JP5979657B2 - Primers and detection kits for detection of Escherichia coli causing food poisoning - Google Patents

Description

  The present invention relates to a method for detecting Escherichia coli causing food poisoning having an eae gene encoding intimin by a loop-mediated isothermal amplification (LAMP) method, and a primer for use in the method It relates to sets.

  The pathogenic Escherichia coli that causes food poisoning has been classified into several categories. Among them, enterohaemorrhagic Escherichia coli (hereinafter referred to as “EHEC”) isolated from patients with hemorrhagic colitis is a major food poisoning. It is a very important bacterial species in terms of pathogenicity. EHEC possesses a protein called intimin for adhering to the intestinal tract of humans and host animals as a major pathogenic factor in addition to a toxin called shiga toxin produced by some Shigella (Non-patent Document 1).

The intimin molecule is encoded by a gene called eae. EHEC injects another protein called Tir (translocated intimin receptor) expressed from another gene in the vicinity of the eae gene into the host cell by the bacterial secretion mechanism called the type III secretion apparatus. To do. Tir moves from the inside of the host cell to the cell surface and functions as an intimin receptor.
Intimin has a barrel structure called β-barrel in its 938 amino acid, and the N-terminal region forms a dimer of intimin and pierces the outer membrane of the fungus. A state in which the region protrudes outside the cells is taken. This C-terminal 280 amino acid region has a function of binding to a Tir molecule. Thus, EHEC adheres strongly to cells by binding to Tir in addition to receptors on the host cell surface, and produces the above-mentioned Shiga toxin in a fixed state (Non-patent Document 1).

  Shiga toxin is a so-called AB type toxin consisting of one molecule of A subunit and five molecules of B subunit. The A subunit is a catalytic subunit having RNA-N-glycosidase activity, and this activity inactivates 60S ribosome, resulting in inhibition of cellular protein synthesis. The B subunit is a cell binding unit that accepts Gb3, a glycolipid on the cell surface, as a receptor. As a result, protein synthesis inhibition of intestinal epithelial cells and vascular endothelial cells occurs, and as a result, it exhibits cytotoxic activity that kills the cells and enterotoxic activity that exhibits liquid retention in the intestinal tract due to the death of intestinal epithelial cells (non- Patent Document 2).

  Intimin, on the other hand, strengthens the adhesion between bacteria and intestinal epithelial cells, and also adds the growth of bacteria, creating a state in which toxins are produced continuously. Lesions with missing villi are observed.

EHEC is further classified into several types according to the serotype, and the most common cause of food poisoning is O157, and in addition, O26, O111, O103, O145, etc. have been confirmed.
The main habitat of EHEC is in the intestine of mammals and birds. In addition to being isolated from cattle, sheep, goats, pigs, chickens, cats, dogs, etc., well water, river mud, insects (flies), etc. It was also separated from. Contamination of meat and internal organs from intestinal contents is a major source of food contamination in the livestock demolition and meat processing processes at slaughterhouses. The preservation state of the internal organs is also a factor that increases contamination (Non-patent Document 1).

EHEC is a gram-negative facultative anaerobic bacterium with no spores. Conventionally, to identify EHEC, acid and gas production by glucose fermentation, indole production test, methyl red against bacteria that grow on selective separation media such as MacConkey medium, dezoxycholate medium, DHL agar medium, etc. After conducting biochemical property tests such as test, Voges-Proskauer test, citrate utilization test, hydrogen sulfide production test, motility lysine decarboxylase test, lactose and mannitol fermentation test, and determined that it is Escherichia , O, K, and H serotypes have been determined. Furthermore, it is judged by performing a cigar toxin production test by an immunochromatography method or a reverse passive latex agglutination reaction. Based on these principles, an O157 detection kit, an O157 and O26 detection kit, etc. that combine a culture medium for separation culture and an immunochromatography method are also commercially available (Non-patent Document 1).
However, such a method of performing various biochemical tests through enrichment or isolation culture requires time until results are obtained. For this reason, gene-based test methods have attracted attention. For example, a PCR (Polymerase Chain Reaction) method for amplifying and confirming an EHEC-specific gene region (for example, VT, VT1, or VT2 for O157, etc.) Widely used (Non-Patent Document 3).

