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A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichiacoli
Journal of Biotechnology 81 (2000) 15 – 25 www.elsevier.com/locate/jbiotec
A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli Isao Ohiso a,*, Makoto Tsuruoka b, Tetsuya Iida c, Takeshi Honda c, Isao Karube d a
Di6ersified Products Di6ision, Nishikawa Rubber Co., Ltd., 2 -2 -8 Misasa-cho, Nishi-ku, Hiroshima 733 -8510, Japan b Ad6anced Science and Technology Laboratory, Hiroshima City, 151 -366 Ozuka, Numata-cho, Asaminami-ku, Hiroshima 731 -3162, Japan c Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka Uni6ersity, 3 -1 Yamadagaoka, Suita, Osaka 565 -0871, Japan d Research Center for Ad6anced Science and Technology, Uni6ersity of Tokyo, 4 -6 -1 Komaba, Meguro-ku, Tokyo 153 -8904, Japan Received 6 July 1999; received in revised form 7 March 2000; accepted 20 March 2000
Abstract A hybridization assay using fluorescence polarization was combined with the asymmetric polymerase chain reaction (PCR) in a method for the detection of the verotoxin type 2 gene of verotoxin-producing Escherichia coli. Six oligonucleotide probes labeled with FITC were designed and evaluated. One of these gave a detection limit of 103 colony forming units per assay, and assay results could be obtained within 5 min after PCR. It appears that the detection limit was restricted mainly by the extent and fidelity of PCR amplification, rather than by the sensitivity of the fluorescence polarization technique, indicating that good probe design facilitates the rapid detection of the PCR product. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid and sensitive detection of oligonucleotide sequences. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence polarization; Verotoxin; Escherichia coli; Verotoxin-producing E. coli; VT2; DNA probe
1. Introduction Verotoxin-producing Escherichia coli (VTEC) is identical to enterohemorrhagic E. coli. When a * Corresponding author. Fax: +81-82-8750610. E-mail address: [email protected] (I. Ohiso).
human is infected with VTEC, the bacterium induces hemorrhagic colitis and hemolytic uremic syndrome (HUS) upon production of verotoxin (Karmali et al., 1983; Karmali, 1989). VTEC infection has now become a worldwide problem. In Japan, outbreaks and sporadic cases have occurred frequently since May 1996. According to a
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report from the Food Sanitation Division of the Ministry of Health and Welfare, reported cases of VTEC infection numbered 9451; including 1808 hospitalizations and 12 deaths in 1996 alone (Infectious Disease Surveillance Center, Japan, 1997). Verotoxin (VT) is also termed Shiga-like toxin (SLT), and two immunologically distinct types, known as type 1 (VT1 or SLT-I) and type 2 (VT2 or SLT-II), exist. These have homologies of 56% in the amino acid sequences and 58% in the nucleotide sequences (Jackson et al., 1987). Although there are genetic and structural similarities between the two toxins, VT2 has an LD50 value approximately 400 times lower than that of VT1 when injected intravenously or intraperitoneally into mice (Tesh et al., 1993). VT2 is 1000 times more potent a cytotoxic agent than VT1 toward human renal microvascular endothelial cells (Louise and Obrig, 1995). A total of 3021 strains of VTEC, comprising 28 different serotypes, were isolated in 1996. Of these, 2352 strains (77.9%) produced both VT1 and VT2, while 359 strains (11.9%) produced VT2 only, and 307 strains (10.2%) produced only VT1 (Infectious Disease Surveillance Center, Japan, 1997). It has been reported that the VT2 production of VTEC was related to HUS (Ostroff et al., 1989; van de Kar et al., 1996; de Mena et al., 1997). Therefore, we considered that the investigation of detection of the VT2 gene should precede that of the less toxic VT1 gene. In this study, we describe a method, which involves a hybridization assay based on fluorescence polarization using fluorescent-labeled oligonucleotides, for the rapid detection of the VT2 gene and, hence, for the detection of about 90% of the total amount of VTEC. The technique of fluorescence polarization itself is well established, and relates the change in the effective volume of a fluorophore to a change in the fluorescence polarization (Perrin, 1926; Weber, 1953). This method is convenient for examining the interactions between molecules, and thus its application to immunoassays, which measure antigen – antibody interactions, has been well studied (Dandliker and Feigen, 1961; Dandliker and De Saussure, 1970; Dandliker et al., 1973; Tsuruoka et al., 1991). Equilibrium determinations of protein – DNA and protein–protein interactions have been performed using fluorescence polarization (Lund-
blad et al., 1996), and fluorescence polarization has been used to detect the hybridization of DNA in solution (Murakami et al., 1991), and to study the kinetics and sequence specificity of DNA hybridization (Herning et al., 1991; Tsuruoka et al., 1996a). Generally, DNA hybridization assays are used to distinguish binding between the target DNA and a probe (previously labeled complementary DNA) based on the response of the probe. Consequently, the separation of bound and free probe is usually required after incubation. This separation process is tedious and troublesome, and has hindered the acceleration and automation of the assay. However, measurements of fluorescence polarization are made in solution, so no separation of bound and free materials is required. The method is capable of monitoring the hybridization directly and rapidly in homogeneous solution. Therefore, several fluorescence polarization-based methods for monitoring hybridization have been investigated as diagnostic methods for the detection of particular genes (Devlin et al., 1993; Tamiya and Karube, 1993; Walker et al., 1996; Gibson et al., 1997; Spears et al., 1997). In previous studies, fluorescence polarization detection was combined with the polymerase chain reaction (PCR) for the amplification of DNA. Moreover, it was demonstrated that optimization of the reaction conditions greatly enhanced the rate and extent of DNA hybridization, while combination with the asymmetric PCR technique made the assay rapid and sensitive (Tsuruoka et al., 1997, 1998). In this study, the VT2 gene was targeted for the detection of VTEC using fluorescence polarization under the reaction conditions previously described (Tsuruoka et al., 1996b). Six oligonucleotide probes and six pairs of primers were designed and evaluated in view of the rapidity of hybridization with the amplified DNA.
