Multiplex primer-extension assay for identification of six pathogenic vibrios

https://doi.org/10.1016/j.ijfoodmicro.2008.10.029Get rights and content

Abstract

A multiplex Primer-Extension Reaction (PER) assay, was specifically designed for the identification, of the major human pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and V. fluvialis) in fishery products. The assay, directed towards the rpoA gene, was tested on a total of 287 samples representing six Vibrio species and ten non-Vibrio species. The primers used in the preliminary PCR, designed in well conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 284 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of these human pathogenic species in seafood samples and to evaluate the potential hazard for consumers.

Introduction

In the last few years, a considerable rise of Vibrio diseases in many developed countries has been recorded. Possible causes are the expansion of the international trade of fishery products and/or the increase in their consumption after an insufficient purification procedure during processing (Tantillo et al., 2004). The Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) estimated a 126% increase in the incidence of Vibrio associated infection between 1996 and 2002, in spite of the repeated actions the Authorities undertake in order to make people more aware of the hazard connected with the consumption of raw seafood (CDC, 2003).

The Vibrio genus is characterized by a large number of species; some of these are human pathogens causing gastrointestinal and wound infections through the ingestion or manipulation of contaminated fishes and shellfish (Thompson et al., 2004). V. cholerae is the etiological agent of cholera, which is spread by contaminated food, water or direct faecal contact with food handlers. Shellfish are mainly responsible for its transmission (Tarr et al., 2007). The presence of V. vulnificus has been well documented in water and molluscs and infections have been associated with consumption of raw oysters and contact with shellfish (Croci et al., 2001). V. parahaemolyticus has been recognized as an important cause of foodborne disease due to the consumption of raw or undercooked seafood in Asia and North America. In Europe, disease caused by V. parahaemolyticus have rarely been reported and little information regarding the prevalence in the environment is available (Croci et al., 2001, Robert-Pillot et al., 2004). Besides these well known species, other vibrios (V. fluvialis, V. alginolyticus, V. mimicus) responsible for sporadic infections, have been sometimes isolated in many countries (Oliver and Kaper, 2001). In particular, V. fluvialis is usually associated with the consumption of raw or improperly cooked seafood (Kothary et al., 2003), while V. alginolyticus and V. mimicus are infrequently reported as cause of foodborne disease (Wei et al., 2008).

Since the epidemiological studies of pathogenic species depend on their unmistakable identification, accurate methods are needed. A great number of traditional microbiological techniques have been developed; they are based on the culture on selective media. However they require confirmatory assays based on biochemical, serological and biomolecular test. Recent advances in molecular biology have introduced new techniques for the detection of microbial species such as the amplification of species-specific fragments (Di Pinto et al., 2005, Nhung et al., 2007, Tarr et al., 2007) or the analysis of the diagnosis sites (Reen and Boyd, 2005, Saha et al., 2006). When closely related species are considered, the analysis of Single Nucleotide Polymorphism (SNP) seems to be the best approach. In these cases biomolecular assays such as the use of restriction enzymes (Saha et al., 2006), TaqMan Minor Groove Binding (MGB) probes (Easterday et al., 2005, U et al., 2005) and sequencing primers associated with fluorescently labelled dideoxy-nucleotide ([F]ddNTPs) (Scott et al., 2007) have been successfully applied. In particular, this latter technique is based on the simultaneous analysis of several diagnosis sites by means of multiplex Primer-Extension Reaction (PER) in conjunction with a genetic analyser. The PER reaction is based on the addition of a [F]ddNTPs to the 3′ end of an unlabeled oligonucleotide (sequencing primer) in the absence of dNTPs in the reaction. Each sequencing primer is designed immediately adjacent to the diagnosis sites and their length is modified by the addition of non-homologous tails at the 5′ end. The extended sequencing primers used to interrogate the diagnosis sites differ by size and colour which is specific to diagnosis site (Quintans et al., 2004).

The aim of the study was the development of the above described assay for differentiation of V. cholerae, V. parahaemolyticus, V. vulnificus, V. fluvialis, V. alginolyticus and V. mimicus. This test could represent a valid tool for the study of the risk factors connected with the main pathogenic Vibrio in seafood.

Section snippets

Samples

All reference species of Vibrio (V. cholerae, V. parahaemolyticus, V. vulnificus, V. fluvialis, V. alginolyticus and V. mimicus) used to develop the assay are listed in Table 1. Bacterial strains belonging to phylogenetically related genera were also tested to verify possible cross-reactions (Table 1). Finally, in order to evaluate the possible applicability of the proposed assay, 245 wild isolates (WI) (55 V. cholerae, 70 V. parahaemolyticus, 2 V. vulnificus, 3 V. fluvialis, 114 V.

Results and discussion

The increase of Vibrio infections due to the consumption and the manipulation of contaminated fish and shellfish has made necessary the precise identification of Vibrio spp.

A correct monitoring of Vibrio species in seafood and in fish products could open new interesting perspectives for epidemiological studies on the principal risk factors associated with these microorganisms.

The aim of our study was to develop an assay for the specific identification of the six Vibrio species (V. cholerae, V.

Acknowledgement

This work was supported by a grant of the Italian Ministry of University and Research.

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