Journal of Microbiological Methods 36 (1999) 215225

Journal
of
Microbiological
Methods

Detection of total and hemolysin-producing Vibrio parahaemolyticus

in shellfish using multiplex PCR amplification of tl, tdh and trh
a,
a
a
a
Asim K. Bej *, Donald P. Patterson , Cynthia W. Brasher , Michael C.L. Vickery ,
a
b
Daniel D. Jones , Charles A. Kaysner
a

Department of Biology, The University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294 -1170, USA
b
Seafood Products Research Center, U.S. Food and Drug Administration, Bothell, WA 98041 -3012, USA

Abstract
Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or
partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus
was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and
thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and
trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains
obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR
amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of
the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh
gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted
in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was
successfully used to detect various strains of V. parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of
detection for all three target gene segments was at least between 10 1 10 2 cfu per 10 g of alkaline peptone water enriched
seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is
well within the action level (10 4 cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline.
Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for
accomplishing a comprehensive detection of V. parahaemolyticus in shellfish. 1999 Elsevier Science B.V. All rights
reserved.
Keywords: Vibrio parahemolyticus; Multiplex PCR; Microorganism; Shellfish; Hemolysin

1. Introduction
Vibrio parahaemolytcus is an enteric pathogen that
causes acute gastroenteritis in humans primarily

*Corresponding author. Tel.: 11-205-934-9857; fax: 11-205975-6097.

E-mail address: abej@uab.edu (A.K. Bej)

when consumed in raw, undercooked or mishandled

seafood (DePaola et al., 1990). This pathogen, like
other members of the genus Vibrio, is a Gramnegative halophilic bacterium distributed worldwide
in the estuarine environment (Janda et al., 1988;
Joseph et al., 1983; Kaysner et al., 1992). Recently,
two outbreaks of V. parahemolyticus-related illnesses
in the US resulting from raw oyster consumption
have heightened the need to develop a rapid and

0167-7012 / 99 / $ see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S0167-7012( 99 )00037-8

216

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

reliable method to detect this pathogen in shellfish

(Nishibuchi and Kaper, 1985). The pathogenic effect
for humans following infection has been associated
with those strains that produce thermostable direct
hemolysin (TDH) (Miyamoto et al., 1969; Honda
and Iida, 1993). Production of TDH by a strain of
this pathogen is referred to as Kanagawa phenomenon positive and can be identified by b-type
hemolysis on Wagatsuma blood agar (Wagatsuma,
1968; Takeda, 1982; Kaysner et al., 1992; Morbidity
and Mortality Weekly Report, 1998). However, the
testing for Kanagawa phenomenon is time consuming; the agar medium is cumbersome to prepare, and
the test may not be determinative since many clinical
isolates are Kanagawa negative and do not produce
TDH (Miyamoto et al., 1969; Tada et al., 1992;
Okuda and Nishibuchi, 1998). Similarly, immunological methods, although shown to be highly sensitive for detecting TDH, fail to detect clinical strains
that lack TDH (Miyamoto et al., 1969; Honda et al.,
1980; Tada et al., 1992; Lee and Pan, 1993; Okuda
and Nishibuchi, 1998). Furthermore, DNA probes
and oligonucleotide probes specific for the gene
encoding for thermostable hemolysin (TDH) and a
tdh-related gene encoding for TRH (Honda et al.,
1991; Kishishita et al., 1992) were shown to react
positively for TDH-negative and several nonparahaemolyticus strains such as V. hollisae, V.
mimicus, and V. cholerae non-O1 (Tada et al., 1992;
Lee et al., 1995). Subsequent studies using tdh or trh
gene probes on V. parahaemolyticus strains showed a
strong correlation between clinically significant
strains and presence of either of these genes, suggesting that both tdh and trh genes are virulence
factors in V. parahaemolyticus (Miyamoto et al.,
1969; Shirai et al., 1990). Presently, there is no in
vitro test that will demonstrate the production of
TRH in this pathogen. Genetic analysis showed that
the trh gene is closely related to the tdh gene with
approximately 68% nucleotide sequence homology
(Honda et al., 1991; Tada et al., 1992; Honda and
Iida, 1993). Moreover, variability in nucleotide
sequences within the trh gene isolated from various
clinical strains leads to the establishment of two
subdivisions of this gene, trh1 and trh2 (Kishishita et
al., 1992). In a PCR amplification-based study, a
number of virulent strains were tdh positive but trh

