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Journal of Food Protection, Vol. 68, No. 1, 2005, Pages 150–153
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
VITO TONY FORTE1
AND
1Dipartimento
di Sanità e Benessere degli Animali, Facoltà di Medicina Veterinaria, Università 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 alginolyticus, 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 consequence 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 extraintestinal infections are more limited and some have been reported only recently (4). Vibrios have acquired a great epidemiological importance because of the increasing occurrence 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 diseases than those induced by V. cholerae O1 (15). In recent
microbiological and epidemiological studies (4, 6, 13), several clinical extraintestinal infections and gastroenteritis
cases caused by toxigenic strains of Vibrio parahaemolyticus and Vibrio alginolyticus have reported.
Identification of these microorganisms from both clinical and food samples is predicated upon an accurate and
specific analytical approach. Conventional standard microbiological 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: [email protected].
ventional phenotypic assays are characterized by low sensitivity and may fail to detect strains of bacteria present in
the samples at low concentrations or with unusual phenotypic 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 (1–3). 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 molecular test based on the detection of the tdh and/or trh
genes (encoding thermostable direct hemolysin and thermostable-related hemolysin, respectively) has been applied
for identification of V. parahaemolyticus (10–12, 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 targeting the gyrB gene, encoding the B subunit of DNA gyrase essential for DNA replication, and the regulatory toxR
gene have been used for the specific detection of V. parahaemolyticus (9, 18). The ctxAB and tcpA genes, known to
play a cardinal role in maintaining virulence in Vibrio cholerae, 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
purposes, rRNA sequence homologies between Vibrio species has also been used (5, 7), although these sequences do
not appear to be suitable for species discrimination.
In this study, the use of V. alginolyticus, V. cholerae,
and V. parahaemolyticus collagenase gene sequences as an
alternative genetic marker for species identification of vibrios was investigated. Because the V. parahaemolyticus
vppC gene encoding metalloprotease shares 77% identity
with V. alginolyticus collagenase and does not show any
extensive sequence homology with other Vibrio metalloproteases (8), three primer pairs on the respective collagenase sequences have been designed and used to perform a
collagenase-targeted multiplex PCR assay for the specific
detection of V. alginolyticus, V. cholerae, and V. parahaemolyticus.
MATERIALS AND METHODS
Bacterial strains. Bacterial reference strains from the American Type Culture Collection (ATCC) and the Istituto Superiore
di Sanità (ISS) collection and environmental and food strains from
seawater and shellfish samples were used in this study (Table 1).
The ATCC strains were processed according to the producer’s instructions. The environmental and food strains were subjected to
enrichment in alkaline-peptone-salt broth containing 3% NaCl, incubated at 378C for 16 h, and then streaking onto thiosulfate citrate bile salts agar plates (Oxoid, Basingstoke, Hampshire, UK)
at 378C for 24 h. Presumptive colonies were subcultured on Trypticase soy agar (Oxoid) and analyzed microscopically and biochemically with API 20E (bioMérieux, Hazelwood, Mo.). The
bacterial strains that were biochemically identified as Vibrio were
grown in Trypticase soy broth (Oxoid) containing 2.5% NaCl and
incubated at 378C for 24 h. The broth cultures were then transferred into 1.5-ml tubes and centrifuged at 13,000 3 g at room
temperature. The resulting pellet was used for nucleic acid extraction.
DNA extraction. The DNA was extracted using the QIAamp
DNA Mini Kit (Qiagen, Hilden, Germany). The pellet was suspended in 180 ml of a solution containing 20 mg/ml lysozyme
(Sigma Chemical Co., St. Louis, Mo.) and incubated for 30 min
at 378C. Then, 200 ml of lysis buffer (Qiagen) and 20 ml of Proteinase K (20 mg/ml) were added, and the suspension was incubated at 568C for 30 min and for a further 15 min at 958C. After
adding 200 ml of ethanol, the resulting mixture was applied to the
QIAamp DNA spin column (Qiagen). The DNA bound to the
column was washed in two centrifugation steps using two different wash buffers to improve the purity of the eluted DNA. The
purified DNA was then eluted from the column in 80 ml of deionized water. The DNA concentration and the purity of the eluate
were measured by absorbance at 260 nm and by calculating the
ratio of absorbance at 260 nm to absorbance at 280 nm using a
spectrophotometer (DU-600, Beckman, Fullerton, Calif.). The
eluted DNA was used as a template in the PCR assay.
Oligonucleotide primers. The oligonucleotide primer pairs
used in this study were designed from GenBank accession nos.
E03106, AF326572, and AE004243 for V. alginolyticus, V. parahaemolyticus, and V. cholerae, respectively. The design and analysis of the primers were carried out with respect to self-complementarity, interprimer annealing, and optimum annealing temperatures. All primer sequences were compared with the GenBank
database for sequence similarity using the BLAST program. Computer analysis indicated that all oligonucleotide primer pairs had
TABLE 1. Reference type strains used in this study
Strain
Aeromonas hydrophila
A. hydrophila
A. hydrophila
A. hydrophila
A. hydrophila
A. hydrophila
A. hydrophila
A. hydrophila
Escherichia coli
Listeria spp.
