KR101402279B1 - Rapid isolation method of enterohaemorrhagic Escherichia coli - Google Patents

Abstract

The present invention relates to a detection method of enterohemorrhagic escherichia coli and, more specifically, to a detection method using an antibody against a verotoxin which is produced by colon bacillus. By using the detection method or a kit for detection according to the present invention, the enterohemorrhagic escherichia coli are rapidly and accurately detected from a specimen. Therefore, it is possible to immediately cope with contagion by diagnosing the infection of the enterohemorrhagic escherichia coli rapidly and accurately.

Description

Rapid isolation method of enterohaemorrhagic Escherichia coli using magnetic beads {Rapid isolation method of enterohaemorrhagic Escherichia coli}

The present invention relates to a method for detecting intestinal haemorrhagic E. coli, and specifically, to a method for rapidly and accurately detecting intestinal haemorrhagic E. coli from a sample using an antibody against verotoxin, a toxin produced by these E. coli, and magnetic beads.

Since Enterohemorrhagic Escherichia coli (EHEC) produces verotoxin (VT), it is known as a serious pathogenic strain that causes hemolytic uremic syndrome during infection. In addition, it can be transmitted not only through skin contact, but also through drinking water, and the mortality rate at the time of infection reaches 10% for infants and 50% for the elderly, making it a very dangerous strain.

As is the case with most infectious infections, it is very important to promptly determine whether the infection is infected and reduce the damage caused by subsequent transmission, but the existing methods of diagnosing infection with intestinal haemorrhagic E. coli have a problem that the test period takes a long time or the accuracy is poor. In particular, the infection of these infectious pathogens requires quarantine measures of the infected person to prevent further transmission, and there is a problem that people who are not infected can be quarantined if a misdiagnosis is made, and development of a rapid and accurate diagnosis method is required.

Traditionally, the feces of diarrhea patients are pre-cultured and then cultured on MacConkey agar medium to select each colony, and then toxin Shiga by polymerase chain reaction (PCR) method. A method of determining the presence or absence of a gene (Shiga toxin gene) was used, and recently, a method of examining 300 colonies at once through a colony southern blot has also been used.

The above methods require a test period of at least 3 to 5 days, and are expensive and manpower consuming methods for separating intestinal hemorrhagic E. coli from a sample of one patient. Such time, manpower, and cost are caused because it is difficult to distinguish between non-pathogenic E. coli and pathogenic E. coli in a stool sample.

The present inventors have made diligent research efforts to develop a more rapid and accurate new method by improving the problems of the existing diagnosis method as described above.

As a result, in the case of using detection particles in the form of a combination of an antibody against verotoxin and magnetic particles, only intestinal hemorrhagic E. coli can be quickly and accurately separated from the sample. It was confirmed that it can be effectively detected, and it was confirmed that it was possible to quickly and accurately diagnose the presence of intestinal hemorrhagic E. coli infection using this detection method, and the present invention was completed.

The main object of the present invention is to provide a method for quickly and accurately detecting intestinal hemorrhagic E. coli.

Another object of the present invention is to provide a kit for detecting intestinal haemorrhagic E. coli that can quickly and accurately detect intestinal haemorrhagic E. coli.

According to one aspect of the present invention, the present invention is characterized by detecting enterohemorrhagic Escherichia coli (EHEC) vero toxin and an antigen-antibody binding reaction of an antibody against vero toxin to detect enterohemorrhagic Escherichia coli cells. It provides a method for detecting intestinal hemorrhagic E. coli.

Vero toxin is also called shiga like toxin because of its structural similarity to shiga toxin.

Previously, antibodies against verotoxin were mainly used for the purpose of neutralizing toxicity. However, the present inventors confirmed that this vero toxin is present in the outer membrane of intestinal hemorrhagic E. coli, and thought that using an antibody against this vero toxin would be able to detect not only toxins but also bacteria. In addition, by binding magnetic particles to this antibody, it was thought that it would be possible to more easily detect the intestinal haemorrhagic E. coli cells, and this was actually confirmed through an experiment.

It is known to date that intestinal hemorrhagic Escherichia coli produces one or two protein toxins of either Verotoxin 1 or Verotoxin 2. Each verotoxin consists of two types of protein subunits (A and B).