On the other hand, a group called enterophathogenic Eshcherichia coli (hereinafter referred to as “EPEC”), which belongs to a different category from EHEC, does not have shiga toxin but also has similar intimin and has characteristic intestinal adhesion. Presents with a pathogenic microvilli disappearing lesion (Non-patent Document 1).
Regardless of the presence of the eae gene, a group of bacteria that produce Shiga toxin is called Shiga-toxin-producing Escherichia coli (hereinafter referred to as “STEC”). The distinction is not clear.
EHEC and STEC with the eae gene also bind to host cells via intimin and exert toxicity as in EHEC. Therefore, if intimin or eae gene is detected, food poisoning occurs with the same mechanism of action. It is possible to collectively detect E. coli causing

  However, intimin is an outer membrane protein consisting of 938 amino acid residues and having a molecular weight of about 94,000, and the C-terminal 280 amino acid region has a remarkable amino acid sequence diversity. To date, there are at least 10 types (α, β, γ, etc.), but there is more sequence diversity within each type, and each subtype (α1, α2, etc.) (Non-Patent Document 4 and Non-Patent Document 5). Therefore, it is difficult to detect by immunological detection methods such as immunochromatography using antibodies.

  Thus, it is conceivable to detect the eae gene by the PCR method. However, the bacteria detection method using the PCR method requires special equipment and a skilled technique to perform the reaction. In addition, much time and labor are required for agarose gel electrophoresis, protein fluorescent labeling, detection, and the like, which are one of the final determination methods. Furthermore, although the PCR method for confirming the presence of the eae gene has been reported for research purposes, it has not been conducted in general tests because there is no commercially available kit.

  By the way, in recent years, a method using the LAMP reaction has been developed as one of new gene amplification methods (Patent Document 1, Non-Patent Document 6, Non-Patent Document 7). This method is an isothermal nucleic acid amplification method, has high specificity and amplification efficiency, and can be performed in about 1 hour from reaction to detection.

The LAMP method prepares four primers (generally called Forward Inner Primer (FIP), Backward Inner Primer (BIP), F3 and B3) based on the sequence of the six regions of the target nucleic acid, and uses a strand displacement reaction. This is a technique for amplifying nucleic acids isothermally.
First, for the target DNA, the three regions F3c, F2c, F1c from the 3 ′ end side, and the regions B1, B2, B3 toward the 5 ′ end side of the target DNA, respectively, for these six regions, Four types of primers are designed: FIP, F3, BIP and B3. Here, regions complementary to the F3c, F2c, and F1c regions are referred to as F3, F2, and F1, respectively, and regions complementary to the B1, B2, and B3 regions are referred to as B1c, B2c, and B3c, respectively.
FIP is a primer designed to have an F2 region complementary to the F2c region of the target DNA on the 3 ′ end side and the same sequence as the F1c region of the target DNA on the 5 ′ end side.
F3 is a primer designed to have an F3 region complementary to the F3c region of the target DNA.
BIP is a primer designed to have a B2 region complementary to the B2c region of the target DNA on the 3 ′ end side and the same sequence as the B1c region of the target gene on the 5 ′ end side.
B3 is a primer designed to have a B3 region complementary to the B3c region of the target DNA.
When a restriction enzyme site is introduced into FIP and BIP, the amplified product can be observed as one band after electrophoresis by treating the amplified product with the restriction enzyme after the reaction. If the target DNA has a restriction enzyme site, the same effect can be obtained without artificially introducing a restriction enzyme site into the primer.
Amplify the target DNA isothermally (about 60-65 ° C) with extremely high amplification efficiency and extremely high specificity by reacting the target DNA with 4 primers, strand displacement DNA polymerase, and substrate (dNTPs) in a reaction buffer. Is done. The strand displacement type DNA polymerase is less expensive than the thermostable DNA polymerase used in the PCR method. In addition, the strand displacement type DNA polymerase synthesizes DNA while unwinding the double strand of the template DNA, so the LAMP method does not require double stranded DNA to be single stranded DNA by thermal denaturation unlike the PCR method. .
Since magnesium pyrophosphate is generated as a byproduct of the amplification reaction, the presence or absence of the target gene can also be determined by visually confirming the cloudiness of the reaction buffer.
Furthermore, by using a loop primer having a complementary sequence between the B1 region and the B2 region or between the F1 region and the F2 region (referred to as LB primer and LF primer, respectively), the origin of DNA synthesis is increased. Amplification efficiency can be increased.
The above describes the case where the target is DNA, but when the target is RNA, an amplification reaction can be performed in the same manner as DNA by adding reverse transcriptase to the reaction buffer (Non-Patent Document 7). ).

