Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction

Molecular and Cellular Probes (1992) 6, 477-487

Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction Jun Tada,' Tetsuo Ohashi, Naoyuki Nishimura,' Yoshinari Shirasaki, 1 Hiroko Ozaki,1 Shigeru Fukushima,'* Jun Takano, Mitsuaki Nishibuchi 2 2 and Yoshifumi Takeda ' Central Research Laboratory, Shimadzu Corporation, 1 Nishinokyo- Kuwabaracho, Nakagyo-ku, Kyoto 604, Japan and 2 Department of Microbiology, Faculty of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606, Japan (Received 8 June 1992, Accepted 23 June 1992) Polymerase chain reaction (PCR) protocols were established for specific detection of the tdh and trh genes, the virulence marker genes of Vibrio parahaemolyticus encoding two related hemolysins. The tdh and trh genes are known to have sequence divergence of up to 3 . 3% and 16%, respectively . Attempts were made to find suitable primer pairs and annealing temperatures to detect each gene without fail . DNAs extracted from 36 representative strains of V . parahaemolyticus were used in the initial screening with various combinations of primer pairs and annealing temperatures . The combinations of primer pairs and annealing temperatures selected were then tested with DNAs extracted from 227 more strains of V . parahaemolyticus and from 133 bacterial strains belonging to 40 species other than V . parahaemolyticus . PCR protocols (primer pairs and annealing temperatures) were established that gave identical results to those obtained with the tdh- and trh-specific polynucleotide probes . These protocols established for the tdh and trh genes could detect 400 fg (100 cells) of cellular DNA carrying the respective gene . Spike experiments demonstrated that the sensitivities of the established PCRs were reduced by a factor of 10 4-105 by an inhibitor(s) present in a normal faecal sample, indicating the need for either DNA extraction or enrichment of the faecal sample in alkaline peptone water for 4 h before the PCR of faecal samples .

KEYWORDS : Vibrio parahaemolyticus, thermostable direct hemolysin gene, PCR .

INTRODUCTION Vibrio parahaemolyticus, a natural inhabitant of estuarine and marine environments, often causes seafood-borne gastroenteritis .' , ' Although the mechanism of its pathogenic effect is still not clearly understood, epidemiological studies have suggested a very strong association of thermostable direct hemolysin (TDH) with disease .` Production of TDH is responsible for a defined characteristic called the

Kanagawa phenomenon,' which is manifested as beta-type hemolysis on a special blood agar called Wagatsuma agar.' Since the Kanagawa phenomenon reaction is difficult to judge, immunological methods have been developed to identify TDH-producing strains specifically,$ and of these, bead-enzymelinked immunosorbent assay' has been shown to be highly sensitive for detecting TDH . A DNA probe

* Author to whom correspondence should be addressed .

0890-8508/92/060477 + 11 $08 .00/0

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© 1992 Academic Press Limited



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J . Tada et alL

method to detect the gene (tdh) encoding TDH is also sensitive and has been used successfully to demonstrate that not only all Kanagawa phenomenonpositive strains but also some Kanagawa phenomenon-negative strains, mostly from clinical sources, have the tdh gene .' Subsequently, the tdh genes cloned from Kanagawa phenomenon-negative strains were found to encode biologically active TDHs,10 12 indicating that all strains having the tdh gene are potentially virulent . The nucleotide sequences of the tdh V. parahaemolyticus far genes of so reported", " , "` show sequence variations of up to 3-3% . Recently, a Kanagawa phenomenon-negative clinical strain not possessing the tdh gene was found to produce a TDH-related hemolysin (TRI-1) ." Like the tdh gene, the gene (trh) encoding TRH has a 567 by coding region, but is only 68% homologous to the tdh gene . 17 A molecular epidemiologic study with tdh- and trh-specific DNA probes demonstrated a strong association of the two genes with clinical strains, suggesting that TRH as well as TDH is an important virulence factor ." Furthermore, significant sequence variation was very recently found in the trh genes of various strains . The trh probe-positive strains manifesting various intensities of hybridization signals could be subdivided into two sub-groups represented by trh 1 (formerly trh) and trh 2 genes ." The trh 1 and trh 2 genes show 84% homology and the trh 2 genebearing strains are also considered potentially virulent ." . The polymerase chain reaction (PCR) method is usually more rapid and sensitive than a DNA or oligonucleotide probe-based hybridization method for detecting a target gene in bacteria . But in using the PCR method for detecting the tdh and trh genes at a high detection rate, possible mismatch(es) of the PCR primers due to the existence of natural variations in their sequences'" 1,13-15,19 must be avoided . The primary aim of this study was to establish PCR protocols giving identical, or nearly identical results to those obtained by the DNA (=polynucleotide) probe method . For this purpose, we first screened various combinations of primer pairs and annealing temperatures using DNAs extracted from representative strains . The selected PCR protocols were screened further with DNAs extracted from a much larger number of organisms. Some enteropathogenic bacteria present in faecal samples could be detected by the PCR method with 20 or without 21 prior DNA extraction from the faecal samples . To assess the practical applicability of the established PCR protocols to faecal samples, we carried out a spike experiment to evaluate the need for DNA extraction from faecal samples .

