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Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification: application to detection of the tdh gene of Vibrio parahaemolyticus
Molecular and Cellular Probes (1992) 6, 48 9-494
Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification : application to detection of the tdh gene of Vibrio
parahaemolyticus Jun Tada, 1 Tetsuo Ohashi,' Naoyuki Nishimura,' Hiroko Ozaki,' Shigeru Fukushima,l * Jun Takano, l Mitsuaki Nishibuchi 2 and Yoshifumi Takeda 2
'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 28 July 1992) A non-isotopic microtitre plate-based assay method was devised for detection of products of the polymerase chain reaction . This assay involves affinity immobilization of the biotinylated amplification products in microtitre plate wells and their fluorescence detection by their hybridization with an oligonucleotide probe linked to alkaline phosphatase . An advantage of this procedure is that the immobilization and hybridization are carried out simultaneously in the wells, thus shortening the assay time. The assay method was applied to the detection of the tdh gene of Vibrio parahaemolyticus . Seven copies of the target chromosome could be detected in about 45 min after 35 cycles of amplification .
Vibrio parahaemolyticus, thermostable direct hemolysin gene, PCR, microtitre plate, alkaline-phosphatase labelled oligonucleotide .
KEYWORDS :
INTRODUCTION Recently we employed the polymerase chain reaction (PCR) for detection of the virulent strains of Vibrio parahaemolyticus,' and established PCR protocols for specific detection of virulence marker genes of this organism, i .e . the tdh and trh genes encoding, respectively, thermostable direct hemolysin and thermostable direct hemolysin-related hemolysin .2 In the previous work,' the PCR products (amplicons) were detected by agarose gel electrophoresis, which is a cumbersome and time-consuming procedure . We wished to improve the procedure for detecting amplicons so that our PCR method could be used easily in clinical laboratories . A non-isotopic method combined with an automation system employing a 96-
well microtitre plate seems promising for this purpose .' In the present study, we developed a rapid, nonisotopic method using microtitre plates to detect amplicons and . demonstrated its utility by applying it to the PCR for detection of the tdh gene of V. parahaemolyticus .
MATERIALS AND METHODS Bacterial strains The 23 strains of V. parahaemolyticus used in this
* Author to whom correspondence should be addressed .
0890-8508/92/060489 + 06 $08 .00/0
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© 1992 Academic Press Limited
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J . Tada et al. Table 1 . Detection by fluorescence of PCR-amplified sequences of the tdh genes of various strains of Vibrio parahaemolyticus Strain
WP1 AQ3115 AQ3948 AQ3158 GH-266 AQ3640 AQ3521 GH-284 AQ3465 GH-286 AQ3242 AQ3776 AQ3853 AQ4050 GH-257 AQ3295 AQ4037 AT-4 AQ3960 AQ3592 AQ3626 BG-31 BG-56
Presence or absence of* tdh gene trh gene
+ + + + + + + + + + + + + + + + -
-
+ + + + + + + + + + -
Detection by fluorescence of PCR-amplified tdh gene sequence' 213 . 12 332 . 70 334 . 79 320 . 44 143 . 87 356 . 41 181 . 54 321 . 00 162 .82 345 . 33 363 . 20 153. 60 349-47 158.88 349. 17 353.95 5 . 65 6 . 91 8 .44 7 . 63 10. 45 8. 31 6.75
* Detected by DNA colony blot hybridization with specific gene probes ." t The supernatants of heated broth cultures (prepared as described in the text) were used as PCR templates. Amplicons were detected after an immobilization-hybridization time of 15 min .
study are listed in Table 1 . These strains are laboratory stock strains . The presence or absence of the tdh and trh genes in these strains were determined by DNA colony blot hybridization tests with specific DNA probes for the tdh and trh genes in previous studies.','
fuged at 5000 rev/min for 1 min, and 3 p1 of the supernatant (corresponding to about 3 X 10 4 cfu of the original culture) was used as a template for PCR amplification .
Biotinylation of a primer and PCR amplification Preparation of DNA template from bacterial cells Vibrio parahaemolyticus strains were grown overnight in L broth at 37 ° C . Cellular DNA of strain WP1 was extracted and purified by standard procedures using phenol and chloroform .' Specified amounts of purified DNA dissolved in 10 mm Tris-1 mm EDTA solution (pH 8. 0) were used as templates for PCR amplification . For heat extraction of DNA from the cells, a 10 µl portion of the broth culture (c .108 cfu ml - ') was added to 90 pl of 10 mm Tris1 mm EDTA solution (pH &0) and the mixture was heated at 95 ° C for 5 min . The mixture was centri-
The pair of primers designated D5 (5'-GGTACTAAATGGCTGACATC-3') and D3 (5'-CCACTACCACTCTCATATGC-3'), which were shown to specifically amplify a 251 by region of the tdh gene' were employed . Primer D5 (sense primer) was obtained and biotinylated at the 5' end as follows . Twenty-five nanomoles of D5 oligonucleotide conjugated with hexylamine at the 5' end (Toray Research Center, Kamakura, Japan) was dissolved in 150µl of 0 . 5 m phosphate buffered saline (PBS, pH 7 . 4) and mixed with 75 pl of 30 mm biotin-N-hydroxysuccinimide ester (GIBCO BRL, Gaithersburg, MD, USA) solution in N, N-dimethylformamide . After overnight incubation at room temperature, the reaction mixture was
New method for detecting products of PCR amplification passed through a PD-10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden), swollen with 20% ethanol . The first 3 ml fraction was collected and lyophilized . Primer D3 was synthesized by a R-cyanoethyl phosphoramidite method in an automated DNA synthesizer (Cyclone plus" DNA synthesizer, Milligen, Burlington, MA, USA) and purified in a highperformance liquid chromatography apparatus (LC6A, Shimadzu Co ., Kyoto, Japan) equipped with a reversed phase column (Cosmosil C18, Nacalai Tesque, Kyoto, Japan) . Thirty-five cycles of amplification with the D3 primer and the biotinylated D5 primer at an annealing temperature of 55 ° C was carried out as described previously .'