The amino acid sequence of Verotoxin 1 and 2 and the nucleotide sequence of the related gene may appear slightly different depending on the strain, but representatively, in the case of Verotoxin 1, subunit A is the amino acid sequence of SEQ ID NO: 1, and subunit B is SEQ ID NO: 2 It may be represented by the amino acid sequence of, and their gene base sequence may be represented by SEQ ID NOs: 3 and 4, respectively. In the case of Verotoxin 2, subunit A may be represented by the amino acid sequence of SEQ ID NO: 5, subunit B is the amino acid sequence of SEQ ID NO: 6, and each gene nucleotide sequence may be represented by SEQ ID NO: 7, 8.

Antibodies against verotoxin used in the present invention can be prepared according to an antibody preparation method using a conventional immune reaction. Verotoxin can be obtained by administering to mammals or birds as an antigen to induce an immune response and then purifying the generated antibody. In the present invention, it is preferable that it is immunoglobulin G (IgG).

According to another aspect of the present invention, the present invention is a) Enterohemorrhagic Escherichia coli , EHEC) contacting a sample with a detection particle formed by binding of an antibody against verotoxin to a magnetic particle; And b) obtaining a conjugate of intestinal haemorrhagic Escherichia coli and the antibody of the detection particle bound by using a magnetic force with a vero toxin interposed therebetween. This is a method of introducing a magnetic particle system to facilitate detection of intestinal hemorrhagic E. coli.

In the present invention, magnetic particles mean micro- or nano-unit (diameter) particles that have a binding force with an antibody and exhibit magnetism. As such magnetic particles, the surface of the magnetic core particle _{is coated with a functional group such as amine (NH 2} ), and various types of magnetic particles with different components or surface functional groups of the core particle are commercially available. It has become, and these can be used. When manufacturing a detection particle by combining this magnetic particle with a verotoxin antibody, it is preferable to follow the manual of each magnetic particle manufacturer, but when using magnetic particles having an amine functional group, the binding reaction temperature is 2 ~ 6℃ (optimum 4°C), it is preferable to set the time to 8 to 24 hours (optimum 18 hours). The lower the reaction temperature is, the better it is to maintain the activity of the antibody, but the binding efficiency with magnetic particles decreases, and the higher the reaction time is, the problem is that the antibody may lose activity. If the reaction time is too short, sufficient binding reaction cannot be achieved, and thus detection particles are generated. Even if the efficiency decreases and the time is prolonged, there may be no effect of increasing the efficiency, or the efficiency may be lowered.

In the present invention, the sample is preferably used by suspending it in a buffer solution (pH 6.5 ~ 8).

When the detection particle of the present invention is added to a sample containing enterohemorrhagic E. coli, a conjugate of the detection particle and vero toxin is generated through an antigen (verotoxin)-antibody binding reaction. Is created.

In the detection method of the present invention, in order to further increase the detection efficiency of intestinal hemorrhagic E. coli, incubating for 2 to 18 hours at 35 to 39° C. in an E. coli culture medium between steps a) and b). It is desirable. This is to increase the binding rate with the antibody by culturing intestinal hemorrhagic E. coli that may exist in the sample to increase the number of cells, and the culture temperature is a temperature suitable for the growth of E. coli, and the incubation time is the increase in the number of E. coli cells and the antibody binding reaction. This is the time considering the efficiency of The optimal time conditions for VT1 and VT2 are slightly different, such as 8 hours and 4 hours, respectively, but in the above range, it is determined that the binding reaction will be sufficiently performed in both cases. However, a more appropriate range for each is 2 to 8 hours for VT1 and 4 to 18 hours for VT2. As a medium for culturing E. coli, TSB, LB, or the like can be used.

In addition, in the detection method of the present invention, a polymerase chain reaction (PCR) is performed using a primer set capable of amplifying some of the verotoxin genes of enterohemorrhagic Escherichia coli targeting enterohemorrhagic Escherichia coli of the conjugate. The step of doing; And analyzing the product amplified through the polymerase chain reaction. If such a PCR method is additionally applied, it is possible to more quickly and accurately determine whether intestinal hemorrhagic E. coli is detected.

The primer set includes a primer set (primer set for VT1) comprising an oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 10; And a primer set comprising an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 11 and an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 12 (primer set for VT2).

In addition, by culturing the conjugate intestinal hemorrhagic Escherichia coli in an Escherichia coli culture medium (especially a solid medium), cells can be directly obtained.