MATERIALS AND METHODS Bacterial strains The bacterial strains used in this study are listed in Table 1 . The strains belonging to the genus Vibrio were described previously ." Other strains were obtained from the American Type Culture Collection, Japan Collection of Microorganisms and the Institute for Fermentation, Osaka. The presence or absence of the tdh and trh genes in Vibrio strains was determined in previous studies by DNA colony blot hybridization tests with DNA probes specific to the tdh and trh genes ."' Strains that gave weak or very weak hybridization signals with a trh gene probe" are included in the trh-positive group in Table 1, but when necessary (see below), the trh genes in various strains were classified into trh 1 and trh 2 genes by DNA colony blot and Southern blot hybridization analyses as reported elsewhere ."

DNA extraction from bacterial cells Pure cultures of the test organisms grown on appropriate agar media were harvested and used for DNA extraction . Cellular DNA was extracted by standard procedures using phenol and chloroform ."

PCR primers Oligonucleotide (19- or 20-mer) primers were designed based on the reported nucleotide sequence of the tdh 2 gene cloned from strain WP1 11 and the trh (=trh 1) gene cloned from strain AQ4037 . 17 The designations and nucleotide sequences of the primers are listed in Table 2 . The primers were synthesized by the (3-cyanoethyl phosphoramidite method with an automated DNA synthesizer (Cyclone plusTM DNA synthesizer, Milligen, Burlington, MA, USA) and purified using a high-performance liquid chromatography apparatus (LC-6A, Shimadzu Co ., Kyoto, Japan) equipped with a reversed phase column (Cosmosil C18, Nacalai Tesque, Kyoto, Japan) .

PCR amplification PCR amplification was performed in a 30 pl mixture composed of 10 mm Tris-HCI (pH 9 . 0), 50 mm KCI, 1-5 mm MgCl2, 0-01% gelatin, 0 . 6µM each of the primers, 02 mm each of the four deoxynucleoside triphosphates (Ultrapure dNTP Set, Pharmacia LKB Biotechnology, Uppsala, Sweden), 0 . 05% Tween 20,



PCR for tdh and trh genes of

Table 1 . Detections of the other organisms

tdh

Organism

Vibrio

parahaemolyticus

Vibrio cholerae, 01 Vibrio cholerae, non-OI Vibrio

mimicus

Vibrio furnissii Vibrio fluvialis Vibrio metschnikovii Bacillus cereus Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis Salmonella typhimurium Salmonella enteritidis Clostridium perfringens Campylobacter jejuni Campylobacter coli Escherichia coli Yersinia enterocolitica Shigella dysenteriae Shigella flexneri Shigella sonnei

Bacteroides fragilis Bacteroides vulgatus Enterococcus faecalis Klebsiella pneumoniae Proteus mirabilis Proteus vulgaris Citrobacter freundii Streptococcus pyogenes Streptococcus pneumoniae

Haemophilus influenzae Neisseria gonorrhoeae Neisseria meningitidis Listeria monocytogenes Lactobacillus acidophilus Bifidobacterium adolescentis Fusobacterium nucleatum Propionibacterium acnes

Veillonella atypica Pseudomonas aeruginosa Corynebacterium diphtheriae Peptostreptococcus anaerobius

Vibrio

parahaemolyticus

and trh genes by PCR assay in DNAs extracted from

No . of strains 25 110 56 72 10 1 73

Presence or absence* of gene' trh gene$

tdh

+ + -

+ + -

-

+

-

-

-

+

-

2

-

-

4 5

-

-

1 1 1 1 1 1 1

NT NT NT NT NT NT NT NT NT NT NT NT NT

NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT

and

Results§ of PCR assay for tdh gene¶ trh gene I + + -

+

NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT

Vibrio parahaemolyticus

+ +

2

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 1 1 1

479

+ -

-

-

-

-

-

-

NT NT

-

NT NT NT NT NT NT NT NT NT NT NT NT NT NT

-

-

-

-

-

-

-

-

-

+, present . -, absent . NT, not tested . j Determined by the DNA colony blot hybridization test with a tdh gene probe in previous studies ."' Determined by the DNA colony blot hybridization test with a trh gene probe in a previous study ." Strains exhibiting weak or very weak hybridization signalst 8 were included in the gene-positive (+) group in order to include both the trh 1 and trh 2 sub-group." § +, only specific amplicons of expected sizes (see Table 3) were detected by gel electrophoresis . -, no amplicons were detected by gel electrophoresis . ¶ Primer pairs D1-D2, D5-D2 and D5-D3 were used at an annealing temperature of 55 ° C. The results obtained with the three primer pairs were identical . II Primer pair R2-R6 was used at an annealing temperature of 55 ° C.

J . Tada et al .