Oligonucleotide probe A 19-mer oligonucleotide probe, which detects the biotinylated amplicons (sense strands) by hybridization at a position 52 bases from the 3' end of the amplicons, was synthesized . The sequence of this probe was identical with that of one of the primers (designated D2 ; 5'-CCAAATACATTTTACTTGG-3') which we used previously for PCR amplification of the tdh gene sequence, and the results of the PCR showed that the D2 sequence is well conserved in the tdh gene.' This oligonucleotide was linked at the 5' end to alkaline phosphatase from calf intestine via a crosslinker and a spacer by the method reported previously .'
Preparation of microtitre plates The wells of microtitre plates (Nunc, Roskilde, Denmark) were coated with streptavidin as follows . Fiftytwo milligrams of bovine serum albumin (BSA, fraction V, Sigma Chemical Co ., St . Louis, MO, USA) dissolved in 600 µl of 0 . 25 M PBS was mixed with 100 µl of 15 mm s-amidocaproatebiotin-N-hydroxysuccinimide (GIBCO BRL) solution in N, N-dimethylformamide . After overnight incubation at room temperature, the reaction mixture was passed through a PD-10 column swollen with 40 mm PBS and the first 2 ml fraction was collected for use as biotinylated BSA . The microtitre wells were treated with 200 p1 of biotinylated BSA in 015 M PBS (0 . 5 OD at 280 nm per well) overnight at 4°C and then with 1 % BSA in 0 . 15 M PBS overnight at 4°C . The wells were then washed twice with wash solution (05% Tween 20, 015 M PBS) . The biotinylated BSA surfaces were saturated with 20 gg ml - ' streptavidin (GIBCO BRL) in 0 . 5% Tween 20, 015% BSA and 0-15m PBS by incubation for at least 1 night at 4 ° C and then were
491
washed twice with 200[d of 0-15m PBS containing 0 . 5% Tween 20 for 1 min .
Detection of biotinylated amplicons by hybridization in the microtitre plate A 2 µl portion of the PCR amplification reaction mixture was dissolved in 50 µl of 10 mm Tris-1 mm EDTA solution . This solution was heated at 95 °C for 5 min, cooled on ice and centrifuged (3000 rev/min) for 30 s . The supernatant (45 µp was transferred rapidly (within c . 5 min) to the streptavidin-coated microtitre plate containing 150 Jul of reaction buffer composed of 13 nM alkaline phosphatase-labelled oligonucleotide probe, 0 . 4 x SSC [1 x SSC is 015 M NaCl plus 0-015m sodium citrate (pH 7 .0)l, 0. 2% Tween 20, 0 . 03% BSA, 3 mm Tris and 0-3 mm EDTA . Immobilization of the amplicons in the well and hybridization with the probe were carried out simultaneously for the specified periods at room temperature . The well was washed sequentially with 2 .00 pl volumes of 0 . 15 M PBS containing 0 . 5% Tween 20 at room temperature for 1 min twice, at 50 ° C for 'I min and at room temperature for 1 min . Then the well was rinsed first with 0 . 15 M PBS, then with 0 . 2 M TrisHCI (pH 9. 5) containing 1 MM MgCI 2 . A volume of 200 µl of enzyme reaction buffer composed of 02 M Tris-HCI (pH 9 .0), 50 mm MgCI 2 , and 001 mm 4methylumbelliferyl phosphate (Sigma Chemical Co .) was added and the microtitre plate was incubated at 37° C for 15 min . The enzyme reaction was stopped by transferring the supernatant to a microtube containing 400 µl of 50 mm EDTA . Fluorescence of the solution was measured with a. fluorescence spectrophotometer (Model RF-5000, Shimadzu Co ., Kyoto, Japan) at 450 nm with excitation at 360 nm .
RESULTS AND DISCUSSION The assay method used in this study is shown schematically in Fig . 1 . The first step was PCR amplification using two primers, one of which was biotinylated at the 5'-end, and then heat denaturation of the arnplicons . The second step was simultaneous immobilization of the amplicons in the well of a microtitre plate and their hybridization with the oligonucleotide probe . The well of the microtitre plate had previously been coated with streptavidin and filled with a large amount of alkaline phosphatase-labelled oligonucleotide probe. After hybridization and immobilization, excess reagents, unhybridized oligonucleotide probe and untrapped amplicons were removed by
J . Tada et al .
49 2
PCR-amplified sequences L I
Oligonucleotide probe 100
111nl
I ` APjniiiir 80 d
cm
60-
U (A
0
40-
U-
Immobilization and hybridization in a microtitre well
20 -
30
60
120
90
Time (min)
Fig.
2.
Effect of the time of simultaneous immobilization
and hybridization on detection of the amplicons . PCR amplification was performed with 1-6 pg (c . 400 chromosomal copies) of cellular DNA extracted from Vibrio parahaemolyticus WP1 . The immobilization-
hybridization reaction was carried out for the indicated times and the hybridization signal expressed as fluorescence was measured . The fluorescence after a time of 120 min was taken as 100% . Results are mean f standard deviation for triplicated determinations .
Enzyme reaction (Addition of substrate and fluorometry)
Fig. 1 . Schematic diagram of the detection of PCR-amplified sequences (amplicons) . One of the PCR primers was biotinylated (B). Amplicons were denatured and added to a streptavidin (SA)-coated well of a microtitre plate . An oligonucleotide probe labelled with alkaline phosphatase (AP) was also added to the well . Immobilization of the amplicons by affinity between biotin and streptavidin and hybridization with the probe
104
were carried out simultaneously in the well . Activity of the alkaline phosphatase was measured fluorometrically .
