Rapid Detection of Vibrio harveyi in Seawater by Real-Time PCR

Fukui, Youhei; Sawabe, Tomoo

doi:10.1264/jsme2.23.172

Cited by 15 publications

(13 citation statements)

References 15 publications

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“…2007). Real‐time PCR assays recently developed for the rapid identification of V. harveyi (Fukui and Sawabe 2008). They are not routinely used because of their requirement for an expensive thermal cycler with a fluorescence detector.…”

Section: Introductionmentioning

confidence: 99%

“…Although PCR assays provide more rapid identification of V. harveyi than conventional assays, this assay requires post-PCR electrophoresis to visually detect the amplified products, and this can be labour-intensive and time-consuming (Goto et al 2007). Real-time PCR assays recently developed for the rapid identification of V. harveyi (Fukui and Sawabe 2008). They are not routinely used because of their requirement for an expensive thermal cycler with a fluorescence detector.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish

Cao

Jian

et al. 2010

Letters in Applied Microbiology

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Aims: The purpose of this study was to establish a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. Methods and Results: A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65°C for 60 min. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10−5 dilution level (equivalent to 17·2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. Conclusions: The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. Significance and Impact of the Study: This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow‐out pond, to prevent the disease outbreak.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish

Cao

Jian

et al. 2010

Letters in Applied Microbiology

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“…Most of scientific publications dealing with rapid, sensitive and specific molecular identification of Vibrio species use PCR technique. Concerning Vibrio harveyi, several PCR assays have been developed (Conejero and Hedreyda, 2003, Fukui and Sawabe, 2008, Pang et al, 2006 aiming to facilitate disease prevention and surveillance in cultured marine animals. More recently Cao et al (2010) successfully developed a novel nucleic acid amplification diagnostic method based on loop-mediated isothermal amplification (LAMP) to detect V. harveyi infection in the cultured gastropod Babylonia areolata.…”

Section: Discussionmentioning

confidence: 99%

“…Vibrio vulnificus (Takahashi et al, 2005a), Vibrio parahaemolyticus (Qin et al, 2008, Takahashi et al, 2005b and V. cholerae (Gubala, 2006). Fukui and Sawabe (2008) developed a real-time PCR assay on V. harveyi targeting the 16S rRNA gene. Nevertheless, the high conservation of nucleotidic sequences of this gene in Vibrionacea resulted in the failure for some phylogenetically closed Vibrio species, V. rotiferianus in particular, to find divergent nucleotidic sequence for PCR primer annealing.…”

Section: Discussionmentioning

confidence: 99%

Development of TaqMan real-time PCR assays for monitoring Vibrio harveyi infection and a plasmid harbored by virulent strains in European abalone Haliotis tuberculata aquaculture

et al. 2013

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International audienceThe Gram-negative bacterium Vibrio harveyi is known to be highly pathogenic for the European abalone Haliotis tuberculata, which is a gastronomically important marine gastropod with a high commercial value. Since 1998, some particular bacterial strains are described as implicated in recurrent mortality outbreaks in French farm and field stocks of abalone. Recently, a 9.6 kb plasmid named pVCR1, was shown to be harbored by one highly V. harveyi virulent ORM4 strain suggesting its involvement in virulence phenotype. Thus, we have developed in the present study two TaqMan real-time PCR assays allowing to (i) rapidly and specifically detect, by a duplex procedure and in less than 2 h, both V. harveyi and the presence of plasmid pVCR1 from unidentified bacterial colony and to (ii) quantify both V. harveyi and the plasmid pVCR1 in the hemolymph of abalone or its surrounding seawater. Quantification curves of V. harveyi or ORM4 strain seeded in hemolymph or artificial sea water samples were equivalent showing excellent qPCR efficacies and detection level as low as 18 V. harveyi cell-equivalent genomic DNA in a PCR reaction well. This qPCR allowed us to monitor V. harveyi ORM4 strain in experimentally infected H. tuberculata. These diagnosis assays could provide powerful and useful tools to better understand the epidemiology of vibriosis caused by V. harveyi in different cultured marine species including H. tuberculata

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“…FISHFC has advantages over many rapid detection methods such as PCR (2,8), quantitative PCR (9,15,16), RT-PCR (13), enzyme-linked immunosorbent assay (ELISA) (5) and FISH (14,20,22). PCR and quantitative PCR are faster but detect both live and dead cells.…”

mentioning

confidence: 99%

Specific and Rapid Quantification of Viable Listeria monocytogenes Using Fluorescence in situ Hybridization in Combination with Filter Cultivation

Fuchizawa

Shimizu

Ootsubo³

et al. 2009

Microb. Environ.

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We developed a new method that rapidly and specifically enumerates only viable Listeria monocytogenes in food using fluorescence in situ hybridization in combination with filter cultivation (FISHFC). Viable L. monocytogenes could be specifically quantified within 16 h using an Alexa647-labeled mRL-2 probe. The coefficient of the correlation between the new method and the conventional plating method was 0.959.

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Rapid Detection of Vibrio harveyi in Seawater by Real-Time PCR

Cited by 15 publications

References 15 publications

Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish

Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish

Development of TaqMan real-time PCR assays for monitoring Vibrio harveyi infection and a plasmid harbored by virulent strains in European abalone Haliotis tuberculata aquaculture

Specific and Rapid Quantification of Viable Listeria monocytogenes Using Fluorescence in situ Hybridization in Combination with Filter Cultivation

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