Download using polymerase chain reaction for detection of enterotoxigenic

USING POLYMERASE CHAIN REACTION FOR DETECTION OF
ENTEROTOXIGENIC VIBRIO CHOLERAE IN SEAFOOD
Huynh Ngoc Thanh Tam, Truong Trong Ngon, Nguyen Duc Cuong, Tran Nhan Dung
and Nguyen Van Ba
Biotechnology Research and Development Institute
ABSTRACT
Vibrio cholerae, Gram–negative, is often found in seawater, brackish water. This
bacterium can produce cholera toxin (one kind of enterotoxin). Cholera toxin is a product
of ctx genes whose structure includes one A subunit and five B subunits. ctxA gene
encodes the A subunit of the toxin, and ctxB gene encodes the B subunit. PCR technique
and specific ctxA primer were used to detect enterotoxigenic. Vibrio cholerae cells were
treated by SDS 10%, or by boiling to release DNA. Using the PCR technique could not
detect directly Vibrio cholerae in seafood sample solutions. When the density of Vibrio
cholerae is more than 107 cells/ml, it can be detected successfully by the PCR technique.
Key words: Enterotoxigenic Vibrio cholerae, enterotoxin, cholera toxin, polymerase
chain reaction (PCR)
INTRODUCTION
Cholera has been one of the most feared diseases for human. It was recognized that two biotypes
were agglutinated by a single antiserum designated O1 in the 1930s. Other strains of Vibrio
cholerae do not react with this antiserum and are termed non-agglutinatable, or more correctly nonO1 strains. Some of these strains produce cholera toxin. In 1992, a new serotype O139, caused
cholera epidemic in some countries, was also isolated from cholera patients in Thailand. The
development of quick and accurate procedures for detecting and characterizing micro-organisms is
needed in many research areas, especially in biological safety. Microbiological quality control is
now being considered in the food industry. To supplement classical methods, PCR is now the most
widely applied method. The experiment of “Using Polymerase chain reaction (PCR) for detection of
Enterotoxigenic Vibrio cholerae in seafood” was carried out in Can Tho University in order to meet
the requirement for controlling food safety.
MATERIALS AND METHODS
Materials
Seafood samples were obtained from seafood materials or from semi-products, finished products
including salt-water fish, fresh-water fish, fresh-water shrimp, salt-water shrimp, clam, cuttle-fish.
Methods
Plate count to determine total aerobic bacteria at 37oC
The protocol for culturing bacteria was followed by Nordic Committee on food analysis (NKML),
1999. Total aerobic bacteria density was counted as the following formula:
N=
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ΣC
(n1 + 0.1n2) d
(According to Vietnamese standard 6404:1998, 1998).
Where: N: a number of colonies present in 1g of sample; ΣC: colonies counted in all Petri dishes; d:
level of dilution in which colonies counted from 25 < x < 250; n1: a number of Petri dishes selected
for counting at the first proper dilution level (n1 = 1 or n1 = 2); n2: a number of Petri dishes selected
for counting at the second proper dilution level (n2 = 1 or n2 = 2).
DNA isolation from Gram-negative bacteria
Bacteria DNA was extracted according to the protocol described by the molecular biology practical
textbook (2004). After culturing in APW medium, Vibrio cholerae was centrifuged at 13.000 r/m
for 5 minutes to pellet cells, and resuspend in 250µL TE pH 8.0. Add 50µL 10 % SDS, 5µL
proteinase K (20mg/L). Incubate at 65oC for 20 minutes. Add 400µL 10% CTAB 0.7M NaCl,
regular mix. Incubate at 65oC for 20 minutes. Then, add 600µL chloroform and isoamyl alcohol
with ratio 24:1. Regular mix and centrifuge at 12.000 r/m for 10 minutes. Transfer top layer to new
tube and add 1mL isopropanol, regular mix, keep at -20oC at least 30 minutes. Centrifuge at 13.000
r/m for 10 minutes. Wash with 1ml ethanol 70%, centrifuge at 12.000 r/m for 5 minutes. Doing
again 2 times to eliminate mixture. Place in vacuum centrifuge at 45oC for 15 minutes to eliminate
ethanol. Pellet is DNA of Vibrio cholerae, dissolve in 100µL 2 times distilled water to utilize.