According to another aspect of the present invention, the present invention is enterohemorrhagic Escherichia (Enterohemorrhagic Escherichia coli , EHEC) provides a kit for detecting intestinal haemorrhagic Escherichia coli including particles for detection obtained by binding an antibody against verotoxin of a magnetic particle. This kit is provided in order to very easily perform the method of detecting intestinal hemorrhagic E. coli of the present invention.

In addition to the kit, it is preferable to further include a primer set capable of amplifying some of the verotoxin genes of intestinal hemorrhagic Escherichia coli, wherein the primer set is an oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 9 and a base of SEQ ID NO: 10 A primer set consisting of an oligonucleotide comprising a sequence; And a primer set comprising an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 11 and an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 12.

If the detection method or the detection kit according to the present invention is used, it is possible to quickly and accurately detect intestinal haemorrhagic E. coli from a sample, and accordingly, it is possible to quickly and accurately diagnose whether intestinal hemorrhagic E. Becomes possible.

1 is a schematic diagram showing a process of detecting intestinal hemorrhagic E. coli according to an embodiment of the present invention.
Figure 2 is a result of confirming the cross-reaction using antibodies against VT1 and VT2 targeting the entire protein of EHEC.
3 is a graph showing the degree of adhesion of magnetic particles and antibodies to VT1 or VT2 over time under a reaction condition of 4°C.
4 is a graph showing detection efficiency according to incubation time by adding detection particles to a sample and adding LB medium.
5 is a graph showing the results of an experiment of cross-reaction with non-EHEC strains. EHEC strains selected from EHEC(VT1), EHEC(VT2), EHEC(VT1 & VT2), VT1 non-expressed EHEC, and VT2 non-expressed EHEC and 9 non-pathogenic E. coli , Salmonella typhimurium , Vibrio Cholera O1 , Shigella It shows the detection rate of EHEC strains targeting a sample mixed with non-EHEC strains such as sonneri.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example One. Verotoxin Anti-antibody preparation

1-1. Administration of antigen to mice ( Antigen boosting )

VT1 and VT2 to be used as antigens were prepared using the E. coli expression system.

As the first immunization with Verotoxin as an antigen, mix with complete Freund's adjuvant (Sigma) of the same amount (about 250µl each) and make an emulsion to make an emulsion in a 6-week-old female BALB/c mouse intraperitoneal injection (ip). Injected into. Each animal was injected with 50 ng of antigen, and 6 rats were administered.

The second immunization proceeded after 2 weeks and was administered in the same manner as above. The third immunization was also carried out in the same way as the second immunization process, and finally, two weeks later, the antigen (the same amount as the third administration) was mixed with PBS and injected into the vein of the tail (intravenous injection, iv). I did. The last administration is done through the caudal vein to induce highly specific B lymphocytes into the spleen. The cell fusion process was performed 3 days after the last immunization.

1-2. Splenocytes and Fusion of myeloma cells ( Cell fusion )

As for the cell fusion method, PEG (polyethylene glycol, Sigma) was used as a fusogen according to the Kohler and Milstein method. Myeloma cells (myeloma, V653, if the number of cells is 7 ~ 8 × 10 ⁵ /㎖), which has been passaged from 2 weeks before cell fusion, is diluted to 1/2 and used for the next day's experiment) and antibody titer is high. After preparing the splenocytes of the mice shown in advance, they were mixed and washed with a cell culture medium not added with FBS, and then slowly stirred over a period of 1 minute using 1 ml of pre-heated (37° C.) PEG. Fused. The fusion process was performed while slowly adding 1X HAT medium (20% FBS, RPMI-1640) over several minutes after fusion. The whole process was performed using a 37°C water bath, and the cells were diluted with HAT-added medium and dispensed at a concentration sufficient to generate one colony per well in a 96 well plate. Dispensed 96 well plates were placed in a 37°C CO ₂ incubator and incubated for 7 days, and in principle, the incubator was not opened until the screening step was entered.

1-3. Specificity test target well Screening of

After 1 week (7 days) after cell fusion, each 96 well plate was taken out and placed on inverted microscopy to observe the formation and extent of cell colony (the size of the colony). Among the marked wells, only those whose colony size occupied around 25% of the total well area were selected for specificity test. The morphological appearances of the colonies formed were classified in consideration of the cell density of each morphology as various forms such as complete floating, clustering, partial adhesion, and complete adhesion appeared.

