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Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction.

Kim JY, Lee JL - J. Sci. Food Agric. (2014)

Bottom Line: The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers.Freshness and quality of fishery products also depend on the total bacterial populations present.Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Ecology, Food Science, College of Agriculture and Related Sciences, Delaware State University, Dover, DE 19901, USA.

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Standard curves of four targets: (A) 16S, (B) tlh, (C) toxR, and (D) vvhA for quantification, plotting Ct values and Log CFU. Pure cultured Vibrio anguillarum, V. parahaemolyticus, and V. vulnificus were used ranges from Log 0 CFU to Log 5 CFU and total bacteria was used ranged from Log 0.6 CFU to Log 5.6 CFU.
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fig02: Standard curves of four targets: (A) 16S, (B) tlh, (C) toxR, and (D) vvhA for quantification, plotting Ct values and Log CFU. Pure cultured Vibrio anguillarum, V. parahaemolyticus, and V. vulnificus were used ranges from Log 0 CFU to Log 5 CFU and total bacteria was used ranged from Log 0.6 CFU to Log 5.6 CFU.

Mentions: In order to verify the sensitivity of the method and the consistency of quantitative measurement, DNA was prepared from a 10-fold diluted pure cultures of Vibrio spp. and in total bacteria to be analyzed using the multiplex real-time PCR. The three standard curves of specific targets obtained from each Vibrio strain in the range of log 0 to log 5 CFU per PCR assay and the standard curve of 16S was generated in the range of log 0.6 to log 5.6 CFU per reaction (Fig. 2). The standard curves of specific targets showed excellent correlations of linear regression with r2 coefficients: tlh, toxR, and vvhA were 0.990, 0.997, and 0.993, respectively, from three independently performed experiments and the coefficient for the 16S standard curve was 0.978.


Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction.

Kim JY, Lee JL - J. Sci. Food Agric. (2014)

Standard curves of four targets: (A) 16S, (B) tlh, (C) toxR, and (D) vvhA for quantification, plotting Ct values and Log CFU. Pure cultured Vibrio anguillarum, V. parahaemolyticus, and V. vulnificus were used ranges from Log 0 CFU to Log 5 CFU and total bacteria was used ranged from Log 0.6 CFU to Log 5.6 CFU.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368387&req=5

fig02: Standard curves of four targets: (A) 16S, (B) tlh, (C) toxR, and (D) vvhA for quantification, plotting Ct values and Log CFU. Pure cultured Vibrio anguillarum, V. parahaemolyticus, and V. vulnificus were used ranges from Log 0 CFU to Log 5 CFU and total bacteria was used ranged from Log 0.6 CFU to Log 5.6 CFU.
Mentions: In order to verify the sensitivity of the method and the consistency of quantitative measurement, DNA was prepared from a 10-fold diluted pure cultures of Vibrio spp. and in total bacteria to be analyzed using the multiplex real-time PCR. The three standard curves of specific targets obtained from each Vibrio strain in the range of log 0 to log 5 CFU per PCR assay and the standard curve of 16S was generated in the range of log 0.6 to log 5.6 CFU per reaction (Fig. 2). The standard curves of specific targets showed excellent correlations of linear regression with r2 coefficients: tlh, toxR, and vvhA were 0.990, 0.997, and 0.993, respectively, from three independently performed experiments and the coefficient for the 16S standard curve was 0.978.

Bottom Line: The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers.Freshness and quality of fishery products also depend on the total bacterial populations present.Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Ecology, Food Science, College of Agriculture and Related Sciences, Delaware State University, Dover, DE 19901, USA.

Show MeSH