480

Table 2. Target gene

PCR primers used in this study

Designation

tdh

trh

D1 D2 D3 D4 D5 D6 R1 R2 R3 R4 R5 R6

Nucleotide base sequence* 82-CCATCTGTCCCTTTTCCTGC-101 454-CCAAATACATTTTACTTGG -436 506-CCACTACCACTCTCATATGC-487 534-GTTGGATATACACATTACC -516 256-GGTACTAAATGGCTGACATC-275 275-GATGTCAGCCATTTAGTACC-256 82-CCATCCATACCTTTTCCTTC-101 256-GGCTCAAAATGGTTAAGCG -275 256-GCCTCAAAATGGTTAAGCGC-276 461-GTTTCATCCAAATACGTTAC-442 465-TGGCGTTTCATCCAAATACG-446 505-CATTTCCGCTCTCATATGC -486

Calculated Td (°C)1 62 56 60 52 58 58 58 56 60 54 58 56

* Sequence in the 5' to 3' (left to right) direction . The primer sequences of the tdh and trh genes are identical to those of the tdh 2 gene" and the trh (=trh1) gene", respectively . The numerals at both ends correspond to the nucleotide positions of the coding regions of the respective genes, numbered from the bases (=position 1) of the N termini of the mature proteins . j' The Td value, temperature at which 50% of the primer is dissociated was calculated by the method of Suggs et al .26

(Perkin-Elmer Cetus Corp .) ; one cycle consisted of

followed by neutralization with 0-5 M Tris-HCI (pH 8-0) containing 1 . 5 M NaCl and 1 mm EDTA at room temperature for 30 min . The amplicons were transferred to a nylon membrane (Hybond TM , Amersham International, Buckinghamshire, UK) in 10 X SSC over-

denaturation at 94 ° C for 1 min, primer annealing at a

night [1 x SSC is 0-15 rn NaCl, 15 mm trisodiumcitrate

specified temperature for 1 min, and extension at

(pH 7 . 0)1 . After washing with 2 X SSC, the membrane was dried with 3 MM paper (Whatman Biosystems, Maidstone, Kent, UK). Probe DNAs specific to the tdh and the trh genes were prepared from recombinant plasmids pCVD518 9 and pKTN101,' 8 respectively, as described previously . The probes were radiolabelled with 32 P in a multiprime DNA labelling system (Amersham International). The specific activities of the labelled probes were 3 X 108 to 6 X 108 cpm µg'. Hybridization was performed in 5 X SSPE [1 X SSPE is

0 . 05% Nonidet P-40, 0 . 75 U of Taq polymerase (Perkin-Elmer Cetus Corp ., Norwalk, CT, USA), and template DNA (1 ng, unless otherwise specified) . The PCR was carried out for 35 cycles in a DNA thermal cycler

72 ° C for 1 min .

Detection of amplified DNAs PCR-amplified

DNAs (amplicons) were usually detected by agarose gel electrophoresis . A 5 µl portion the PCR-amplified mixture was separated in 3% agarose (Agarose-ME, Nacalai Tesque) gel by electrophoresis at 3 to 4 V cm"' for 1 h in TAE buffer [40 mm Tris-acetate (pH 8. 0), 20 mm EDTAI containing 0 . 5 gg ml-' ethidium bromide . After electrophoresis, the gel was examined with a u .v. (at 302 nm) transilluminator (UVP Inc ., San Gabriel, CA, USA) and photographed . The target-specific amplicons were identified by their size, which was determined by their mobility relative to those of molecular weight markers (Hin cli-digested phage OX174 DNA) . To confirm the specificity of the amplicons and to assess the sensitivity of the PCR assay, we examined

0 . 15 m NaCl, 18 mm sodium phosphate, 1 mm EDTA, (pH 8.0)], 1 % SDS, 5 X Denhardt's mixture (1 X Denhardt's mixture is 0-02% Ficoll, 0-02% polyvinylpyrrolidone, 0-02% bovine serum albumin) supplemented with 0-1 mg ml"' poly A (Boehringer Mannheim Bio-

chemicals, Indianapolis, IN, USA) and a radiolabelled probe (7 . 5 X 106-1 . 5 X 10' cpm) at 65 ° C overnight . After hybridization, the membrane was washed with 1 X SSPE and 0. 1 % SDS twice at 65°C for 20 min, airdried and exposed to X-ray film overnight (Fuji RX, Fuji Film Co ., Kanagawa, Japan) .

selected samples by Southern blot hybridization . For Southern blotting, 5 µl of 500-fold diluted or undiluted (sensitivity test) PCR-amplified mixture was subjected to agarose gel electrophoresis as described

Examination of spiked faeces by PCR

above. The gel was treated with 0-5 M NaOH containing 1 . 5 M NaCl at room temperature for 30 min,

A 0. 1 g portion of normal human faeces was suspended in 10 ml of phosphate-buffered saline