Whether the LAMP method is successful depends on the design of the primer. In primer design, the distance between primers, Tm value of each primer region, terminal stability of each primer region, GC content, secondary structure, etc. are important, although design support software for designing this also exists, A primer designed in software does not always amplify a target nucleic acid with high efficiency and high specificity. In particular, when the LAMP method is used for detecting microorganisms, an optimal target sequence must first be identified and a primer capable of specifically amplifying that sequence must be prepared.
So far, no target sequence and primer for specifically detecting the eae gene of Escherichia coli causing food poisoning by the LAMP method have been found.

JP 2002-330796 A

Dictionary of Food Safety 2009. Japanese Society for Food Hygiene II-1.2.5-6. P. 114-122, Asakura Shoten Japanese Bacteriological Journal 2010. Vol. 65, No. 2, 3, 4. 297-308. Journal of Clinical Microbiology 2004. Vol. 42. No. 2, 645-651. Infection and Immunity 2000. Vol.68 No.1 64-71. Infection and Immunity 2005. Vol.73 No.1 18-29. Notomi, T. et al., Nucleic Acids research, 2000, 28 (12), e63 Hiroshi Ushikubo, “Principle of the LAMP Method: Simple and Rapid Amplification of Genes”, Virus 54, No. 1, p. 107-112, 2004

  An object of the present invention is to provide a method for detecting E. coli causing food poisoning by the LAMP method, a primer set and a kit used in the method, and the like.

As a result of repeated studies to solve the above problems, the present inventor has identified a region on the eae gene necessary for specifically detecting E. coli having a food poisoning having the eae gene, and the region by the LAMP method. Primers that can be amplified were designed and synthesized, and it was confirmed that the region could actually be specifically amplified, thereby completing the present invention.
That is, the present invention
[1] Primer set for detecting food poisoning-causing Escherichia coli having an eae gene by the LAMP method, including a set of oligonucleotides of any of the following (a) to (d):
(A) a set of four oligonucleotides each consisting of a base sequence represented by SEQ ID NOs: 14, 50, 65 and 89;
(B) a set of four oligonucleotides that are the complementary strands of each of the four oligonucleotides of (a);
(C) a set of four oligonucleotides that hybridize with the four oligonucleotides according to (a) or (b), respectively, under stringent conditions; and (d) any of the oligonucleotides of (a) to (c) A set of oligonucleotides in which 1 to several bases are deleted, added or substituted in at least one of the oligonucleotides included in the set of nucleotides and function as a primer set for the LAMP method;
[2] The primer set according to the above [1], further comprising the oligonucleotide of any one of (e) to (h):
(E) an oligonucleotide having the base sequence represented by SEQ ID NO: 99;
(F) an oligonucleotide that is the complementary strand of the oligonucleotide of (e); and (g) an oligonucleotide that hybridizes under stringent conditions with the oligonucleotide of (e) or (f); and (h) ( an oligonucleotide in which 1 to several bases are deleted, added or substituted in any of the oligonucleotides e) to (g) and function as a primer for the LAMP method;
[3] The food poisoning-causing Escherichia coli is any Escherichia coli selected from the group consisting of enterohemorrhagic Escherichia coli (EHEC), enteropathogenic Escherichia coli (EPEC), and visual sight producing toxin-producing Escherichia coli (STEC) [1] or the primer set according to [2];
[4] A kit for detecting food poisoning-causing Escherichia coli having an eae gene, comprising the primer set according to any one of [1] to [3] above;
[5] A method for detecting Escherichia coli causing food poisoning having the eae gene,
Extracting a nucleic acid in a sample;
According to the LAMP method, GenBank Accession No. Detecting a region included in positions 642 to 877 of AF530557;
And [6] The method according to [5], wherein the primer set according to any one of [1] to [3] is used in the LAMP method.