1-4. Enzyme Immunoassay ( ELISA Specificity test using)

The antigen solution was dispensed at a concentration of 10 μg/ml into a 96 well plate at a concentration of 100 μl and reacted at 37° C. for 60 minutes. 0.05M sodium bicarbonate solution was used as a diluent. The reaction solution was removed, 200 μl of washing solution (phosphate buffer, 0.05% Tween 20) was added per well, and the removal process was repeated three times. After washing, 200 µl of a blocking solution (a phosphate buffer containing 1% BSA) was added to each well and reacted at 37° C. for 30 minutes. After removing the blocking solution and the reaction solution, 200 μl of the washing solution was added per well, and the process of removing again was repeated 3 times. 100 μl of monoclonal antibody (mAb) solution was dispensed per well, 100 μl of the diluted solution was dispensed into the well for negative control, and then reacted at 37° C. for 30 minutes, the reaction solution was removed, and 200 μl of the washing solution was added per well. The process of removing again was repeated three times. A reagent in which HRP (Horse reddish peroxidase) was fused to anti-mouse IgG was dispensed per well by 100 µl and reacted at 37°C for 30 minutes. The reaction solution was removed, 200 μl of the washing solution was added per well, and the process of removing again was repeated three times. An enzyme substrate solution (o-phenylenediamine 0.4 mg/ml in phosphate citrate buffer pH 5.0) was dispensed at 100 μl and reacted for 30 minutes at room temperature in a dark room. The absorbance value was checked at 492 nm (yellow), and the absorbance value 3 times or more of the absorbance of the negative control group was determined as negative or positive based on the minimum value of the positive reaction.

1-4. Western blotting Specificity test using method

The purified recombinant proteins were electrophoresed on SDS-PAGE, and then the recombinant proteins were electrotransfered to a nitrocellulose membrane (0.45㎛; Bio-Rad). Towbin et al. After transferring by the method of (1979), the membrane was blocked overnight at room temperature with a blocking buffer (0.01M PBS, 3% skim milk, Sigma), and an antibody solution (hybridoma cell culture solution) verified through ELISA was titrated to the blocking solution. It was diluted to concentration and reacted at room temperature for 1 hour.

Each reaction step was washed 5 times with PBS-Tween 20 (0.05%), and Alkaline phosphatase-conjugated anti-mouse IgG (Sigma) was diluted to an appropriate concentration in a blocking solution and reacted for 1 hour. A fresh substrate solution (NBT/BCIP, sigma) was prepared and reacted at room temperature for 10 minutes. Distilled water was added to stop the reaction, and the result was visually read.

1-5. Seed cell line And management of production cell lines

The fusion cell line judged to secrete the desired antibody was subjected to primary and secondary cloning by the limiting dilution method, and the fusion cell population was treated so that only clones derived from one cell were induced. The established monoclonal antibody cell line was suspended in a medium _{and grown in a 37°C, CO 2} cell incubator for 3 to 4 days, and the obtained cell suspension was centrifuged at 1,300 rpm for 10 minutes. Add 10% DMSO and 20% FBS to the culture solution), ^{dispense 1 ml (1×10 6} cells/ml) into an ampoule for cryopreservation, record the cell line name, passage number, and passage date, and put it in a freezing container. After storing overnight in a deep freezer (-70℃), it was frozen and preserved at -196℃ (liquid nitrogen) to be used as a protozoan cell line.

Thereafter, the produced original monoclonal antibody secreting cell line was taken out, quickly thawed in a 37°C constant temperature water bath, suspended in a medium, and allowed to grow in a 37°C CO ₂ cell incubator for 3 to 4 days, and then the proliferated cell suspension was centrifuged at 1,300 rpm for 10 minutes. The precipitated cells are suspended in a cryoprotectant, and then ^{dispensed into ampoules for cryopreservation by 1 ml (1×10 6} cells/ml), record the cell line name, passage number, and passage date, and record at -196℃ (liquid nitrogen). And used as a monoclonal antibody-secreting cell line for production.