negative, whereas some showed amplification for trh

but not for tdh (Lee and Pan, 1993). DNADNA
hybridization, using tdh- or trh-specific probes
(Nishibuchi et al., 1985; Nishibuchi et al., 1986;
Yamamoto et al., 1992), or use of restriction fragment length polymorphism (RFLP) of the tdh and
trh genes (Suthienkul et al., 1996) produced results
comparable to the PCR study. In addition, DNA
DNA hybridization using the tdh gene as a probe
showed positive hybridization with the tdh-like gene
in non-parahaemolyticus strains of vibrios such as V.
hollisae, V. mimicus and V. cholerae non-O1
(Nishibuchi et al., 1985; Nishibuchi et al., 1986;
Yamamoto et al., 1992; Honda and Iida, 1993).
Therefore, using the tdh gene exclusively as a probe
for DNADNA hybridization is not reliable for
specific detection of V. parahaemolyticus. In separate
studies, PCR amplification methodology was used to
detect V. parahaemolyticus isolates by targeting only
the tdh gene fragment (Lee and Pan, 1993) or a
0.71-kbp cloned segment of the chromosomal DNA,
pR72H, of unknown function (Lee et al., 1995). A
separate gene, thermolabile hemolysin (tl), has been
characterized in this pathogen which does not seem
to cause hemolysis on Wagatsuma agar (Taniguchi et
al., 1985, 1986). Also, the thermolabile hemolysin
has not been reported to cause virulence in humans
as all human isolates are found to be either positive
for thermostable direct hemolysin or thermostablerelated hemolysin or both. However, this gene was
shown to be present in all of the V. parahaemolyticus
strains tested previously (Taniguchi et al., 1985,
1986; A. Bej, unpublished). Although in previously
reported studies, the tdh or the trh genes were used
as targets, the PCR- and gene probe-based detection
of this pathogen as a species specific target has not
been sought so far. Therefore, a multiplex PCRbased detection system targeting tl gene for total, and
both tdh and trh genes for hemolysin-producing
pathogenic strains of V. parahaemolyticus is necessary for comprehensive detection of this pathogen in
shellfish. In this study, we describe development of
such a multiplex PCR-amplification approach to
detect total and hemolysin-producing pathogenic
strains of V. parahaemolyticus in shellfish by simultaneously targeting the tl, tdh and trh genes in a
single PCR reaction.

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

2. Materials and methods

2.1. Bacterial strains and growth media

V. parahaemolyticus strains and other bacterial
species used in this study are listed in Tables 1 and
2. All V. parahaemolyticus strains were grown on
nutrient agar or broth (Difco) supplemented with 3%
(w / v) NaCl or on trypticase soy agar (Difco) supplemented with 5% (v / v) defibrinated sheep blood
(Colorado Serum) (Atlas, 1993) at 358C. Other
Vibrio spp. were grown on either marine agar or in
marine broth (Difco) or LB agar or LB broth (10 g
bacto tryptone, 5 g yeast extract, 10 g NaCl per liter;
for LB agar, LB broth supplemented with 14 g bacto
agar per liter) at 358C.

2.2. Determination of Kanagawa phenomenon

All V. parahaemolyticus strains used in this study
were grown in alkaline peptone water [10 g Bacto
peptone (Difco), 10 g NaCl (pH 8.5) per liter
(APW)] (Atlas, 1993) at 358C on a rotatory shaker
set at 200 rpm (New Brunswick Scientific) until the
OD 450 reached between 0.5 and 0.6. A 10-ml aliquot
of each of the cultures was spotted aseptically on
Wagatsuma blood agar (Wagatsuma, 1968) and
grown overnight at 358C. The Kanagawa phenomenon was determined for those V. parahaemolyticus
strains which showed a characteristic halo surrounding the growth due to b-hemolysis.

2.3. DNA purification

Total genomic DNA from all pure cultures of
bacterial strains was purified by following the procedure described by (Ausubel et al., 1987). Briefly, 2
ml of an overnight grown culture from each strain of
V. parahaemolyticus were collected by centrifugation, and cell pellets resuspended in 567 ml of Tris
EDTA buffer [10 mM Tris ? Cl (pH 8.0), 1 mM
EDTA]. Next, 30 ml of 10% (w / v) sodium dodecyl
sulfate and 3 ml proteinase K (Sigma) (20 mg / ml)
were added, and the mixture was incubated for 1 h at
378C. The samples were treated with 100 ml of 5 M
NaCl and 80 ml of hexadecyltrimethyl ammonium
bromide (CTAB) / NaCl, and incubated at 658C for