PCR for tdh and trh genes of Vibrio parahaemolyticus (pH 7 . 0) . After vigorous mechanical shaking (3000 rpm) for 60 s, insoluble particulate matter was removed by low-speed centrifugation and aliquots of the suspension were mixed with an equal volume (100 µl) of serially diluted bacterial culture (overnight culture of WP1 or AQ4037 in LB broth with 1 NaCI23) . The numbers of viable cells in the cultures were determined by a plate count method . The spiked sample was subjected to PCR without (direct PCR) or with a prior treatment ; i .e . extraction of DNA from the spiked faeces or enrichment culture of the spiked faeces . For direct PCR of the spiked faeces, the sample was heated at 95 ° C for 5 min and a 5 tl portion was subjected to PCR . For extraction of DNA from the spiked faeces, the sample (200 µl) was incubated first with 400 pl of lysozyme solution (1 mg ml - ' at 37° C for 10 min and then with 20 .tl of proteinase K solution (20 mg ml - ') and 20 .tl of 20% sodium dodecylsulfate solution at room temperature for 1 h . DNA was extracted from the lysate by a series of phenol-chloroform treatments [400 .d of 10 mm Tris-1 mm EDTA (pH 80-saturated phenol, 400 µl of chloroform-isoamyl alcohol (24 :1 v/v), and 400 lal of chloroform] and precipitated with 40 µl of 3 M sodium acetate solution and 800 µl of cold ethanol . Then it was rinsed with 70% ethanol, dried, and dissolved in 200 gl of 10 mm Tris-1 m m EDTA solution (pH 8 . 0) . A 5 µl portion of the DNA solution was subjected to PCR . For the enrichment method, the spiked sample (200 µl) was inoculated into 1 ml of alkaline peptone water 24 and incubated with shaking at 100 rpm at 37°C for 4 h . The culture was heated at 95°C for 5 min and a 5 pl portion was subjected to PCR . PCR was performed as described above except that the total volume of the reaction mixture was increased to 50 µl . A 5 µl portion of the PCR-amplified mixture was examined by gel electrophoresis as described above to detect the specific amplicons .

RESULTS Screening of PCR primers and annealing temperatures The nucleotide sequences of five representative tdh (tdh 1-tdh 5) genes"," and the trh (=trh 1) gene" of V . parahaemolyticus were compared . The tdh gene group and the trh (trh 1) gene differed by as much as 31-32%, sequence heterogeneity being observed throughout the coding regions . Therefore, it was impossible to design a PCR primer pair for simultaneous detections of the tdh and trh genes. However, five regions containing sequences that were well conserved among the five tdh genes but were signi-

481

ficantly different from those of the trh 1 gene were detected . We thus decided to design primers targeting these regions for detecting the tdh and trh genes separately . The tdh 2 gene is one of the two tdh gene copies carried by Kanagawa phenomenon-positive strains and is the gene mainly responsible for production of extracellular TDH . ",'3,25 Therefore, we selected) this gene as a representative of the tdh genes . Six PCR primers for the tdh gene targeted to the tdh 2 genespecific sequences within the five conserved regions were prepared (D1-D6 in Table 2) . Primers D11, D2 and D4 did not have any mismatch with the four other tdh genes . Primer D3 had 1 by and 2 by mismatches with two and one other tdh gene, respectively . Primer D5 had a 1 by mismatch with the four other tdh genes . Seven pairs of the tdh primers (Table 3) were used for amplification of the DNAs extracted from 36 selected strains of V. parahaernolyticus . Of the 36 strains, 17 strains had both the tdh and trh genes, 17 the tdh gene only, and two neither gene . The PCR products were assayed by gel electrophoresis. All seven primer pairs gave specific arnplicons of the expected sizes (Table 3) at annealing temperatures of 50°C and below . But small amounts of additional non-specific amplicons (differing in size from the specific amplicons) were also observed in many cases . When the annealing temperature was raised to 55 ° C, three primer sets, D1-D2, D5-D2 and D5-D3, gave the specific amplicons only and these amplicons were detected only with the 34 tdhcarrying strains . But results with the four other primer pairs were unsuccessful at 55 ° C, no specific amplicons being obtained with some tdh-bearing strains and non-specific amplicons with some other strains . The D1-D3 pair gave no specific amplicons with one tdh-positive strain and a non-specific amplicon with one strain . The D1-D4 pair gave non-specific amplicons with three tdh-positive strains . The D1-D6 pair gave non-specific amplicons with two strains. The D5-D4 pair gave non-specific amplicons with one strain . Therefore, the three successful primer pairs and an annealing temperature of 55 ° C were chosen for more extensive tests with DNAs extracted from 227 more strains of V. parahaemolyticus . The results obtained with the three primer pairs were identical and that the results completely agreed with those obtained with the tdh gene probe . The results obtained with these three probes for all 263 tested strains of V. parahaemolyticus are summarized in Table 1 . Initially, four PCR primers were designed for detection of the trh (=trh 1) gene (R1-R5 in Table 2) . Experiments with DNAs extracted from the 36 selected strains were carried out with four primer



4 82

J . Tada et al. Table 3.

Pairs

Target gene tdh

trh

of

PCR primers used in this study

Designation*

Amplified region'

Length (bp) of amplicon

D1-D2 D1-D3 D1-D4 D1-D6 D5-D2 D5-D3 D5-D4 R1-R4 R1-R5 R3-R4 R3-R5 R2-R6

82-454 82-506 82-534 82-275 256-454 256-506 256-534 82-461 82-465 256-461 256-465 256-505

373 425 453 194 199 251 279 380 384 206 210 250

* Designations of primers are the same as in Table 2. The numerals correspond to the nucleotide positions of the coding regions of the respective genes, numbered from the bases (= position 1) of the N termini of the mature proteins . See legend to Table 2 for detailed explanation .