Example 2. Manufacture of particles for detection and using the same Intestinal bleeding E. coli detection

2-1. VT1 & VT2 Monoclonal Of antibodies With outer membrane protein Cross-reaction confirmation

The cross-reaction of antibodies against VT1 & VT2 with other proteins of EHEC was confirmed. As a result of this, it was confirmed that, as shown in FIG. 2, only VT1 and VT2 were reacted with the total proteins of EHEC, but did not cross-react with other proteins.

2-2. Manufacture of particles for detection and magnetic particles-antibody Adhesion Confirm

Chemicell's SiMAG-Protein A and SiMAG-Protein G products (magnetic particles having an amine functional group) were used as magnetic particles, and the manual provided by the manufacturer was followed.

In order to confirm the optimal time conditions for attaching the antibody to these magnetic particles, 100 µg of antibody and 500 µg of magnetic particles were reacted at 4°C for each hour, and then the amount of protein of the antibody that failed to adhere to the magnetic particles was quantified. .

As a result of this, as shown in FIG. 3, it was found that the coupling efficiency gradually decreased after 4 hours. SiMAG-Protein G bonded gently until 4 hours, but after 4 hours, the bonding strength was 5-10% higher than that of SiMAG-Protein A. However, this difference is seen as an experimental error, and it is judged that the two magnetic particles can be used without distinction. In summarizing these results, it is considered that the most ideal conditions for the binding reaction between the magnetic particles and the antibody should be 18 hours at 4°C. In addition, it is judged that the ratio of the antibody and magnetic particles during the binding reaction should be 1: 5 ~ 1: 15 (based on weight).

2-3. VT1 Production strains and VT2 Confirmation of detection of production strain

The detection efficiency of the detection particles by incubation time was confirmed using an LB medium in which a strain secreting VT1 and VT2 toxins and non-pathogenic E. coli were mixed.

The detection was confirmed by the polymerase chain reaction (PCR) method using the following primers.

VT1 primer F: ATAAATCGCCATTCGTTGACTAC

VT1 primer R: AGAACGCCCACTGAGATCATC

VT2 primer F: GGCACTGTCTGAAACTGCTCC

VT2 primer R: TCGCCAGTTATCTGACATTCTG

The bacterial solution was suspended in sterile water, then boiled at 100° C. for 5 minutes, and 1 μl of the supernatant obtained by centrifuging at 15,000 g for 5 minutes was used as a template. 1 µl (10 pmole) of each of the four primers and 1 µl of a template were added to adjust the volume of the PCR reaction solution to a total of 20 µl. Taq polymerase used Intron's Maxime PCR PreMix (i-StarMAXII) Cat.No.25281. PCR conditions were 94°C for 5 minutes, followed by 94°C for 30 seconds, 55°C for 60 seconds, and 72°C for 30 seconds, for a total of 30 times, followed by reaction at 72°C for 5 minutes. By electrophoresis after PCR, the product size of VT1 was 180bp and VT2 was 255bp.

EHEC (VT1) showed the best detection rate when incubated for 8 hours, but EHEC (VT2) showed the best result in 4 hours incubation (see FIG. 4). As a result of synthesizing these results, it is judged that it would be better to set the incubation time to 4 hours for quick and easy separation of EHEC.

2-4. Check the detection limit

To confirm the detection limit of EHEC, an EDL933 strain secreting both VT1 and VT2 was used. In the state where the non-pathogenic E. coli is at ^{a concentration of 10 8} CFU, add 50 µl of detection particles to the LB medium each mixed with EDL933 to a concentration of 1 CFU to 10 ⁴ CFU, and shake culture for 4 hours and wash 5 times with PBS. To isolate the bacteria. As a result, as shown in Table 1, an error occurred due to dilution of the cells in the results of ^{1 CFU and 10 1} CFU, but a detection rate of 80% or more was observed in ^{10 2 CFU.}

1 CFU 10 ¹ CFU 10 ² CFU 10 ³ CFU 10 ⁴ CFU EDL933 68 189 1049 20066 38333 non-pathogenic E. coli 137 96 169 188 159 EHEC isolation% 33% 66% 86% 99% 99%

* Result value: The number of colonies confirmed by culturing the isolated bacteria in solid medium.

* In the process of bacterial isolation, since the number of cells grows and the number of cells increases during shaking culture, the number of colonies identified after detection is larger than the number of cells before detection.