Salmonella spp.
Vibrio alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. alginolyticus
V. anguillarum
V. carchariae
V. cholerae non-O1
V. cholerae O1 Inaba
V. cholerae O1 NAG
V. cholerae O1 Ogawa
V. cholerae non-O1
V. cholerae non-O1
V. cholerae non-O1
V. cholerae non-O1
V. cholerae non-O1
V. damsela
V. fluvialis
V. fluvialis
V. fluvialis
V. hollisae
V. metschinikovii
V. mimicus
V. mimicus
V. mimicus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. parahaemolyticus
V. vulnificus
V. vulnificus
V. vulnificus
Source
ATCC 23213
ATCC 21763
ATCC 23213
ATCC 21763
ISS collection
ISS collection
ISS collection
ISS collection
ATCC 35421
ATCC 7646
ATCC 35664
ATCC 33839
ATCC 33787
ATCC 17749
ISS collection
ISS collection
ISS collection
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
ATCC 43306
ATCC 35084
ATCC 25872
ATCC 9459
ATCC 25872
ATCC 9458
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
ATCC 33536
ATCC 33810
ISS collection
ISS collection
ATCC 33565
ATCC 7708
ATCC 33653
ISS collection
ISS collection
ATCC 17802
ATCC BAA-242
ATCC BAA-238
ATCC 33845
ATCC 43996
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
Shellfish
ATCC 27562
Shellfish
Shellfish
151
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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. parahaemolyticus, 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, positions 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
Table 1. The PCR assay was performed in a total volume of 25
ml using 12.5 ml of HotStarTaq Master Mix (Qiagen), which provides 2.5 units per reaction of DNA polymerase, 0.2 mM of each
deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), 13
PCR buffer (with 1.5 mM MgCl2), 0.5 mM of each primer, and
1ml of DNA. The mixture was processed in a Mastercycler (Eppendorf, Milan, Italy) with an initial activation step at 958C for
15 min, followed by 35 cycles of denaturation at 948C for 30 s,
annealing at 578C for 30 s, and extension at 728C for 60 s, and a
final extension at 728C for 5 min. The template-free reactions were
included in the PCR setup as negative controls.
Multiplex PCR. After checking that all bacteria strains reported in Table 1 amplified specifically and efficiently in separate
reactions using the same PCR program, a multiplex PCR was set
up. The reaction was performed in a total volume of 25 ml using
12.5 ml of HotStarTaq Master Mix. The multiplex PCR was performed with 2 ml (50 ng/ml) of template.
FIGURE 1. Agarose gel electrophoresis showing the results of
multiplex PCR of three target gene segments from purified DNA
of V. alginolyticus, V. cholerae, and V. parahaemolyticus. Lane 1,
100-bp DNA ladder; lane 2, 737-bp amplicon from V. alginolyticus; lane 3, 389-bp amplicon from V. cholerae; lane 4, 271-bp
amplicon from V. parahaemolyticus; lane 5, negative control.
by sequence analysis carried out with ABI PRISM 3100
(Applied Biosystems, Rome, Italy). The sequence analysis
corresponded to the published sequence of the tested reference strains. The negative controls subjected to multiplex
PCR produced negative results. There was no significant
variability among the results of 10 replicate experiments.
Amplified product detection. The amplified products were
analyzed by electrophoresis on a 1.5% (wt/vol) agarose NA gel
(Pharmacia, Uppsala, Sweden) in 13 Tris-borate-EDTA buffer
(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
a UV transilluminator. A Gene Ruler 100-bp DNA Ladder Plus
(MBI Fermentas, Vilnius, Lithuania), consisting of DNA fragments ranging in size from 3,000 to 100 bp, was used as a molecular weight marker.
Multiplex PCR. The collagenase-based multiplex PCR
performed with simultaneous use of the three pairs of primers targeting V. alginolyticus, V. cholerae, and V. parahaemolyticus collagenase genes allowed successful detection
and discrimination of the three Vibrio species (Fig. 1). The
different sizes of the amplification products allowed rapid
and specific discrimination of V. cholerae, V. parahaemolyticus, and V. alginolyticus by gel electrophoresis. The annealing temperature, extension time, and primer concentration used in the multiplex PCR were optimal. The negative
controls subjected to multiplex PCR produced negative results.
RESULTS
DISCUSSION
Oligonucleotide primers. The three pairs of oligonucleotide primers that were designed to be complementary
to the V. cholerae, V. parahaemolyticus, and V. alginolyticus 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
The multiplex PCR based on the diversity of the collagenase gene sequences between V. alginolyticus and V.
parahaemolyticus and the lack of significant homology with
V. cholerae collagenase sequence (8) represents a valid alternative molecular approach for specific and rapid detection of these three Vibrio species, which are important pathogens 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 differentiation 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 species identification of Vibrios to complement more conventional phenotypic identification systems.
