Multiplex PCR for detection of the Vibrio genus and five pathogenic Vibrio species with primer sets designed using comparative genomics

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Abstract

Background

The genus Vibrio is clinically significant and major pathogenic Vibrio species causing human Vibrio infections are V. cholerae, V. parahaemolyticus, V. vulnificus, V. alginolyticus and V. mimicus. In this study, we screened for novel genetic markers using comparative genomics and developed a Vibrio multiplex PCR for the reliable diagnosis of the Vibrio genus and the associated major pathogenic Vibrio species.

Methods

A total of 30 Vibrio genome sequences were subjected to comparative genomics, and specific genes of the Vibrio genus and five major pathogenic Vibrio species were screened. The designed primer sets from the screened genes were evaluated by single PCR using DNAs from various Vibrio spp. and other non- Vibrio bacterial strains. A sextuplet multiplex PCR using six primer sets was developed to enable detection of the Vibrio genus and five pathogenic Vibrio species.

Results

The designed primer sets from the screened genes yielded specific diagnostic results for target the Vibrio genus and Vibrio species. The specificity of the developed multiplex PCR was confirmed with various Vibrio and non- Vibrio strains. This Vibrio multiplex PCR was evaluated using 117 Vibrio strains isolated from the south seashore areas in Korea and Vibrio isolates were identified as Vibrio spp., V. parahaemolyticus, V. vulnificus and V. alginolyticus, demonstrating the specificity and discriminative ability of the assay towards Vibrio species.

Conclusions

This novel multiplex PCR method could provide reliable and informative identification of the Vibrio genus and major pathogenic Vibrio species in the food safety industry and in early clinical treatment, thereby protecting humans against Vibrio infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0577-3) contains supplementary material, which is available to authorized users.

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DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae

John Heidelberg, Jonathan A. Eisen, William C. Nelson … (2000)

Here we determine the complete genomic sequence of the Gram negative, γ-Proteobacterium Vibrio cholerae El Tor N16961 to be 4,033,460 base pairs (bp). The genome consists of two circular chromosomes of 2,961,146 bp and 1,072,314 bp that together encode 3,885 open reading frames. The vast majority of recognizable genes for essential cell functions (such as DNA replication, transcription, translation and cell-wall biosynthesis) and pathogenicity (for example, toxins, surface antigens and adhesins) are located on the large chromosome. In contrast, the small chromosome contains a larger fraction (59%) of hypothetical genes compared with the large chromosome (42%), and also contains many more genes that appear to have origins other than the γ-Proteobacteria. The small chromosome also carries a gene capture system (the integron island) and host ‘addiction’ genes that are typically found on plasmids; thus, the small chromosome may have originally been a megaplasmid that was captured by an ancestral Vibrio species. The V. cholerae genomic sequence provides a starting point for understanding how a free-living, environmental organism emerged to become a significant human bacterial pathogen. Supplementary information The online version of this article (doi:10.1038/35020000) contains supplementary material, which is available to authorized users.

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Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae.

Kozo MAKINO, Kenshiro Oshima, Ken Kurokawa … (2003)

Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. V parahaemolyticus strains of a few specific serotypes, probably derived from a common clonal ancestor, have lately caused a pandemic of gastroenteritis. The organism is phylogenetically close to V cholerae, the causative agent of cholera. The whole genome sequence of a clinical V parahaemolyticus strain RIMD2210633 was established by shotgun sequencing. The coding sequences were identified by use of Gambler and Glimmer programs. Comparative analysis with the V cholerae genome was undertaken with MUMmer. The genome consisted of two circular chromosomes of 3288558 bp and 1877212 bp; it contained 4832 genes. Comparison of the V parahaemolyticus genome with that of V cholerae showed many rearrangements within and between the two chromosomes. Genes for the type III secretion system (TTSS) were identified in the genome of V parahaemolyticus; V cholerae does not have these genes. The TTSS is a central virulence factor of diarrhoea-causing bacteria such as shigella, salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or intimately interacting with intestinal epithelial cells. Our results suggest that V parahaemolyticus and V cholerae use distinct mechanisms to establish infection. This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections, which are generally associated with non-inflammatory diarrhoea.

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Epidemiology of seafood-associated infections in the United States.

Martha Iwamoto, Tracy Ayers, Barbara Mahon … (2010)

Seafood is part of a healthful diet, but seafood consumption is not risk-free. Seafood is responsible for an important proportion of food-borne illnesses and outbreaks in the United States. Seafood-associated infections are caused by a variety of bacteria, viruses, and parasites; this diverse group of pathogens results in a wide variety of clinical syndromes, each with its own epidemiology. Some seafood commodities are inherently more risky than others, owing to many factors, including the nature of the environment from which they come, their mode of feeding, the season during which they are harvested, and how they are prepared and served. Prevention of seafood-associated infections requires an understanding not only of the etiologic agents and seafood commodities associated with illness but also of the mechanisms of contamination that are amenable to control. Defining these problem areas, which relies on surveillance of seafood-associated infections through outbreak and case reporting, can lead to targeted research and help to guide control efforts. Coordinated efforts are necessary to further reduce the risk of seafood-associated illnesses. Continued surveillance will be important to assess the effectiveness of current and future prevention strategies.

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Author and article information

Contributors

Hyun-Joong Kim: ritww001@gmail.com

Ji-Oh Ryu: badawldh@naver.com

Shin-Young Lee: sinyoung0442@naver.com

Ei-Seul Kim: a20080600@gmail.com

Hae-Yeong Kim: hykim@khu.ac.kr

Journal

Journal ID (nlm-ta): BMC Microbiol

Journal ID (iso-abbrev): BMC Microbiol

Title: BMC Microbiology

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2180

Publication date (Electronic): 26 October 2015

Publication date PMC-release: 26 October 2015

Publication date Collection: 2015

Volume: 15

Electronic Location Identifier: 239

Affiliations

Institute of Life Sciences & Resources and Graduate School of Biotechnology, Kyung Hee University, Yongin, 446-701 Republic of Korea

Article

Publisher ID: 577

DOI: 10.1186/s12866-015-0577-3

PMC ID: 4624192

PubMed ID: 26502878

SO-VID: 5ca7824b-6280-4b88-9429-b5dc3a86d60c

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 8 January 2015

Date accepted : 19 October 2015

Custom metadata

ScienceOpen disciplines: Microbiology & Virology

Keywords: identification,pcr,vibrios,v. cholerae,v. parahaemolyticus,v. vulnificus,v. alginolyticus,v. mimicus,comparative genomics

Data availability:

ScienceOpen disciplines: Microbiology & Virology

Keywords: identification, pcr, vibrios, v. cholerae, v. parahaemolyticus, v. vulnificus, v. alginolyticus, v. mimicus, comparative genomics

Multiplex PCR for detection of the Vibrio genus and five pathogenic Vibrio species with primer sets designed using comparative genomics

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Abstract

Background

Methods

Results

Conclusions

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Most cited references 34

DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae

Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae.

Epidemiology of seafood-associated infections in the United States.

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