (HPeVs) was used to identify two isolates from two Japanese children's stool specimens. Molecular analysis revealed that these isolates belonged to HPeV type 4, and their nucleotide identity in the P1 region was 85.0%.Human parechoviruses (HPeVs), members of the Parechovirus genus of the Picornaviridae family, are genetically classified into six types. HPeVs have been associated with gastrointestinal and respiratory symptoms and also with severe symptoms such as transient paralysis and neonatal sepsis in young children (3,5). HPeV type 1 (HPeV1) and HPeV3 infections are common worldwide (5, 6); however, other HPeV infections, especially HPeV4 infections, are less common. HPeV4 strains have been isolated from young children with fever, TORCH syndrome, and lymphadenitis in a few instances (2, 10, 11).This paper describes the identification and characterization of HPeV4 strains using molecular and immunological techniques. In 2001 and 2005, we isolated viruses from stool specimens from a 1-year-old girl with herpangina and from a 3-year-old boy with acute gastroenteritis, respectively, in Fukuoka City, Japan. The stool specimens were cultured weekly at 37°C for three passages on Caco-2, RD-18S, VeroE6, and HEp-2 cells. Picornaviridae-like cytopathic effects were observed in the Caco-2, RD-18S, and VeroE6 cell lines. To identify these isolates, a molecular typing method based on reverse transcription-PCR (RT-PCR) and direct sequencing was carried out. Viral RNAs were extracted from cell culture supernatants by using a QIAamp viral RNA mini kit (Qiagen, Germany). RT-PCRs were performed with specific primers (187/011, 188/011, and 189/011 for the enterovirus VP1 gene and E23P1/HPV-N1 for the HPeV VP0 gene) (5, 9). Table 1 shows the molecular typing results from the cell culture samples. In case 1, the Caco-2 and RD-18S cell culture samples were positive by RT-PCR for enterovirus, and human coxsackievirus group A type 2 was identified by direct sequencing of the VP1 amplicon. In both case 1 and 2, all of the Caco-2, RD-18S, and VeroE6 culture samples were positive for HPeV by RT-PCR. The VeroE6 culture sample from case 1 and the Caco-2 culture sample from case 2 were designated Fuk2001-282 and Fuk2005-123, respectively. These amplicon sequences were compared with sequences from HPeV reference strains HPeV1 Harris, HPeV2 Williamson, HPeV3 A308-99, HPeV4 K251176-02, HPeV4 T75-4077, HPeV5 T92-15, and HPeV6 NII561-2000.Both Fuk2001-282 and Fuk2005-123 showed high similarities to the VP0 gene of the HPeV4 reference strains (positions 710 to 1,339 on HPeV1 Harris); the nucleotide identities of Fuk2001-282 to HPeV4 K251176-02 and HPeV4 T75-4077 were 84.9% and 89.5%, respectively, and those of Fuk2005-123 to K251176-02 and T75-4077 were 85.4% and 85.2%, respec-

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