JPH04320675A - Oligonucleotide for detecting enterotoxigenic escharichia coli and detection using the same oligonucleotide - Google Patents

Abstract

PURPOSE:To detect enterotoxigenic Escherichia coli, capable of producing LT. CONSTITUTION:An oligonucleotide (sequence no. 1-5) to be selectively hybridized with LT gene produced by enterotoxigenic Escherichia coli is prepared and the oligonucleotide is used as a primer in gene amplification to selectively detect only enterotoxigenic Escherichia coli to cause food poisoning symptoms.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the detection of Escherichia coli producing heat-labile enterotoxins (LT) in clinical tests, particularly food poisoning tests or food tests.

0002

[Prior art] Test materials include patient's vomit, feces, food,
In the case of wipes, enrichment culture, isolation culture, pure culture, and confirmation culture must be performed before identifying toxigenic E. coli. Continuing further,
It is only through serological tests, intestinal toxin production tests, and other biochemical tests that it is identified as toxigenic E. coli. The time required for each culture stage is 18 to 24 hours, and the total time required for subsequent tests requires a long time of one week.

For the purpose of detecting heat-labile enterotoxin (hereinafter referred to as LT), enterotoxin detection kits using reverse passive latex agglutination reactions are commercially available; CT) and LT are known to have immunological common antigens with each other; therefore, CT and LT
It is difficult to distinguish between and detect.

[0004] In addition, the specimen needs to be pure cultured and, furthermore, must have been narrowed down to some extent to identify the bacterial species. The operations up to this stage of narrowing down are complicated and take a long time. Moreover, this kit alone requires 20 to 24 hours.

[0005]

[Problems to be Solved by the Invention] As mentioned above, all of the conventional methods require extremely complicated operations and a long time to identify toxigenic E. coli. I didn't go for any tests.

On the other hand, recently, DNA probe methods or hybridization methods using oligonucleotides have been attempted. However, when hybridization is performed on a membrane or other support using a probe in which oligonucleotides are labeled and then detected, it is difficult to obtain sufficient detection sensitivity and selectivity in bacterial testing.

[0007] The present invention detects nucleic acids derived from toxigenic Escherichia coli using gene amplification technology in which oligonucleotides function as primers for nucleic acid synthesis reactions.
The purpose is to provide a rapid and highly sensitive detection method for testing food poisoning bacteria.

[0008]

[Means for Solving the Problems] The present invention produces oligonucleotides that selectively hybridize with nucleic acids derived from toxigenic Escherichia coli, and uses these oligonucleotides as primers for gene amplification to produce toxins that cause food poisoning symptoms. It is characterized by selectively detecting only Escherichia coli.

[0009] The oligonucleotide used in the present invention targets the nucleotide sequence encoding the heat-labile toxin (LT) gene produced by toxigenic Escherichia coli present in the sample, and An oligonucleotide chemically synthesized to be complementary, the synthetic nucleotides having the following sequence group, (5') d-CCCAGATGAAATAAA
ACGT-(3')...(a) (5')d-CC
TGAGATATATTGTGCTC-(3')...
・(b) (5')d-ACAAACCGGCTTTG
TCAGATAT-(3')・(c) (5')d-G
TTATATATGTCAACCTCTGAC-(3'
)・(d) (5')d-ACCGGTATTACAG
It is characterized by consisting of AAATCTGA-(3')...(e) or a corresponding complementary sequence.

[0010] Here, gene amplification is performed using Polymerase Chain Reac developed by Saiki et al.
tion method (hereinafter abbreviated as PCR method; Science
230, 1350 (1985)). When detecting a specific nucleotide sequence region (in the case of the present invention, the LT gene of toxigenic E. coli), this method recognizes the positive strand at one end of the region and the negative strand at the other end. Prepare an oligonucleotide that can hybridize, use it as a primer for a template-dependent nucleotide polymerization reaction against a sample nucleic acid that has been made into a single-stranded state by heat denaturation, and separate the generated double-stranded nucleic acid into single strands again. and cause the same reaction to occur again. By repeating this series of operations, the copy number of the region sandwiched between the two primers increases to the point where it can be detected.

The specimen may be clinical test materials such as feces, urine, blood, tissue homogenate, etc., or food materials.

[0012] In order to use these materials as samples for PCR, an operation to release nucleic acid components from bacterial cells present in the materials is required as a pretreatment. However, PCR will proceed if there are several to tens of molecules of nucleic acids that can be hybridized with the primers, so a short treatment of the test material with lytic enzyme, surfactant, alkali, etc. is sufficient for PCR to proceed. A sample solution with a certain amount of nucleic acid can be prepared.

The oligonucleotide used as a primer in the present invention is a nucleotide fragment having a length of 10 bases or more, preferably 15 bases or more, and is either chemically synthesized or natural, in terms of selectivity, detection sensitivity, and reproducibility. either will do. Further, the primer does not need to be specifically labeled for detection.

[0014] The amplified region in the nucleotide sequence of the specific gene of Vibrio parahaemolyticus defined by the primer is
The number may be from 50 bases to 2,000 bases, preferably from 100 bases to 1,000 bases.