  According to the present invention, by using the LAMP method, E. coli having an eae gene encoding intimin can be specifically detected, that is, distinguished from other E. coli and other intestinal bacteria and easily and rapidly. Can do. The present invention is useful for detecting contamination due to food poisoning-causing E. coli in various food samples, environmental samples, clinical samples, and the like.

FIG. 1 shows the results of detection of the eae gene by the LAMP method using a high-concentration sample solution using the primer set of the present invention (primer set 32, no LB primer). FIG. 2 shows the results of detection of the eae gene by the LAMP method using a high-concentration sample solution using the primer set of the comparative example (primer set 25, no LB primer). FIG. 3 shows the result of detecting the eae gene by the LAMP method using the primer set of the present invention (primer set 32, with LB primer) using a high-concentration sample solution (upper stage), and the primer of the comparative example The result (bottom) of detecting the eae gene by the LAMP method using a set (primer set 25, with LB primer) using a high-concentration sample solution is shown. FIG. 4 shows the result of detecting the eae gene by the LAMP method using a high-concentration sample solution using the primer set of the present invention (primer set 32, with LB primer). FIG. 5 shows the results of detecting the eae gene by the LAMP method using a low-concentration sample solution using the primer set of the present invention (primer set 32, with LB primer).

The primer set according to the present invention is a set for detecting food poisoning-causing Escherichia coli having an eae gene by the LAMP method.
The present inventor collected all the base sequences of the eae gene using international gene databases (GenBank, EMBL and DDBJ), and arranged different ones even if they differ by one base. Got the sequence. The base sequence of this eae gene can be obtained from the GenBank database, for example, and has registration numbers such as AF530555, AF530557, AJ579305, and the like. These were compared to identify regions where the sequences were conserved 100% and 95% or more. Among these regions, a region having a length of about 250 bases, 4/5 or more of which was 100% conserved, and the remaining 1/5 being conserved by 95% or more was identified. A plurality of such regions could be identified. Specific sequences were identified from them, and LAMP primers were designed.

Based on the base sequence of the eae gene, the present inventor designed a primer targeting a region encoding intimin (for example, the 642th to 877th positions of registration number AF530557).
In designing the primer, the region where the Tm value of each region was about 58 to 65 ° C. was selected targeting the above base sequence, and the region between the F2 and B2 regions was about 120 bp. Primers for eae gene detection were set by narrowing down regions that satisfy these several conditions, which are important points in designing LAMP primers.

  When 33 kinds of designed primers were synthesized and amplified by LAMP method using various E. coli strains, a primer set consisting of the base sequences represented by SEQ ID NOs: 14, 50, 65 and 89 was used. Thus, it was confirmed that amplified acid was always obtained from E. coli having intimin, and amplified acid was not obtained from E. coli and other intestinal bacteria without intimin.

The first embodiment of the primer set of the present invention includes the primers shown in the table below.
This primer set was designed targeting the region from position 642 to position 877 in GenBank accession number AF530557. The region amplified by this primer set was confirmed to be a portion showing no homology as compared with the base sequences of other bacterial species available from GenBank and the like.

The primer set of the present invention may include a complementary strand of each oligonucleotide in the first aspect described above.
For example, a set comprising four oligonucleotides consisting of a sequence complementary to the base sequence represented by SEQ ID NOs: 14, 50, 65 and 89 is also specific for the region from position 642 to position 877 in GenBank accession number AF530557. Can be detected.

In addition, the primer set of the present invention may include four oligonucleotides that hybridize with each oligonucleotide in the first aspect described above under stringent conditions.
Here, stringent conditions include, for example, 5% Denhardt's Solution (containing 0.1% Ficoll (Pharmacia), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 0.5% SDS, and 100 μg / ml salmon sperm. This refers to conditions for washing at 65 ° C. in a 6 × SSC solution containing DNA (1 × SSC is 0.15 M NaCl, 15 mM sodium citrate). Stringency can be controlled by salt concentration (ionic strength), temperature, etc. Under conditions of higher stringency, ie lower salt concentration and higher temperature, only DNA with sufficiently high homology will hybridize. Soybeans. Therefore, under such conditions, the four oligonucleotides that hybridize with each of the oligonucleotides in the first aspect under stringent conditions function as primers for the LAMP method, as in the primer set in the first aspect. To do. A person skilled in the art can appropriately select stringent conditions by adjusting the temperature and the salt concentration.

