Abstract
Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria 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 Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria 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 rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria 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 Listeria 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 Listeria 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.
Highlights
Listeria spp. is a non-sporulating, gram-positive bacterium [1]
L. seeligeri isolated from the environment showed a specific pattern; typestrain of L. seeligeri was grouped with L. monocytogenes strains
Tm values of the 5 strains of L. monocytogenes ranged from 82.5760.30±83.2560.32uC, and Tm of the 2 L. welshimeri strains were 83.8860.15 and 84.0860.12uC, respectively (Table 3)
Summary
Listeria spp. is a non-sporulating, gram-positive bacterium [1]. The Listeria genus consists of L. monocytogenes, L. innocua, L. seeligeri, L. ivanovii, L. grayi, and L. welshimeri [1], as well as the recently classified L. marthii [2], L. rocourtiae [3], L. fleischmannii [4], and L. weihenstephanensis [5]. Of the Listeria species, L. monocytogenes can be transmitted among humans and animals, and it is the cause of listeriosis [1]. It can cause sporadic food poisoning±for which it is known in various Western countries [8],[9]±through milk products such as cheese [9] and processed meats such as sausages and salami [10]. In the U.S.A., FDA standards have zero tolerance for L. monocytogenes contamination in processed foods, but with certain exceptions [11]. The contamination level of L. monocytogenes in processed foods is strictly regulated in the EU at ,100 cfu/g [12]. To analyze the route of contamination of food products, environments such as farms [9], [15] and fish farms [16] have been examined
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