217

Table 1
Specificity of the multiplex PCR amplification of tdh, trh and tl
target genes in V. parahaemolyticus. List of bacterial strains and
distribution of the tdh, trh, and tl genes from multiplex PCR
amplification
Strain

Target Genes
tl

tdh

Kanagawa
reaction
trh

Vibrio parahaemolyticus isolated from human patients

47583
1
1
1
1
47977
1
1
1
1
48256
1
2
2
2
47978
1
1
2
1
48057
1
1
1
1
48215
1
1
1
1
48275
1
1
1
1
48432
1
1
1
1
48262
1
1
1
1
48291
1
1
2
1
901128
1
2
2
2
41977
1
1
1
1
9401392
1
1
1
1
9401416
1
1
1
1
9401078
1
1
1
1
SAK5
1
2
2
1
SAK8
1
1
2
1
SAK11
1
1
2
1
ATCC17802
1
1
2
1
T3937
1
1
2
1
T3979
1
1
2
1
T3980
1
1
2
1
KCHD613
1
2
2
2
8657
1
1
2
1
8659
1
1
2
1
8700
1
1
2
1
553-14
1
2
2
1
V. parahaemolyticus
F113A
F25-1B
CRAB
M35OA
8338335
5C-1C
832850
9200713
2A17J
4A35J
5A35J
5A63J
10A102J
12A7C
13A15J
14B21K
13A17J

isolated from various seafood samples

1
1
1
1
1
1
1
1
1
2
2
2
1
1
1
1
1
1
1
1
1
1
2
nd
1
1
2
1
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2
1
2
2
2

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

218
Table 1. Continued
Strain

855329-2
855330-5B
855330-1A
9736165
95046527
95046528
18V10B
3V0C
G181
WR5
83320964
CO5-1A
33V1C
6V-1A
8G5
96736341
27V10B
30V10A
M25OB
AOC1
AOC2
AOC3
AOC4
AOC5
AOC6
AOC7

Table 1. Continued
Target Genes

Kanagawa
reaction

tl

tdh

trh

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

2
2
1
2
1
2
1
1
1
2
2
2
1
1
2
2
2
1
2
2
2
2
2
2
2
2

2
2
1
2
1
2
2
1
1
2
2
2
2
1
2
2
2
1
2
2
2
2
2
2
2
2

2
2
1
2
1
2
2
1
1
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2

V. parahaemolyticus isolated from environmental waters

W9OA
1
1
1
1
WR1
1
2
2
2
WR2
1
2
2
1
VP89
1
1
1
1
VP89-1B
1
2
2
2
OR152
1
1
1
1
VP5G
1
1
1
1
VP60
1
1
1
1
VP35
1
1
1
1
VP43-1A
1
1
1
1
35V0A
1
2
2
2
VP53
1
2
2
2
VP99
1
2
2
2
96Q
1
2
2
2
D3 OA
1
2
2
2
V. parahaemolyticus isolated from oyster plants
B1A
1
2
2
C2 1A
1
2
2
JJ2J1C
1
1
2
C3 1A
1
1
1
W3
1
1
1
MM3
1
2
2
C4-3B
1
1
1
J4
1
1
1

2
2
1
1
1
2
1
1

Strain

JJ41B1
JJ41B2
MMX4
BB5
FF5
JJ51A
MMX5
B71B
M7
N7
B8

Target Genes

Kanagawa
reaction

tl

tdh

trh

1
1
1
1
1
1
1
1
1
1
1

1
1
2
1
1
1
1
1
1
1
2

1
1
2
1
1
1
1
1
1
1
2

V. parahaemolyticus obtained from various

14D1
1
1
14D10
1
1
14D13
1
2
14D14
1
2
15A17J
1
2
NY 477
1
1
AQ4037
1
2

1
1
2
1
1
1
1
1
1
1
2

laboratories
1
1
2
1
2
2
2
2
2
2
2
1
1
2

Non-V. parahaemolyticus strains

Vibrio hollisae
89A1960
2
89A1961
2
89A7503
2
89A4206
2

1
2
1
2

2
2
2
2

2
2
2
2

Vibrio cholerae
89A4555
O145B
0138
C153PY
154 (O1)
569B (O1)

2
2
2
2
2
2

2
2
2
2
2
2

2
2
2
2
2
2

2
2
2
2
2
2

V. mimicus
V. vulnificus
V. alginolyticus
V. damsela
V. fluvialis
V. furnissii
V. metschnikovii
V. proteolyticus
Plesiomonas spp.