2. Materials and methods
2.1. Synthetic oligonucleotides All oligonucleotides were synthesized using a DNA synthesizer (Model 392; Perkin-Elmer,
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USA) using the automated phosphoramidite coupling method. Oligonucleotide sequences were designed according to the sequence of the gene for VT2 given by Jackson et al. (1987). Their sequences are shown in Tables 1 and 2. Melting temperatures (Tm) of oligonucleotides were calculated using the formula Tm =[(A + T) × 2]+ [(G +C)×4]. Six oligonucleotide probes were designed to hybridize with an antisense strand of VT2 DNA, thus their sequences are a part of the sense strand. The probes (PB-1 to PB-6) were labeled with fluorescein at their 5% termini. The probes were dissolved in TE buffer (10 mM Tris – HCl and 1 mM ethylenediamine tetraacetic acid, pH 8.0) containing 0.8 M NaCl (Tsuruoka et al., 1996b). Six complementary oligonucleotides (C-1 to C-6) were also synthesized and dissolved in TE buffer.
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Six pairs of PCR primers (PM-1 to PM-6) were also synthesized, which were designed to amplify the regions of the VT2 gene including the target sequences PB-1 to PB-6, respectively (Fig. 1).
2.2. Template DNA Template DNA for PCR was prepared from a VTEC O157 strain carrying both VT1 and VT2 genes, which was a cause of the outbreak in Okayama, Japan in June, 1996 (Watanabe et al., 1996). The bacterium was verified using a standard immunological method involving bacterial cultivation and electrophoresis of the PCR products. The strain was grown overnight, producing a full density culture in LB (Luria–Bertani) medium. The cell density was confirmed by mea-
Fig. 1. Locations of the primers and the probes on the VT2 gene. The amplified region between the upstream primer () and the downstream primer () is indicted as ( — ). The position of the probe () is indicated on the corresponding target region. Table 1 Sequences of probes and their complementary oligonucleotides Probe Sequencea
Tm (°C)b
Complementary Sequence oligonucleotide
PB-1 PB-2 PB-3 PB-4 PB-5 PB-6
70 70 64 70 72 70
C-1 C-2 C-3 C-4 C-5 C-6
a b
5%-F 5%-F 5%-F 5%-F 5%-F 5%-F
TCAGGGGGCGCGTTCTGTTCG-3% ACGTGTCGCAGCGCTGGAACG-3% CAGGCGCGTTTTGACCATCTT-3% CCATCATCAGGGGGCGCGTTC-3% CGCCGGGAGACGTGGACCTCA-3% TGGCGGCGGATTGTGCTAAAGG-3%
FITC labeling abbreviated as F. Tm calculated using [(A+T)×2]+[(G+C)×4].
5%-CGAACAGAACGCGCCCCCTGA-3% 5%-CGTTCCAGCGCTGCGACACGT-3% 5%-AAGATGGTCAAAACGCGCCTG-3% 5%-GAACGCGCCCCCTGATGATGG-3% 5%-TGAGGTCCACGTCTCCCGGCG-3% 5%-CCTTTAGCACAATCCGCCGCCA-3%
Tm calculated using [(A+T)×2]+[(G+C)×4].
5%-TTATACTGAATTGCCATCATC-3% 5%-ATACGAGGGCTTGATGTCTAT-3% 5%-GTTTTTCTTCGGTATCCTATT-3% 5%-AGTATCGGGGAGAGGATG-3% 5%-TCGACCCCTCTTGAACATA-3% 5%-TAATACGGCAACAAATACTTTCTA-3%
PM-1u PM-2u PM-3u PM-4u PM-5u PM-6u a
Sequence
Upstream primer
Table 2 Primer sequences
56 60 56 56 56 62
Tm (°C)a
PM-1d PM-2d PM-3d PM-4d PM-5d PM-6d
Downstream primer
5%-TCCTTTATTTACCCGTTGTA-3% 5%-AACTCCATTAACGCCAGATA-3% 5%-GAAAGTATTTGTTGCCGTAT-3% 5%-AAATAAAACCGCCATAAACAT-3% 5%-AACTCCTTTATTTACCCGTTGTAT-3% 5%-CCGTCAACCTTCACTGTAA-3%
Sequence
54 56 54 54 64 56
Tm (°C)a
189 275 290 304 829 791
Product length (base pair)
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suring the light absorbance at 610 nm. Cells from this culture were harvested by centrifugation (12 000 rpm, 10 min) and resuspended in 0.2 ml sterile distilled water. The suspension was then heated at 100°C for 10 min to extract DNA from the cells, and recentrifuged (12 000 rpm, 5 min). The supernatant was collected. Based on the cell density of the original medium, the supernatant was diluted to 104 colony forming units (cfu) ml − 1 in TE buffer. When 1 ml of this DNA template was used for PCR, it was found to correspond to 104 cfu per assay. In the subsequent experiments for determining the detection limit of the assay, serial dilutions of this solution were used. Salmon sperm DNA (Funakoshi, Japan) was used as a control DNA sample.
2.3. Polymerase chain reaction PCR amplification was performed in a total volume of 100 ml, containing 2.5 U Taq polymerase (TaKaRa Ex Taq; Takara Shuzo, Japan), the PCR buffer, 0.2 mM dNTPs, 1 ml DNA template sample, 10 pmol upstream primer and 100 pmol downstream primer. The concentration of the downstream primer was ten times greater than that of the upstream primer in order to perform asymmetric PCR, aiming to generate mainly single-stranded target DNA (Tsuruoka et al., 1997, 1998). The amplification procedure was as follows. The reaction mixture was incubated at 94°C for 1 min. Next, it was subjected to 40 cycles of incubation at 94°C for 30 s to denature the DNA, at 45°C for 30 s to anneal the primers, and at 72°C for 30 s to extend the annealed primers. Finally, it was subjected to an additional cycle without extension so as not to form double-stranded DNA, which would mask the target sequence of the single-stranded probe during hybridization. After the final cycle, it was kept in the thermal cycler at 4°C. Following amplification, the samples were subjected to electrophoresis on a 2% agarose gel and the amplification products were visualized by ethidium bromide staining. A 100 base pair DNA ladder (Takara Shuzo, Japan) was used as the marker.
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2.4. Apparatus Fluorescence polarization measurements were performed using a fluorescence spectrophotometer Model FP-777 (JASCO, Japan) equipped with a Peltier temperature controller. The excitation wavelength was 485 nm with a bandwidth of 5 nm, and the emission wavelength was 525 nm with a bandwidth of 10 nm. The samples were measured in a quartz cell (1× 1 cm2). DNA amplification was performed using a PCR thermal cycler Model 9600 (Perkin-Elmer, USA).
