150

Journal of Food Protection, Vol. 68, No. 1, 2005, Pages 150153

Copyright Q, International Association for Food Protection

Research Note

A Collagenase-Targeted Multiplex PCR Assay for Identification

of Vibrio alginolyticus, Vibrio cholerae, and
Vibrio parahaemolyticus
ANGELA DI PINTO,1* GIUSEPPINA CICCARESE,2 GIUSEPPINA TANTILLO,1 DOMENICO CATALANO,3 AND
VITO TONY FORTE1

1Dipartimento di Sanita e Benessere degli Animali, Facolta di Medicina Veterinaria, Universita degli Studi di Bari, Valenzano (Ba), Italy; 2Istituto
Zooprofilattico di Puglia e Basilicata, 73012 Campi Salentina, Lecce, Italy; and 3Istituto di Tecnologie Biomediche Sezione di Bari, 168/5-70126
Bari, Italy

MS 04-114: Received 12 March 2004/Accepted 10 July 2004

ABSTRACT
A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio algino-
lyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three
primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches
for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important.
The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to
discriminate among the three Vibrio species.

The genus Vibrio includes human pathogenic species
commonly associated with outbreaks of diarrheal diseases
in humans due to the consumption of raw or improperly
cooked seafood. Vibrio infections may also occur as a con-
sequence of exposures of skin lesions, such as cuts, open
wounds, or abrasions, to aquatic environments and marine
animals. The gastrointestinal Vibrio diseases attributable to
seafood ingestion are well known, but data on extraintes-
tinal infections are more limited and some have been re-
ported only recently (4). Vibrios have acquired a great ep-
idemiological importance because of the increasing occur-
rence of foodborne disease outbreaks, particularly due to
noncholera Vibrio species. Particular attention is currently
being focused on Vibrio cholerae O139, which has been
implicated in numerous outbreaks of seafood-associated
gastroenteritis producing classic cholera symptoms, and on
non-O1 strains that cause less severe gastrointestinal dis-
eases than those induced by V. cholerae O1 (15). In recent
microbiological and epidemiological studies (4, 6, 13), sev-
eral clinical extraintestinal infections and gastroenteritis
cases caused by toxigenic strains of Vibrio parahaemoly-
ticus and Vibrio alginolyticus have reported.
Identification of these microorganisms from both clin-
ical and food samples is predicated upon an accurate and
specific analytical approach. Conventional standard micro-
biological methods for the detection of Vibrio spp., based
on the traditional analysis of phenotypic profile, are slow
and laborious, often requiring several days to perform. Con-
* Author for correspondence. Tel: 1390805443970; Fax: 1390805443855;
E-mail: a.dipinto@veterinaria.uniba.it.
ventional phenotypic assays are characterized by low sen-
sitivity and may fail to detect strains of bacteria present in
the samples at low concentrations or with unusual pheno-
typic profiles (17). Advances in molecular technology have
led to a shift from conventional phenotypic methods for the
identification of microorganisms to molecular methods,
which are more sensitive and specific for the detection of
low numbers of bacteria and of viable but not culturable
microrganisms (13). Different molecular targets have been
used to identify the presence of Vibrio spp. both in clinical
samples and in seafood. The genes encoding the virulence
determinants and their expression regulator genes have
been used to characterize numerous Vibrio species. A mo-
lecular test based on the detection of the tdh and/or trh
genes (encoding thermostable direct hemolysin and ther-
mostable-related hemolysin, respectively) has been applied
for identification of V. parahaemolyticus (1012, 16). Lee
et al. (11) developed a molecular approach based on the
amplification of a DNA fragment that is highly conserved
in all strains of V. parahaemolyticus. PCR procedures tar-
geting the gyrB gene, encoding the B subunit of DNA gyr-
ase essential for DNA replication, and the regulatory toxR
gene have been used for the specific detection of V. para-
haemolyticus (9, 18). The ctxAB and tcpA genes, known to
play a cardinal role in maintaining virulence in Vibrio chol-
erae, are believed to be exclusively associated with clinical
strains of the O1 and O139 serogroups. Rivera et al. (14)
described a multiplex PCR assay based on the detection of
the two main virulence-associated factors, cholera toxin and
toxin coregulated pilus, that can be used to quickly detect
V. cholerae O1 and V. cholerae O139. For identification
J. Food Prot., Vol. 68, No. 1 MULTIPLEX PCR ASSAY FOR VIBRIO SPP. IDENTIFICATION 151

