You are on page 1of 4


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

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

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 ventional phenotypic assays are characterized by low sen-
commonly associated with outbreaks of diarrheal diseases sitivity and may fail to detect strains of bacteria present in
in humans due to the consumption of raw or improperly the samples at low concentrations or with unusual pheno-
cooked seafood. Vibrio infections may also occur as a con- typic profiles (17). Advances in molecular technology have
sequence of exposures of skin lesions, such as cuts, open led to a shift from conventional phenotypic methods for the
wounds, or abrasions, to aquatic environments and marine identification of microorganisms to molecular methods,
animals. The gastrointestinal Vibrio diseases attributable to which are more sensitive and specific for the detection of
seafood ingestion are well known, but data on extraintes- low numbers of bacteria and of viable but not culturable
tinal infections are more limited and some have been re- microrganisms (13). Different molecular targets have been
ported only recently (4). Vibrios have acquired a great ep- used to identify the presence of Vibrio spp. both in clinical
idemiological importance because of the increasing occur- samples and in seafood. The genes encoding the virulence
rence of foodborne disease outbreaks, particularly due to determinants and their expression regulator genes have
noncholera Vibrio species. Particular attention is currently been used to characterize numerous Vibrio species. A mo-
being focused on Vibrio cholerae O139, which has been lecular test based on the detection of the tdh and/or trh
implicated in numerous outbreaks of seafood-associated genes (encoding thermostable direct hemolysin and ther-
gastroenteritis producing classic cholera symptoms, and on mostable-related hemolysin, respectively) has been applied
non-O1 strains that cause less severe gastrointestinal dis- for identification of V. parahaemolyticus (1012, 16). Lee
eases than those induced by V. cholerae O1 (15). In recent et al. (11) developed a molecular approach based on the
microbiological and epidemiological studies (4, 6, 13), sev- amplification of a DNA fragment that is highly conserved
eral clinical extraintestinal infections and gastroenteritis in all strains of V. parahaemolyticus. PCR procedures tar-
cases caused by toxigenic strains of Vibrio parahaemoly- geting the gyrB gene, encoding the B subunit of DNA gyr-
ticus and Vibrio alginolyticus have reported. ase essential for DNA replication, and the regulatory toxR
Identification of these microorganisms from both clin- gene have been used for the specific detection of V. para-
ical and food samples is predicated upon an accurate and haemolyticus (9, 18). The ctxAB and tcpA genes, known to
specific analytical approach. Conventional standard micro- play a cardinal role in maintaining virulence in Vibrio chol-
biological methods for the detection of Vibrio spp., based erae, are believed to be exclusively associated with clinical
on the traditional analysis of phenotypic profile, are slow strains of the O1 and O139 serogroups. Rivera et al. (14)
and laborious, often requiring several days to perform. Con- described a multiplex PCR assay based on the detection of
the two main virulence-associated factors, cholera toxin and
* Author for correspondence. Tel: 1390805443970; Fax: 1390805443855; toxin coregulated pilus, that can be used to quickly detect
E-mail: V. cholerae O1 and V. cholerae O139. For identification

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
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.

Oligonucleotide primers. The three pairs of oligonu- The multiplex PCR based on the diversity of the col-
cleotide primers that were designed to be complementary lagenase gene sequences between V. alginolyticus and V.
to the V. cholerae, V. parahaemolyticus, and V. alginoly- parahaemolyticus and the lack of significant homology with
ticus collagenase genes produced specific amplicons of the V. cholerae collagenase sequence (8) represents a valid al-
expected sizes. In particular, the V. alginolyticus primers ternative molecular approach for specific and rapid detec-
produced a specific 737-bp amplicon in all V. alginolyticus tion of these three Vibrio species, which are important path-
strains tested, the V. cholerae primers produced a specific ogens associated with seafood poisoning in different areas
389-bp amplicon in all V. cholerae strains, including O1 of the world. The procedure may be used for the molecular
and non-O1, and the V. parahaemolyticus primers amplified confirmation of biochemically identified Vibrio strains from
a fragment of 271 bp. Comparing total DNA from different seafood samples implicated in outbreaks of food poisoning.
Vibrio species and related bacterial strains, the primer pair Strain culture methods allow only ambiguous differentia-
sets investigated provided species-specific identification of tion of Vibrio species and others that are genetically related
V. cholerae, V. parahaemolyticus, and V. alginolyticus. The such as Aeromonas hydrophila, which is often misidentified
other strains failed to yield positive amplification with these as a Vibrio species by conventional biochemical methods.
primers. The specificity of the PCR products was confirmed The results indicate that the collagenase gene may be a