2.5. Fluorescence polarization The measurement conditions adopted were the optimal conditions described in a previous study of the rate of DNA hybridization using fluorescence polarization (Tsuruoka et al., 1996b). Measurements were performed at 46°C in solutions of TE buffer containing 0.8 M NaCl and 10 − 9 M probe. Samples used in measurements contained either 80 ml complementary oligonucleotide (10 − 8 M final concentration), distilled water, salmon sperm DNA (800 mg l − 1), or post-PCR mixture. Fluorescence polarization measurements were taken every 1 min for at least 15 min after addition of the sample. The equation describing fluorescence polarization is:
1 1 1 1 RTt = + − P P0 P0 3 Vh where the polarization P depends on the effective volume V of the fluorescent substance (molecular mass multiplied by molar volume), the coefficient of viscosity h of the solvent, the absolute temperature T, and the lifetime t of the excited state of the fluorescent substance (Weber, 1953). R is the gas constant and P0 is the value of P at 0 K. Given that T, t and h are constant, it can be seen that polarization increases with increasing V, which is a property of the fluorescent-labeled substance. Thus, DNA hybridization can be monitored when one strand of DNA is labeled with a fluorescent substance and the labeled DNA binds with the target DNA.
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3. Results
3.1. Hybridization with complementary oligonucleotide The time courses of polarization of the probes (PB-1 to PB-6) in the presence and absence of their complementary oligonucleotides (C-1 to C-6) are shown in Fig. 2. The polarization value (P) of any probe alone was approximately 0.050. This value indicated that the probe was not forming double-stranded DNA (Tsuruoka et al., 1996b). In the presence of the respective complementary oligonucleotide, an increase in P was observed, which indicated an increase in the effective volume of the probe due to hybridization. Apart from PB-2, the increase in P was observed immediately after addition of the sample. For PB-2, P rose more slowly than with other probes, reaching a plateau approximately of 0.090 after 60 min. In some cases, the value of P observed in the initial stage of the measurement gradually decreased. This is probably due to the lower temperature of the sample. During the measurements, the temperature of the cell holder was set to 46°C. However, when we commenced measurements for a sample of low temperature, it would occasionally take a few minutes for the temperature of the sample to reach 46°C. When the temperature is low, P should be large. When the experiments shown in Fig. 2 were performed, we did not pay sufficient attention to this point. The temperatures of the samples ranged from 4°C to room temperature.
3.2. Hybridization with DNA amplified by PCR The results of electrophoresis of the PCR products are shown in Fig. 3. For each pair of primers, the DNA fragment of the VT2 gene was amplified to the expected size, although the band of PM-1 was weaker than the others. No amplification products were found for the negative control template (salmon sperm DNA) using any of the primer pairs (data not shown). The time courses of polarization of the probes (PB-1 to PB-6) in the presence of the post-PCR
mixtures, amplified by corresponding primer pairs (PM-1 to PM-6), and in the presence of salmon sperm DNA are shown in Fig. 2. All measurements using salmon sperm DNA as the control gave values equal to that of the probe (ca. 0.050), which indicated that the probes were not hybridized nonspecifically. In the presence of the post-PCR mixture, results were not consistent. For PB-1, no significant change of P was observed. For PB-2, P showed a gradual rise from approximately 0.060 and did not reach a plateau after 60 min (data from 25 to 60 min are omitted from the figure). The increase of P for PB-2 with the post-PCR mixture is almost indiscernible; however, the value of P after 60 min was 0.073, which was certainly above the value of PB-2 alone. For PB-2, the increase of P in the presence of the post-PCR mixture was lower than in the presence of C-2. If a longer time was allowed for hybridization (more than 60 min), the P value of the PCR mixture might have reached a similar or larger value than that with the complementary oligonucleotide. For PB-3, P increased to the same extent as in the presence of C-3. For PB-4, although P rose more slowly than in the presence of C-4, it reached a value of 0.100, just as in the presence of C-4. For PB-5, P rose above the value of the plateau observed in the presence of C-5. For PB-6, P was approximately 0.120; larger than in the presence of C-6, reaching a plateau in less than 5 min. When the same experiments for PB-6 were repeated (n= 20), the average of P, measured at 10 min, was 0.121, and its standard deviation was 0.0082. The varying of these results illustrates the differing behavior of the probes in hybridizing with the amplified DNA fragment. It is evident that the hybridization rates of the six different probes with the complementary DNA of the PCR products are quite diverse.
3.3. Detection limit of the assay The limits of detection for primer PM-6 and probe PB-6 were evaluated under the experimental conditions already mentioned. Template DNA samples corresponding to 104, 103, 102 and 101 cfu per assay were used. The electrophorograms of
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Fig. 2. Hybridization of probes as monitored by fluorescence polarization: ( ) probe alone; () with complementary oligonucleotide; (2) with salmon sperm DNA; ( ) with post-PCR mixture.
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4. Discussion
Fig. 3. Electrophoresis of amplified products of VT2 gene using various primer pairs: lanes M, 100 base pair ladder DNA marker; lane 1, PM-1; lane 2, PM-2; lane 3, PM-3; lane 4, PM-4; lane 5, PM-5; lane 6, PM-6.
Fig. 4. Electrophoresis of VT2 DNA fragments amplified using PM-6: lanes M, 100 base pair ladder DNA marker; lane 1, 104 cfu per assay; lane 2, 103 cfu per assay; lane 3, 102 cfu per assay; lane 4, 101 cfu per assay.
the amplified products are shown in Fig. 4. A band of DNA of the expected size was clearly visible in samples of 104 and 103 cfu per assay. The time courses of fluorescence polarization of PB-6 with the post-PCR mixture are shown in Fig. 5. Similar significantly higher P values were observed using samples of 104 and 103 cfu per assay. Additionally, the 102 cfu per assay sample, which appeared as a faint band upon electrophoresis, had a polarization lower than those of the 104 and 103 cfu per assay samples but much higher than those of the 101 cfu per assay sample or probe alone.
In the presence of the complementary oligonucleotide, fluorescence polarization of the probes always increased (Fig. 2). In all cases, the effective volume of the probe was increased upon hybridization with its complementary oligonucleotide, as previously reported (Herning et al., 1991; Murakami et al., 1991; Tsuruoka et al., 1996b). The time course data for all six probes in the presence of complementary oligonucleotides, PCR products or salmon sperm DNA clearly demonstrated that the increase of P was due to the hybridization of the probes with their target DNAs, although the rates of hybridization were very different (Fig. 2). The conditions for measurements of hybridization (temperature, concentrations of the probe and the complementary oligonucleotide, and concentration of NaCl) were the optimal conditions determined in a previous study (Tsuruoka et al., 1996b). For all probes except PB-2, P instantly increased to an approximately constant value upon adding the complementary oligonucleotide. This plateauing of P indicated the equilibration of hybridization. In the case of PB-2, P rose gradually over 60 min, eventually reaching a plateau of approximately 0.090. This was probably due to the ability of the oligonucleotide (and, therefore, C-2) to form a dimer as follows:
Fig. 5. Time course of VT2 gene detection: ( ) probe alone; () 101 cfu per assay; ( ) 102 cfu per assay; () 103 cfu per assay; (") 104 cfu per assay.
