A multiplatform real-time polymerase chain reaction detection assay for Vibrio cholerae

Koskela, Katja A.; Matero, Pirjo; Blatny, Janet Martha; Fykse, Else Marie; Olsen, Jaran Strand; Nuotio, L.; Nikkari, Simo

doi:10.1016/j.diagmicrobio.2009.07.009

Cited by 26 publications

(14 citation statements)

References 15 publications

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“…All other non-O1/non-O139 serotypes are usually associated with less-severe gastrointestinal, blood, wound, and ear infections (3). V. cholerae has been classified as a potential category B terrorism agent by the U.S. Centers for Disease Control and Prevention (4). Detection and quantification of V. cholerae, especially of serogroup O1/O139 strains in environmental samples, are still difficult tasks, and no international standard is available.…”

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confidence: 99%

“…New molecular biological methods based on real-time quantitative PCR (qPCR) technology have the ability to provide bacterial genome equivalent estimates in as little as 3 h (10,11). Several qPCR protocols have been developed for V. cholerae in the past decade, but all of them have limitations when applied to the quantification of toxigenic and nontoxigenic V. cholerae in environmental water samples (4,(12)(13)(14)(15)(16). The published qPCR methods did not perform as a multiplex qPCR (which is necessary to distinguish between toxigenic and nontoxigenic V. cholerae in one assay), or they lacked an internal amplification control (IC) or no standards for quantification were used, or the method could not separate between toxigenic and nontoxigenic V. cholerae (4,(12)(13)(14)(15)(16).…”

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confidence: 99%

“…Several qPCR protocols have been developed for V. cholerae in the past decade, but all of them have limitations when applied to the quantification of toxigenic and nontoxigenic V. cholerae in environmental water samples (4,(12)(13)(14)(15)(16). The published qPCR methods did not perform as a multiplex qPCR (which is necessary to distinguish between toxigenic and nontoxigenic V. cholerae in one assay), or they lacked an internal amplification control (IC) or no standards for quantification were used, or the method could not separate between toxigenic and nontoxigenic V. cholerae (4,(12)(13)(14)(15)(16). Moreover, the application of qPCR to environmental samples has often been limited by the presence of high concentrations of substances with negative effects on DNA extraction or PCR efficiency, such as salts or humic substances (17,18).…”

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A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Bliem

Schauer

Plicka³

et al. 2015

Appl Environ Microbiol

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Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 ؋ 10 2 to 2.3 ؋ 10 4 cell equivalents liter ؊1 , whereas GR-corrected abundances ranged from 4.7 ؋ 10 3 to 1.6 ؋ 10 6 cell equivalents liter ؊1 . GR-corrected qPCR results were in good agreement with an independent cellbased direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs. Vibrio cholerae is a waterborne bacterium found worldwide in brackish water, coastal areas, and estuarine environments (1). Although the species V. cholerae comprises more than 200 serotypes, only serotypes O1 and O139 are currently able to cause epidemic and pandemic cholera outbreaks, with more than 100,000 reported death cases per year (2). All other non-O1/non-O139 serotypes are usually associated with less-severe gastrointestinal, blood, wound, and ear infections (3). V. cholerae has been classified as a potential category B terrorism agent by the U.S. Centers for Disease Control and Prevention (4). Detection and quantification of V. cholerae, especially of serogroup O1/O139 strains in environmental samples, are still difficult tasks, and no international standard is available. Currently accepted cultivation-based methods take at least 48 h to obtain a final result (5) and severely underestimate the presence of O1/O139 serogroups, due to the fact that these strains predominantly enter a viable but nonculturable (VBNC) state once released from the human intestine into the environment (6, 7). Alternatively, fluorescence in situ hybridization (FISH) and the direct fluorescent-antibody assay (DFA) are direct cell-...