useful alternative target for phylogenetic analysis and spe- eny of the genus Vibrio based on 16S rRNA sequences. Int. J. Syst.
Bacteriol. 42:5863.
cies identification of Vibrios to complement more conven-
6. Gomez, J. M., R. Fajardo, J. Patino, and C. A. Arias. 2003. Necro-
tional phenotypic identification systems. tizing fasciitis due to V. alginolyticus in an immunocompetent pa-
The molecular approach allows researchers to over- tient. J. Clin. Microbiol. 41:34273429.
come the disadvantages of culture-based methods (17), 7. Kim, M. S., and H. D. Jeong. 2001. Development of 16S rRNA
which may be misleading, may underestimate the number targeted PCR methods for the detection and differentiation of V.
vulnificus in marine environments. Aquaculture 193:199211.
of the viable cells, and may allow the growing of Vibrio-
8. Kim, S. K., J. Y. Yang, and J. Cha. 2002. Cloning and sequence
like colonies or other species that are not successfully in- analysis of a novel metalloprotease gene from Vibrio parahaemo-
hibited, thus invalidating the results. The results of this lyticus 04. Gene 283:277286.
study have confirmed the utility of molecular tools for rou- 9. Kim, Y. B., J. Okuda, C. Matsumoto, N. Takahashi, S. Hashimoto,
tine identification of pathogens in food and rapid charac- and M. Nishibuchi. 1999. Identification of Vibrio parahaemolyticus
strains at the species level by PCR targeted to the toxR gene. J. Clin.
terization of bacteria with particular growth requirements Microbiol. 37:11731177.
or unusual biochemical patterns. Further studies are re- 10. Lee, C. Y., and S. F. Pan. 1993. Rapid and specific detection of the
quired to confirm the use of collagenase gene as an alter- thermostable direct haemolysin gene in Vibrio parahaemolyticus by
native genetic marker for vibrios. In particular, the speci- the polymerase chain reaction. J. Gen. Microbiol. 139:32253231.
ficity of the primer pairs may be tested on a wide number 11. Lee, C. Y., S. F. Pan, and C. H. Chen. 1995. Sequence of a cloned
pR72H fragment and its use for detection of Vibrio parahaemolyti-
of strains of each target organisms. The reliability of the cus in shellfish with the PCR. Appl. Environ. Microbiol. 61:1311
method for the detection of the three Vibrio species should 1317.
also be evaluated in mixed cultures. 12. Lee, C. Y., S. F. Pan, Y. S. Lee, and C. L. Lee. 1994. Detection and
identification of Vibrio parahaemolyticus in faecal samples of out-
ACKNOWLEDGMENTS break patients by in vitro amplification of thermostable direct hae-
molysin gene fragment. J. Chin. Agric. Chem. Soc. 32:103112.
The authors thank Dr. Aureli (Istituto Superiore di Sanita) and Dr. 13. Levin, W. C., and P. M. Griffin. 1993. Vibrio infection on the Gulf
Cancellotti (Istituto Zooprofilattico Sperimentale delle Venezie) for pro- Coast: results of four years of regional surveillance. Gulf Coast Vib-
viding bacteria strains. rio working group. J. Infect. Dis. 167:479483.
14. Rivera, I. N. G., E. K. Lipp, A. Gil, N. Choopun, A. Huq, and R.
REFERENCES R. Colwell. 2003. Method of DNA extraction and application of
multiplex polymerase chain reaction to detect toxigenic Vibrio chol-
1. Aono, E., H. Sugita, J. Kawasaki, H. Sakakibara, T. Takahashi, K. erae O1 and O139 from aquatic ecosystems. Environ. Microbiol. 5:
Endo, and Y. Deguchi. 1997. Evaluation of the polymerase chain 599606.
reaction method for identification of Vibrio vulnificus isolated from 15. Sack, D. A., R. B. Sack, G. B. Nair, and A. K. Siddique. 2004.
marine environments. J. Food Prot. 60:8183. Cholera. Lancet 363:223233.
2. Arias, C. R., M. J. Pujalte, E. Garay, and R. Aznar. 1998. Genetic 16. Tada, J., T. Ohash, N. Nishimura, Y. Shirasaki, H. Ozaki, S. Fukish-
relatedness among environmental, clinical and diseased-eel Vibrio ima, J. Takano, M. Nishibuchi, and Y. Takeda. 1992. Detection of
vulnificus isolates from different geographic regions by ribotyping the thermostable direct haemolysin gene (tdh) and the thermostable
and randomly amplified polymorphic DNA PCR. Appl. Environ. Mi- direct haemolysin-related hemolysin gene (trh) of Vibrio parahae-
crobiol. 64:34033410. molyticus by polymerase chain reaction. Mol. Cell. Probes 6:477
3. Coleman, S. S., and J. D. Oliver. 1996. Optimization of conditions 487.
for the polymerase chain reaction amplification of DNA from cul- 17. Tang, Y., N. M. Ellis, M. K. Hopkins, D. H. Smith, D. E. Dodge,
turable and nonculturable cells of Vibrio vulnificus. FEMS Micro- and D. H. Persing. 1998. Comparison of phenotypic and genotypic
biol. Ecol. 19:127132. techniques for identification of unusual aerobic pathogenic gram-
4. Daniels, N. A., L. MacKinnon, R. Bishop, S. Altekruse, B. Ray, R. negative bacilli. J. Clin. Microbiol. 36:36743679.
M. Hammond, S. Thompson, S. Wilson, N. H. Bean, P. M. Griffin, 18. Venkateswaran, K., N. Dohmoto, and S. Harayama. 1998. Cloning
and L. Slutsker. 2000. Vibrio parahaemolyticus infections in the and nucleotide sequence of the gyrB gene of Vibrio parahaemoly-
United States, 19731998. J. Infect. Dis. 181:16611666. ticus and its application in detection of this pathogen in shrimp. Appl.
5. Dorsch, M., D. Lane, and E. Stackebrandt. 1992. Towards a phylog- Environ. Microbiol. 64:681687.