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For other probes, possible homodimer forming sequences comprised four or less consecutive base pairs. Such interactions could delay the hybridization of PB-2 or C-2 with their targets. No hybridization with salmon sperm DNA was detected using any of the probes (Fig. 2). A previous study reported that hybridization could be measured when the sequence of sample DNA had three or less mismatches within a 24-mer probe (Tsuruoka et al., 1996a). Apart from PB-3, the time courses of P in the presence of post-PCR mixture for the six probes were very different from those in the presence of complementary oligonucleotides (Fig. 2). No hybridization was observed for PB-1, probably because of the weak amplification of PM-1 (Fig. 3). Although hybridization of PB-2 may be slow for the reasons already mentioned, even slower hybridization than in the presence of C-2 was observed. The other four probes, PB-3 to PB-6, showed clear differences between P of the probe alone and in the presence of salmon sperm DNA. Using these four probes, measurements of hybridization with the post-PCR products of VT2 were possible. Any one of these four probes could be used in VT2 detection to distinguish between negative and positive samples. In particular, PB-6 in the presence of post-PCR product showed the most rapid hybridization and displayed the largest difference in P between that of the probe alone. PB-6 in the presence of post-PCR product also displayed the largest difference in P between that in the presence of the complementary oligonucleotide. The differing shape of the curves corresponding to the hybridization of the PCR product and its complementary oligonucleotide are difficult to explain. The calculated Tm values of the probes are all approximately 70°C, although that of PB-3 is slightly lower (64°C) (Table 1). It was supposed that the time courses of hybridization would be related to the conformation of the amplified DNA fragment, which should exist predominantly as single-stranded DNA due to asymmetric amplifi-
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cation. Depending on its sequence, this singlestranded DNA may have some secondary structure due to intramolecular complementary base pairing. We suspect that the hybridization of the probe to the PCR product could be being hindered by the formation of some secondary conformation of the PCR product. Consequently, it is difficult to predict the detailed interactions of the probes and the amplified DNA fragments. Nonetheless, we know that the conformation of the target DNA fragment as well as that of the probe are important factors in hybridization reactions, and it is only through experiment that we can determine which are the most efficient probes. The limit of detection as determined using PM6 and PB-6 (Figs. 3 and 4) was 103 cfu per assay in a sample prior to PCR. It was demonstrated that the sensitivity of this assay was almost equal to gel electrophoresis. For the detection of VT2 gene in food samples, an enrichment step prior to the PCR would be required to produce samples above the detection limit. These results show that the detection limit was determined mainly by the efficiency of amplification, rather than the sensitivity of the fluorescence polarization measurement technique. In addition, the sensitivity of the fluorescence polarization is significantly influenced by the choice of the probe and the primer. In the case of PM-1 and PB-1, the sensitivity was less than that of gel electrophoresis (Fig. 2). On the other hand, the sensitivity using PM-6 and PB-6 was almost equal to that of gel electrophoresis (Figs. 4 and 5). This method could be a very useful tool for detecting specific sequences in the amplified products of nucleic acid samples. Thus, optimization of the primer sequence and PCR conditions should improve the detection limit of 103 cfu per assay and further decrease the assay time to less than 5 min as in this system. Several methods for the monitoring of DNA hybridization in homogeneous solution using fluorophore-labeled oligonucleotide probes have been reported, based on fluorescence energy transfer (Morrison and Stols, 1993; Tyagi and Kramer, 1996), and fluorescence excimer formation (Masuko et al., 1998). Compared with these methods, the advantage of this fluorescence polarization
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method is that the probe architecture is very simple.
5. Conclusions Detection of the VT2 gene of verotoxin-producing E. coli using our primers and probes was successful except for the set of PM-1 and PB-1. The detection limit using PM-6 and PB-6 was 103 cfu per assay, and results could be obtained within 5 min after PCR amplification, using a fluorescence polarization assay. Moreover, detection of the VT1 gene using the same methodology is in progress. The final goal of our study is the rapid detection of VTEC and typing of its VT genes. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid, sensitive and selective detection of oligonucleotide sequences. The design of appropriate primers should enable the selective amplification of target sequences, while the design of the probe should facilitate the rapid detection of the post-PCR product. A combination of both would greatly assist in the rapid and specific detection of the gene. Using this technique, it should be possible to perform assays for virtually any organism or virus, providing a suitable target oligonucleotide sequence can be isolated.
Acknowledgements We would like to thank Dr Scott McNiven for assistance in preparing this manuscript.