The primer set of the present invention is an oligonucleotide that functions as a primer set for the LAMP method in which at least one of the oligonucleotides in the first aspect described above is deleted, added, or substituted from 1 to several bases. It may be a set.
The number from 1 to several can be selected, for example, from 1, 2, 3, 4, or 5, and the site to be deleted, added or substituted is the 5 ′ end of the oligonucleotide or 3 'A terminal may be sufficient, and parts other than a terminal may be sufficient.
For example, a restriction enzyme site may be introduced into each primer. When a restriction enzyme site is introduced, the amplified product can be detected as a single band after electrophoresis by treating the amplified product with the restriction enzyme after the reaction of the LAMP method.

  In this specification, food poisoning-causing Escherichia coli having an eae gene is not particularly limited as long as it is an Escherichia coli that causes food poisoning regardless of its extent and has the eae gene, for example, O157, O26, O111, O103, O145, MN157 (Journal of Veterinary Medical Sciences 62 (11): 1151-1155, 2000.), A29, B31 and the like.

The primer set of the first aspect may further include any of the following (i) to (iv).
(I) an oligonucleotide having the base sequence represented by SEQ ID NO: 99;
(Ii) an oligonucleotide which is the complementary strand of the oligonucleotide of (i);
(Iii) an oligonucleotide that hybridizes with the oligonucleotide according to (i) or (ii) under stringent conditions; and (iv) one to several oligonucleotides in any one of the oligonucleotides (i) to (iii) An oligonucleotide having a base deleted, added or substituted and functioning as a primer for the LAMP method.
The oligonucleotide consisting of the base sequence represented by SEQ ID NO: 99 functions as an LB primer that is a loop primer in the LAMP method. Loop primer is a primer that has a complementary sequence to the single-stranded part of the loop on the 5 'end side of the dumbbell structure (between the B1 and B2 regions or between the F1 and F2 regions) in the LAMP method. is there.
The LAMP method can be performed with four primers, FIP, BIP, F3, and B3. By using a loop primer, it is possible to increase the starting point of DNA synthesis and increase the amplification efficiency.
1 in oligonucleotides (iii) and (i) to (iii) which hybridize with oligonucleotide (ii), oligonucleotide (i) or (ii) which are complementary strands of such LB primer under stringent conditions Oligonucleotide (iv) from which several bases are deleted, added or substituted and functions as a primer for the LAMP method also functions as an LB primer.

  The primer set of the present invention may further contain an LF primer that is a loop primer.

(kit)
In the present specification, the food poisoning cause Escherichia coli detection kit includes the primer set according to the present invention and is suitable for the detection of food poisoning cause Escherichia coli having the eae gene. Such a kit may contain, for example, a primer set, a reaction solution, a strand displacement DNA polymerase, distilled water, a positive control, a negative control, an instruction manual, and the like. Furthermore, reagents and instruments for extracting nucleic acids from the sample, various reaction tubes, chips, and the like may be provided.
When intestinal hemorrhagic Escherichia coli in food is cultured and measured, a medium for enrichment may be provided.
By using such a kit, for example, amplification and detection of amplification products can be efficiently performed by adding a sample to a reaction tube and setting the sample in a real-time turbidity measurement device for LAMP.

(Detection method)
In the present specification, the method for detecting food poisoning-causing Escherichia coli having the eae gene is included in positions 642 to 877 in GenBank accession number AF530557 among eae genes by the step of extracting nucleic acid in the sample and the LAMP method. Detecting a region.
Samples include, for example, various food samples such as meat, vegetables, fruits, dairy products, and seafood, environmental samples such as soil, river and lake water, tissues derived from animals such as humans, cows, and horses, samples such as feces It can be.
The step of extracting nucleic acid from these samples can be performed by a known method or a method analogous thereto, and for example, can be performed by a method of extracting DNA with alkali heat. In this case, for example, the sample is heated in an alkaline solution, centrifuged, and then the supernatant is used for the LAMP method. The nucleic acid may be concentrated before being subjected to the LAMP method.
Nucleic acid may be extracted by a method in which cells are treated with Lysozyme or Proteinase K and then heated. Further purification may be performed by phenol / chloroform treatment, ethanol precipitation or centrifugation.
Prior to nucleic acid extraction, enrichment culture, concentration of bacteria, separation, and the like may be performed. Specifically, enrichment culture is performed on an agar medium, and DNA is extracted from the formed colonies. Bacteria can be concentrated and separated according to a known method using filtration, centrifugation, or the like.