purposes, rRNA sequence homologies between Vibrio spe- TABLE 1. Reference type strains used in this study
cies has also been used (5, 7), although these sequences do Strain Source
not appear to be suitable for species discrimination.
In this study, the use of V. alginolyticus, V. cholerae, Aeromonas hydrophila ATCC 23213
and V. parahaemolyticus collagenase gene sequences as an A. hydrophila ATCC 21763
alternative genetic marker for species identification of vib- A. hydrophila ATCC 23213
rios was investigated. Because the V. parahaemolyticus A. hydrophila ATCC 21763
A. hydrophila ISS collection
vppC gene encoding metalloprotease shares 77% identity
A. hydrophila ISS collection
with V. alginolyticus collagenase and does not show any A. hydrophila ISS collection
extensive sequence homology with other Vibrio metallo- A. hydrophila ISS collection
proteases (8), three primer pairs on the respective collage- Escherichia coli ATCC 35421
nase sequences have been designed and used to perform a Listeria spp. ATCC 7646
collagenase-targeted multiplex PCR assay for the specific Salmonella spp. ATCC 35664
detection of V. alginolyticus, V. cholerae, and V. parahae- Vibrio alginolyticus ATCC 33839
molyticus. V. alginolyticus ATCC 33787
V. alginolyticus ATCC 17749
MATERIALS AND METHODS V. alginolyticus ISS collection
V. alginolyticus ISS collection
Bacterial strains. Bacterial reference strains from the Amer-
V. alginolyticus ISS collection
ican Type Culture Collection (ATCC) and the Istituto Superiore
V. alginolyticus Shellfish
di Sanita (ISS) collection and environmental and food strains from
V. alginolyticus Shellfish
seawater and shellfish samples were used in this study (Table 1).
V. alginolyticus Shellfish
The ATCC strains were processed according to the producers in-
V. alginolyticus Shellfish
structions. The environmental and food strains were subjected to
V. alginolyticus Shellfish
enrichment in alkaline-peptone-salt broth containing 3% NaCl, in-
V. alginolyticus Shellfish
cubated at 378C for 16 h, and then streaking onto thiosulfate cit-
V. anguillarum ATCC 43306
rate bile salts agar plates (Oxoid, Basingstoke, Hampshire, UK)
V. carchariae ATCC 35084
at 378C for 24 h. Presumptive colonies were subcultured on Tryp-
V. cholerae non-O1 ATCC 25872
ticase soy agar (Oxoid) and analyzed microscopically and bio-
V. cholerae O1 Inaba ATCC 9459
chemically with API 20E (bioMerieux, Hazelwood, Mo.). The
V. cholerae O1 NAG ATCC 25872
bacterial strains that were biochemically identified as Vibrio were
V. cholerae O1 Ogawa ATCC 9458
grown in Trypticase soy broth (Oxoid) containing 2.5% NaCl and
V. cholerae non-O1 Shellfish
incubated at 378C for 24 h. The broth cultures were then trans-
V. cholerae non-O1 Shellfish
ferred into 1.5-ml tubes and centrifuged at 13,000 3 g at room
V. cholerae non-O1 Shellfish
temperature. The resulting pellet was used for nucleic acid ex-
V. cholerae non-O1 Shellfish
traction.
V. cholerae non-O1 Shellfish
DNA extraction. The DNA was extracted using the QIAamp V. damsela ATCC 33536
DNA Mini Kit (Qiagen, Hilden, Germany). The pellet was sus- V. fluvialis ATCC 33810
pended in 180 ml of a solution containing 20 mg/ml lysozyme V. fluvialis ISS collection
(Sigma Chemical Co., St. Louis, Mo.) and incubated for 30 min V. fluvialis ISS collection
at 378C. Then, 200 ml of lysis buffer (Qiagen) and 20 ml of Pro- V. hollisae ATCC 33565
teinase K (20 mg/ml) were added, and the suspension was incu- V. metschinikovii ATCC 7708
bated at 568C for 30 min and for a further 15 min at 958C. After V. mimicus ATCC 33653
adding 200 ml of ethanol, the resulting mixture was applied to the V. mimicus ISS collection
QIAamp DNA spin column (Qiagen). The DNA bound to the V. mimicus ISS collection
column was washed in two centrifugation steps using two differ- V. parahaemolyticus ATCC 17802
ent wash buffers to improve the purity of the eluted DNA. The V. parahaemolyticus ATCC BAA-242
purified DNA was then eluted from the column in 80 ml of de- V. parahaemolyticus ATCC BAA-238
ionized water. The DNA concentration and the purity of the eluate V. parahaemolyticus ATCC 33845
were measured by absorbance at 260 nm and by calculating the V. parahaemolyticus ATCC 43996
ratio of absorbance at 260 nm to absorbance at 280 nm using a V. parahaemolyticus Shellfish
spectrophotometer (DU-600, Beckman, Fullerton, Calif.). The V. parahaemolyticus Shellfish
eluted DNA was used as a template in the PCR assay. V. parahaemolyticus Shellfish
V. parahaemolyticus Shellfish
Oligonucleotide primers. The oligonucleotide primer pairs V. parahaemolyticus Shellfish
used in this study were designed from GenBank accession nos. V. parahaemolyticus Shellfish
E03106, AF326572, and AE004243 for V. alginolyticus, V. par- V. parahaemolyticus Shellfish
ahaemolyticus, and V. cholerae, respectively. The design and anal- V. parahaemolyticus Shellfish
ysis of the primers were carried out with respect to self-comple- V. parahaemolyticus Shellfish
mentarity, interprimer annealing, and optimum annealing temper- V. vulnificus ATCC 27562
atures. All primer sequences were compared with the GenBank V. vulnificus Shellfish
database for sequence similarity using the BLAST program. Com- V. vulnificus Shellfish
puter analysis indicated that all oligonucleotide primer pairs had
152 DI PINTO ET AL. J. Food Prot., Vol. 68, No. 1

