Specific and Rapid Quantification of Viable Listeria monocytogenes Using Fluorescence in situ Hybridization in Combination with Filter Cultivation

Fuchizawa, I.; Shimizu, Satoru; Ootsubo, Masashi; Kawai, Yuji; Yamazaki, Koji

doi:10.1264/jsme2.me09102

Cited by 18 publications

(6 citation statements)

References 22 publications

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“…Shimizu et al (2009) showed that FISHFC was an equally accurate yet faster method than the conventional plate count method when enumerating viable C. perfringens in ground beef. Fuchizawa et al (2009) also showed that FISHFC could be used to enumerate viable L. monocytogenes in smoked salmon and Camembert cheese.…”

Section: Discussionmentioning

confidence: 97%

Rapid Quantitative Detection of Salmonella enterica Using Fluorescence In Situ Hybridization with Filter-cultivation (FISHFC) Method

Shimizu

Aoi

Osanai

et al. 2013

FSTR

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Specific detection and enumeration of Salmonella enterica in food using conventional culture-based methods (CCBM) are time consuming and labor intensive. This study was conducted to develop a rapid S. enterica detection and enumeration method by combining fluorescence in situ hybridization (FISH) with micro-colony formation culture (FISHFC). Specificity tests of the SAL343 probe for S. enterica detection revealed that SAL343-associated fluorescent micro-colonies were observed specifically for S. enterica, but not for any other organisms. This finding suggests that SAL343 is highly specific for detecting S. enterica using FISHFC. For validation, FISHFC with SAL343 was compared to CCBM, with multiple selective agar, using spiked food samples; no significant differences in enumeration were found between FISHFC and CCBM (p > 0.05). The FISHFC method allowed enumeration of S. enterica within 10 h while CCBM allowed enumeration within 5 days. Therefore, the FISHFC method has potential application for more rapid and specific enumeration of S. enterica in food samples compared to other available methods.Keywords: Salmonella enterica, fluorescence in situ hybridization, FISHFC, rapid detection *To whom correspondence should be addressed. E-mail: yamasaki@fish.hokudai.ac.jp IntroductionSalmonella are Gram-negative, facultatively anaerobic, rod-shaped bacteria that exist ubiquitously in the environment and are commonly found in water, animal intestine, reptilian skin and food. Salmonella also causes salmonellosis in humans worldwide due to the consumption of contaminated food such as fresh fruits and vegetables (Matthews, 2006). The genus Salmonella currently consists of two species: S. enterica and S. bongori. S. enterica is comprised of six subspecies: enterica, salamae, arizonae, diarizonae, houtenae and indica, which are further subdivided into more than 2,500 serotypes (Agbaja et al., 2011;Su and Chiu, 2007). Most of the Salmonella responsible for disease in humans and animals are members of S. enterica. S. enterica serotype Enteritidis and Typhimurium are two of the main sources of human food poisoning. Although S. bongori has been reported to infect humans, it is generally associated with cold-blooded animals (Giammanco et al., 2002). Therefore, S. enterica are clinically important pathogens of humans.Routine methods for enumerating Salmonella are traditional microbiological methods based on plating using selective agar and biochemical identification. However, the biochemical characteristics of some isolates may differ from the common phenotypic properties of typical strains. Moreover, it is recommended that multiple selective agars be used for Salmonella detection in food samples (Nye et al., 2002). Thus, the conventional methods for detecting Salmonella are time-consuming and labor-intensive, taking at least several days to obtain the final results (Kim et al., 2007). To overcome technical problems associated with Salmonella detection, a number of molecular-based methods, such as PCR, quantitative PCR (qPCR), enzy...

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Section: Discussionmentioning

confidence: 97%

Rapid Quantitative Detection of Salmonella enterica Using Fluorescence In Situ Hybridization with Filter-cultivation (FISHFC) Method

Shimizu

Aoi

Osanai

et al. 2013

FSTR

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“…There are already FISH procedures developed for Listeria spp. detection, but only a few probes are specific for L. monocytogenes (Almeida et al, 2011;Fuchizawa et al, 2009;Moreno et al, 2011;Wang et al, 1991;Zhang et al, 2012) (Table S1 of supplementary material). Most of the existing probes are not simultaneously specific and sensitive because of the high number of non-target strains or the limited coverage of the target strains.…”

Section: Evaluation Of the L Monocytogenes Probes Described In Litermentioning

confidence: 99%

“…Several 16S or 23S rRNA probes have been developed for the detection of Listeria spp. by FISH methods (Almeida et al, 2011;Brehm-Stecher et al, 2005;Fuchizawa et al, 2008Fuchizawa et al, , 2009Moreno et al, 2011;Schmid et al, 2003;Wang et al, 1991;Zhang et al, 2012) but only a few of them are able to specifically detect L. monocytogenes (Almeida et al, 2011;Fuchizawa et al, 2009;Moreno et al, 2011;Wang et al, 1991;Zhang et al, 2012). These methods have been described as being highly specific and sensitive but there is no comparison between the probes.…”

Section: Introductionmentioning

confidence: 99%

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes

Rocha

Sousa²,

Cerqueira

et al. 2019

Food Microbiology

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Listeria monocytogenes is one of the most important foodborne pathogens due to the high hospitalization and mortality rates associated to an outbreak. Several new molecular methods that accelerate the identification of L. monocytogenes have been developed, however conventional culture-based methods still remain the gold standard. In this work we developed a novel Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) method for the specific detection of L. monocytogenes. The method was based on an already existing PNA probe, LmPNA1253, coupled with a novel blocker probe in a 1:2 ratio. The method was optimized for the detection of L. monocytogenes in food samples through an evaluation of several rich and selective enrichment broths. The best outcome was achieved using One Broth Listeria in a two-step enrichment of 24 h plus 18 h. For validation in food samples, ground beef, ground pork, milk, lettuce and cooked shrimp were artificially contaminated with two ranges of inoculum: a low level (0.2-2 CFU/25 g or mL) and a high level (2-10 CFU/25 g or mL). The PNA-FISH method performed well in all types of food matrices, presenting an overall accuracy of ≈99% and a detection limit of 0.5 CFU/25 g or mL of food sample.

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“…They need efficient tools to control this pathogen, in order to comply with food safety regulations while minimizing economic losses. Different rapid methods have been developed for detection of L. monocytogenes such as immunoassays, fluorescent in situ hybridization, amplification methods or impedanciometry (Cho & Irudayaraj, 2013;Fuchizawa, Shimizu, Ootsubo, Kawai, & Yamazaki, 2009;Labrador, Rota, Pérez, Herrera, & Bayarri, 2018;Rodriguez-Lazaro, Gonzalez-Garcia, Gattuso, Gianfranceschi, & Hernandez, 2014). The impedance method…”

Section: Introductionmentioning

confidence: 99%

Comparative evaluation of impedanciometry combined with chromogenic agars or RNA hybridization and real-time PCR methods for the detection of L. monocytogenes in dry-cured ham

et al. 2018

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Specific and Rapid Quantification of Viable Listeria monocytogenes Using Fluorescence in situ Hybridization in Combination with Filter Cultivation

Cited by 18 publications

References 22 publications

Rapid Quantitative Detection of Salmonella enterica Using Fluorescence In Situ Hybridization with Filter-cultivation (FISHFC) Method

Rapid Quantitative Detection of Salmonella enterica Using Fluorescence In Situ Hybridization with Filter-cultivation (FISHFC) Method

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes

Comparative evaluation of impedanciometry combined with chromogenic agars or RNA hybridization and real-time PCR methods for the detection of L. monocytogenes in dry-cured ham

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