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A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Bliem

Schauer

Plicka³

et al. 2015

Appl Environ Microbiol

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“…The exploitation of the two RT-qPCR techniques either routine or high speed protocol could assess the sensitivity and accuracy of FMD diagnosis of the field samples Using of variety of samples and tissues isolated from different animal species in Egypt was considered an important need and good tool for detection of a real-life situation of FMDV observed in areas of endemic infections (Hole et al, 2010). Suitable laboratory methods are so-called rapid-cycle or high-speed PCR assays; results are provided in less than 1 h (Wittwer et al, 2001), those PCR systems have been described recently for various pathogens such as Vibrio cholera, group B Streptococcus bacteria, Influenza virus or adenoviruses (Fujimoto et al, 2010;Wilson et al, 2010;Sakurai et al, 2011;Koskela et al, 2009;Molsa et al, 2012). Modifications are processing to improve the routinely used machines of RT-PCR for achieving rapid processing of the samples, like our used machine (Eco TM Real-Time PCR system) which showed an excellent high-speed PCR results using a short thermal pro le in the test confirming the TM possibility for an effective speeding up of PCR protocols in general.…”

Section: Discussionmentioning

confidence: 99%

Rapid Molecular Detection of Genetically Diverted Foot and Mouth Disease Virus Serotype O During the Outbreak of 2012 in Egypt

Rahman¹,

Hoffmann²,

Haas³

et al. 2015

International J. of Virology

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Increasing the international trade of animals and their products and the continuous changing of Foot and Mouth Disease Virus (FMDV) lead to the rise of disease introductions and create an urgent need for laboratory methods facilitating a swift and sensitive confirmation of suspect outbreaks and fast characterization of new isolates. As FMDV is extremely contagious and affects all cloven-hoofed animals, it provides a continuous burden and risk to the livestock industries of the developing world, in particular due to mortality in young animals, weight and milk loss, lameness and the trade restrictions necessary to control the disease. The control of FMD in Egypt requires an accurate analysis of the newly introduced viral strains and an analysis of their relationship with the current circulating strains and the routinely used vaccines. This study evaluates a recently developed rapid Reverse-Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) for the identification of FMDV in samples from suspect cases. Samples were collected from gum, tongue epithelium, vesicular fluid, buccal and gum swabs, as well as saliva of infected cattle, buffalo and sheep and examined using two RT-qPCR of routine and high-speed formats using "IRES1" and "3D-OIE" primers. In addition, partial VP1 sequencing of some selected isolates was carried out for phylogenetic analysis. The high-speed RT-qPCR allowed a reliable diagnosis of FMDV in less than half an hour and can be used as a fast and valuable method for the monitoring and controlling of the foot and mouth disease. A new variant genotype (FMD-O/Eg/Mans/14) was circulating in Egypt during the outbreak of 2012 showing 98.5% identity to the isolated strain in Sudan (SUD_6_2008).

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“…To date, a plethora of real-time PCR assays for the detection of V. cholerae have been published. Three examples are the assays published by Huang et al (2009), Koskela et al (2009) and Mehrabadi et al (2012) While the elaborate multiplex assays typically published in the literature are technologically impressive, almost none of these include a single process control which is carried through both nucleic acid extraction and PCR amplification steps. In addition, most target several virulence genes which may introduce unnecessary complexity when the principal concern is to detect toxigenic V. cholerae in the simplest way possible.…”

Section: Introductionmentioning

confidence: 99%

Development and implementation of a rapid real-time polymerase chain reaction assay for the detection of toxigenic <i>Vibrio cholerae</i> in water

Leat

Grundlingh

2013

WSA

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Assays which use real-time polymerase chain reaction (PCR) technology can be developed for the rapid identification of genetic sequences carried by waterborne pathogens. Rand Water has established facilities within which a selection of PCR assays will be developed. This paper reports on the optimisation and validation of the first assay to be implemented. This assay facilitates the detection of the ctxA gene of toxigenic Vibrio cholerae (V. cholerae) strains. The assay also includes an internal process control in the form of an Escherichia coli (E. coli) strain carrying a single genomic copy of the gfp gene from Aequorea victoria. Establishment of the assay required the selection of suitable PCR primers and probes for both the ctxA and gfp genes. This was followed by an optimisation phase where ideal PCR cycling conditions and primer/probe concentrations were established. A validation phase established the performance parameters of the assay. Parameters assessed included: limit of detection, sensitivity, specificity, reproducibility and robustness. The validation was conducted using potable water, surface water and sewage effluent matrices. The process has resulted in the establishment of a robust assay for the detection of toxigenic V. cholerae strains within 24 hours after samples are received.

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A multiplatform real-time polymerase chain reaction detection assay for Vibrio cholerae

Cited by 26 publications

References 15 publications

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Rapid Molecular Detection of Genetically Diverted Foot and Mouth Disease Virus Serotype O During the Outbreak of 2012 in Egypt

Development and implementation of a rapid real-time polymerase chain reaction assay for the detection of toxigenic <i>Vibrio cholerae</i> in water

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