significant affinity for only the target genes. The primers were
designed so that the predicted amplification product sizes would
be different. The primer pairs used for V. alginolyticus, V. para-
haemolyticus, and V. cholerae, respectively, were VA-F (59-cga
gta cag tca ctt gaa agc c-39, positions 1526 through 1547) and
VA-R (59-cac aac aga act cgc gtt acc-39, positions 2242 through
2263), producing a 737-bp fragment (GenBank accession no.
E03106), VP-F (59-gaa agt tga aca tca tca gca cga-39, positions
93 through 116) and VP-R (59-ggt cag aat caa acg ccg-39, posi-
tions 347 through 364), which amplify a 271-bp region (GenBank
accession no. AF326572), and VC-F (59-cgg cgt ggc tgg ata cat
tg-39, positions 1956 through 1976) and VC-R (59-gtc aca ctt aaa
tag tag cgt cc-39, positions 2226 through 2249), which amplify a
389-bp region (GenBank accession no. AE004243). The primers
were synthesized by MWG Biotech (Milan, Italy).
Specificity of primer pairs. To evaluate the specificity of
each oligonucleotide primer pair to its target gene, a PCR assay
was carried out by testing all the reference strains reported in
FIGURE 1. Agarose gel electrophoresis showing the results of
Table 1. The PCR assay was performed in a total volume of 25
multiplex PCR of three target gene segments from purified DNA
ml using 12.5 ml of HotStarTaq Master Mix (Qiagen), which pro-
of V. alginolyticus, V. cholerae, and V. parahaemolyticus. Lane 1,
vides 2.5 units per reaction of DNA polymerase, 0.2 mM of each
100-bp DNA ladder; lane 2, 737-bp amplicon from V. alginoly-
deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), 13
ticus; lane 3, 389-bp amplicon from V. cholerae; lane 4, 271-bp
PCR buffer (with 1.5 mM MgCl2), 0.5 mM of each primer, and
amplicon from V. parahaemolyticus; lane 5, negative control.
1ml of DNA. The mixture was processed in a Mastercycler (Ep-
pendorf, Milan, Italy) with an initial activation step at 958C for
15 min, followed by 35 cycles of denaturation at 948C for 30 s, by sequence analysis carried out with ABI PRISM 3100
annealing at 578C for 30 s, and extension at 728C for 60 s, and a (Applied Biosystems, Rome, Italy). The sequence analysis
final extension at 728C for 5 min. The template-free reactions were corresponded to the published sequence of the tested ref-
included in the PCR setup as negative controls. erence strains. The negative controls subjected to multiplex
Multiplex PCR. After checking that all bacteria strains re- PCR produced negative results. There was no significant
ported in Table 1 amplified specifically and efficiently in separate variability among the results of 10 replicate experiments.
reactions using the same PCR program, a multiplex PCR was set
up. The reaction was performed in a total volume of 25 ml using
Multiplex PCR. The collagenase-based multiplex PCR
12.5 ml of HotStarTaq Master Mix. The multiplex PCR was per- performed with simultaneous use of the three pairs of prim-
formed with 2 ml (50 ng/ml) of template. ers targeting V. alginolyticus, V. cholerae, and V. parahae-
molyticus collagenase genes allowed successful detection
Amplified product detection. The amplified products were and discrimination of the three Vibrio species (Fig. 1). The
analyzed by electrophoresis on a 1.5% (wt/vol) agarose NA gel
different sizes of the amplification products allowed rapid
(Pharmacia, Uppsala, Sweden) in 13 Tris-borate-EDTA buffer
and specific discrimination of V. cholerae, V. parahaemo-
(0.89 M Tris, 0.89 M boric acid, 0.02 M EDTA, pH 8.0; USB,
Cleveland, Ohio) and visualized by ethidium bromide staining and lyticus, and V. alginolyticus by gel electrophoresis. The an-
a UV transilluminator. A Gene Ruler 100-bp DNA Ladder Plus nealing temperature, extension time, and primer concentra-
(MBI Fermentas, Vilnius, Lithuania), consisting of DNA frag- tion used in the multiplex PCR were optimal. The negative
ments ranging in size from 3,000 to 100 bp, was used as a mo- controls subjected to multiplex PCR produced negative re-
lecular weight marker. sults.