B
103
washing the well . Finally a fluorescent substrate was then added, and the fluorescence of the supernatant was measured with a fluorescence spectropho-
U U
N
1 02
0
U-
tometer . Various properties of the assay were investigated using purified cellular DNA of V .
WP1 (tdh + )'
parahaemolyticus
as a template . First, PCR amplification 10
was performed using 1-6 pg of template DNA (corresponding to c . 400 chromosomal copies) . The reaction time for the simultaneous immobilization and hybridization was varied to determine the time needed to obtain the maximal hybridization signal (Fig . 2) . About 25% of the maximal hybridization signal was observed after 15 min reaction, and nearly all the maximal signal produced after 60 min reaction .
I i ud 0 (
I
i iimd l0
i unJ i uund i nuni 102
10 3
104
i nui~ 105
Amount of template DNA (no . of chromosomal copies)
The sensitivity of the assay was examined next (Fig . 3). Various amounts of template DNA, ranging from 4 fg to 80 pg (equivalent to 1-2
X
104 chromoso-
mal copies), were subjected to PCR amplification and the amplicons were detected after immobilizationhybridization times of 15 min (Fig . 3, curve A) and 60 min (Fig . 3, curve B) . The assay curves were linear
Fig. 3 Sensitivity of the assay. PCR amplification was performed with various amounts of template DNA (purified cellular DNA of Vibrio parahaemolyticus WP1) . The amplicons were detected after immobilizationhybridization times of 15 min (curve A) and 60 min (curve B) . Results are expressed as mean ± standard deviation for triplicate determinations .
New method for detecting products of PCR amplification for the template DNA in the range of 4 fg-1 . 6 pg (1-
400 chromosomal copies) with immobilization-hybridization times of both 15 and 60 min . The assay curves seem to be saturated with more than 1 . 6 pg of template DNA . Further study is needed to determine the reason for this saturation, but it seems most likely to be competition between the hybridization probe and the amplified antisense strand for the target sequence and/or saturation of the PCR reaction . When the detection cut-off value was set at the mean of the background signal (no template DNA) plus five times the standard deviation, the cut-off values were 21 . 46 (=8-36+5x2-65) and 81 . 17 (=58 . 67+5 x 4. 50) for 15 min and 60 min immobilization-hybridization, respectively. On the basis of these values, the detection limits for 15 min and 60 min immobilization-hybridizations were determined to be 7 and 2 chromosomal copies, respectively, from the assay curves . The results indicate that 15 min immobilization-hybridization provides sufficient sensitivity and that 60 min immobilizationhybridization leads mainly to an increase in the background signal . Therefore, we concluded that 15 min immobilization-hybridization has more practical value than 60 min immobilization-hybridization . The PCR procedure (the primer pair and the annealing temperature) employed in this study was previously shown to amplify the tdh gene but not the trh gene, which is c . 68% homologous to the tdh gene.' One of the primers was biotinylated at its 5' end in the present assay . In addition, in the present assay the amplicons are detected by their hybridization with an oligonucleotide probe linked to alkaline phosphatase. To confirm that the specificity of the PCR is maintained in the present assay and to investigate the performance of this assay, V . parahaemolyticus strains carrying the tdh and/or trh gene were examined . In this experiment, a new variable was introduced to assess the applicability of our assay to enrichment culture-based sample treatment ;' instead of phenol-chloroform extraction of DNA template from the sample, template DNA was extracted by simply heating the broth culture of the test strain and then used for PCR amplification . PCR amplicons were detected with an immobilizationhybridization time of 15 min . A total of 23 strains were tested (Table 1) . All 16 tdh gene-positive strains including WP1 gave strongly positive fluorescence readings (143 . 87-363 . 20) . All 7 tdh gene-negative strains including 5 trh gene-positive strains gave fluorescence readings (5 . 65-10 . 45) below the cutoff value described above (21 .46). The difference between the fluorescence readings for the tdh genepositive and negative strains was highly significant (P < 0 . 001) . Thus the presence of the trh gene in the
493
test strains did not appear to influence the results of the assay, indicating that this assay is very specific for the tdh gene . The fluorescence reading of WP1 (Table 1) corresponded to that given by c . 1 . 6 x 103 chromosomal copies extracted from WP1 by the phenol-chloroform method (determined from Fig . 3, curve A) . This result indicated that the template DNA obtained by heat extraction gave 1 log lower sensitivity than DNA extracted by the phenol-chloroform method . This may be due in part to the inefficiency of preparing the template DNA by the simple heating method or to inhibition of the immobilization, hybridization or enzyme reaction by impurities carried over from the bacterial culture . In a previous spike experiment,' we found that 4 h alkaline-peptone enrichment of a faecal sample followed by template DNA extraction by heating was a successful alternative to DNA extraction by the phenol-chloroform method for preparing PCR template for detection of the tdh gene .' Since the enrichment culture-heat treatment method yielded more (c . 1 log higher) amplicons than the phenol-chloroform extraction method,' the observed 1 log reduction in the sensitivity in the present assay due to DNA template extraction by heating would be compensated for if the present assay method were combined with enrichment culture-based treatment of the faecal samples. The assay method developed in this study is similar to that of Dahlen et al.' in that the amplicons are immobilized in microtitre wells by biotin-streptavidin affinity and that they are detected by hybridization with a non-isotopically labelled oligonucleotide probe . But in our method affinity-based immobilization of the amplicons and their hybridization with the probe are carried out simultaneously, thus shortening the detection time . In addition, unlike in the sandwich hybridization format of Dahlen et a!., 8 biotinylated PCR primers are employed in our method . This obviates the need to include a biotinylated oligonucleotide for immobilization of the amplicons . Furthermore, we employed the sensitive method of fluorometric detection in solution with the oligonucleotide probe linked to alkaline phosphatase .' In this work, we established a sensitive, rapid, nonisotopic, and microtitre-based assay for detection of the PCR product . The rapidity of our assay is attributable to the very high binding constant of the biotinavidin complex, to solution hybridization with a short oligonucleotide probe and to immobilization and hybridization of the amplicons simultaneously . Thus, by this procedure, a few copies of the target chromosome can be detected specifically in about 45 min (including 15 min for the immobilization-hybridization step) after 35 cycles of PCR amplification . As far
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J . Tada et al.
