Simultaneous detection of Pathogenic Vibrio species using multiplex real-time PCR

Kim, Hye-Jin; Lee, Hyunjung; Lee, Kyu‐Ho; Cho, Jae-Chang

doi:10.1016/j.foodcont.2011.08.019

Cited by 25 publications

(9 citation statements)

References 47 publications

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“…This novel assay could overcome the limitations of culturedependent methods and common molecular methods (Josefsen et al 2010;Kim et al 2012;Garrido et al 2013;Ma et al 2014;Zhang et al 2015), which not only saved time but also quantified viable cells in food matrixes and, therefore, can be served as a reliable tool to improve food safety.…”

Section: Discussionmentioning

confidence: 99%

“…Traditional culture-based methods for detecting and enumerating these two pathogens in food products include enrichment, selective plating, and biochemical confirmation and take between 5 and 7 days (Kim et al 2012;Garrido et al 2013;Ma et al 2014;Zhang et al 2015). Real-time PCR is a faster, more sensitive, less labor-intensive quantitative detection method, and has been widely used for detecting and enumerating many pathogens in food (Feng 2007;Willenburg and Divol 2012;Schnetzinger et al 2013;Xiao et al 2015).…”

Section: Introductionmentioning

confidence: 99%

“…Real-time PCR is a faster, more sensitive, less labor-intensive quantitative detection method, and has been widely used for detecting and enumerating many pathogens in food (Feng 2007;Willenburg and Divol 2012;Schnetzinger et al 2013;Xiao et al 2015). For maximum efficiency, multiplex real-time PCR was developed as an automated highthroughput technique for simultaneous and direct detection of more than two pathogens in the same reaction (Kim et al 2012;Garrido et al 2013;Ma et al 2014;Zhang et al 2015). However, one of the limitations of real-time PCR and multiplex real-time PCR methods for the detection of bacteria in food samples is that both viable and dead cells are detected (Kramer et al 2009;Schnetzinger et al 2013;Macé et al 2013;Salam et al 2014).…”

Section: Introductionmentioning

confidence: 99%

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Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Zhang

Liu

Lou

et al. 2015

Appl Microbiol Biotechnol

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A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10(8) CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10(1)-10(2) CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes, respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Zhang

Liu

Lou

et al. 2015

Appl Microbiol Biotechnol

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“…Six genetic loci, pilF (pilus-type IV assembly gene), vcg (ORF no. VV0401, virulence-correlated gene of strain YJ016), viuB (vulnibactin gene), vuuA (ferric vulnibactin receptor), vvhA (VV hemolysin gene), and CPS (capsular polysaccharide) alleles were analyzed according to the methods of Roig et al [ 30 ], Rosche et al [ 71 ], Panicker et al [ 72 ], Kim et al [ 73 ], Kaysner and DePaola [ 74 ], and Han et al [ 75 ], respectively. Associations between genotypic characteristics and strain origin (environmental or clinical), as well as associations among the genotypic characteristics themselves, were evaluated with the chi-square (χ 2 ) test and Fisher's exact test (α = 0.05).…”

Section: Methodsmentioning

confidence: 99%

Genotypic Diversity and Population Structure of Vibrio vulnificus Strains Isolated in Taiwan and Korea as Determined by Multilocus Sequence Typing

Kim¹,

Cho²

2015

PLoS ONE

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The genetic diversity and population structure of Vibrio vulnificus isolates from Korea and Taiwan were investigated using PCR-based assays targeting putative virulence-related genes and multilocus sequence typing (MLST). BOX-PCR genomic fingerprinting identified 52 unique genotypes in 84 environmental and clinical V. vulnificus isolates. The majority (> 50%) of strains had pathogenic genotypes for all loci tested; moreover, many environmental strains had pathogenic genotypes. Although significant (p < 0.05) inter-relationships among the genotypes were observed, the association between genotype and strain source (environmental or clinical) was not significant, indicating that genotypic characteristics alone are not sufficient to predict the isolation source or the virulence of a given V. vulnificus strain and vice versa. MLST revealed 23–35 allelic types per locus analyzed, resulting in a total of 44 unique sequence types (STs). Two major monophyletic groups (lineages A and B) corresponding to the two known lineages of V. vulnificus were observed; lineage A had six STs that were exclusively environmental, whereas lineage B had STs from both environmental and clinical sources. Pathogenic and nonpathogenic genotypes predominated in MLST lineages B and A, respectively. In addition, V. vulnificus was shown to be in linkage disequilibrium (p < 0.05), although two different recombination tests (PHI and Sawyer’s tests) detected significant evidence of recombination. Tajima’s D test also indicated that V. vulnificus might be comprised of recently sub-divided lineages. These results suggested that the two lineages revealed by MLST correspond to two distinct ecotypes of V. vulnificus.

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“…Compared to conventional PCR, real-time PCR offers better options for multiplexing several targets in a single reaction. Recently, there has been an increase in the development of real-time multiplex PCRs for the detection of multiple pathogens including foodborne pathogens (41)(42)(43)(44)(45)(46). In one such study, Hu et al developed a molecular beaconbased multiplex real-time PCR for the detection of various foodborne pathogens, namely S. enterica subsp.…”

Section: Real-time Pcrmentioning

confidence: 99%

Molecular Tools To Study Preharvest Food Safety Challenges

Kumar

Thakur

2018

Microbiol Spectr

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Preharvest food safety research and activities have advanced over time with the recognition of the importance and complicated nature of the preharvest phase of food production. In developed nations, implementation of preharvest food safety procedures along with strict monitoring and containment at various postharvest stages such as slaughter, processing, storage, and distribution have remarkably reduced the burden of foodborne pathogens in humans. Early detection and adequate surveillance of pathogens at the preharvest stage is of the utmost importance to ensure a safe meat supply. There is an urgent need to develop rapid, cost-effective, and point-of-care diagnostics which could be used at the preharvest stage and would complement postmortem and other quality checks performed at the postharvest stage. With newer methods and technologies, more efforts need to be directed toward developing rapid, sensitive, and specific methods for detection or screening of foodborne pathogens at the preharvest stage. In this review, we will discuss the molecular methods available for detection and molecular typing of bacterial foodborne pathogens at the farm. Such methods include conventional techniques such as endpoint PCR, real-time PCR, DNA microarray, and more advanced techniques such as matrix-assisted layer desorption ionization-time of flight mass spectrometry and whole-genome sequencing.

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Simultaneous detection of Pathogenic Vibrio species using multiplex real-time PCR

Cited by 25 publications

References 47 publications

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Genotypic Diversity and Population Structure of Vibrio vulnificus Strains Isolated in Taiwan and Korea as Determined by Multilocus Sequence Typing

Molecular Tools To Study Preharvest Food Safety Challenges

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