  The step of detecting the region indicated by the region contained in positions 642 to 877 in GenBank accession number AF530557 by the LAMP method involves the above supernatant, primer set, strand displacement DNA polymerase, substrate (dNTPs), and reaction buffer. It can be performed by mixing and reacting. When the nucleic acid is RNA, the reaction can be carried out in one pot by adding reverse transcriptase.

  The region included in positions 642 to 877 in GenBank registration number AF530557 means not only the region from positions 642 to 877, but also any area included between them, for example, continuous from positions 642 to 877 A region of 180 bases or more, a continuous region of 200 bases or more, and the like can be selected.

The reaction can be performed, for example, at 65 ° C. for 60 minutes, and then the enzyme is inactivated by a method such as raising the temperature. By performing the reaction at 65 ° C. for 60 minutes, it is possible to amplify the nucleic acid 10 ⁹ to 10 ¹⁰ times.
As the LAMP reaction reagent, for example, a Loopamp DNA amplification reagent kit (excluding a primer set) commercially available from Eiken Chemical Co., Ltd. may be used. In this case, examples of the reaction liquid are as follows.
Double concentration buffer for reaction: 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH ₄ ) ₂ SO ₄ , 16 mM MgSO ₄ , 0.2% Tween 20, 1.6 M Betaine;
DATP, dCTP, dGTP, dTTP each at a final concentration of 2.8 mM;
DNA polymerase: Bst DNA Polymerase 8 units / μl;
Each primer (final concentration) of the present invention: FIP and BIP 40 μM each, F3 and B3 5 μM each, LB 20 μM.
As the LAMP reaction solution, for example, 4.5 μl of sterilized distilled water, 12.5 μl of double buffer solution, 1 μl of each primer of FIP, BIP, F3, B3, LB, 1 μl of DNA polymerase, sample solution (template) Add 2 μl of DNA to a total volume of 25 μl.

Alternatively, a commercially available DNA polymerase (for example, Bst DNA polymerase Large fragment manufactured by New England BioLab) may be used, and a reaction buffer solution (10-fold concentration) attached thereto may be used. In this case, examples of the reaction liquid are as follows.
Buffer for 10-fold concentration reaction: 200 mM Tris-HCl (pH 8.8), 100 mM KCl, 100 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 1% Triton X-100; 50 mM MgSO ₄ ;
DATP, dCTP, dGTP, dTTP each having a final concentration of 25 mM (for example, dNTPs Mixture (25 mM each) manufactured by Nippon Gene);
5M Betaine (eg Sigma);
DNA polymerase: Bst DNA polymerase Large fragment 8units / μl;
Each primer (final concentration) of the present invention: FIP and BIP 40 μM each, F3 and B3 5 μM each, LB 20 μM.
In this case, as the LAMP reaction solution, for example, 2.1 μl of sterilized distilled water, 2.5 μl of 10 × concentration buffer for reaction, 3.0 μl of MgSO 4, 8.0 μl of Betaine, 1.4 μl of dNTPs Mixture, FIP, BIP, F3 Add 1 μl of each of the B3 and LB primers, add 1 μl of DNA polymerase and 2 μl of sample solution (template DNA) to make a total volume of 25 μl.

If the target sequence is present in the sample, that is, if food poisoning-causing Escherichia coli having the eae gene is contained, white turbidity is generated by magnesium pyrophosphate as a byproduct of the amplification reaction. Therefore, by confirming the increase in turbidity of the reaction solution, it is possible to confirm whether or not the food poisoning-causing E. coli having the eae gene was contained in the sample. The increase in turbidity may be confirmed by visual observation, or may be measured using a dedicated turbidity measuring device. If necessary, the presence or absence of DNA fragments may be detected using agarose gel electrophoresis or the like.
Use positive and / or negative controls to compare changes in control turbidity with changes in sample turbidity to ensure that the reaction has progressed properly and improve the accuracy of the test. Can do.