pairs, R1-R4, R1-R5, R3-R4 and R3-R5 (Table 3) . Of the four primer pairs, R3-R5 gave the most successful results at an annealing temperature of 55 ° C, but not below, in detecting the trh gene (found to be trh 1 later) of the 36 test strains . At this point in our study, the sequence of the trh 2 gene and the distributions of the trh 1 and trh 2 genes in the test strains of V . parahaemolyticus were determined in a separate study ." The primers R1, R2, R3, R4 and R5 were found to have 1 bp, 2 bp, 2 bp, 4 by and 3 by mismatches, respectively, with the trh 2 gene. Therefore, the PCR

primer pair gave varying results, particularly with trh 2-positive strains, at an annealing temperature of 55 ° C (results not shown) . The R2-R6 primer pair detected both the trh 1 and the trh 2 genes and nonspecific amplicons were not observed at this annealing temperature (Tables 1 and 4) . The R3-R5 primer pair detected trh 1-positive strains with very high specificity when the annealing temperature was increased to 60 ° C (Table 4) .

with the primer pair R3-R5 was considered specific for the trh 1 gene, but not for the trh 2 gene . Accordingly, an additional primer R6 derived from the trh 1 sequence but having no mismatch with the

Specificity of PCR The above screening test suggested that the primer pairs D1-D2, D5-D2 and D5-D3 for the tdh gene and the primer pair R2-R6 for the trh (both trh 1 and trh 2) gene, both used at an annealing temperature of 55°C, would be successful . The specificities of these PCR protocols were further assessed by amplifying the DNA templates extracted from organisms other than

2 gene was designed (Table 2) . The primer pairs R3-R5 and R2-R6, the latter being designed to detect both the trh 1 and the trh 2 gene, were then tested with DNAs extracted from 263 strains, including the above 36 strains, of V. parahaemolyticus . The R3-R5 trh

Results of PCR assay for detection of the trh1 extracted from 263 strains of Vibrio parahaemolyticus . Table 4 .

and trh2 genes in DNA

Results of PCR assay with primer Presence or absence* of pair / annealing temperature No . of strains 39 40 2 182

trh1 gene

trh2 gene

R2-R6 / 55 ° C

R3-R5 / 60°C

+ +

*+, present; -, absent . Determined by DNA colony blot and Southern blot hybridization analyses with the trh 1- and trh 2-specific DNA probes in a separate study ."



PCR for tdh and trh genes of Vibrio parahaemolyticus V. parahaemolyticus . These included 98 strains belonging to 5 species of the genus Vibrio and 35 strains belonging to 35 species spanning over 25 other genera (Table 1) . The three pairs of the primers for the tdh gene gave identical results . The PCR results obtained for the Vibrio species agreed with those obtained with the tdh and trh gene probes; only rare strains of Vibrio cholerae non-O1 and Vibrio mimicus possessing the tdh gene211 were positive by the PCR for the tdh gene . All the organisms other than Vibrio gave negative results (no amplicons at all) with any of the primer pairs . The specificities of the amplicons were confirmed by Southern blot hybridization analyses with the tdh and trh (=trh 1) gene probes (Fig . 1) . The DNAs extracted from representative strains of V. parahaemolyticus were used as templates : tdh-positive and trh 1-negative strains (lanes 2-5), tdh-negative and trh 1-positive strains (lanes 6-9), tdh- and trh 1positive strains (lanes 11-14), and tdh- and trh 1negative strains (lanes 15 and 16) . Hybridization was carried out under high-stringency conditions to distinguish tdh- and trh 1-specific hybridizations . The trh 2 gene sequences produce weak or no hybridization signals with the trh 1-specific probe under this condition in Southern blot analysis ." Therefore, to avoid confusion, trh 2 gene-positive strains were not in-

(a) I

483

cluded . The DNA fragments amplified from the tdh (Figs la and 1c, lanes 2-5 and 11-14) and trh (Figs lb and 1d, lanes 6-9 and 11-14) genes hybridized only with the respective probes . No other PCR products were detected either by gel electrophoresis or by Southern blot hybridization .

Sensitivity of PCR The sensitivities of the PCR methods established for the tdh and trh genes were evaluated with the DNAs extracted from strains WP1 and AQ4037, respectively . WP1, a representative Kanagawa phenomenonpositive strain, carries two tdh gene copies" but has no trh gene ." AQ4037 carries the trh 1 gene, which was used for nucleotide sequencing, 17 but does not have the tdh gene ." The D5-D3 and R2-R6 primer pairs for the tdh and trh gene, respectively, were both employed at an annealing temperature of 55 ° C . The PCR-amplified mixtures were serially diluted and examined by gel electrophoresis and Southern blot analysis . The limits of detection of the tdh and trh genes were both 400 fg DNA (corresponding to c . 100 cells) by gel electrophoresis (Figs 2a and 2b) and 40 fg DNA (c . 10 cells) by Southern blot hybridization analysis (Figs 2c and 2d) .