2-5. Normal bacterial flora And cross-reaction with other pathogenic bacteria

We tried to confirm the cross-reaction with other bacteria than EHEC. In general, in the stool sample, in addition to EHEC, various kinds of microorganisms including non-pathogenic E. Therefore, even if other microorganisms other than EHEC to be detected exist together, the detection rate must be high. In addition to E. coli, Salmonella and Vibrio are relatively easy to identify, but non-pathogenic E. coli is not, so it is particularly important to confirm cross-reaction with non-pathogenic E. coli.

9 Non-pathogenic E. coli , other Salmonella typhimurium , Vibrio , obtained through Chungbuk Institute of Health and Environment Cholera O1 , Shigella sonneri was used as a non-EHEC strain. In addition, one of the strains in which the VT1 and VT2 toxins of EHEC were negative by the RPLA method (for convenience, they are labeled as VT1 non-expressed EHEC and VT2 non-expressed EHEC) were selected for cross-reaction. Was confirmed.

EHEC (VT1), EHEC (VT2), EHEC (VT1 & VT2), VT1 non-expressed EHEC, VT2 non-expressed EHEC. A method of checking was used.

As a result of cross-reaction confirmation, as shown in FIG. 5, it was confirmed that the selection separation rate of EHEC (VT1), EHEC (VT2), and EHEC (VT1 & VT2) from 9 weeks of non-pathogenic E. coli or other pathogenic bacteria was an average of 80% or more. On the other hand, as a result of confirming the same cross-reaction with VT1 non-expressed EHEC or VT2 non-expressed EHEC instead of EHEC, the cells were isolated in all experiments, although there was a slight difference in the case of VT1 non-expressed EHEC.

VT1 or VT2 non-expressed EHECs were thought to not produce verotoxin, but are still harmful strains. While this strain showed a negative result in the RPLA method, as confirmed through this example, when the detection particles of the present invention were used, the detection was confirmed, although the detection rate was relatively low compared to EHEC.

This is considered to be because these VT1 or VT2 non-expressed EHECs do not produce VT1 or VT2 at all, but produce in trace amounts, and the detection particles of the present invention efficiently bind to VT1 or VT2 produced in trace amounts.