  In the above detection method, the primer set of the present invention may be used. According to the primer set of the present invention, the eae gene can be specifically detected, and the presence or absence of Escherichia coli causing food poisoning can be accurately confirmed.

  The disclosures of all patent and non-patent documents cited herein are hereby incorporated by reference in their entirety.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to this at all. Those skilled in the art can change the present invention into various modes without departing from the meaning of the present invention, and such changes are also included in the scope of the present invention.

1) Primer design
The eae gene of EHEC, STEC, and EPEC was compared with the base sequences of other E. coli, and a LAMP primer specific to the eae gene was designed. The primer sequences in the designed primer set are as follows. A primer purified by HPLC was used.

The following table shows the positions (positions) of F2, F1c, B1c, B2, F3, and B3 for each primer set. The position is represented by the position of the base corresponding to GenBank Accession No. 530557. In order to match other sequences with variations, a primer including a portion not completely matched with AF530557 is also included.

2) LAMP reaction
The concentration of each reagent used in the LAMP reaction is as follows. The LAMP reaction reagent is a Loopamp DNA amplification reagent kit manufactured by Eiken Chemical Co., Ltd. The experiment was conducted according to
Buffer solution for double concentration: 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH ₄ ) ₂ SO ₄ , 16 mM MgSO ₄ , 0.2% Tween 20, 1.6M Betaine.
Each final concentration of 2.8 mM dATP, dCTP, dGTP, dTTP.
DNA polymerase: Bst DNA Polymerase 8 units / μl.
Primer (final concentration): FIP, BIP 40 μM each, F3, B3 5 μM each, LB 20 μM each.

  LAMP reaction solution is 4.5 μl of sterile distilled water, 12.5 μl of buffer solution for double concentration reaction, 1 μl of each primer of FIP, BIP, F3, B3, and LB, 1 μl of DNA polymerase, 2 μl of sample solution (template DNA) Was added to prepare a total of 25 μl of reaction solution.

  The sample solution (template DNA) was adjusted as follows. First, overnight culture at 37 ° C on Luria-Bertani (LB) agar medium (trypton 10g, Yeast extract 5g, NaCl 5g, Agar 15g dissolved in 1 liter of distilled water and sterilized in a petri dish) The E. coli is scraped with a platinum loop, suspended in 500 μl of saline-EDTA solution (0.15 M NaCl, 0.1 M Sodium EDTA, pH 8.0), added with 25 μl of 20% SDS solution, and heated at 60 ° C. for 10 minutes. Then lysed. To this, 500 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1) (PCI) was added and stirred vigorously to denature the protein. This was centrifuged at 8,000 rpm for 10 minutes, and the supernatant was collected. This PCI addition / stirring / centrifugation was repeated at least three times, and 2.5 times the amount of 99% ethanol was added to the resulting supernatant and mixed. The supernatant was discarded, leaving a DNA precipitate precipitated with ethanol. The sediment was rinsed at least three times with 70% ethanol, and finally with 99% ethanol to remove as much of the supernatant as possible. The obtained sediment was dissolved in 100 μl of distilled water to obtain a sample stock solution. A solution obtained by diluting this with distilled water was used as a sample solution (template DNA).

E. coli strain EHEC11 (O157A, O157B, 87-1, MN157, 4-2, 1-1, 17-2, 54-1, A29, B31 and 14-5) carrying the eae gene is used as the E. coli control 7 strains (9-1, 13-4, sample E, 18-1, FU-1, MC-1, KR-1, IW-1, 34-105) that do not have the eae gene were used. .
These strains are all stored in the inventor's laboratory, and O157A, O157B (names used in the inventor's laboratory), A29 and B31 are actually food poisoning. Is the stock that caused 87-1, 4-2, 1-1, 17-2, 54-1, and 14-5 have been isolated from healthy cattle feces and confirmed to possess the stx and eae genes.
Of the strains used as controls, 9-1, 13-4, and 18-1 were isolated from feces of healthy cattle and confirmed that they have the stx gene but no eae gene. . Samples E and 34-105 are strains isolated from the beef surface by meat inspection and have been confirmed to have the stx gene but not the eae gene. FU-1, MC-1, KR-1, and IW-1 are strains isolated from swine that developed E. coli disease in swine called edema disease, and the Shiga toxin gene stx- specific for the edema disease strain Has e but not eae gene.

