Development of a PCR Assay Targeting the rpoA Gene for the Screening of Vibrio Genus
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Development of a PCR Assay Targeting the rpoA Gene for the Screening of Vibrio Genus Alessandra Dalmasso & Fabio La Neve & Elisabetta Suffredini & Luciana Croci & Laura Serracca & Maria Teresa Bottero & Tiziana Civera
Received: 24 November 2008 / Accepted: 14 May 2009 / Published online: 4 June 2009 # Springer Science + Business Media, LLC 2009
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, A. Dalmasso (*) : F. La Neve : M. T. Bottero : T. Civera Dipartimento di Patologia Animale, Facoltà di Medicina Veterinaria, Università degli Studi di Torino, Via Leonardo da Vinci 44, 10095 Grugliasco, Turin, Italy e-mail: [email protected] F. La Neve e-mail: [email protected] M. T. Bottero e-mail: [email protected] T. Civera e-mail: [email protected] E. Suffredini : L. Croci Istituto Superiore di Sanità, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Viale Regina Elena 299, 00161 Rome, Italy E. Suffredini e-mail: [email protected] L. Croci e-mail: [email protected] L. Serracca Istituto Zooprofilattico Sperimentale del Piemonte, Valle d’Aosta e Liguria, Via degli Stagnoni 96, 19136 La Spezia, Italy e-mail: [email protected]
in order to eliminate any false-negative results, bacteriumspecific 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 wildtype 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. Keywords rpoA . Vibrio spp. . PCR
Introduction Various gram-negative heterotrophic bacteria may be isolated in seafood, such as Aeromonas, Pseudomonas, Alcaligenes, Moraxella, Acinetobacter, Flavobacterium, Cytophaga, Chromobacterium, Photobacterium, Lucibacterium, and Vibrio (Cavallo et al. 2009). Vibrio species have been extensively studied for their implication in related public health hazards. The Vibrio genus comprises 74 species including ten species pathogenic to humans: Vibrio cholerae, Vibrio mimicus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cincinnatiensis, Vibrio vulnificus, Vibrio furnissii, Vibrio metschnikovii,
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