<110> Korea Center for Disease Control and Prevention <120> Rapid isolation method of enterohaemorrhagic Escherichia coli <130> PA140307-C01 <160> 12 <170> KopatentIn 2.0 <210> 1 <211> 315 <212> PRT <213> Escherichia coli O157:H7 str. EDL933 <400> 1 Met Lys Ile Ile Ile Phe Arg Val Leu Thr Phe Phe Phe Val Ile Phe 1 5 10 15 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 25 30 Lys Thr Tyr Val Asp Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr 35 40 45 Pro Leu Gln Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp 50 55 60 Ser Gly Thr Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp 65 70 75 80 Pro Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn 85 90 95 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100 105 110 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly 130 135 140 Ile Ser Arg Thr Gly Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser 145 150 155 160 Tyr Leu Asp Leu Met Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val 165 170 175 Ala Arg Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 Phe Arg Gln Ile Gln Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser 225 230 235 240 Val Arg Val Gly Arg Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly 245 250 255 Ser Val Ala Leu Ile Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 Arg Met Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285 Val Arg Gly Ile Thr His Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu 290 295 300 Gly Ala Ile Leu Met Arg Arg Thr Ile Ser Ser 305 310 315 <210> 2 <211> 89 <212> PRT <213> Escherichia coli O157:H7 str. EDL933 <400> 2 Met Lys Lys Thr Leu Leu Ile Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 15 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 20 25 30 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 35 40 45 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln 50 55 60 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly 65 70 75 80 Gly Gly Phe Ser Glu Val Ile Phe Arg 85 <210> 3 <211> 948 <212> DNA <213> Escherichia coli O157:H7 str. EDL933 <400> 3 tcaactgcta atagttctgc gcatcagaat tgcccccaga gtggatgaat cccacaatat 60 tttattgtgc gtaatcccac ggactcttcc atctgccgga cacatagaag gaaactcatc 120 agatgccatt ctggcaactc gcgatgcatg atgatgacaa ttcagtatta atgccacgct 180 tcccagaatt gcattaatgc ttccaaaaga aattcttcct acacgaacag agtcttgtcc 240 atgataatca ggcaggacac tactcaacct tccccagttc aatgtaagat caacatcttc 300 agcagtcatt acataagaac gcccactgag atcatccagt gttgtacgaa atcccctctg 360 tatttgccga aaacgtaaag cttcagctgt cacagtaaca aaccgtaaca tcgctcttgc 420 cacagactgc gtcagtgagg ttccactatg cgacattaaa tccagataag aagtagtcaa 480 cgaatggcga tttatctgca tccccgtacg actgatccct gcaacacgct gtaacgtggt 540 atagctactg tcaccagaca atgtaaccgc tgttgtacct ggaaaggtaa catgtgaaaa 600 atcagcaaag cgataaaaaa cattatttgt cctgttaaca aatcctgtca catataaatt 660 atttcgttca acaataagcc gtagattatt aaaccgccct tcctctggat ctatccctct 720 gacatcaact gcaaacaaat tatcccctgt gccactatca atcatcagta aagacgtacc 780 tcctgatgaa atagtctgta atggagtacc tattgcagag cgaatgacat tcagcgaatc 840 tacatacgtc tttgcagtcg agaagtctaa ggtaaattcc ttcgcaacca cattaactga 900 aaagataaca aagaaaaaag ttagcactct aaaaataatt attttcat 948 <210> 4 <211> 270 <212> DNA <213> Escherichia coli O157:H7 str. EDL933 <400> 4 tcaacgaaaa ataacttcgc tgaatccccc tccattatga caggcattag ttttaatggt 60 tacagtcatc cccgtaattt gcgcactgag aagaagagac tgaagattcc atctgttggt 120 aaataattct ttatcaccca ctttaactgt aaaggtatcg tcatcattat attttgtata 180 ctccaccttt ccagttacac aatcaggcgt cgccagcgca cttgctgaaa aaaatgaaag 240 cgatgcagct attaataatg tttttttcat 270 <210> 5 <211> 319 <212> PRT <213> Escherichia coli O157:H7 str. EDL933 <400> 5 Met Lys Cys Ile Leu Phe Lys Trp Val Leu Cys Leu Leu Leu Gly Phe 1 5 10 15 Ser Ser Val Ser Tyr Ser Arg Glu Phe Thr Ile Asp Phe Ser Thr Gln 20 25 30 Gln Ser Tyr Val Ser Ser Leu Asn Ser Ile Arg Thr Glu Ile Ser Thr 35 40 45 Pro Leu Glu His Ile Ser Gln Gly Thr Thr Ser Val Ser Val Ile Asn 50 55 60 His Thr Pro Pro Gly Ser Tyr Phe Ala Val Asp Ile Arg Gly Leu Asp 65 70 75 80 Val Tyr Gln Ala Arg Phe Asp His Leu Arg Leu Ile Ile Glu Gln Asn 85 90 95 Asn Leu Tyr Val Ala Gly Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr 100 105 110 Arg Phe Ser Asp Phe Thr His Ile Ser Val Pro Gly Val Thr Thr Val 115 120 125 Ser Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Ala 130 135 140 Leu Glu Arg Ser Gly Met Gln Ile Ser Arg His Ser Leu Val Ser Ser 145 150 155 160 Tyr Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp Ala 165 170 175 Ser Arg Ala Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 Phe Arg Gln Ile Gln Arg Glu Phe Arg Gln Ala Leu Ser Glu Thr Ala 195 200 205 Pro Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp 210 215 220 Gly Arg Ile Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val 225 230 235 240 Arg Val Gly Arg Ile Ser Phe Asn Asn Ile Ser Ala Ile Leu Gly Thr 245 250 255 Val Ala Val Ile Leu Asn Cys His His Gln Gly Ala Arg Ser Val Arg 260 265 270 Ala Val Asn Glu Glu Ser Gln Pro Glu Cys Gln Ile Thr Gly Asp Arg 275 280 285 Pro Val Ile Lys Ile Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala 290 295 300 Ala Phe Leu Asn Arg Lys Ser Gln Phe Leu Tyr Thr Thr Gly Lys 305 310 315 <210> 6 <211> 89 <212> PRT <213> Escherichia coli O157:H7 str. EDL933 <400> 6 Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Val Asn 1 5 10 15 Ala Met Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr 20 25 30 Asn Glu Asp Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp 35 40 45 Thr Ser Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr 50 55 60 Gly Met Thr Val Thr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly 65 70 75 80 Phe Ala Glu Val Gln Phe Asn Asn Asp 85 <210> 7 <211> 960 <212> DNA <213> Escherichia coli O157:H7 str. EDL933 <400> 7 atgaagtgta tattatttaa atgggtactg tgcctgttac tgggtttttc ttcggtatcc 60 tattcccggg agtttacgat agacttttcg acccaacaaa gttatgtctc ttcgttaaat 120 agtatacgga cagagatatc gacccctctt gaacatatat ctcaggggac cacatcggtg 180 tctgttatta accacacccc accgggcagt tattttgctg tggatatacg agggcttgat 240 gtctatcagg cgcgttttga ccatcttcgt ctgattattg agcaaaataa tttatatgtg 300 gccgggttcg ttaatacggc aacaaatact ttctaccgtt tttcagattt tacacatata 360 tcagtgcccg gtgtgacaac ggtttccatg acaacggaca gcagttatac cactctgcaa 420 cgtgtcgcag cgctggaacg ttccggaatg caaatcagtc gtcactcact ggtttcatca 480 tatctggcgt taatggagtt cagtggtaat acaatgacca gagatgcatc cagagcagtt 540 ctgcgttttg tcactgtcac agcagaagcc ttacgcttca ggcagataca gagagaattt 600 cgtcaggcac tgtctgaaac tgctcctgtg tatacgatga cgccgggaga cgtggacctc 660 actctgaact gggggcgaat cagcaatgtg cttccggagt atcggggaga ggatggtgtc 720 agagtgggga gaatatcctt taataatata tcagcgatac tggggactgt ggccgttata 780 ctgaattgcc atcatcaggg ggcgcgttct gttcgcgccg tgaatgaaga gagtcaacca 840 gaatgtcaga taactggcga caggcctgtt ataaaaataa acaatacatt atgggaaagt 900 aatacagctg cagcgtttct gaacagaaag tcacagtttt tatatacaac gggtaaataa 960 960 <210> 8 <211> 270 <212> DNA <213> Escherichia coli O157:H7 str. EDL933 <400> 8 atgaagaaga tgtttatggc ggttttattt gcattagctt ctgttaatgc aatggcggcg 60 gattgtgcta aaggtaaaat tgagttttcc aagtataatg aggatgacac atttacagtg 120 aaggttgacg ggaaagaata ctggaccagt cgctggaatc tgcaaccgtt actgcaaagt 180 gctcagttga caggaatgac tgtcacaatc aaatccagta cctgtgaatc aggctccgga 240 tttgctgaag tgcagtttaa taatgactga 270 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VT1 <400> 9 ataaatcgcc attcgttgac tac 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VT1 <400> 10 agaacgccca ctgagatcat c 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VT2 <400> 11 ggcactgtct gaaactgctc c 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VT2 <400> 12 tcgccagtta tctgacattc tg 22