(b) 2 3 4 5 6 7 8 9

(c) 1 2 3 4 5 6 7 8 9

10 II 12 13 14 15 16

10 II 12 13 14 15 16

1 2 3 4 5 6 7 8 9

( d ) 1 2 3 4 5 6 7 8 9

10 I I

10

12 13 14 15 16

1I 12 13 14 15 16

Fig. 1 . Analysis of PCR products of DNAs extracted from 14 representative strains of V. parahaemolyticus . DNA samples were extracted from the following strains: lanes 2-5, tdh-positive and trh-negative strains ; lanes 6-9, tdh-negative and trh-positive strains ; lanes 11-14, tdh-positive and trh-positive strains ; lanes 15 and 16, tdh-negative and trh-negative strains . (a) and (c), PCR amplification of the tdh gene with primer pair D5-D3 annealed at 55 ° C . (b) and (d), PCR amplification of the trh gene with primer R2-R6 annealed at 55°C . (a) and (b), Analysis by agarose gel electrophoresis . A 5 pl portion of the PCR-amplified mixture was separated in 3% agarose gel containing ethidium bromide and the amplicons were located under u .v . light . (c) and (d), Analysis by Southern blot hybridization with specific DNA probes . A 5lal portion of 500-fold diluted PCR-amplified mixture was separated by 3% agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with a 32 P-labelled DNA probe specific to the tdh gene (c) or the trh gene (d). The expected positions of the specific amplicons relative to those of the molecular weight markers (Hin cll-digested OX174 DNA, lanes 1 and 10) are indicated by arrowheads .



J . Tada et al .

48 4

2

3

4

5

6

7

8

(c) I

2

3

4

5

6

7

5

6

7

(d ) 2

3

4

5

6

7

8

I

2

3

4

8

Fig. 2. Analysis of PCR products to assess the sensitivities of PCR for the tdh and trh genes. (a) and (c), PCR amplification of the tdh gene with primer pair D5-D3 annealed at 55 ° C. DNA extracted from V . parahaemolyticus strains WP1 carrying the tdh (tdh 1 and tdh 2) genes was amplified . (b) and (d), PCR amplification of the trh gene with primer pair R2-R5 annealed at 55 ° C . DNA extracted from AQ4037 carrying the trh 1 gene [(b) and (d)1 was amplified . The estimated amounts of cellular DNA present in each of the initial PCR mixtures were zero (DNA not added, lane 2), 4 fg (lane 3), 40 fg (lane 4), 400 fg (lane 5), 4 pg (lane 6), 40 pg (lane 7), and 400 pg (lane 8) . Samples (5 µl) of the PCR-amplified mixture were analyzed by gel electrophoresis(see legend to Fig . 1) [(a) and (b)] and samples (5 µp of the PCR products (undiluted) were analysed by Southern blot hybridization (see legend to Fig . 1) [(c) and (d)1 . The blots were hybridized with the DNA probes specific to the tdh gene (c) and the trh gene (d) . Molecular weight markers (Hin cll-digested OX174 DNA) were loaded in lane 1 . The expected positions of the specific amplicons relative to those of the molecular weight marker are indicated by arrowheads .

Examination of spiked faeces by PCR

WP1 and the trh gene of AQ4037, respectively . The limits of detection (minimum numbers of spiked cells

The need for pretreatment of faecal samples was

needed to obtain a positive result) after the different

investigated to apply the established PCR protocol for

treatments were determined (Fig . 3) . For suspension

detection of tdh- and/or trh-positive organisms in

of a normal faecal sample, spiking with at least the

spiked faecal samples . Suspensions of a normal faecal

order of 10 5 cells was necessary to detect the tdh and

sample were mixed with known numbers of viable

trh genes by the PCR (Fig . 3a, lane 8 and Fig . 3b, lane

cells of WP1 (tdh + , trh - ) or AQ4037 (tdh - , trh + ) . The

8) . When the DNA extraction method was employed,

spiked samples were subjected to PCR with or with-

the detection limits for the tdh And trh genes were

out treatment (by DNA extraction or enrichment in

reduced by factors of 10 5 and 10", respectively, (Fig .

alkaline peptone water) prior to PCR . The primer pairs

3c, lane 3 and Fig . 3d, lane 4) . The enrichment

D5-D3 and R2-R6 were both used at an annealing

method also reduced the detection limits drastically,

temperature of 55 ° C for detection of the tdh gene of

resulting in higher sensitivities than the DNA extrac-

PCR for tdh and trh genes of Vibrio parahaemolyticus

3

3

4

5

6

7

8

4

5 6

485

8

9

9

.4-

Fig . 3 . Detections of the tdh and trh genes in spiked faeces by PCR. (a), (c) and (e), PCR for detection of the tdh gene in faeces spiked with strain WP1 . The primer pair D5-D3 was employed at an annealing temperature of 55 ° C . The estimated numbers of viable cells of WP1 in the initial PCR mixtures were 0 (not spiked, lane 2), 1 . 5 (lane 3), 1 . 5 x 10 (lane 4), 1 . 5 x 10 2 (lane 5), 1 .5 X 10 3 (lane 6), 1 . 5 X 10^ (lane 7), 1 . 5 x 10 5 (lane 8) and 1 . 5 x 105 (lane 9) . (b), (d) and (f), PCR for detection of the trh gene in faeces spiked with strain AQ4037 . The primer pair R2-R6 was employed at an annealing temperature of 55°C . The estimated numbers of viable cells of AQ4037 in the initial PCR mixtures were 0 (not spiked, lane 2), 1 . 2 (lane 3), 1 . 2 x 10 (lane 4), 12 x 102 (lane 5), 12 x 10 3 (lane 6), 12 x 10° (lane 7), 12 x 10 5 (lane 8), 12 x 106 (lane 9) . PCR amplification was performed on the spiked samples directly ((a) and (b)], on the DNA extracted from the spiked samples [(c) and (d)], and on enriched cultures of the spiked samples [(e) and (f)] . The expected positions of the specific amplicons relative to those of the molecular weight markers (Hin cll-digested 0X174 DNA, lanes 1) are indicated by arrows .