as we know, this assay method is faster than any other microtitre-based non-isotopic methods for detection of PCR products reported so far .'-' 8 Our assay method was designed for PCR amplification of template DNA extracted from clinical samples . This detection method could thus be applied to other PCR protocols than that for the tdh gene unless the DNA template extracted from the clinical sample contains a substantial amount of an inhibitor(s) of PCR amplification and/or amplicon detection . We are now trying to develop an automated system using our assay method .
ACKNOWLEDGEMENTS We are indebted to Tetsuichi Hontani, Motosada Kiri and Tatsuo Sato of Shimadzu Corporation of Japan for their continuous encouragement and suggestions . We are also grateful to Akira Murakami of Kyoto Institute of Technology for useful suggestions and discussions, and to Kumiko Akioka and Tomoko Nakayama for typing this manuscript . This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 . Tada, J., Ohashi, T ., Nishimura, N ., Shirasaki, Y ., Ozaki, H ., Fukushima, S ., Takano, J ., Nishibuchi, M . & Takeda, Y. (1992) . Detection of thermostable direct hemolysin (TDH) gene (tdh) and TDH-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction . Molecular and Cellular Probes 6, 477-87 . 2 . Shirai, H ., Ito, H ., Hirayama, Y ., Nakamoto, Y ., Nakabayashi, N ., Kumagai, K., Takeda, Y . & Nishibuchi, M. (1990) . Molecular epidemiologic evidence for association of thermostable direct hemolysin (TDH) and TDHrelated hemolysin of Vibrio parahaemolyticus with gastroenteritis . Infection and Immunity 58, 3568-73 . 3 . Persing, D . H . (1991) . Polymerase chain reaction : trenches to benches . Journal of Clinical Microbiology 29,1281-5 . 4 . 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 . 5 . Maniatis, T ., Fritsch, E . F . & Sambrook, J . (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, N .Y . : Cold Spring Harbor Laboratory . 6. Murakami, A ., Tada, J ., Yamagata, K . & Takano, J . (1990) . Highly sensitive detection of DNA using enzyme-linked DNA-probe . 1 . Colorimetric and fluorometric detection . Nucleic Acids Research 17, 5587-95 .
7 . Nishibuchi, M . & Kaper, J . B . (1990). Duplication and variation of the thermostable direct haemolysin Ztdh) gene in Vibrio parahaemolyticus . Molecular Microbiology 4, 87-99 . 8 . Dahlen, P . 0 ., litia, A . J ., Skagius, G ., Frostell, A ., Nunn, M. F . & Kwiatkowski, M . (1991). Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescencebased hybridization assay . Journal of Clinical Microbiology 29, 798-804. 9 . Bobo, L., Coutlee, F ., Yolken, R . H ., Quinn, T . & Viscidi, R . P . (1990) . Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay . Journal of Clinical Microbiology 28,1968-73 . 10 . Coutlee, F ., Saint-Antoine, P ., Olivier, C ., Voyer, H ., Kessous-Elbaz, A ., Berrada, F ., Begin, P ., Giroux, L . & Viscidi, R. (1991) . Evaluation of infection with human immunodeficiency virus type 1 by using non-isotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA . Journal of Clinical Microbiology 29, 2461-7 . 11 . Homes, E ., Wasteson, Y . & Olsvik, O . (1991) . Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimen by an immobilized, colorimetric, nested polymerase chain reaction . Journal of Clinical Microbiology 29, 2375-9 . 12 . Keller, G . H ., Huang, D .-P . & Manak, M. M. (1989) . A sensitive non-isotopic hybridization assay for HIV-1 DNA . Analytical Biochemistry 177, 27-32 . 13 . Keller, G . H ., Huang, D .-P. & Manak, M . M . (1991) . Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtitre wells . Journal of Clinical Microbiology 29, 638-41 . 14 . Keller, G . H ., Huang, D .-P., Shin, J . W .-K . & Manak, M . M . (1990) . Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization . Journal of Clinical Microbiology 28, 1441-6. 15 . Kemp, D . J ., Smith, D . B., Foote, S . J ., Samaras, N . & Peterson, M . G . (1989) . Colorimetric detection of specific DNA segments amplified by polymerase chain reactions . Proceedings of the National Academy of Sciences, USA . 86, 2423-7 . 16 . Landgraf . A ., Reckmann, B . & Pingoud, A . (1991) . Direct analysis of polymerase chain reaction products using enzyme-linked immunosorbent assay techniques. Analytical Biochemistry 198, 86-91 . 17. Mantero, G ., Zonaro, A ., Albertini, A ., Bertolo, P. & Primi, D . (1991) . DNA enzyme immunoassay : general method for detecting products of polymerase chain reaction . Clinical Chemistry 37, 422-9 . 18 . Nickerson, D . A ., Kaiser, R., Lappin, S ., Stewart, J ., Hood, L . & Landegren, U . (1990). Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay . Proceedings of the National Academy of Sciences, USA 87, 8923-7.
Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification : application to detection of the tdh gene of Vibrio
parahaemolyticus Jun Tada, 1 Tetsuo Ohashi,' Naoyuki Nishimura,' Hiroko Ozaki,' Shigeru Fukushima,l * Jun Takano, l Mitsuaki Nishibuchi 2 and Yoshifumi Takeda 2
'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 28 July 1992) A non-isotopic microtitre plate-based assay method was devised for detection of products of the polymerase chain reaction . This assay involves affinity immobilization of the biotinylated amplification products in microtitre plate wells and their fluorescence detection by their hybridization with an oligonucleotide probe linked to alkaline phosphatase . An advantage of this procedure is that the immobilization and hybridization are carried out simultaneously in the wells, thus shortening the assay time. The assay method was applied to the detection of the tdh gene of Vibrio parahaemolyticus . Seven copies of the target chromosome could be detected in about 45 min after 35 cycles of amplification .
Vibrio parahaemolyticus, thermostable direct hemolysin gene, PCR, microtitre plate, alkaline-phosphatase labelled oligonucleotide .
KEYWORDS :
INTRODUCTION Recently we employed the polymerase chain reaction (PCR) for detection of the virulent strains of Vibrio parahaemolyticus,' and established PCR protocols for specific detection of virulence marker genes of this organism, i .e . the tdh and trh genes encoding, respectively, thermostable direct hemolysin and thermostable direct hemolysin-related hemolysin .2 In the previous work,' the PCR products (amplicons) were detected by agarose gel electrophoresis, which is a cumbersome and time-consuming procedure . We wished to improve the procedure for detecting amplicons so that our PCR method could be used easily in clinical laboratories . A non-isotopic method combined with an automation system employing a 96-
well microtitre plate seems promising for this purpose .' In the present study, we developed a rapid, nonisotopic method using microtitre plates to detect amplicons and . demonstrated its utility by applying it to the PCR for detection of the tdh gene of V. parahaemolyticus .
MATERIALS AND METHODS Bacterial strains The 23 strains of V. parahaemolyticus used in this
* Author to whom correspondence should be addressed .
0890-8508/92/060489 + 06 $08 .00/0
489
© 1992 Academic Press Limited
490
J . Tada et al. Table 1 . Detection by fluorescence of PCR-amplified sequences of the tdh genes of various strains of Vibrio parahaemolyticus Strain
WP1 AQ3115 AQ3948 AQ3158 GH-266 AQ3640 AQ3521 GH-284 AQ3465 GH-286 AQ3242 AQ3776 AQ3853 AQ4050 GH-257 AQ3295 AQ4037 AT-4 AQ3960 AQ3592 AQ3626 BG-31 BG-56
Presence or absence of* tdh gene trh gene
+ + + + + + + + + + + + + + + + -
-
+ + + + + + + + + + -
Detection by fluorescence of PCR-amplified tdh gene sequence' 213 . 12 332 . 70 334 . 79 320 . 44 143 . 87 356 . 41 181 . 54 321 . 00 162 .82 345 . 33 363 . 20 153. 60 349-47 158.88 349. 17 353.95 5 . 65 6 . 91 8 .44 7 . 63 10. 45 8. 31 6.75
* Detected by DNA colony blot hybridization with specific gene probes ." t The supernatants of heated broth cultures (prepared as described in the text) were used as PCR templates. Amplicons were detected after an immobilization-hybridization time of 15 min .
study are listed in Table 1 . These strains are laboratory stock strains . The presence or absence of the tdh and trh genes in these strains were determined by DNA colony blot hybridization tests with specific DNA probes for the tdh and trh genes in previous studies.','
fuged at 5000 rev/min for 1 min, and 3 p1 of the supernatant (corresponding to about 3 X 10 4 cfu of the original culture) was used as a template for PCR amplification .
Biotinylation of a primer and PCR amplification Preparation of DNA template from bacterial cells Vibrio parahaemolyticus strains were grown overnight in L broth at 37 ° C . Cellular DNA of strain WP1 was extracted and purified by standard procedures using phenol and chloroform .' Specified amounts of purified DNA dissolved in 10 mm Tris-1 mm EDTA solution (pH 8. 0) were used as templates for PCR amplification . For heat extraction of DNA from the cells, a 10 µl portion of the broth culture (c .108 cfu ml - ') was added to 90 pl of 10 mm Tris1 mm EDTA solution (pH &0) and the mixture was heated at 95 ° C for 5 min . The mixture was centri-
The pair of primers designated D5 (5'-GGTACTAAATGGCTGACATC-3') and D3 (5'-CCACTACCACTCTCATATGC-3'), which were shown to specifically amplify a 251 by region of the tdh gene' were employed . Primer D5 (sense primer) was obtained and biotinylated at the 5' end as follows . Twenty-five nanomoles of D5 oligonucleotide conjugated with hexylamine at the 5' end (Toray Research Center, Kamakura, Japan) was dissolved in 150µl of 0 . 5 m phosphate buffered saline (PBS, pH 7 . 4) and mixed with 75 pl of 30 mm biotin-N-hydroxysuccinimide ester (GIBCO BRL, Gaithersburg, MD, USA) solution in N, N-dimethylformamide . After overnight incubation at room temperature, the reaction mixture was
New method for detecting products of PCR amplification passed through a PD-10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden), swollen with 20% ethanol . The first 3 ml fraction was collected and lyophilized . Primer D3 was synthesized by a R-cyanoethyl phosphoramidite method in an automated DNA synthesizer (Cyclone plus" DNA synthesizer, Milligen, Burlington, MA, USA) and purified in a highperformance liquid chromatography apparatus (LC6A, Shimadzu Co ., Kyoto, Japan) equipped with a reversed phase column (Cosmosil C18, Nacalai Tesque, Kyoto, Japan) . Thirty-five cycles of amplification with the D3 primer and the biotinylated D5 primer at an annealing temperature of 55 ° C was carried out as described previously .'