2
2
2
2
2
2
2
2
2

2
2
2
1
2
2
2
2
2

2
2
2
2
2
2
2
2
2

2
2
2
2
2
2
2
2
2

1 5presence.
2 5absence.
nd5not determined

10 min. Following incubation, the sample was first

extracted with an equal volume of chloroformisoamyl alcohol (24:1, v / v) and then with phenol

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

219

Table 2
Summary of distribution and occurence of tl, tdh and trh genes among V. parahaemolyticus and non-V. parahaemolyticus isolates tested by
multiplex PCR amplification
Target
genes

Sources of V. parahaemolyticus strains isolates

Clinical
isolates

Food
isolates

Percent distribution of individual genes

tl
27 / 27
43 / 43
(100%)
(100%)
tdh
22 / 27
14 / 43
(81.48%)
(32.56%)
trh
11 / 27
10 / 43
(40.74%)
(2.3%)
Percent distribution of genes in various combinations
tl 1 tdh 1 trh 1
tl 1 tdh 1 trh 2
tl 1 tdh 2 trh 1
tl 1 tdh 2 trh 2
a

11 / 27
(40.74%)
11 / 27
(40.74%)
0 / 27
(0%)
5 / 27
(18.51%)

10 / 43
(23.25%)
4 / 43
(9.3%)
0 / 43
(0%)
29 / 43
(67.44%)

Environmental
isolates

Oyster plant
isolates

Obtained from
various labs.

Total a

15 / 15
(100%)
7 / 15
(46.66%)
7 / 15
(46.66%)

19 / 19
(100%)
14 / 19
(73.68%)
13 / 19
(68.4%)

7/7
(100%)
3/7
(42.8%)
2/7
(28.57%)

111 / 111
(100%)
60 / 111
(54%)
43 / 111
(38.73%)

7 / 15
(46.66%)
0 / 15
(0%)
0 / 15
(0%)
8 / 15
(53.33%)

13 / 19
(68.4%)
1 / 19
(5.2%)
0 / 19
(0%)
5 / 19
(26.3%)

1/7
(14.28%)
2/7
(28.5%)
1/7
(14.28%)
3/7
(42.85%)

42 / 111
(37.8%)
18 / 111
(16.2%)
1 / 111
(0.9%)
50 / 111
(45%)

Distribution of individual genes or in combinations in all 111 V. parahaemolyticus isolates tested in this study.

chloroformisoamyl alcohol (25:24:2, v / v). The

DNA was precipitated with 0.6 volume of 100% cold
isopropanol and washed with 1 ml of 70% cold ethyl
alcohol. The DNA pellet was dried in a DNA 120
speedvac (Savant) for 10 min and resuspended in
Tris ? EDTA buffer (pH 8.0). An aliquot (typically
12 ml) of the sample was subjected to spectrophotometric analysis at optical densities of 260 and
280 nm wavelengths to determine the purity and
efficiency of recovery.

2.4. Oligonucleotide primers

Nucleotide sequences, locations, melting temperatures (T m ) of oligonucleotide primers specific for the
tl, tdh, and trh genes, and size of the amplicons
following PCR amplification are described in Table
3. The T m value for each of the primers was
estimated by using the equation, T m (8C)52(A1
T)14(G1C) (Suggs et al., 1981). All oligonucleotide primers were custom-synthesized by Integrated
DNA Technology, Inc., Coralville, IA.

2.5. Multiplex PCR amplifications

Multiplex PCR amplification was optimized in a
50-ml reaction consisting of 0.5 mg of purified
genomic DNA from V. parahaemolyticus F113A
strain, 1 mM of each of the oligonucleotide primers
for tl, tdh, trh (2.5 ml of each of the primers from a
20 mM stock suspension), 5 ml of a 103 PCR
reaction buffer (103 buffer consisted of 500 mM
Tris?Cl, pH 8.9, 500 mM KCl and 25 mM, 30 mM
or 40 mM MgCl 2 ; final concentration of 13), 200
mM of each of the dNTPs (8 ml from a 5 mM stock
dNTP) (Pharmacia), 2.5 units AmpliTaq DNA polymerase (Perkin Elmer) and an appropriate volume of
sterile MilliQ water (Millipore).
All multiplex PCR amplifications were performed
in a DNA thermal cycler (Perkin Elmer, Model 480)
using the following temperature-cycling parameters:
initial denaturation at 948C for 3 min followed by 30
cycles of amplification; each cycle consisted of
denaturation at 948C for 1 min, primer annealing at
588C for 1 min, and primer extension at 728C for 1
min. Following the amplification cycles, samples