Determinate Vibrio cholerae by PCR method
Vibrio cholerae standard or after isolation was boiled for 5 minutes to release DNA. DNA samples
were utilized for PCR reaction. Mix solution for one PCR reaction includes compositions and
volume to obtain total 50µL including PCR buffer 10X, dNTP 10mM, forward and reverse primers
10pmol/µL, Tag polymerase 5U/µL, miliQ water and DNA samples. PCR cycling conditions are as
following: heating of the mixture at 94°C for 3 minutes to break cells and release DNA (1 cycle),
DNA amplification was carried out for 35 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C
for 1 minute, store samples at 4oC for 30 minutes (1 cycle).
Electrophoresis. Mix 8-10µL portions of PCR reactions with gel loading buffer and load into
sample wells of 1.5% agarose gel submerged in 1X TE containing 0.02%o ethidium bromide (load
1.2µL with standard band DNA 200bp). The appropriate migration was run with a constant voltage
of 25-50 Volt and electrophoresis for 2-3 hours.
Read results. The agarose gel with a UV transilluminator was illuminated and visualized bands
relative to molecular weight marker migration.
Determination name and sequence of primers. Software BLAST at website
http://www.ncbi.nlm.nih.gov/BLAST/ was utilized to determinate name and sequence of primers.
At the same time, DNA club software was added and transferred from sequence of simple strand to
supplementary strand.
RESULTS AND DISCUSSIONS
Determination name and nucleotide sequence of target gene
Results of primers are 5’-TGAAATAAAGCAGTCAGGTG-3’ (forward primer), and 5’GGTATTCTGCACAAATCAG -3’ (reverse primer). The primer sequence at location 237 -256 of
target gene with length 777 bp coding for peptide chain of subunit A1 of ctxA gene, is the most
important composition in cholera toxin structure of Vibrio cholerae. In addition, the sequence of
primers and length of target gene was in good agreement with previous studies of Koch et al.
(1995), Charles et al., (2004). They utilized this primer pair in PCR reaction to amplify the target
gene to detect Vibrio cholerae which produced cholera toxin in food. In this experiment, we utilized
name of target gene to name for ctxA primer.
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Similarly, the primer pair 5’-CGGGCAGATTCTAGACCTCCTG-3’(forward primer),
5’CGATGATCTTGGAGCATTCCCAC-3’ (reverse primer) was to detect Vibrio cholerae O1 caused
cholera toxin, this result indicated that sequence of gene was at location 1-22 of target gene with
length 564 bp of ctxA gene. Sequence of primers and length of target gene were consistent with the
previous studies of Olsvik et al., (1993) and the name of this primer is ctxA’.
Study specificity of primer pairs ctxA
Specificity of primer pair ctxA for detecting Vibrio cholerae toxin was evaluated by PCR technique.
Solution of some pure culture strains such as Vibrio cholerae from different sources, Vibrio
parahaemolyticus, Escherichia coli, Staphylococcus aureus, Samonella spp, Shigella spp and some
other bacteria were treated by heating. Results were presented in Table 1.
Table 1 indicates that two strains Vibrio cholerae from Center of preventive medicine-Ca Mau City
and Branch of Aquatic product 6 had positive results, and they were shown at DNA band with size
777 bp. Two strains have presence of ctxA gene and capacity to belong to Vibrio cholerae type
serum O1 or O139 produced cholera toxin. Other bacteria (sometimes including Vibrio cholerae
strain) maybe did not have ctxA gene, so the result was negative. The above data was confirmed by
observation of Chow et al., (2001) and Albert (1994). Not all of strains of Vibrio cholerae contain
pathogen cholera toxin but some strains have type serum O1 and O139. That was the reason why
Agency of Sanitary of Japan required change some contents confirm about Vibrio cholerae in health
certificate accompany with sea food container import to Japan is “having no Vibrio cholerae (O1,
O139) in 25 gram product” from 31/05/1999, not required Vibrio cholerae certificate.
In brief, our preliminary results revealed that primer pair ctxA was specific primer to detect Vibrio
cholerae producing cholera toxin by PCR method.