References Dandliker, W.B., De Saussure, V.A., 1970. Fluorescence polarization in immunochemistry. Immunochemistry 7, 799– 828. Dandliker, W.B., Feigen, G.A., 1961. Quantification of the antigen-antibody reaction by the polarization of fluorescence. Biochem. Biophys. Res. Commun. 5, 299–304. Dandliker, W.B., Kelly, R.J., Dandliker, J., Farquhar, J.,
Levin, J., 1973. Fluorescence polarization immunoassay. Theory and experimental method. Immunochemistry 10, 219 – 227. de Mena, M.F., Tous, M., Miliwebsky, E., Chillemi, G., Rivas, M., 1997. Differential kinetic patterns for Shiga toxin production by Escherichia coli. Rev. Argent. Microbiol. 29, 167 – 175. Devlin, R., Studholme, R.M., Dandliker, W.B., Fahy, E., Blumeyer, K., Ghosh, S.S., 1993. Homogeneous detection of nucleic acids by transient-state polarized fluorescence. Clin. Chem. 39, 1939 – 1943. Gibson, N.J., Gillard, H.L., Whitcombe, D., Ferrie, R.M., Newton, C.R., Little, S., 1997. A homogeneous method for genotyping with fluorescence polarization. Clin. Chem. 43, 1336 – 1341. Herning, T., Tamiya, E., Karube, I., Kobayashi, S., 1991. Specific liquid DNA hybridization kinetics measured by fluorescence polarization. Anal. Chim. Acta 244, 207 – 213. Infectious Disease Surveillance Center, Japan, 1997. Verocytotoxin-producing Escherichia coli (enterohemorrhagic E. coli ) infections, Center for the Promotion of Preventative Medicine, Toyoko, Japan, 1996 – June 1997. Infectious Agents Surveillance Report 18, No. 7. Jackson, M.P., Neill, R.J., O’Brien, A.D., Holmes, R.K., Newland, J.W., 1987. Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933. FEMS Microbiol. Lett. 44, 109 – 114. Karmali, M.A., Steele, B.T., Petric, M., Lim, C., 1983. Sporadic cases of hemolytic-uraemic syndrome associated with faecal cytotoxin and cytotoxin-producing Escherichia coli in stools. Lancet 1, 619 – 620. Karmali, M.A., 1989. Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2, 15 – 38. Louise, C.B., Obrig, T.G., 1995. Specific interaction of Escherichia coli O157:H7-derived Shiga-like toxin II with human renal endothelial cells. J. Infect. Dis. 172, 1397 – 1401. Lundblad, J.R., Laurance, M., Goodman, R.H., 1996. Fluorescence polarization analysis of protein – DNA and protein – protein interactions. Mol. Endocrinol. 10, 607 – 612. Masuko, M., Ohtani, H., Ebata, K., Shimadzu, A., 1998. Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore. Nucleic Acids Res. 26, 5409 – 5416. Morrison, L.E., Stols, L.M., 1993. Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution. Biochemistry 32, 3095 – 3104. Murakami, A., Nakaura, M., Nakatsuji, Y., Nagahara, S., Tran-Cong, Q., Makino, K., 1991. Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy. Nucleic Acids Res. 19, 4097 – 4102. Ostroff, S.M., Tarr, P.I., Neill, M.A., Lewis, J.H., HargrettBean, N., Kobayashi, J.M., 1989. Toxin genotypes and plasmid profiles as determinants of systemic sequelae in
A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli Isao Ohiso a,*, Makoto Tsuruoka b, Tetsuya Iida c, Takeshi Honda c, Isao Karube d a
Di6ersified Products Di6ision, Nishikawa Rubber Co., Ltd., 2 -2 -8 Misasa-cho, Nishi-ku, Hiroshima 733 -8510, Japan b Ad6anced Science and Technology Laboratory, Hiroshima City, 151 -366 Ozuka, Numata-cho, Asaminami-ku, Hiroshima 731 -3162, Japan c Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka Uni6ersity, 3 -1 Yamadagaoka, Suita, Osaka 565 -0871, Japan d Research Center for Ad6anced Science and Technology, Uni6ersity of Tokyo, 4 -6 -1 Komaba, Meguro-ku, Tokyo 153 -8904, Japan Received 6 July 1999; received in revised form 7 March 2000; accepted 20 March 2000
Abstract A hybridization assay using fluorescence polarization was combined with the asymmetric polymerase chain reaction (PCR) in a method for the detection of the verotoxin type 2 gene of verotoxin-producing Escherichia coli. Six oligonucleotide probes labeled with FITC were designed and evaluated. One of these gave a detection limit of 103 colony forming units per assay, and assay results could be obtained within 5 min after PCR. It appears that the detection limit was restricted mainly by the extent and fidelity of PCR amplification, rather than by the sensitivity of the fluorescence polarization technique, indicating that good probe design facilitates the rapid detection of the PCR product. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid and sensitive detection of oligonucleotide sequences. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence polarization; Verotoxin; Escherichia coli; Verotoxin-producing E. coli; VT2; DNA probe
1. Introduction Verotoxin-producing Escherichia coli (VTEC) is identical to enterohemorrhagic E. coli. When a * Corresponding author. Fax: +81-82-8750610. E-mail address: [email protected] (I. Ohiso).
human is infected with VTEC, the bacterium induces hemorrhagic colitis and hemolytic uremic syndrome (HUS) upon production of verotoxin (Karmali et al., 1983; Karmali, 1989). VTEC infection has now become a worldwide problem. In Japan, outbreaks and sporadic cases have occurred frequently since May 1996. According to a
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report from the Food Sanitation Division of the Ministry of Health and Welfare, reported cases of VTEC infection numbered 9451; including 1808 hospitalizations and 12 deaths in 1996 alone (Infectious Disease Surveillance Center, Japan, 1997). Verotoxin (VT) is also termed Shiga-like toxin (SLT), and two immunologically distinct types, known as type 1 (VT1 or SLT-I) and type 2 (VT2 or SLT-II), exist. These have homologies of 56% in the amino acid sequences and 58% in the nucleotide sequences (Jackson et al., 1987). Although there are genetic and structural similarities between the two toxins, VT2 has an LD50 value approximately 400 times lower than that of VT1 when injected intravenously or intraperitoneally into mice (Tesh et al., 1993). VT2 is 1000 times more potent a cytotoxic agent than VT1 toward human renal microvascular endothelial cells (Louise and Obrig, 1995). A total of 3021 strains of VTEC, comprising 28 different serotypes, were isolated in 1996. Of these, 2352 strains (77.9%) produced both VT1 and VT2, while 359 strains (11.9%) produced VT2 only, and 307 strains (10.2%) produced only VT1 (Infectious Disease Surveillance Center, Japan, 1997). It has been reported that the VT2 production of VTEC was related to HUS (Ostroff et al., 1989; van de Kar et al., 1996; de Mena et al., 1997). Therefore, we considered that the investigation of detection of the VT2 gene should precede that of the less toxic VT1 gene. In this study, we describe a method, which involves a hybridization assay based on fluorescence polarization using fluorescent-labeled oligonucleotides, for the rapid detection of the VT2 gene and, hence, for the detection of about 90% of the total amount of VTEC. The technique of fluorescence polarization itself is well established, and relates the change in the effective volume of a fluorophore to a change in the fluorescence polarization (Perrin, 1926; Weber, 1953). This method is convenient for examining the interactions between molecules, and thus its application to immunoassays, which measure antigen – antibody interactions, has been well studied (Dandliker and Feigen, 1961; Dandliker and De Saussure, 1970; Dandliker et al., 1973; Tsuruoka et al., 1991). Equilibrium determinations of protein – DNA and protein–protein interactions have been performed using fluorescence polarization (Lund-
blad et al., 1996), and fluorescence polarization has been used to detect the hybridization of DNA in solution (Murakami et al., 1991), and to study the kinetics and sequence specificity of DNA hybridization (Herning et al., 1991; Tsuruoka et al., 1996a). Generally, DNA hybridization assays are used to distinguish binding between the target DNA and a probe (previously labeled complementary DNA) based on the response of the probe. Consequently, the separation of bound and free probe is usually required after incubation. This separation process is tedious and troublesome, and has hindered the acceleration and automation of the assay. However, measurements of fluorescence polarization are made in solution, so no separation of bound and free materials is required. The method is capable of monitoring the hybridization directly and rapidly in homogeneous solution. Therefore, several fluorescence polarization-based methods for monitoring hybridization have been investigated as diagnostic methods for the detection of particular genes (Devlin et al., 1993; Tamiya and Karube, 1993; Walker et al., 1996; Gibson et al., 1997; Spears et al., 1997). In previous studies, fluorescence polarization detection was combined with the polymerase chain reaction (PCR) for the amplification of DNA. Moreover, it was demonstrated that optimization of the reaction conditions greatly enhanced the rate and extent of DNA hybridization, while combination with the asymmetric PCR technique made the assay rapid and sensitive (Tsuruoka et al., 1997, 1998). In this study, the VT2 gene was targeted for the detection of VTEC using fluorescence polarization under the reaction conditions previously described (Tsuruoka et al., 1996b). Six oligonucleotide probes and six pairs of primers were designed and evaluated in view of the rapidity of hybridization with the amplified DNA.
