A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli

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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.

Introduction

Verotoxin-producing Escherichia coli (VTEC) is identical to enterohemorrhagic E. coli. When a 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 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 (Lundblad 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, Tsuruoka et al., 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.

Section snippets

Synthetic oligonucleotides

All oligonucleotides were synthesized using a DNA synthesizer (Model 392; Perkin-Elmer, 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 Table 1, Table 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

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

Discussion

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

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

Acknowledgements

We would like to thank Dr Scott McNiven for assistance in preparing this manuscript.

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