Oligonucleotide probe A 19-mer oligonucleotide probe, which detects the biotinylated amplicons (sense strands) by hybridization at a position 52 bases from the 3' end of the amplicons, was synthesized . The sequence of this probe was identical with that of one of the primers (designated D2 ; 5'-CCAAATACATTTTACTTGG-3') which we used previously for PCR amplification of the tdh gene sequence, and the results of the PCR showed that the D2 sequence is well conserved in the tdh gene.' This oligonucleotide was linked at the 5' end to alkaline phosphatase from calf intestine via a crosslinker and a spacer by the method reported previously .'
Preparation of microtitre plates The wells of microtitre plates (Nunc, Roskilde, Denmark) were coated with streptavidin as follows . Fiftytwo milligrams of bovine serum albumin (BSA, fraction V, Sigma Chemical Co ., St . Louis, MO, USA) dissolved in 600 µl of 0 . 25 M PBS was mixed with 100 µl of 15 mm s-amidocaproatebiotin-N-hydroxysuccinimide (GIBCO BRL) solution in N, N-dimethylformamide . After overnight incubation at room temperature, the reaction mixture was passed through a PD-10 column swollen with 40 mm PBS and the first 2 ml fraction was collected for use as biotinylated BSA . The microtitre wells were treated with 200 p1 of biotinylated BSA in 015 M PBS (0 . 5 OD at 280 nm per well) overnight at 4°C and then with 1 % BSA in 0 . 15 M PBS overnight at 4°C . The wells were then washed twice with wash solution (05% Tween 20, 015 M PBS) . The biotinylated BSA surfaces were saturated with 20 gg ml - ' streptavidin (GIBCO BRL) in 0 . 5% Tween 20, 015% BSA and 0-15m PBS by incubation for at least 1 night at 4 ° C and then were
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washed twice with 200[d of 0-15m PBS containing 0 . 5% Tween 20 for 1 min .
Detection of biotinylated amplicons by hybridization in the microtitre plate A 2 µl portion of the PCR amplification reaction mixture was dissolved in 50 µl of 10 mm Tris-1 mm EDTA solution . This solution was heated at 95 °C for 5 min, cooled on ice and centrifuged (3000 rev/min) for 30 s . The supernatant (45 µp was transferred rapidly (within c . 5 min) to the streptavidin-coated microtitre plate containing 150 Jul of reaction buffer composed of 13 nM alkaline phosphatase-labelled oligonucleotide probe, 0 . 4 x SSC [1 x SSC is 015 M NaCl plus 0-015m sodium citrate (pH 7 .0)l, 0. 2% Tween 20, 0 . 03% BSA, 3 mm Tris and 0-3 mm EDTA . Immobilization of the amplicons in the well and hybridization with the probe were carried out simultaneously for the specified periods at room temperature . The well was washed sequentially with 2 .00 pl volumes of 0 . 15 M PBS containing 0 . 5% Tween 20 at room temperature for 1 min twice, at 50 ° C for 'I min and at room temperature for 1 min . Then the well was rinsed first with 0 . 15 M PBS, then with 0 . 2 M TrisHCI (pH 9. 5) containing 1 MM MgCI 2 . A volume of 200 µl of enzyme reaction buffer composed of 02 M Tris-HCI (pH 9 .0), 50 mm MgCI 2 , and 001 mm 4methylumbelliferyl phosphate (Sigma Chemical Co .) was added and the microtitre plate was incubated at 37° C for 15 min . The enzyme reaction was stopped by transferring the supernatant to a microtube containing 400 µl of 50 mm EDTA . Fluorescence of the solution was measured with a. fluorescence spectrophotometer (Model RF-5000, Shimadzu Co ., Kyoto, Japan) at 450 nm with excitation at 360 nm .
RESULTS AND DISCUSSION The assay method used in this study is shown schematically in Fig . 1 . The first step was PCR amplification using two primers, one of which was biotinylated at the 5'-end, and then heat denaturation of the arnplicons . The second step was simultaneous immobilization of the amplicons in the well of a microtitre plate and their hybridization with the oligonucleotide probe . The well of the microtitre plate had previously been coated with streptavidin and filled with a large amount of alkaline phosphatase-labelled oligonucleotide probe. After hybridization and immobilization, excess reagents, unhybridized oligonucleotide probe and untrapped amplicons were removed by
J . Tada et al .
49 2
PCR-amplified sequences L I
Oligonucleotide probe 100
111nl
I ` APjniiiir 80 d
cm
60-
U (A
0
40-
U-
Immobilization and hybridization in a microtitre well
20 -
30
60
120
90
Time (min)
Fig.
2.
Effect of the time of simultaneous immobilization
and hybridization on detection of the amplicons . PCR amplification was performed with 1-6 pg (c . 400 chromosomal copies) of cellular DNA extracted from Vibrio parahaemolyticus WP1 . The immobilization-
hybridization reaction was carried out for the indicated times and the hybridization signal expressed as fluorescence was measured . The fluorescence after a time of 120 min was taken as 100% . Results are mean f standard deviation for triplicated determinations .
Enzyme reaction (Addition of substrate and fluorometry)
Fig. 1 . Schematic diagram of the detection of PCR-amplified sequences (amplicons) . One of the PCR primers was biotinylated (B). Amplicons were denatured and added to a streptavidin (SA)-coated well of a microtitre plate . An oligonucleotide probe labelled with alkaline phosphatase (AP) was also added to the well . Immobilization of the amplicons by affinity between biotin and streptavidin and hybridization with the probe
104
were carried out simultaneously in the well . Activity of the alkaline phosphatase was measured fluorometrically .