2. Materials and methods
2.1. Synthetic oligonucleotides All oligonucleotides were synthesized using a DNA synthesizer (Model 392; Perkin-Elmer,
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USA) using the automated phosphoramidite coupling method. Oligonucleotide sequences were designed according to the sequence of the gene for VT2 given by Jackson et al. (1987). Their sequences are shown in Tables 1 and 2. Melting temperatures (Tm) of oligonucleotides were calculated using the formula Tm =[(A + T) × 2]+ [(G +C)×4]. Six oligonucleotide probes were designed to hybridize with an antisense strand of VT2 DNA, thus their sequences are a part of the sense strand. The probes (PB-1 to PB-6) were labeled with fluorescein at their 5% termini. The probes were dissolved in TE buffer (10 mM Tris – HCl and 1 mM ethylenediamine tetraacetic acid, pH 8.0) containing 0.8 M NaCl (Tsuruoka et al., 1996b). Six complementary oligonucleotides (C-1 to C-6) were also synthesized and dissolved in TE buffer.
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Six pairs of PCR primers (PM-1 to PM-6) were also synthesized, which were designed to amplify the regions of the VT2 gene including the target sequences PB-1 to PB-6, respectively (Fig. 1).
2.2. Template DNA Template DNA for PCR was prepared from a VTEC O157 strain carrying both VT1 and VT2 genes, which was a cause of the outbreak in Okayama, Japan in June, 1996 (Watanabe et al., 1996). The bacterium was verified using a standard immunological method involving bacterial cultivation and electrophoresis of the PCR products. The strain was grown overnight, producing a full density culture in LB (Luria–Bertani) medium. The cell density was confirmed by mea-
Fig. 1. Locations of the primers and the probes on the VT2 gene. The amplified region between the upstream primer () and the downstream primer () is indicted as ( — ). The position of the probe () is indicated on the corresponding target region. Table 1 Sequences of probes and their complementary oligonucleotides Probe Sequencea
Tm (°C)b
Complementary Sequence oligonucleotide
PB-1 PB-2 PB-3 PB-4 PB-5 PB-6
70 70 64 70 72 70
C-1 C-2 C-3 C-4 C-5 C-6
a b
5%-F 5%-F 5%-F 5%-F 5%-F 5%-F
TCAGGGGGCGCGTTCTGTTCG-3% ACGTGTCGCAGCGCTGGAACG-3% CAGGCGCGTTTTGACCATCTT-3% CCATCATCAGGGGGCGCGTTC-3% CGCCGGGAGACGTGGACCTCA-3% TGGCGGCGGATTGTGCTAAAGG-3%
FITC labeling abbreviated as F. Tm calculated using [(A+T)×2]+[(G+C)×4].
5%-CGAACAGAACGCGCCCCCTGA-3% 5%-CGTTCCAGCGCTGCGACACGT-3% 5%-AAGATGGTCAAAACGCGCCTG-3% 5%-GAACGCGCCCCCTGATGATGG-3% 5%-TGAGGTCCACGTCTCCCGGCG-3% 5%-CCTTTAGCACAATCCGCCGCCA-3%
Tm calculated using [(A+T)×2]+[(G+C)×4].
5%-TTATACTGAATTGCCATCATC-3% 5%-ATACGAGGGCTTGATGTCTAT-3% 5%-GTTTTTCTTCGGTATCCTATT-3% 5%-AGTATCGGGGAGAGGATG-3% 5%-TCGACCCCTCTTGAACATA-3% 5%-TAATACGGCAACAAATACTTTCTA-3%
PM-1u PM-2u PM-3u PM-4u PM-5u PM-6u a
Sequence
Upstream primer
Table 2 Primer sequences
56 60 56 56 56 62
Tm (°C)a
PM-1d PM-2d PM-3d PM-4d PM-5d PM-6d
Downstream primer
5%-TCCTTTATTTACCCGTTGTA-3% 5%-AACTCCATTAACGCCAGATA-3% 5%-GAAAGTATTTGTTGCCGTAT-3% 5%-AAATAAAACCGCCATAAACAT-3% 5%-AACTCCTTTATTTACCCGTTGTAT-3% 5%-CCGTCAACCTTCACTGTAA-3%
Sequence
54 56 54 54 64 56
Tm (°C)a
189 275 290 304 829 791
Product length (base pair)
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suring the light absorbance at 610 nm. Cells from this culture were harvested by centrifugation (12 000 rpm, 10 min) and resuspended in 0.2 ml sterile distilled water. The suspension was then heated at 100°C for 10 min to extract DNA from the cells, and recentrifuged (12 000 rpm, 5 min). The supernatant was collected. Based on the cell density of the original medium, the supernatant was diluted to 104 colony forming units (cfu) ml − 1 in TE buffer. When 1 ml of this DNA template was used for PCR, it was found to correspond to 104 cfu per assay. In the subsequent experiments for determining the detection limit of the assay, serial dilutions of this solution were used. Salmon sperm DNA (Funakoshi, Japan) was used as a control DNA sample.