220

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

Table 3
List of oligonucleotide primers, target genes, amplicon sizes, T m values and sources of gene sequences used for the multiplex PCR
amplification detection of total and hemolysin-producing Vibrio parahaemolyticus
Target
gene

Primer sequence

Tm
(8C)

Amplicon
size (kbp)a

Source

tl b

L-tl: 59-aaa gcg gat tat gca gaa gca ctg-39

R-tl: 59-gct act ttc tag cat ttt ctc tgc-39

58.63
51.11

0.45

(Taniguchi et al., 1985, 1986)

tdh c

L-tdh: 59-gta aag gtc tct gac ttt tgg ac-39

R-tdh: 59-tgg aat aga acc ttc atc ttc acc-39

48.58
53.27

0.269

(Nishibuchi and Kaper, 1985)

trh d

L-trh: 59-ttg gct tcg ata ttt tca gta tct-39

R-trh: 59-cat aac aaa cat atg ccc att tcc g-39

51.37
58.32

0.5

(Honda and Iida, 1993;

Honda et al., 1991)

kbp5kilobase pair.
tl5thermolabile hemolysin.
c
tdh5thermostable direct hemolysin.
d
trh5thermostable-related direct hemolysin.
b

were kept at 728C for 5 min to allow final extension

of the incompletely synthesized DNA.

2.6. Specificity of oligonucleotide primers

The specificity of each set of oligonucleotide
primers for its respective target gene was determined
by PCR amplification of the purified genomic DNA
from 111 V. parahaemolyticus isolates, and 19
bacterial strains other than V. parahaemolyticus listed
in Tables 1 and 2.

2.7. Seeding oyster tissue homogenates with V.

parahaemolyticus
All oysters (Crassostrea virginica) used in this
study were purchased from local seafood restaurants,
transported to the laboratory on ice, shucked and
homogenized following the standard methods of
American Public Health Association (1970). The
oyster tissue homogenates were then stored in a
sterile beaker on ice and exposed to ultraviolet (UV)
light for 8 h with occasional vortexing to eliminate
any naturally occurring V. parahaemolyticus strains.
Following UV treatment, the tissue homogenates
were stored at
2788C until used. V.
parahaemolyticus strains ATCC 48256 (tl 1 tdh 2
trh 2 ), V. parahaemolyticus NY477 (tl 1, tdh1,
trh2), V. parahaemolyticus AQ4037 (tl 1, tdh 2 , trh
1), and V. parahaemolyticus ATCC 47583 (tl
1 tdh 1 trh 1 ) were grown in APW (pH 8.5) until
the OD 450 reached 0.2. The cultures were serially
diluted in APW (pH 8.5) and the number of cells / ml

in each dilution was determined on Wagatsuma blood

agar. Each of the V. parahaemolyticus cultures (1 ml)
was used separately to seed 10 g of UV-treated
oyster tissue homogenate. Subsequently, 350 ml of
APW (pH 8.5) were added to the seeded oyster tissue
homogenates and the V. parahaemolyticus cells enriched at 358C for 6 h on a rotatory shaker set at 200
rpm (Innova 4000, New Brunswick Scientific). Following enrichment, a 0.5-ml aliquot of each of the
cultures was collected in a 1.5-ml microcentrifuge
tube and total DNA was purified. Unseeded oyster
tissue homogenates serving as negative controls were
subjected to treatments parallel to those for seeded
samples.

2.8. Purification of DNA from seeded or unseeded

oyster tissue homogenates
Total DNA from seeded or unseeded oyster tissue
homogenates was purified by modification of the
procedure described by Gannon et al. (1992). In this
procedure, 0.5 ml of each of the enriched V.
parahaemolyticus strains from oyster tissue homogenates was treated with 0.2 ml of a lysis buffer
consisting of 10 mM EDTA, 100 mM Tris?Cl (pH
8.0), 5 mg proteinase K (Sigma) / ml, and 50 mg
sarkosyl (Fisher) / ml at 658C for 10 min. Following
lysis, 100 ml of ice cold 3 M NaOAc (pH 4.6) were
added and the tube was inverted several times to
mix. Then an equal volume (800 ml) of phenol
chloroformisoamyl alcohol (24:24:1, v / v) was
added to the sample and the tube was shaken rapidly
till the sample was cloudy. The sample was cen-

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

trifuged at 10 0003g for 10 min at room temperature and the clear supernatant was transferred to a
new microcentrifuge tube. Total DNA was precipitated with 0.8 ml of ice-cold isopropanol, the tubes
inverted several times to mix and centrifuged at
12 0003g for 10 min. The supernatant was decanted
and the pellet washed with 0.5 ml of ice-cold 70%
(v / v) alcohol, centrifuged, and the pellet dried in a
speedvac DNA 20 (Savant). The DNA sample was
resuspended in 10 ml of TE buffer (pH 8.0) and
incubated at 658C for 30 min before being subjected
to PCR amplification.