B
103
washing the well . Finally a fluorescent substrate was then added, and the fluorescence of the supernatant was measured with a fluorescence spectropho-
U U
N
1 02
0
U-
tometer . Various properties of the assay were investigated using purified cellular DNA of V .
WP1 (tdh + )'
parahaemolyticus
as a template . First, PCR amplification 10
was performed using 1-6 pg of template DNA (corresponding to c . 400 chromosomal copies) . The reaction time for the simultaneous immobilization and hybridization was varied to determine the time needed to obtain the maximal hybridization signal (Fig . 2) . About 25% of the maximal hybridization signal was observed after 15 min reaction, and nearly all the maximal signal produced after 60 min reaction .
I i ud 0 (
I
i iimd l0
i unJ i uund i nuni 102
10 3
104
i nui~ 105
Amount of template DNA (no . of chromosomal copies)
The sensitivity of the assay was examined next (Fig . 3). Various amounts of template DNA, ranging from 4 fg to 80 pg (equivalent to 1-2
X
104 chromoso-
mal copies), were subjected to PCR amplification and the amplicons were detected after immobilizationhybridization times of 15 min (Fig . 3, curve A) and 60 min (Fig . 3, curve B) . The assay curves were linear
Fig. 3 Sensitivity of the assay. PCR amplification was performed with various amounts of template DNA (purified cellular DNA of Vibrio parahaemolyticus WP1) . The amplicons were detected after immobilizationhybridization times of 15 min (curve A) and 60 min (curve B) . Results are expressed as mean ± standard deviation for triplicate determinations .
New method for detecting products of PCR amplification for the template DNA in the range of 4 fg-1 . 6 pg (1-
400 chromosomal copies) with immobilization-hybridization times of both 15 and 60 min . The assay curves seem to be saturated with more than 1 . 6 pg of template DNA . Further study is needed to determine the reason for this saturation, but it seems most likely to be competition between the hybridization probe and the amplified antisense strand for the target sequence and/or saturation of the PCR reaction . When the detection cut-off value was set at the mean of the background signal (no template DNA) plus five times the standard deviation, the cut-off values were 21 . 46 (=8-36+5x2-65) and 81 . 17 (=58 . 67+5 x 4. 50) for 15 min and 60 min immobilization-hybridization, respectively. On the basis of these values, the detection limits for 15 min and 60 min immobilization-hybridizations were determined to be 7 and 2 chromosomal copies, respectively, from the assay curves . The results indicate that 15 min immobilization-hybridization provides sufficient sensitivity and that 60 min immobilizationhybridization leads mainly to an increase in the background signal . Therefore, we concluded that 15 min immobilization-hybridization has more practical value than 60 min immobilization-hybridization . The PCR procedure (the primer pair and the annealing temperature) employed in this study was previously shown to amplify the tdh gene but not the trh gene, which is c . 68% homologous to the tdh gene.' One of the primers was biotinylated at its 5' end in the present assay . In addition, in the present assay the amplicons are detected by their hybridization with an oligonucleotide probe linked to alkaline phosphatase. To confirm that the specificity of the PCR is maintained in the present assay and to investigate the performance of this assay, V . parahaemolyticus strains carrying the tdh and/or trh gene were examined . In this experiment, a new variable was introduced to assess the applicability of our assay to enrichment culture-based sample treatment ;' instead of phenol-chloroform extraction of DNA template from the sample, template DNA was extracted by simply heating the broth culture of the test strain and then used for PCR amplification . PCR amplicons were detected with an immobilizationhybridization time of 15 min . A total of 23 strains were tested (Table 1) . All 16 tdh gene-positive strains including WP1 gave strongly positive fluorescence readings (143 . 87-363 . 20) . All 7 tdh gene-negative strains including 5 trh gene-positive strains gave fluorescence readings (5 . 65-10 . 45) below the cutoff value described above (21 .46). The difference between the fluorescence readings for the tdh genepositive and negative strains was highly significant (P < 0 . 001) . Thus the presence of the trh gene in the
493
test strains did not appear to influence the results of the assay, indicating that this assay is very specific for the tdh gene . The fluorescence reading of WP1 (Table 1) corresponded to that given by c . 1 . 6 x 103 chromosomal copies extracted from WP1 by the phenol-chloroform method (determined from Fig . 3, curve A) . This result indicated that the template DNA obtained by heat extraction gave 1 log lower sensitivity than DNA extracted by the phenol-chloroform method . This may be due in part to the inefficiency of preparing the template DNA by the simple heating method or to inhibition of the immobilization, hybridization or enzyme reaction by impurities carried over from the bacterial culture . In a previous spike experiment,' we found that 4 h alkaline-peptone enrichment of a faecal sample followed by template DNA extraction by heating was a successful alternative to DNA extraction by the phenol-chloroform method for preparing PCR template for detection of the tdh gene .' Since the enrichment culture-heat treatment method yielded more (c . 1 log higher) amplicons than the phenol-chloroform extraction method,' the observed 1 log reduction in the sensitivity in the present assay due to DNA template extraction by heating would be compensated for if the present assay method were combined with enrichment culture-based treatment of the faecal samples. The assay method developed in this study is similar to that of Dahlen et al.' in that the amplicons are immobilized in microtitre wells by biotin-streptavidin affinity and that they are detected by hybridization with a non-isotopically labelled oligonucleotide probe . But in our method affinity-based immobilization of the amplicons and their hybridization with the probe are carried out simultaneously, thus shortening the detection time . In addition, unlike in the sandwich hybridization format of Dahlen et a!., 8 biotinylated PCR primers are employed in our method . This obviates the need to include a biotinylated oligonucleotide for immobilization of the amplicons . Furthermore, we employed the sensitive method of fluorometric detection in solution with the oligonucleotide probe linked to alkaline phosphatase .' In this work, we established a sensitive, rapid, nonisotopic, and microtitre-based assay for detection of the PCR product . The rapidity of our assay is attributable to the very high binding constant of the biotinavidin complex, to solution hybridization with a short oligonucleotide probe and to immobilization and hybridization of the amplicons simultaneously . Thus, by this procedure, a few copies of the target chromosome can be detected specifically in about 45 min (including 15 min for the immobilization-hybridization step) after 35 cycles of PCR amplification . As far
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as we know, this assay method is faster than any other microtitre-based non-isotopic methods for detection of PCR products reported so far .'-' 8 Our assay method was designed for PCR amplification of template DNA extracted from clinical samples . This detection method could thus be applied to other PCR protocols than that for the tdh gene unless the DNA template extracted from the clinical sample contains a substantial amount of an inhibitor(s) of PCR amplification and/or amplicon detection . We are now trying to develop an automated system using our assay method .
