Dataset Information

Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae.

ABSTRACT: Vibrio cholerae O1 and O139 has been known for its ability to cause epidemics. These strains produce cholera toxin which is the main cause of secretory diarrhea. V. cholerae non-O1 and non-O139 strains are also capable of causing gastroenteritis as well as septicemia and peritonitis. It has been proven that virulence factors such as T6SS, hapA, rtxA, and hlyA are present in almost all V. cholerae strains. It is imperative that viable but non-culturable cells of V. cholerae are also detected since they are also known to cause diarrhea. Thus, the aim of this study was to develop an assay that detects all V. cholerae regardless of their serotype, culturable state, and virulence genes present, by targeting the species specific conserved ompW sequence. The developed assay meets these goals with 100% specificity and is capable of detecting as low as 5.46 copy number of V. cholerae. Detection is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24-1.32) and remarkable reproducibility (CV%: 1.08-3.7). Amplification efficiencies in the 89-100% range were observed. The assay is more economical than Taqman-based multiplex real-time PCR assays. Compared to other real-time assays, the ompW assay is specific and sensitive, has better repeatability and reproducibility, and is more economical.

SUBMITTER: Rashid RB

PROVIDER: S-EPMC5437123 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae.

Rashid Ridwan Bin RB Ferdous Jannatul J Tulsiani Suhella S Jensen Peter Kjaer Mackie PKM Begum Anowara A

Frontiers in public health 20170519

Vibrio cholerae O1 and O139 has been known for its ability to cause epidemics. These strains produce cholera toxin which is the main cause of secretory diarrhea. V. cholerae non-O1 and non-O139 strains are also capable of causing gastroenteritis as well as septicemia and peritonitis. It has been proven that virulence factors such as T6SS, hapA, rtxA, and hlyA are present in almost all V. cholerae strains. It is imperative that viable but non-culturable cells of ...[more]

PMID: 28580353

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Project description:The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.

Dataset Information

Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae.

Publications

Development and Validation of a Novel Real-time Assay for the Detection and Quantification of <i>Vibrio cholerae</i>.

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