Simultaneous Enrichment of Salmonella spp, Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes by Single Broth and Screening of the Pathogens by Multiplex Real-time PCR

Kobayashi, Hiroshi; Kubota, Jun; Fujihara, Kumiko; Honjoh, Ken–ichi; M, Iio; Fujiki, Naomi; Nakabe, Mika; Oda, S.; Satoyama, Toshiya; Takasu, Kazushige; Nakanishi, Hisao; Miyamoto, Takahisa

doi:10.3136/fstr.15.427

Cited by 26 publications

(18 citation statements)

References 27 publications

(20 reference statements)

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“…Several attempts have already been made to obtain recovery of multiple pathogens concurrently (Omiccioli et al 2009;Kim and Bhunia 2008;Kobayashi et al 2009) through the development or improvement of single enrichment broths. Such advances represent a promising alternative for future food quality analysis.…”

Section: Discussionmentioning

confidence: 99%

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

et al. 2010

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In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.

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Section: Discussionmentioning

confidence: 99%

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

et al. 2010

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“…These results were obtained by using the precipitates of sonicated samples and Biacore J. O157 at 1.8 × 10 6 CFU/mL, SE at 0.9 × 10 7 CFU/mL and LM at 1.8 × 10 7 CFU/mL produced positive signal changes of 39, 65, and 22 RU, respectively, and were significantly higher than those of the control samples. It has been reported that the cell concentration of O157, SE, and LM was less than 10 8 CFU/mL after the simultaneous enrichment of six foodborne pathogens, including these three pathogens (Kobayashi et al, 2009). Hence, the lower limit of detection using an SPR biosensor with specific PAbs for O157, SE, and LM was determined to be 1.8 × 10 6 , 0.9 × 10 7 , and 1.8 × 10 7 CFU/mL, respectively.…”

Section: Cross-reactivity Test On Supernatants and Precipitatesmentioning

confidence: 99%

Development of a Simultaneous Detection Method for Foodborne Pathogens Using Surface Plasmon Resonance Biosensors

Zhang

Kitaoka

Tsuji

et al. 2014

FSTR

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Surface plasmon resonance (SPR) biosensors were used to develop a rapid and simultaneous detection method for three important foodborne pathogens, i.e., Escherichia coli O157:H7 (O157), Salmonella Enteritidis (SE), and Listeria monocytogenes (LM). Bacterial homogenates prepared by sonication from bacterial suspensions at variouscell concentrations were analyzed using SPR biosensors and sensor chips with polyclonal antibodies specific to each of the target pathogens. The precipitates from the homogenates were demonstrated to be suitable for specific and simultaneous detection of O157, SE, and LM. By using the precipitates and a custom-built multichannel SPR biosensor, the lower detection limit for O157, SE, and LM was determined to be 0.6 × 10 6 , 1.8 × 10 6 , and 0.7 × 10 7 CFU/mL, respectively, in the presence of non-target pathogens at concentrations of 10 5 to 10 8 CFU/mL.

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“…Excellent results were obtained with the modified version of N17 broth with all the bacteria tested, obtaining colony counts higher than 10 6 CFU/ml for all microorganisms tested. Another non-selective enrichment broth recently developed is the simultaneous enrichment broth (SEB) developed by Kobayashi et al SEB was applied for the simultaneous recovery of Salmonella spp., L. monocytogenes, S. aureus, Bacillus cereus, Vibrio parahaemolyticus, and E. coli O157:H7; and its performance was compared to tryptic soy broth (TSB) (Kobayashi et al, 2009). Even though satisfactory results were obtained when compared against TSB, further studies need to be performed with natural and spiked food and environmental samples.…”

Section: Enrichment Brothsmentioning

confidence: 99%

“…Out of these, PrepMan Ultra® and InstaGene® have been extensively applied, especially with Gram negative bacteria. The DNeasy Tissue Kit is one of the most frequently selected kit as it has proven suitable for different types of samples and with different types of micro-organisms (Elizaquivel & Aznar, 2008;Elizaquível et al, 2011;Jofré et al, 2005;Kobayashi et al, 2009;McGuinness et al, 2009). …”

Section: Dna Extraction Protocolsmentioning

confidence: 99%

“…Concerning L. monocytogenes, the most widely used targets for its specific detection are the genes hlyA which encodes for the listeriolisin O (Elizaquível et al, 2011;Kobayashi et al, 2009;Navas et al, 2006;Omiccioli et al, 2009;Rodriguez-Lazaro, Hernández, et al, 2004;Ruiz-Rueda et al, 2010;Suo et al, 2010), and the prfA which is a central virulence regulator of this bacterium (D'Urso et al, 2009;Jofré et al, 2005;Rossmanith, Krassnig, Wagner, & Hein, 2006). Additionally, the iap gene, coding the protein p60 essential for cell viability, has also been applied (Flekna, Stefanic, et al, 2007;Mukhopadhyay & Mukhopadhyay, 2007;Wuenscher, Köhler, Bubert, Gerike, & Goebel, 1993).…”

Section: Listeria Monocytogenesmentioning

confidence: 99%

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Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

Chapela

Garrido‐Maestu

2015

Cogent Food & Agriculture

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Microbiological analysis of food is an integrated part of microbial safety management in the food chain. Monitoring and controlling foodborne pathogens are traditionally carried out by conventional microbiological methods based on culture-dependent approaches in control laboratories and private companies. However, polymerase chain reaction (PCR) has revolutionized microbiological analysis allowing detection of pathogenic microorganisms in food, without the necessity of classical isolation and identification. However, at present, PCR and quantitative polymerase chain reaction (qPCR) are essential analytical tools for researchers working in the field of foodborne pathogens. This manuscript reviews recently described qPCR methods applied for foodborne bacteria detection, serving as economical, safe, and reliable alternatives for application in the food industry and control laboratories. Multiplex qPCR, which allows the simultaneous detection of more than one pathogen in one single reaction, saving considerable effort, time, and money, is emphasized in the article.

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Simultaneous Enrichment of Salmonella spp, Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes by Single Broth and Screening of the Pathogens by Multiplex Real-time PCR

Cited by 26 publications

References 27 publications

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

Development of a Simultaneous Detection Method for Foodborne Pathogens Using Surface Plasmon Resonance Biosensors

Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

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