2.3. Polymerase chain reaction PCR amplification was performed in a total volume of 100 ml, containing 2.5 U Taq polymerase (TaKaRa Ex Taq; Takara Shuzo, Japan), the PCR buffer, 0.2 mM dNTPs, 1 ml DNA template sample, 10 pmol upstream primer and 100 pmol downstream primer. The concentration of the downstream primer was ten times greater than that of the upstream primer in order to perform asymmetric PCR, aiming to generate mainly single-stranded target DNA (Tsuruoka et al., 1997, 1998). The amplification procedure was as follows. The reaction mixture was incubated at 94°C for 1 min. Next, it was subjected to 40 cycles of incubation at 94°C for 30 s to denature the DNA, at 45°C for 30 s to anneal the primers, and at 72°C for 30 s to extend the annealed primers. Finally, it was subjected to an additional cycle without extension so as not to form double-stranded DNA, which would mask the target sequence of the single-stranded probe during hybridization. After the final cycle, it was kept in the thermal cycler at 4°C. Following amplification, the samples were subjected to electrophoresis on a 2% agarose gel and the amplification products were visualized by ethidium bromide staining. A 100 base pair DNA ladder (Takara Shuzo, Japan) was used as the marker.
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2.4. Apparatus Fluorescence polarization measurements were performed using a fluorescence spectrophotometer Model FP-777 (JASCO, Japan) equipped with a Peltier temperature controller. The excitation wavelength was 485 nm with a bandwidth of 5 nm, and the emission wavelength was 525 nm with a bandwidth of 10 nm. The samples were measured in a quartz cell (1× 1 cm2). DNA amplification was performed using a PCR thermal cycler Model 9600 (Perkin-Elmer, USA).
2.5. Fluorescence polarization The measurement conditions adopted were the optimal conditions described in a previous study of the rate of DNA hybridization using fluorescence polarization (Tsuruoka et al., 1996b). Measurements were performed at 46°C in solutions of TE buffer containing 0.8 M NaCl and 10 − 9 M probe. Samples used in measurements contained either 80 ml complementary oligonucleotide (10 − 8 M final concentration), distilled water, salmon sperm DNA (800 mg l − 1), or post-PCR mixture. Fluorescence polarization measurements were taken every 1 min for at least 15 min after addition of the sample. The equation describing fluorescence polarization is:
1 1 1 1 RTt = + − P P0 P0 3 Vh where the polarization P depends on the effective volume V of the fluorescent substance (molecular mass multiplied by molar volume), the coefficient of viscosity h of the solvent, the absolute temperature T, and the lifetime t of the excited state of the fluorescent substance (Weber, 1953). R is the gas constant and P0 is the value of P at 0 K. Given that T, t and h are constant, it can be seen that polarization increases with increasing V, which is a property of the fluorescent-labeled substance. Thus, DNA hybridization can be monitored when one strand of DNA is labeled with a fluorescent substance and the labeled DNA binds with the target DNA.
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3. Results
3.1. Hybridization with complementary oligonucleotide The time courses of polarization of the probes (PB-1 to PB-6) in the presence and absence of their complementary oligonucleotides (C-1 to C-6) are shown in Fig. 2. The polarization value (P) of any probe alone was approximately 0.050. This value indicated that the probe was not forming double-stranded DNA (Tsuruoka et al., 1996b). In the presence of the respective complementary oligonucleotide, an increase in P was observed, which indicated an increase in the effective volume of the probe due to hybridization. Apart from PB-2, the increase in P was observed immediately after addition of the sample. For PB-2, P rose more slowly than with other probes, reaching a plateau approximately of 0.090 after 60 min. In some cases, the value of P observed in the initial stage of the measurement gradually decreased. This is probably due to the lower temperature of the sample. During the measurements, the temperature of the cell holder was set to 46°C. However, when we commenced measurements for a sample of low temperature, it would occasionally take a few minutes for the temperature of the sample to reach 46°C. When the temperature is low, P should be large. When the experiments shown in Fig. 2 were performed, we did not pay sufficient attention to this point. The temperatures of the samples ranged from 4°C to room temperature.
3.2. Hybridization with DNA amplified by PCR The results of electrophoresis of the PCR products are shown in Fig. 3. For each pair of primers, the DNA fragment of the VT2 gene was amplified to the expected size, although the band of PM-1 was weaker than the others. No amplification products were found for the negative control template (salmon sperm DNA) using any of the primer pairs (data not shown). The time courses of polarization of the probes (PB-1 to PB-6) in the presence of the post-PCR
mixtures, amplified by corresponding primer pairs (PM-1 to PM-6), and in the presence of salmon sperm DNA are shown in Fig. 2. All measurements using salmon sperm DNA as the control gave values equal to that of the probe (ca. 0.050), which indicated that the probes were not hybridized nonspecifically. In the presence of the post-PCR mixture, results were not consistent. For PB-1, no significant change of P was observed. For PB-2, P showed a gradual rise from approximately 0.060 and did not reach a plateau after 60 min (data from 25 to 60 min are omitted from the figure). The increase of P for PB-2 with the post-PCR mixture is almost indiscernible; however, the value of P after 60 min was 0.073, which was certainly above the value of PB-2 alone. For PB-2, the increase of P in the presence of the post-PCR mixture was lower than in the presence of C-2. If a longer time was allowed for hybridization (more than 60 min), the P value of the PCR mixture might have reached a similar or larger value than that with the complementary oligonucleotide. For PB-3, P increased to the same extent as in the presence of C-3. For PB-4, although P rose more slowly than in the presence of C-4, it reached a value of 0.100, just as in the presence of C-4. For PB-5, P rose above the value of the plateau observed in the presence of C-5. For PB-6, P was approximately 0.120; larger than in the presence of C-6, reaching a plateau in less than 5 min. When the same experiments for PB-6 were repeated (n= 20), the average of P, measured at 10 min, was 0.121, and its standard deviation was 0.0082. The varying of these results illustrates the differing behavior of the probes in hybridizing with the amplified DNA fragment. It is evident that the hybridization rates of the six different probes with the complementary DNA of the PCR products are quite diverse.
3.3. Detection limit of the assay The limits of detection for primer PM-6 and probe PB-6 were evaluated under the experimental conditions already mentioned. Template DNA samples corresponding to 104, 103, 102 and 101 cfu per assay were used. The electrophorograms of
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Fig. 2. Hybridization of probes as monitored by fluorescence polarization: ( ) probe alone; () with complementary oligonucleotide; (2) with salmon sperm DNA; ( ) with post-PCR mixture.
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4. Discussion
Fig. 3. Electrophoresis of amplified products of VT2 gene using various primer pairs: lanes M, 100 base pair ladder DNA marker; lane 1, PM-1; lane 2, PM-2; lane 3, PM-3; lane 4, PM-4; lane 5, PM-5; lane 6, PM-6.
