Real-Time
PCR Analysis of Vibrio vulnificus from
Oysters

ABSTRACT Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (102 to 108 CFU ml−1), with a lower limit of 72 fg of genomic DNA μl of PCR mixture−1 or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r 2 = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood.

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Application of the rpoS Gene for Species-Specific Detection of Vibrio vulnificus by Real-Time PCR

Journal of Microbiology and Biotechnology ◽

10.4014/jmb.0800.176 ◽

2008 ◽

pp. 1841-1847 ◽

Cited By ~ 2

Author(s):

In-Soo Kong

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Vulnificus ◽

Specific Detection ◽

Rpos Gene ◽

Species Specific

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Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5702-5709.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5702-5709 ◽

Cited By ~ 64

Author(s):

Gitika Panicker ◽

Asim K. Bej

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Vulnificus ◽

Pcr Amplification ◽

Tissue Homogenate ◽

Oyster Tissue ◽

Initial Inoculum ◽

Oligonucleotide Primers ◽

Pure Cultures ◽

Taqman Pcr

ABSTRACT We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (CT ) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 103 V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 103 CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.

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Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

Journal of Medical Microbiology ◽

10.1099/jmm.0.46759-0 ◽

2007 ◽

Vol 56 (1) ◽

pp. 56-65 ◽

Cited By ~ 22

Author(s):

Dobryan M. Tracz ◽

Paul G. Backhouse ◽

Adam B. Olson ◽

Joanne K. McCrea ◽

Julie A. Walsh ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Rapid Detection ◽

Vibrio Vulnificus ◽

Comparative Sequence Analysis ◽

Molecular Techniques ◽

Array Technology ◽

Species Specific ◽

Pcr Targeting

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.

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Application of mitochondrial DNA analysis for microbial source tracking purposes in shellfish harvesting waters

Water Science & Technology ◽

10.2166/wst.2010.767 ◽

2010 ◽

Vol 61 (1) ◽

pp. 1-7 ◽

Cited By ~ 17

Author(s):

Craig Baker-Austin ◽

Rachel Rangdale ◽

James Lowther ◽

David N. Lees

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Real Time Pcr ◽

Dna Analysis ◽

Pcr Amplification ◽

Source Tracking ◽

Pcr Analysis ◽

Taqman Probes ◽

Faecal Matter ◽

Species Specific

We present a method for the reliable detection and source characterisation of faecal pollution in water and shellfish matrices, utilising real-time PCR analysis of mitochondrial DNA targets. In this study we designed real-time PCR (TaqMan) probes to target human, bovine, ovine and swine mtDNA. PCR amplification using species-specific TaqMan probes on faecal matter and mixed effluent slurries revealed no cross-reactions between species of interest and other vertebrate faecal matter. Performed as a single blind experiment we were able to correctly identify faecal material in 17/20 effluents (85% correct). mtDNA degrades relatively quickly in faecally-spiked water samples (∼2 weeks), a similar timeframe of environmental persistence to several bacterial faecal indictors, highlighting its applicability. The procedure described here is specific, rapid (<5 hours) and sensitive. These results confirm the suitability of using species-specific mtDNA as an indicator in source tracking studies in surface waters, shellfish harvesting areas and shellfish matrices.

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Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species

Parasitology ◽

10.1017/s0031182018001361 ◽

2018 ◽

Vol 145 (13) ◽

pp. 1733-1738 ◽

Cited By ~ 6

Author(s):

Lucas J. Cunningham ◽

J. Russell Stothard ◽

Mike Osei-Atweneboana ◽

Samuel Armoo ◽

Jaco J. Verweij ◽

...

Keyword(s):

High Resolution ◽

Real Time ◽

Real Time Pcr ◽

Strongyloides Stercoralis ◽

Curve Analysis ◽

Pcr Analysis ◽

High Resolution Melt ◽

Middle Income ◽

Ancylostoma Duodenale ◽

Species Specific

AbstractWith the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato–Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.

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