2.9. Sensitivity of the multiplex PCR amplification

detection in seeded oysters

221

achieved with equimolar quantities of each of the

oligonucleotide primers (2.5 mM of each of the
primers), 25 mM MgCl 2 in PCR reaction buffer
(103), and a primer annealing temperature of 558C
(Fig. 1).

3.2. Distribution of tl, tdh, and trh genes in V.

parahaemolyticus isolates and correlation with the
Kanagawa reaction
The results from the multiplex PCR showed
positive amplification of the tl gene segment in all
111 (100%) V. parahaemolyticus isolates whereas,
genomic DNAs from four V. hollisae, six V. cholerae,
eight Vibrio spp., and one Plesiomonas sp. did not

V. parahaemolyticus F113A (tl 1 tdh1 trh1) was

grown exponentially (OD 600 50.17) and serially
diluted. The number of cells per ml of culture was
determined by viable plate count on Wagatsuma
blood agar plates. An aliquot (1 ml) of each of the
serially diluted V. parahaemolyticus F113A cultures
consisting of 10 4 to 10 0 cells was added to 10 g of
individual oyster tissue homogenates and pre-enriched at 358C for 6 h. Following pre-enrichment, 0.5
ml of each of the samples was subjected to DNA
purification by the procedure described above.

2.10. Detection of the PCR amplified DNAs

PCR-amplified DNAs (10-ml aliquot) were separated in a 1% (w / v) NuSieve 3:1 agarose gel (FMC
Bioproducts) in which 2310 24 mg per ml of
ethidium bromide were added. Electrophoresis was
performed using 13 TAE buffer [40 mM Tris?Cl
(pH 8.0), 1.18 ml acetic acid, 2 mM Na 2 EDTA per
liter] (Taniguchi et al., 1985) and a constant voltage
of 5 V/ cm. Following electrophoretic separation,
DNA fragments in the gels were visualized under a
Photoprep I UV transilluminator (Fotodyne) and
photographed using Type 55 polaroid film.

3. Results and discussion

3.1. Optimization of the multiplex PCR

In multiplex PCR, the amplification of all three
genes, tl, tdh, and trh, with equal intensities was

Fig. 1. Agarose gel electrophoresis showing the results from PCR

amplification of genomic DNA from V. parahaemolyticus F113A.
Lane 1, 123-bp DNA ladder (Gibco) as size marker enhanced by
PRORFLP (Nashville, TN, USA) computer software; lane 2, multiplex PCR DNA ProScan amplification using oligonucleotide primers specific for the tl, tdh and the trh genes showing three bands
of DNA with expected molecular weights of 0.450 kbp, 0.269
kbp, and 0.5 kbp, respectively; lane 3, PCR amplification using
oligonucleotide primers specific for the trh gene; lane 4, PCR
amplification using oligonucleotide primers specific for the tl
gene; lane 5, PCR amplification using oligonucleotide primers
specific for the tdh gene.

222

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

show PCR amplification of this gene suggesting that

the L-TL and R-TL oligonucleotide primers and the
tl gene are specific for the detection of total V.
parahaemolyticus (Tables 1 and 2). However, only
60 (54%) of the total 111 V. parahaemolyticus
isolates showed positive PCR amplification of the
tdh gene segment, and only 43 (38.73%) isolates
showed amplification of the trh gene segment,
suggesting that not all isolates tested in this study
were b-hemolytic. Also, 42 (37.8%) isolates showed
positive PCR amplification of both tdh and trh genes
(Tables 1 and 2), suggesting that some V.
parahaemolyticus strains can carry both hemolytic
genes. Whether the presence of both tdh and trh
genes in these strains increases their virulence to
humans is not known. Interestingly, three isolates (V.
parahaemolyticus SAK5, 553-14, and WR2) showed
no PCR amplification of the tdh gene segment but
were Kanagawa positive. All three of these isolates
showed negative PCR amplification of both tdh and
trh gene segments. Among these three isolates, two
(SAK5 and 553-14) were human-patient isolates and
the third (WR2) was an environmental isolate. In
contrast, three other isolates (V. parahaemolyticus
33V1C, 30V10A, and 18V10B) showed positive
amplification of the tdh gene segment but produced a
negative Kanagawa reaction. Among these latter
three isolates, PCR amplification results showed the
presence of both tdh and trh gene segments in V.
parahaemolyticus 30V10A whereas, 18V10B and
33V1C showed positive PCR amplification of only
the tdh gene segment. All three of these V.
parahaemolyticus isolates were from seafood samples. The majority of the V. parahaemolyticus strains
exhibited a direct correlation between the presence of
the thermostable hemolysin genes and positive reaction for Kanagawa pehnomenon on Wagatsuma
blood agar. However, the reasons why the six strains
did not show the same correlation is currently not
understood.
The distribution of the tl, tdh, and trh gene
showed the majority (45%) of the 111 isolates were
positive for the tl gene but negative for both tdh and
trh genes. Forty-two (37.8%) isolates were positive
for all three genes (Tables 1 and 2). Also, 16.2% of
the isolates were positive for the tl and the tdh genes,
but not the trh gene. Only one isolate was positive
for tl and trh genes, but not the tdh gene. None of