Fig. 4. Electrophoresis of VT2 DNA fragments amplified using PM-6: lanes M, 100 base pair ladder DNA marker; lane 1, 104 cfu per assay; lane 2, 103 cfu per assay; lane 3, 102 cfu per assay; lane 4, 101 cfu per assay.
the amplified products are shown in Fig. 4. A band of DNA of the expected size was clearly visible in samples of 104 and 103 cfu per assay. The time courses of fluorescence polarization of PB-6 with the post-PCR mixture are shown in Fig. 5. Similar significantly higher P values were observed using samples of 104 and 103 cfu per assay. Additionally, the 102 cfu per assay sample, which appeared as a faint band upon electrophoresis, had a polarization lower than those of the 104 and 103 cfu per assay samples but much higher than those of the 101 cfu per assay sample or probe alone.
In the presence of the complementary oligonucleotide, fluorescence polarization of the probes always increased (Fig. 2). In all cases, the effective volume of the probe was increased upon hybridization with its complementary oligonucleotide, as previously reported (Herning et al., 1991; Murakami et al., 1991; Tsuruoka et al., 1996b). The time course data for all six probes in the presence of complementary oligonucleotides, PCR products or salmon sperm DNA clearly demonstrated that the increase of P was due to the hybridization of the probes with their target DNAs, although the rates of hybridization were very different (Fig. 2). The conditions for measurements of hybridization (temperature, concentrations of the probe and the complementary oligonucleotide, and concentration of NaCl) were the optimal conditions determined in a previous study (Tsuruoka et al., 1996b). For all probes except PB-2, P instantly increased to an approximately constant value upon adding the complementary oligonucleotide. This plateauing of P indicated the equilibration of hybridization. In the case of PB-2, P rose gradually over 60 min, eventually reaching a plateau of approximately 0.090. This was probably due to the ability of the oligonucleotide (and, therefore, C-2) to form a dimer as follows:
Fig. 5. Time course of VT2 gene detection: ( ) probe alone; () 101 cfu per assay; ( ) 102 cfu per assay; () 103 cfu per assay; (") 104 cfu per assay.
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For other probes, possible homodimer forming sequences comprised four or less consecutive base pairs. Such interactions could delay the hybridization of PB-2 or C-2 with their targets. No hybridization with salmon sperm DNA was detected using any of the probes (Fig. 2). A previous study reported that hybridization could be measured when the sequence of sample DNA had three or less mismatches within a 24-mer probe (Tsuruoka et al., 1996a). Apart from PB-3, the time courses of P in the presence of post-PCR mixture for the six probes were very different from those in the presence of complementary oligonucleotides (Fig. 2). No hybridization was observed for PB-1, probably because of the weak amplification of PM-1 (Fig. 3). Although hybridization of PB-2 may be slow for the reasons already mentioned, even slower hybridization than in the presence of C-2 was observed. The other four probes, PB-3 to PB-6, showed clear differences between P of the probe alone and in the presence of salmon sperm DNA. Using these four probes, measurements of hybridization with the post-PCR products of VT2 were possible. Any one of these four probes could be used in VT2 detection to distinguish between negative and positive samples. In particular, PB-6 in the presence of post-PCR product showed the most rapid hybridization and displayed the largest difference in P between that of the probe alone. PB-6 in the presence of post-PCR product also displayed the largest difference in P between that in the presence of the complementary oligonucleotide. The differing shape of the curves corresponding to the hybridization of the PCR product and its complementary oligonucleotide are difficult to explain. The calculated Tm values of the probes are all approximately 70°C, although that of PB-3 is slightly lower (64°C) (Table 1). It was supposed that the time courses of hybridization would be related to the conformation of the amplified DNA fragment, which should exist predominantly as single-stranded DNA due to asymmetric amplifi-
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cation. Depending on its sequence, this singlestranded DNA may have some secondary structure due to intramolecular complementary base pairing. We suspect that the hybridization of the probe to the PCR product could be being hindered by the formation of some secondary conformation of the PCR product. Consequently, it is difficult to predict the detailed interactions of the probes and the amplified DNA fragments. Nonetheless, we know that the conformation of the target DNA fragment as well as that of the probe are important factors in hybridization reactions, and it is only through experiment that we can determine which are the most efficient probes. The limit of detection as determined using PM6 and PB-6 (Figs. 3 and 4) was 103 cfu per assay in a sample prior to PCR. It was demonstrated that the sensitivity of this assay was almost equal to gel electrophoresis. For the detection of VT2 gene in food samples, an enrichment step prior to the PCR would be required to produce samples above the detection limit. These results show that the detection limit was determined mainly by the efficiency of amplification, rather than the sensitivity of the fluorescence polarization measurement technique. In addition, the sensitivity of the fluorescence polarization is significantly influenced by the choice of the probe and the primer. In the case of PM-1 and PB-1, the sensitivity was less than that of gel electrophoresis (Fig. 2). On the other hand, the sensitivity using PM-6 and PB-6 was almost equal to that of gel electrophoresis (Figs. 4 and 5). This method could be a very useful tool for detecting specific sequences in the amplified products of nucleic acid samples. Thus, optimization of the primer sequence and PCR conditions should improve the detection limit of 103 cfu per assay and further decrease the assay time to less than 5 min as in this system. Several methods for the monitoring of DNA hybridization in homogeneous solution using fluorophore-labeled oligonucleotide probes have been reported, based on fluorescence energy transfer (Morrison and Stols, 1993; Tyagi and Kramer, 1996), and fluorescence excimer formation (Masuko et al., 1998). Compared with these methods, the advantage of this fluorescence polarization
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method is that the probe architecture is very simple.
5. Conclusions Detection of the VT2 gene of verotoxin-producing E. coli using our primers and probes was successful except for the set of PM-1 and PB-1. The detection limit using PM-6 and PB-6 was 103 cfu per assay, and results could be obtained within 5 min after PCR amplification, using a fluorescence polarization assay. Moreover, detection of the VT1 gene using the same methodology is in progress. The final goal of our study is the rapid detection of VTEC and typing of its VT genes. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid, sensitive and selective detection of oligonucleotide sequences. The design of appropriate primers should enable the selective amplification of target sequences, while the design of the probe should facilitate the rapid detection of the post-PCR product. A combination of both would greatly assist in the rapid and specific detection of the gene. Using this technique, it should be possible to perform assays for virtually any organism or virus, providing a suitable target oligonucleotide sequence can be isolated.
Acknowledgements We would like to thank Dr Scott McNiven for assistance in preparing this manuscript.
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