RESULTS
Oligonucleotide primers. The three pairs of oligonu-
cleotide primers that were designed to be complementary
to the V. cholerae, V. parahaemolyticus, and V. alginoly-
ticus collagenase genes produced specific amplicons of the
expected sizes. In particular, the V. alginolyticus primers
produced a specific 737-bp amplicon in all V. alginolyticus
strains tested, the V. cholerae primers produced a specific
389-bp amplicon in all V. cholerae strains, including O1
and non-O1, and the V. parahaemolyticus primers amplified
a fragment of 271 bp. Comparing total DNA from different
Vibrio species and related bacterial strains, the primer pair
sets investigated provided species-specific identification of
V. cholerae, V. parahaemolyticus, and V. alginolyticus. The
other strains failed to yield positive amplification with these
primers. The specificity of the PCR products was confirmed
DISCUSSION
The multiplex PCR based on the diversity of the col-
lagenase gene sequences between V. alginolyticus and V.
parahaemolyticus and the lack of significant homology with
V. cholerae collagenase sequence (8) represents a valid al-
ternative molecular approach for specific and rapid detec-
tion of these three Vibrio species, which are important path-
ogens associated with seafood poisoning in different areas
of the world. The procedure may be used for the molecular
confirmation of biochemically identified Vibrio strains from
seafood samples implicated in outbreaks of food poisoning.
Strain culture methods allow only ambiguous differentia-
tion of Vibrio species and others that are genetically related
such as Aeromonas hydrophila, which is often misidentified
as a Vibrio species by conventional biochemical methods.
The results indicate that the collagenase gene may be a
J. Food Prot., Vol. 68, No. 1 MULTIPLEX PCR ASSAY FOR VIBRIO SPP. IDENTIFICATION
useful alternative target for phylogenetic analysis and spe-
cies identification of Vibrios to complement more conven-
tional phenotypic identification systems.
The molecular approach allows researchers to over-
come the disadvantages of culture-based methods (17),
which may be misleading, may underestimate the number
of the viable cells, and may allow the growing of Vibrio-
like colonies or other species that are not successfully in-
hibited, thus invalidating the results. The results of this
study have confirmed the utility of molecular tools for rou-
tine identification of pathogens in food and rapid charac-
terization of bacteria with particular growth requirements
or unusual biochemical patterns. Further studies are re-
quired to confirm the use of collagenase gene as an alter-
native genetic marker for vibrios. In particular, the speci-
ficity of the primer pairs may be tested on a wide number
of strains of each target organisms. The reliability of the
method for the detection of the three Vibrio species should
also be evaluated in mixed cultures.
ACKNOWLEDGMENTS
The authors thank Dr. Aureli (Istituto Superiore di Sanita) and Dr.
Cancellotti (Istituto Zooprofilattico Sperimentale delle Venezie) for pro-
viding bacteria strains.
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