ACKNOWLEDGEMENTS We are indebted to Tetsuichi Hontani, Motosada Kiri and Tatsuo Sato of Shimadzu Corporation of Japan for their continuous encouragement and suggestions . We are also grateful to Akira Murakami of Kyoto Institute of Technology for useful suggestions and discussions, and to Kumiko Akioka and Tomoko Nakayama for typing this manuscript . This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 . Tada, J., Ohashi, T ., Nishimura, N ., Shirasaki, Y ., Ozaki, H ., Fukushima, S ., Takano, J ., Nishibuchi, M . & Takeda, Y. (1992) . Detection of thermostable direct hemolysin (TDH) gene (tdh) and TDH-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction . Molecular and Cellular Probes 6, 477-87 . 2 . Shirai, H ., Ito, H ., Hirayama, Y ., Nakamoto, Y ., Nakabayashi, N ., Kumagai, K., Takeda, Y . & Nishibuchi, M. (1990) . Molecular epidemiologic evidence for association of thermostable direct hemolysin (TDH) and TDHrelated hemolysin of Vibrio parahaemolyticus with gastroenteritis . Infection and Immunity 58, 3568-73 . 3 . Persing, D . H . (1991) . Polymerase chain reaction : trenches to benches . Journal of Clinical Microbiology 29,1281-5 . 4 . 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 . 5 . Maniatis, T ., Fritsch, E . F . & Sambrook, J . (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, N .Y . : Cold Spring Harbor Laboratory . 6. Murakami, A ., Tada, J ., Yamagata, K . & Takano, J . (1990) . Highly sensitive detection of DNA using enzyme-linked DNA-probe . 1 . Colorimetric and fluorometric detection . Nucleic Acids Research 17, 5587-95 .
7 . Nishibuchi, M . & Kaper, J . B . (1990). Duplication and variation of the thermostable direct haemolysin Ztdh) gene in Vibrio parahaemolyticus . Molecular Microbiology 4, 87-99 . 8 . Dahlen, P . 0 ., litia, A . J ., Skagius, G ., Frostell, A ., Nunn, M. F . & Kwiatkowski, M . (1991). Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescencebased hybridization assay . Journal of Clinical Microbiology 29, 798-804. 9 . Bobo, L., Coutlee, F ., Yolken, R . H ., Quinn, T . & Viscidi, R . P . (1990) . Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay . Journal of Clinical Microbiology 28,1968-73 . 10 . Coutlee, F ., Saint-Antoine, P ., Olivier, C ., Voyer, H ., Kessous-Elbaz, A ., Berrada, F ., Begin, P ., Giroux, L . & Viscidi, R. (1991) . Evaluation of infection with human immunodeficiency virus type 1 by using non-isotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA . Journal of Clinical Microbiology 29, 2461-7 . 11 . Homes, E ., Wasteson, Y . & Olsvik, O . (1991) . Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimen by an immobilized, colorimetric, nested polymerase chain reaction . Journal of Clinical Microbiology 29, 2375-9 . 12 . Keller, G . H ., Huang, D .-P . & Manak, M. M. (1989) . A sensitive non-isotopic hybridization assay for HIV-1 DNA . Analytical Biochemistry 177, 27-32 . 13 . Keller, G . H ., Huang, D .-P. & Manak, M . M . (1991) . Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtitre wells . Journal of Clinical Microbiology 29, 638-41 . 14 . Keller, G . H ., Huang, D .-P., Shin, J . W .-K . & Manak, M . M . (1990) . Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization . Journal of Clinical Microbiology 28, 1441-6. 15 . Kemp, D . J ., Smith, D . B., Foote, S . J ., Samaras, N . & Peterson, M . G . (1989) . Colorimetric detection of specific DNA segments amplified by polymerase chain reactions . Proceedings of the National Academy of Sciences, USA . 86, 2423-7 . 16 . Landgraf . A ., Reckmann, B . & Pingoud, A . (1991) . Direct analysis of polymerase chain reaction products using enzyme-linked immunosorbent assay techniques. Analytical Biochemistry 198, 86-91 . 17. Mantero, G ., Zonaro, A ., Albertini, A ., Bertolo, P. & Primi, D . (1991) . DNA enzyme immunoassay : general method for detecting products of polymerase chain reaction . Clinical Chemistry 37, 422-9 . 18 . Nickerson, D . A ., Kaiser, R., Lappin, S ., Stewart, J ., Hood, L . & Landegren, U . (1990). Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay . Proceedings of the National Academy of Sciences, USA 87, 8923-7.