the isolates were negative for the tl gene but positive

for the tdh and / or the trh gene, suggesting the
occurrence of the tdh gene in combination with the tl
gene is more prevalent than with the trh gene
segment. Also, the majority of clinical isolates were
positive for the tdh gene segment, but none were
positive only for the trh gene and negative for the
tdh gene. Therefore, the role of the trh gene in
virulence to humans needs to be investigated.
Tests of these isolates on Wagatsuma blood agar
showed a total of 58 isolates as b-hemolytic, demonstrating absolute correlation with positive PCR amplification of the tdh or the trh gene, or both. These
results suggest the tdh gene is more prevalent than
the trh gene in these isolates. Since several of these
isolates showed positive amplifications of tdh but not
the trh gene, as well as the reverse pattern, it is
important that a multiplex PCR be used to achieve a
rapid but comprehensive analysis of the V.
parahaemolyticus isolates with b-hemolytic properties.

3.3. Detection of V. parahaemolyticus in seeded

oyster tissue homogenates
PCR amplification targeting the tl, tdh, and trh
genes individually or in combination from various
strains of V. parahaemolyticus showed amplifications
of DNA segments of expected molecular weights of
0.45 kbp, 0.269 kbp, and 0.5 kbp, respectively (Fig.
2). This suggests that the oligonucleotide primers
used for the multiplex PCR-based assay were specific for their targeted gene segments enabling
detection in shellfish of V. parahaemolyticus through
one or both of these virulence-associated genes (tdh
and trh) in combination with the genus-specific tl
gene.
Detectable levels of PCR-amplified DNA bands
from all three target genes in agarose gel is evidenced from the oyster tissue homogenates to which
10 2 cells of V. parahaemolyticus were added (Fig. 3).
In addition, positive PCR amplification of the trh and
the tl gene segments was detected in oyster tissue
homogenates in which 10 1 V. parahaemolyticus cells
were added (Fig 3). The entire procedure including
the purification of DNA from oyster tissue homogenate, PCR amplification and detection of the amplified DNA can be finished within 8 h of the

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

Fig. 2. Agarose gel electrophoresis showing the results of multiplex PCR amplification of DNA purified from 10 g of APWenriched oyster tissue homogenate seeded with V.
parahaemolyticus strains with various combinations of the tl, tdh,
and the trh genes. Lane 1, 123-bp DNA ladder (Gibco) as size
marker enhanced by PRORFLP computer software; lane 2, V.
parahaemolyticus F113A showing amplification of all three genes;
lane 3, V. parahaemolyticus 8700 showing amplification of only
the tdh and the tl genes; lane 4, V. parahaemolyticus AQ4037
showing amplification of only the trh and the tl genes; lane 5, V.
parahaemolyticus 48268 showing amplification of only the tl
gene; lane 6, amplification of purified genomic DNA from V.
parahaemolyticus F113A as positive control; lane 7, amplification
of DNA from oyster tissue homogenate in which no V.
parahaemolyticus cells were added as negative control.

pre-enrichment step. It should be possible to improve

the sensitivity of detection of all three amplified
genes by 10-fold, i.e. 1-cell level for the tl and trh,
and 10-cell level for the tdh genes, if DNADNA
hybridization is applied to these amplified DNA
samples. However, detection of #10 2 V.
parahaemolyticus cells per 10 g of oyster tissue
homogenate is well within range ($10 4 cells per 1
g) of the action level recommended in NSSP guidelines (NSSP, 1997). The oyster tissue homogenates
not seeded with V. parahaemolyticus showed no
amplification of the targeted gene segments, suggesting the minimum sensitivity of detection
achieved from seeded samples was not due to the