The LAMP reaction was performed using a real-time turbidity measurement device Realoop-30 sold by Fujitsu System Solutions. An isothermal reaction at 65 ° C. was performed for 60 minutes, followed by enzyme deactivation treatment at 80 ° C. for 2 minutes.
The turbidity measuring device can observe the white turbidity due to magnesium pyrophosphate, a by-product of LAMP reaction, over time, those that increase turbidity are positive for the eae gene (+), and increased turbidity is observed None were negative (-).

3) Results The results using the primer sets 32 and 25 are shown in FIGS. 1-5 and the following table.
Each lane of FIGS. 1-3 and 5 is as follows.
Each lane in FIG. 4 is as follows.

1 to 4 show the results of using a high-concentration sample solution obtained by diluting the above-described sample stock solution 100 times in order to confirm that a nonspecific reaction does not occur in the negative control.
In the primer set 32, when LB was not added (FIG. 1) and when it was added (FIG. 3 upper part and FIG. 4), the cloudiness of magnesium pyrophosphate which is a by-product of the amplification reaction was observed only in the EHEC carrying the eae gene. STEC that does not have the eae gene did not cause white turbidity.
Primer set 25 did not cause white turbidity in STEC that did not have the eae gene, but there was one in which white turbidity was not clear for STEC that had the eae gene (FIG. 2). In particular, when LB was added, there was a tendency that white turbidity did not occur sufficiently (lower part of FIG. 3).
The other primer sets had many false positives and false negatives, and were not suitable for specifically detecting the eae gene.

  FIG. 5 shows the results of using the sample solution obtained by diluting the above-described sample stock solution 10,000 times with respect to the primer set 32. When primer set 32 was used, it was confirmed that even when a DNA sample with a low concentration was used, a white turbidity was clearly produced in the strain carrying the eae gene.

For confirmation, detection was performed by the PCR method (Journal of Clinical Microbiology 2004. Vol. 42. No. 2 645-651.) Which is one of the eae gene detection methods of EHEC, STEC and EPEC. The results are shown in the table below. The results of detection by PCR were completely consistent with the results of LAMP. This indicates that the test according to the present invention gives correct results.

  As shown in the above table, by performing the LAMP method using the primer set according to the present invention, food poisoning-causing E. coli having the eae gene could be specifically confirmed within 1 hour.

SEQ ID NOs: 1-26 and 101 show the DNA sequences of FIP primers.
Sequence number 27-50 shows the DNA sequence of a BIP primer.
SEQ ID NOs: 51-69, 100 and 102-104 show the DNA sequences of F3 primers.
SEQ ID NOs: 70-89 and 105 show the DNA sequence of the B3 primer.
SEQ ID NOs: 90-93 show the DNA sequences of LF primers.
SEQ ID NOs: 94-99 show the DNA sequences of the LB primers.

Claims (4)

A primer set for detecting Escherichia coli causing food poisoning having an eae gene by the LAMP method, comprising the following oligonucleotides (i) to (v) :
(I) an oligonucleotide having the base sequence represented by SEQ ID NO: 14;
(Ii) an oligonucleotide having the base sequence represented by SEQ ID NO: 50;
(Iii) an oligonucleotide having the base sequence represented by SEQ ID NO: 65;
(Iv) an oligonucleotide having the base sequence represented by SEQ ID NO: 89 ; and
(V) An oligonucleotide having the base sequence represented by SEQ ID NO: 99 .   2. The food poisoning causing Escherichia coli is any Escherichia coli selected from the group consisting of enterohemorrhagic Escherichia coli (EHEC), enteropathogenic Escherichia coli (EPEC), and visual observation toxin-producing Escherichia coli (STEC). The primer set described. A kit for detecting food poisoning-causing Escherichia coli having the eae gene, comprising the primer set according to claim 1 or 2 . A method for detecting Escherichia coli causing food poisoning having the eae gene,
Extracting a nucleic acid in a sample;
Using the primer set according to claim 1 or 2 to detect a region contained in positions 642 to 877 of SEQ ID NO: 106 by the LAMP method;
Including methods.