tion method ; a single viable cell in a faecal sample

32% sequence divergence"-' 9) prevented establish-

could be detected by this method (Fig . 3e, lane 3 and Fig . 3f, lane 3) . No amplicon was observed with

ment of a primer pair for simultaneous detections of the tdh and trh genes, but minor sequence variations

unspiked faeces (Figs 3a-3f, lane 2), confirming the

within each gene group (tdh and trh) were overcome

specificity of the established PCR protocol .

by extensive screening of primer pairs and annealing

DISCUSSION The purpose of this study was to establish PCR protocols to detect tdh- and/or trh gene-positive strains of V. parahaemolyticus . The main problem in achieving this goal was variations in the nucleotide sequences of the genes . Considerable differences between the sequences of the tdh and trh genes (31-

temperatures . As in gene probe methods, separate PCR protocols for the tdh and trh genes were established . The nucleotide sequences of the tdh genes of V. parahaemolyticus so far reported",",' 3-15 show relatively small sequence diversity (less than 3 . 3%) . Nevertheless, four of the seven primer pairs targeted at the conserved regions were unsuccessful in an initial screening test at an annealing temperature of 55 ° C . This temperature appeared considerably stringent because it was very close to the calculated Td

486

J. Tada et al.

values (temperatures at which 50% of the primer is expected to be dissociated 26) of most primers (Table 2) . The annealings of the three effective primer pairs for the tdh gene were also carried out at this temperature in the established protocol, and this condition proved suitable . The established PCR protocol for the tdh gene was specific for various tdh genes, including those of V. cholerae non-O1 and V. mimicus having 97.0-98 . 6% homologies with the tdh 2 gene 27,29 (Table 1), and this protocol gave sufficient sensitivity (Fig. 2) . The trh genes are known to have more sequence variations than the tdh genes ; the sequences of the trh 1 and trh 2 genes, representatives of the two subgroups of the trh gene, differ by as much as 16% and show significant strain-to-strain sequence variations in some regions ." This sub-grouping of the trh genes based on hybridization analyses with trh 1- and trh 2specific DNA (=polynucleotide) probes 19 was supported by the present results . The trh 1-specific primer pair R3-R5 employed at an annealing temperature of 60 ° C detected trh 1-positive strains with very high specificity (Table 4) . The fact that two exceptional trh 2-positive strains were detected by this trh 1-specific PCR (Table 4) suggests considerable sequence divergence of the trh 2 gene in these strains . Despite significant sequence variations in the trh gene, the PCR with the primer pair R2-R6 and an annealing temperature of 55 °C specifically detected all trh (trh 1 and trh 2) gene-positive strains (Tables 1 and 4) and this PCR protocol was as sensitive as the PCR protocol for the tdh gene (Fig. 2). The results of spike experiments demonstrated the need for either DNA extraction from faeces or enrichment of the faecal sample in alkaline peptone water for 4 h prior to PCR; the sensitivities of the established PCR protocols were reduced by a factor of 10 4-105 by an inhibitor(s) present in undiluted normal faeces . The sensitivities with DNA extracted from spiked faeces (Fig. 3) were higher than those with DNA extracted from pure bacterial cultures (Fig . 2) when compared on the basis of viable cell numbers (c . 4 fg DNA per cell) . This discrepancy is probably because the cultures used for spiking the faeces contained not only viable cells but also DNA released from autolysed cells. A possible alternative to DNA extraction and enrichment culture is dilution of faecal samples prior to PCR, because PCR reaction is not inhibited when normal faeces are diluted by a factor of more than 104 (Tada, J & Shirasaki, S ., unpublished observation) . If a faecal sample contains 10 8 viable cells per 1 g, as few as 5 cells would be present in the PCR mixture and their DNA could be detected by PCR . But if the faecal samples contain fewer than 10' cells per 1 g, faecal dilution would probably not be a practical

method . Further studies on faecal samples from patients suffering from V. parahaemolyticus infection are necessary to examine this partricular point.