Claims (6)

a) Enterohemorrhagic Escherichia contacting the sample with a detection particle comprising an antibody to berotoxin of E. coli , EHEC bound to magnetic particles; And
and b) obtaining a conjugate in the form of binding between the intestinal hemorrhagic Escherichia coli and the antibody of the detection particles, using a magnetic force. The method according to claim 1,
Wherein the berotoxin is berotoxin 1 (VT1) or berotoxin 2 (VT2). 3. The method of claim 2,
The verotoxin 1 is composed of a subunit A represented by the amino acid sequence of SEQ ID NO: 1 and a subunit B represented by the amino acid sequence of SEQ ID NO: 2,
Wherein the verotoxin 2 comprises a subunit A represented by the amino acid sequence of SEQ ID NO: 5 and a subunit B represented by the amino acid sequence of SEQ ID NO: 6. The method according to claim 1,
Between the steps a) and b)
And culturing in an E. coli culture medium at 35 to 39 DEG C for 2 to 18 hours. The method according to claim 1,
After the step b)
Performing a polymerase chain reaction using a primer set capable of amplifying a part of a berotoxin gene of enterohemorrhagic Escherichia coli against enterohemorrhagic Escherichia coli of the combination; And
And analyzing the product amplified by the polymerase chain reaction. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 6. The method of claim 5,
The primer set
A primer set consisting of an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 10; And
A primer set consisting of an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 11 and an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 12.

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