223

Fig. 3. Agarose gel electrophoresis showing sensitivity of detection of the multiplex PCR-amplified DNA from various concentrations of V. parahaemolyticus F113A cells seeded in 10 g of
oyster tissue homogenate showing the sensitivity of the detection.
Lane 1, 123 bp DNA ladder (Gibco) as size marker enhanced by
4
3
PRORFLP computer software; lane 2, 10 cells; lane 3, 10 cells;
lane 4, 10 2 cells; lane 5, 10 1 cells; lane 6, 10 0 cells; lanes 7 and 8,
0 cells; lane 9, amplification of DNA from oyster tissue homogenate in which no V. parahaemolyticus cells were added as negative
control; lane 10, amplification of purified genomic DNA from V.
parahaemolyticus F113A as positive control; lane 11, PCR
negative control.

presence of any indigenous V. parahaemolyticus

cells.

4. Conclusions
In this study, a multiplex PCR procedure has been
optimized to amplify individually or in different
combinations two virulence-associated genes, tdh
and trh, along with the species-specific tl gene from
various V. parahaemolyticus strains. The selected
oligonucleotide primers were highly specific for their
respective target gene segments and did not show
any amplification when purified genomic DNA from
bacterial species other than V. parahaemolyticus were
subjected to PCR. The minimum level of detection
of this pathogen was $10 2 cells per 10 g of preenriched oyster tissue homogenate for the tdh gene
segment, whereas, the levels for the tl and trh genes
were reduced to $10 1 cells. The 1-fold less sensitivity of detection of the tdh gene segment is not
understood.
The majority (|96%) of the V. parahaemolyticus
strains tested in this study exhibited direct correlation
between the presence of the thermostable hemolysin
gene and the Kanagawa reaction suggesting that if

224

A.K. Bej et al. / Journal of Microbiological Methods 36 (1999) 215 225

necessary, both tests can be performed simultaneously to confirm the presence of potentially virulent
strains of V. parahaemolyticus in shellfish. In previously reported studies, the presence of the Kanagawa
phenomenon has been directly correlated with the
positive detection of urease by urea hydrolysis in this
pathogen (Kaysner et al., 1994; Okuda et al., 1997a
and b; Ghosh and Sehgal, 1998; Iida et al., 1998).
However, all V. parahaemolyticus strains isolated
from infected human patients from recent outbreaks
(Okuda et al., 1997a and b; Morbidity and Mortality
Weekly Report, 1998), exhibited negative reaction
for urease, although positive for the tdh or the trh
genes. Therefore, the test for urease may not be
suitable for correlating the presence of the virulence
strains of this pathogen (Okuda et al., 1997a and b).
PCR amplification of the tl gene segment occurred
in all 111 V. haemolyticus strains tested in this study.
This suggests the tl gene is V. parahaemolyticusspecific, and can be used as a genus-specific marker
for PCR amplification detection of this pathogen.
Since some V. parahaemolyticus strains are trh2 but
tdh1 and others the reverse, targeting these two
genes simultaneously in a single PCR reaction
should provide comprehensive information about the
occurrence and distribution of the virulence-associated genes in this pathogen. The significance of the
presence of the trh gene in a limited number of the V.
parahaemolyticus isolates as compared to the tdh
gene is currently not perceived.
This multiplex PCR method, which targets three
gene segments simultaneously and enables detection
of
total
and
hemolysin-producing
V.
parahaemolyticus, is less time-consuming and more
cost-effective than the conventional PCR approach,
in which each of the three target genes is amplified
in three separate PCR reactions. The multiplex PCRbased detection described in this report could be
completed within 8 h of pre-enrichment with the
sensitivity of detection of 10 1 10 2 cells per 10 g of
shellstock. This result corresponded with the detection of the virulent strains of V. parahaemolyticus
carrying the tdh gene in marine fish (Karunasagar et
al., 1996). The availability of a rapid and reliable
multiplex PCR that comprehensively detects V.
parahaemolyticus in shellfish by simultaneously
amplifying the genus-specific tl gene and hemolysinproducing tdh and trh genes in a single reaction has

the potential to reduce V. parahaemolyticus-associated illnesses in humans consuming seafood.

Acknowledgements
This research was supported by the National
Oceanic and Atmospheric Administration, the Mississippi Alabama Sea Grant Consortium and the
University of Alabama at Birmingham (Grant no.
R-LR 26). We thank A. DePaola for valuable suggestions, and L. Beasely for technical assistance.

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