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Development of a PCR Assay Targeting the rpoA Gene for the Screening of Vibrio Genus

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Abstract

A polymerase chain reaction (PCR) method based on the RNA polymerase alpha subunit (rpoA) gene was developed for the detection of the Vibrio genus. The specific primers were designed aligning the rpoA gene sequences available in GenBank of all Vibrio species. The specificity of the primers was tested against 35 Vibrio species. In addition, 12 species phylogenetically related to the Vibrio genus were used as negative control. Moreover, in order to eliminate any false-negative results, bacterium-specific primers targeting the 16S rRNA gene were introduced in the test as a noncompetitive internal amplification control. The rpoA primers correctly amplified all the Vibrio species considered. No cross-reaction was observed when tested against closely related species. To estimate the applicability of this method, 336 Vibrio wild-type strains isolated from Italian aquaculture products and from imported seafoods were tested. The sensitivity, tested using serial dilutions of different pure cultures of certified strains, resulted to 103 colony-forming units per milliliter. The assay proved to be specific, rapid, and reliable. It can be proposed as a routine screening technique for the confirmation of Vibrio genus in isolated colonies.

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Acknowledgements

The research has been supported by a grant of the Italian Ministry of University and Research (MIUR-PRIN 2004).

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Authors and Affiliations

  1. Dipartimento di Patologia Animale, Facoltà di Medicina Veterinaria, Università degli Studi di Torino, Via Leonardo da Vinci 44, 10095, Grugliasco, Turin, Italy

    Alessandra Dalmasso, Fabio La Neve, Maria Teresa Bottero & Tiziana Civera

  2. Istituto Superiore di Sanità, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Viale Regina Elena 299, 00161, Rome, Italy

    Elisabetta Suffredini & Luciana Croci

  3. Istituto Zooprofilattico Sperimentale del Piemonte, Valle d’Aosta e Liguria, Via degli Stagnoni 96, 19136, La Spezia, Italy

    Laura Serracca

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  1. Alessandra Dalmasso
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  2. Fabio La Neve
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  3. Elisabetta Suffredini
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  4. Luciana Croci
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  5. Laura Serracca
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  6. Maria Teresa Bottero
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  7. Tiziana Civera
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Correspondence to Alessandra Dalmasso.

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Dalmasso, A., La Neve, F., Suffredini, E. et al. Development of a PCR Assay Targeting the rpoA Gene for the Screening of Vibrio Genus. Food Anal. Methods 2, 317–324 (2009). https://doi.org/10.1007/s12161-009-9089-9

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  • DOI: https://doi.org/10.1007/s12161-009-9089-9

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