The molecular approach allows researchers to overcome 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 Vibriolike colonies or other species that are not successfully inhibited, thus invalidating the results. The results of this
study have confirmed the utility of molecular tools for routine identification of pathogens in food and rapid characterization of bacteria with particular growth requirements
or unusual biochemical patterns. Further studies are required to confirm the use of collagenase gene as an alternative genetic marker for vibrios. In particular, the specificity 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.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS
The authors thank Dr. Aureli (Istituto Superiore di Sanità) and Dr.
Cancellotti (Istituto Zooprofilattico Sperimentale delle Venezie) for providing bacteria strains.
13.
14.
REFERENCES
1.
2.
3.
4.
5.
Aono, E., H. Sugita, J. Kawasaki, H. Sakakibara, T. Takahashi, K.
Endo, and Y. Deguchi. 1997. Evaluation of the polymerase chain
reaction method for identification of Vibrio vulnificus isolated from
marine environments. J. Food Prot. 60:81–83.
Arias, C. R., M. J. Pujalte, E. Garay, and R. Aznar. 1998. Genetic
relatedness among environmental, clinical and diseased-eel Vibrio
vulnificus isolates from different geographic regions by ribotyping
and randomly amplified polymorphic DNA PCR. Appl. Environ. Microbiol. 64:3403–3410.
Coleman, S. S., and J. D. Oliver. 1996. Optimization of conditions
for the polymerase chain reaction amplification of DNA from culturable and nonculturable cells of Vibrio vulnificus. FEMS Microbiol. Ecol. 19:127–132.
Daniels, N. A., L. MacKinnon, R. Bishop, S. Altekruse, B. Ray, R.
M. Hammond, S. Thompson, S. Wilson, N. H. Bean, P. M. Griffin,
and L. Slutsker. 2000. Vibrio parahaemolyticus infections in the
United States, 1973–1998. J. Infect. Dis. 181:1661–1666.
Dorsch, M., D. Lane, and E. Stackebrandt. 1992. Towards a phylog-
15.
16.
17.
18.
153
eny of the genus Vibrio based on 16S rRNA sequences. Int. J. Syst.
Bacteriol. 42:58–63.
Gomez, J. M., R. Fajardo, J. Patino, and C. A. Arias. 2003. Necrotizing fasciitis due to V. alginolyticus in an immunocompetent patient. J. Clin. Microbiol. 41:3427–3429.
Kim, M. S., and H. D. Jeong. 2001. Development of 16S rRNA
targeted PCR methods for the detection and differentiation of V.
vulnificus in marine environments. Aquaculture 193:199–211.
Kim, S. K., J. Y. Yang, and J. Cha. 2002. Cloning and sequence
analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04. Gene 283:277–286.
Kim, Y. B., J. Okuda, C. Matsumoto, N. Takahashi, S. Hashimoto,
and M. Nishibuchi. 1999. Identification of Vibrio parahaemolyticus
strains at the species level by PCR targeted to the toxR gene. J. Clin.
Microbiol. 37:1173–1177.
Lee, C. Y., and S. F. Pan. 1993. Rapid and specific detection of the
thermostable direct haemolysin gene in Vibrio parahaemolyticus by
the polymerase chain reaction. J. Gen. Microbiol. 139:3225–3231.
Lee, C. Y., S. F. Pan, and C. H. Chen. 1995. Sequence of a cloned
pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR. Appl. Environ. Microbiol. 61:1311–
1317.
Lee, C. Y., S. F. Pan, Y. S. Lee, and C. L. Lee. 1994. Detection and
identification of Vibrio parahaemolyticus in faecal samples of outbreak patients by in vitro amplification of thermostable direct haemolysin gene fragment. J. Chin. Agric. Chem. Soc. 32:103–112.
Levin, W. C., and P. M. Griffin. 1993. Vibrio infection on the Gulf
Coast: results of four years of regional surveillance. Gulf Coast Vibrio working group. J. Infect. Dis. 167:479–483.
Rivera, I. N. G., E. K. Lipp, A. Gil, N. Choopun, A. Huq, and R.
R. Colwell. 2003. Method of DNA extraction and application of
multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems. Environ. Microbiol. 5:
599–606.
Sack, D. A., R. B. Sack, G. B. Nair, and A. K. Siddique. 2004.
Cholera. Lancet 363:223–233.
Tada, J., T. Ohash, N. Nishimura, Y. Shirasaki, H. Ozaki, S. Fukishima, J. Takano, M. Nishibuchi, and Y. Takeda. 1992. Detection of
the thermostable direct haemolysin gene (tdh) and the thermostable
direct haemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol. Cell. Probes 6:477–
487.
Tang, Y., N. M. Ellis, M. K. Hopkins, D. H. Smith, D. E. Dodge,
and D. H. Persing. 1998. Comparison of phenotypic and genotypic
techniques for identification of unusual aerobic pathogenic gramnegative bacilli. J. Clin. Microbiol. 36:3674–3679.
Venkateswaran, K., N. Dohmoto, and S. Harayama. 1998. Cloning
and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp. Appl.
Environ. Microbiol. 64:681–687.