A dendrogram based on MLTSA and MVLST.

<p>The dendrogram is based on nucleotide sequences in 3 multilocus tandem-repeat sequence analysis (MLTSA) regions (regions 1, 2, and 3) and 6 multi-virulence-locus sequence typing (MVLST) regions. Epidemic clone groupings are shown to the right of the isolates. JF: Japanese food isolate, JC: Japanese clinical isolate.</p

Allelic diversities of MLTSA with 3 regions and MVLST with 6 regions.

<p>Allelic diversities of MLTSA with 3 regions and MVLST with 6 regions.</p

Genetic Characteristics of Japanese Clinical <i>Listeria monocytogenes</i> Isolates

<div><p><i>Listeria monocytogenes</i> causes foodborne illnesses through consumption of ready-to-eat foods. Although 135-201annual listeriosis cases have been estimated in Japan, the details regarding the clinical isolates such as infection source, virulence level, and other genetic characteristics, are not known. In order to uncover the trends of listeriosis in Japan and use the knowledge for prevention measures to be taken, the genetic characteristics of the past human clinical isolates needs to be elucidated. For this purpose, multilocus tandem-repeat sequence analysis (MLTSA) and multi-virulence-locus sequence typing (MVLST) were used in this study. The clinical isolates showed a variety of genetically distant genotypes, indicating they were from sporadic cases. However, the MVLST profiles of 7 clinical isolates were identical to those of epidemic clone (EC) I isolates, which have caused several serious outbreaks in other countries, suggesting the possibility that they have strong virulence potential and originated from a single outbreak. Moreover, 6 Japanese food isolates shared their genotypes with ECI isolates, indicating that there may be risks for listeriosis outbreak in Japan. This is the first investigational study on genetic characteristics of Japanese listeriosis isolates. The listeriosis cases happened in the past are presumably sporadic, but it is still possible that some isolates with strong virulence potential have caused listeriosis outbreaks, and future listeriosis risks also exist.</p></div

Sequence of primers used in this study.

<p>Sequence of primers used in this study.</p

Establishment of a Simple and Rapid Identification Method for <i>Listeria</i> spp. by Using High-Resolution Melting Analysis, and Its Application in Food Industry

<div><p><i>Listeria monocytogenes</i> is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. <i>Listeria</i> spp., of which <i>L. monocytogenes</i> is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying <i>Listeria</i> spp.; however, many of the methods cannot identify newly categorized <i>Listeria</i> spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of <i>Listeria</i> spp. has been presented using the genes <i>rarA</i> and <i>ldh</i> as targets for HRMA. DNA sequences of 9 <i>Listeria</i> species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using <i>rarA</i> gene, including 3 new species. The remaining 2 species were identified by HRMA of <i>ldh</i> gene. The newly developed HRMA method was then used to assess <i>Listeria</i> isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying <i>Listeria</i> spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying <i>Listeria</i> spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.</p></div

Tm value of 33 strains which coud not identify by <i>rarA</i> gene HRMA.

<p>Tm value of 33 strains which coud not identify by <i>rarA</i> gene HRMA.</p

The result of high resolution melting analysis of <i>rarA</i> for 81 strains isolated from food processing plant.

<p>Representative profiles of the high resolution melting curves (normalized and temperature shifted difference plot) of <i>rarA</i> amplicons for 81strains isolated from food processing plant. They were classified for 1: <i>L. innocua</i>, 2: <i>L. monocytogenes</i>/<i>L. welshimeri</i> and 3: <i>L. seeligeri</i> on the basis of HRM curve profiles. One strain colored by brown was unidentified.</p

The result of high resolution melting analysis of <i>rarA</i> for 19 strains of 9 <i>Listeria</i> spp.

<p>Representative profiles of the high resolution melting curves (normalized and temperature shifted difference plot) of <i>rarA</i> amplicons for <i>L. innocua</i> (upper green line), <i>L. welshimeri</i>^T (upper blue line), <i>L. welshimeri</i> 019-3w (blue line in the middle), <i>L. monocytogenes</i> ATCC19114, ATCC19116 (lower blue lines), <i>L. monocytogenes</i> CIP107776, CIP103575 (base line), ATCC19115 (pink lines), <i>L. seeligeri</i>^T (pink line), <i>L. fleischmannii</i>^T (upper brown line), <i>L. seeligeri</i> 2–1 (red line), <i>L. marthii</i>^T (lower green line), <i>L. ivanovii</i>^T (yellow line), <i>L. grayi </i>^T (gray line) and <i>L. rocourtiae </i>^T (lower brown line). T: type strain.</p

Tm value of 5 <i>L. monoytogenes</i> strains and 2 <i>L. welshimeri</i> strains for <i>ldh</i> gene.

<p>Tm value of 5 <i>L. monoytogenes</i> strains and 2 <i>L. welshimeri</i> strains for <i>ldh</i> gene.</p

Comparison of results of identification by HRM and 16S rDNA sequencing Listeria spp. isolated from the food industry.

<p>The sequences of primer region are omitted.</p