ACKNOWLEDGMENTS We are indebted to Tetsuichi Hontani, Motosada Kiri and Tatsuo Sato of Shimadzu Corporation of Japan for their continuous encouragement and suggestions . We also thank Tomoko Nakayama, Kumiko Akioka, Masako Tokushige, Nahomi Yamaguchi and Toshiko Nagai for technical shige, assistance . This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 . Fujino, T., Sakaguchi, G ., Sakazaki, R . & Takeda, Y . (eds) (1974) . International Symposium on Vibrio parahaemolyticus . Tokyo : Saikon Publishing Co . 2 . Joseph, S. W ., Colwell, R. R . & Kaper, J . B . (1982). Vibrio parahaemolyticus and related halophilic vibrios . CRC Critical Reviews in Microbiology 10, 77-124 . 3 . Miyamoto, Y ., Kato, T :, Obara, Y ., Akiyama, S ., Takizawa, K . & Yamai, S . (1969) . In vitro hemolytic characteristics of Vibrio parahaemolyticus: its close correlation with human pathogenicity . Journal of Bacteriology 100,1147-9 . 4. Sakazaki, R ., Tamura, K ., Kato, T ., Obara, Y ., Yamai, S . & Hobo, K . (1968) . Studies on the enteropathogenic, facultatively halophic bacteria, Vibrio parahaemolyticus . III . Enteropathogenicity . Japanese Journal of Medical Science and Biology 21, 325-31 . 5 . Sakurai, J ., Matsuzaki, A ., Takeda, Y . & Miwatani, T . (1974). Existence of two distinct hemolysins in Vibrio parahaemolyticus . Infection and Immunity 9, 777-80. 6 . Honda, T ., Chearskul, S., Takeda, Y . & Miwatani, T . (1980) . Immunological methods for detection of Kanagawa phenomenon of Vibrio parahaemolyticus . Journal of Clinical Microbiology 11, 600-3 . 7 . Honda, T ., Yoh, M ., Kongmuang, U . & Miwatani, T . (1985) . Enzyme-linked immunosorbent assays for detection of thermostable direct hemolysin of Vibrio parahaemolyticus . Journal of Clinical Microbiology 22, 383-6 . 8 . Oku, Y ., Uesaka, Y., Hirayama, T . & Takeda, Y. (1988) . Development of a highly sensitive bead-ELISA to detect bacterial protein toxins . Microbiology and Immunology 32, 807-16 . 9 . Nishibuchi, M ., Ishibashi, M ., Takeda, Y . & Kaper, J . B. (1985) . Detection of the thermostable direct hemolysin gene and related DNA sequence in Vibrio parahaemolyticus and other Vibrio species by DNA colony hybridization test. Infection and Immunity 49, 481-6 . 10 . Baba, K ., Shirai, H ., Terai, A., Takeda, Y . & Nishibuchi, M. (1991) . Analysis of the tdh gene cloned from a tdh gene and trh gene-positive strain of Vibrio parahaemolyticus . Microbiology and Immunology 35, 253-8 . 11 . Nishibuchi, M . & Kaper, J . B. (1990) . Duplication and variation of the thermostable direct haemolysin (tdh) gene in Vibrio parahaemolyticus . Molecular Microbiology 4, 87-99 .



PCR for tdh and trh genes of Vibrio parahaemolyticus

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21 . Shirai, H ., Nishibuchi, M ., Ramamurthy, T ., Blhattacharya, S . K ., Pal, S . C . & Takeda . Y . (1991) . Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae. Journal of Clinical Microbiology 29, 2517-21 . 22 . Maniatis, T., Fritsch, E. F. & Sambrook, J . (1982). Molecular Cloning . A Laboratory Manual. pp . 436-478 Cold Spring Harbor, NY : Cold Spring Harbor Laboratory . 23 . Miller, J . H . (1972) . Experiments in Molecular Genetics. p . 433 . Cold Spring Harbor, NY : Cold Spring Harbor Laboratory . 24 . Leininger, H . V . (1976) . Equipment, media, reagents, routine tests and strains . In : Compendium of Methods for Microbiological Examination of Foods. (Speck, M. L. ed .) pp . 10-94 . Washington, DC : American Public Health Association . 25 . Nishibuchi, M ., Kumagai, K . & Kaper, J . B . (1991) . Contribution of the tdh 1 gene of Kanagawa phenomenon-positive Vibrio parahaemolyticus to production of extracellular thermostable direct hemolysin . Microbial Pathogenesis 11, 453-60 . 26 . Suggs, S . V ., Hirose, T ., Miyake, T., Kawashima, E . H ., Johnson, M . J ., Itakura, K . & Wallace, R . B . (1981) . Use of synthetic oligodeoxyribonucleotides for the isolation of specific cloned DNA sequences . In : Developmental Biology Using Purified Genes . (Brown, D . D . & Fox, C . F ., eds) pp . 683-93 . New York : Academic Press . 27 . Baba, K ., Shirai, H ., Terai, A ., Kumagai, K ., Takeda, Y . & Nishibuchi, M . (1991) . Similarity of the tdh gene-bearing plasmids of Vibrio cholera non-O1 and Vibrio parahaemolyticus . Microbial Pathogenesis 10, 61-70 . 28 . Nishibuchi, M ., Khaeomanee-iam, V ., Honda, T ., Kaper, J. B. & Miwatani, T. (1990) . Comparative analysis of the hemolysin genes of Vibrio cholerae non-O1, V. mimicus and V. hollisae that are similar to the tdh gene of V. parahaemolyticus . FEMS Microbiology Letters 67, 251-6 . 29 . Terai, A ., Shirai, H ., Yoshida, 0 ., Takeda, Y. & Nishibuchi, M . (1990) . Nucleotide sequence of the thermostable direct hemolysin gene (tdh gene) of Vibrio mimicus and its evolutionary relationship with the tdh genes of Vibrio parahaemolyticus . FEMS Microbiology Letters 71, 319-24.