Table 1. Results of PCR reaction of some different strains
Code
Strain
Source
1
Vibrio cholerae
Center of preventive medicine-Ca Mau City
+
2
Vibrio cholerae
Nam Khoa Limited Liability Company
-
3
Vibrio cholerae
Branch of Aquatic product 6
+
4
Vibrio cholerae
Medicine University of Ho Chi Minh City
-
5
Vibrio cholerae
Isolate from sea fish at Kien Giang
-
6
Vibrio cholerae
Isolate from Pangasius hypophthalmus (fish) at An Giang
-
7
Vibrio parahaemolyticus Branch of Aquatic product 6
-
8
Escherichia coli
Branch of Aquatic product 6
-
9
Staphylococcus aureus
Branch of Aquatic product 6
-
10
Samonella spp
Branch of Aquatic product 6
-
11
Shigella spp
Branch of Aquatic product 6
-
12
Some other bacteria
Branch of Aquatic product 6
-
Note: + is detect ctxA gene, - is not detect ctxA gene
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Result
Comparison primer pair ctxA and primer pair ctxA’
To verify result of experiment, PCR was used on pure cultured Vibrio cholerae strain and primer
pair ctxA’ was used to detect Vibrio cholerae having type serum O1 and producing cholera toxin.
Two strains being from Center of preventive medicine-Ca Mau City and Branch of Aquatic product
6 contained ctxA gene. However, electrophoresis gave different results. Sample 1 and 2 were
amplified products of target gene with 777 bp when using ctxA primer. Meanwhile, sample 3 and 4
were amplified products of target gene correlative with 564 bp when using ctxA’ primer. This result
was appropriate with conform of Phung Dac Cam (2003) and other authors. It is Vibrio cholerae
that belongs to type serum O1 having very high cholera toxin (>95%) and is the agent of six big
cholera in the world.
The primer pair established on ctxA gene is very specific to detect Vibrio cholerae producing
cholera toxin by PCR method. As a result, two strains from Center of preventive medicine-Ca Mau
City and Branch of Aquatic product 6 were selected and ctxA primer pair was used for the next
experiments.
DNA releasing condition
Vibrio cholerae was digested to release DNA template by heating directly at 94oC, 3 minutes in
PCR mixture; or at boiling temperature, 5 minutes in waterbath; or by SDS 10%. The PCR products
of bacterial lysing obtained from all treatments were positive.
NaCl concentration in medium of Vibrio cholerae culture for PCR
PCR is easily affected by bacterial culture medium. The suitable culture medium of Vibrio cholerae
was APW medium containing 1 to 3% NaCl. Vibrio cholerae was put in water and APW medium
with 0%, 1%, 2%, and 3% of NaCl for PCR.
The electrophoresis of PCR products showed that samples of Vibrio cholerae in water and APW
medium without NaCl were positive, and the other samples were negative. Ion Na+ has competitive
relation with Mg2+ and K+ of PCR buffer that reduce polymerease activity (Pham Thanh Ho, 2002).
Or NaCl is not pure so it contains some other ions that can affect PCR. Therefore, in other
experiments, we only obtained bacterial lysing from mixture of bacteria and water for PCR.
Minimum cells of Vibrio cholerae for PCR detection
Vibrio cholerae were cultured in TSA medium for overnight, then suspended and diluted with
distilled water. Bacterial cells per every diluted suspension were counted by agar – plate counting
method.
Generally, minimum cells of Vibrio cholerae for PCR is 7.0x106 cells/ml, or 1.4x104 cells/2µl of
bacterial lysing for each PCR. However, PCR product of 777 bp on agarose gel was not clear. This
result is the same results of Olsivk et al., (1993), Koch et al., (1995). Therefore, the minimum cells
of Vibrio cholerae is 7.0x106 cells/ml was proposed for detecting by PCR.
Sensitivity of PCR for direct detection of Vibrio cholerae in aquaculture samples
Six aquaculture samples were prepared previously for PCR. The volume 10 ml of each sample were
mixed in APW medium without NaCl; then incubated in 1ml of Vibrio cholerae with 109 cells/ml.
The final liquid was mixed for 3 minutes and used for PCR.
All PCR products of 6 samples that were incubated with Vibrio cholerae were negative. It
concluded that aquaculture samples still remained undetermined components affecting PCR.
According to Koch et al., (1995), aquaculture samples were incubated in APW medium without
NaCl for 6 to 21 hours to get the lysing which contained DNA templates for PCR. The results
showed that many steps should be carried out to remove affecting chemicals in the samples.
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Sensitivity of PCR for direct detection of Vibrio cholerae in aquaculture samples
with isolation method
Six samples of fish, crustacean, and molluscan species of sea – food industry living in different
areas were chosen. These samples were suspended in APW medium for homogeneous liquid; and
then incubated with Vibrio cholerae at 37oC ± 1oC, 6 to 8 hours before they were streaked on agar
plates of TCBS medium. Pure colonies of Vibrio cholerae were gotten after 16 to 24 hours, then
they were used for PCR. The results showed that all samples were positive.
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