[0015] A thermostable DNA polymerase is used in the template-dependent nucleotide polymerization reaction, but the origin of this enzyme is 90-95°C, the temperature of the annealing operation for hybridizing the primers is 37-65°C, and the temperature of the polymerization reaction is 90-95°C. PCR was performed at 50-75°C, with this as one cycle.
is carried out for 20 to 42 cycles for amplification.

[0016] For detection, the presence of the amplified nucleotide fragment and its length can be confirmed by directly subjecting the reaction solution after PCR to agarose gel electrophoresis. From the results, it can be determined whether a nucleotide with a sequence that the primer should recognize exists in the sample. This determination determines the presence or absence of E. coli that can directly produce LT. Other methods such as electrophoresis, chromatography, and DNA probe methods are also effective for detecting amplified nucleotide fragments.

[0017]

[Operation] The present invention detects LT derived from toxigenic Escherichia coli using gene amplification technology in which oligonucleotides function as primers for nucleic acid synthesis reactions, and enables simple, rapid and highly sensitive detection.

[0018]

EXAMPLES (Experimental Example 1) Preparation of Samples For searching for toxigenic E. coli, 492 strains of E. coli isolated from patients with diarrhea shown in the vertical headings of Tables 1 to 13 were used. These strains were provided by the Department of Microbiology, Faculty of Medicine, Kyoto University (head: Professor Yoshifumi Takeda). Each strain was inoculated into an appropriate enrichment medium and cultured overnight at 37°C under aerobic conditions. Bacterial cells were collected from 5 ml by centrifugation. 10 mm Tris-HCl buffer (pH
7. After washing once with 5), add 1 m of lysozyme to the same buffer.
The solution was dissolved to give a concentration of 0.g/ml. The cells were suspended in 5 ml and lysed at 37°C for 10 minutes. A mixture of phenol and chloroform (mixing ratio 1:1) saturated with the buffer was added to the lysate and stirred well. After centrifugation, the supernatant liquid was collected and treated with ethanol to precipitate nucleic acid components. The precipitate was dissolved in 1 ml of the above buffer solution and used as a sample.

Synthesis of primers Base sequence of the LT gene of Escherichia coli that produces heat-labile enterotoxin (Yamamoto, T., T. Tamura a
nd T. Yokota (1984): Primar
y structure of heat-labile
e enterotoxin produced by
Escherichia coli pathway
nic for humans. J. Biol.
Chem. , 259:5037-5044 and Yamamoto, T. , T. Gojobori
and T. Yokota (1987): Evol.
utionary origin of pathog
enic determinants in ente
rotoxigenic Eshelichia co
liand Vibrio cholerae O1.
J. Bateriol. , 169:1352-1
357) to the sequence shown in claim 1 ((a),
(b), (c), (d), and (e)), and oligonucleotides having the same sequences were chemically synthesized. Chemical synthesis is performed using Cyclone Plus DNA Synthesizer (
Milligen (manufactured by Bioresearch) was used, and the β-cyanoethyl phosphoramidite method was used. The synthesized oligonucleotide was purified by high performance liquid chromatography using a C18 reverse phase column.

[0020] PCR Using 3 μl of the above sample solution, add 16.0 μl of sterile water to it. 05μ
1, 3 μl of 10x reaction buffer, dNTP solution 4. 8
μl, primer (1) 1. 5 μl, primer (
2)1. 5 μl, and 0.0 μl of thermostable DNA polymerase. 15 μl was added to prepare a 30 μl reaction solution. Add mineral oil (SIGMA) to the container containing this reaction solution.
50 µl of 50 μl of 100% of the reaction mixture was added and layered on top of the reaction solution. Contents of each added solution and primers (1) and (2)
The combinations are shown below.

10x reaction buffer: 500mM KCl, 10
0mM Tris-HCl (pH 8.3), 15mM
MgCl2,
0.1% (W/V) Gelatin dNT
P solution: dATP, dCTP, dGTP, dT
The final concentration of each mixture is
1.25mM Primers (1) and (2): Each aqueous solution of the chemically synthesized purified product mentioned above

(Concentration 5 ODU/
ml) Combination of primers: Three sets of primers were prepared as follows using the chemically synthesized purified product described above and used in the experiment. The reaction conditions are as follows.

Heat denaturation: 94°C, 1 minute annealing:
55 ℃, 1 minute polymerization reaction: 72 ℃, 1 minute The process from thermal denaturation to annealing to polymerization reaction is 1.
35 cycles (total time required: about 3 hours). These operations are
DNA thermal cycler (Perkin Elmer
(manufactured by Cetus) by programming the above reaction conditions.

Detection In order to detect the amplified nucleotide fragments from the reaction solution, agarose gel electrophoresis was performed as follows.

[0024] The agarose gel has a gel concentration of 3% (W/V
) containing ethidium bromide (0.5 μl/ml). The electrical conditions for electrophoresis were a constant voltage of 100V,
It took 30 minutes. For operating methods and other conditions, please refer to Man
Molecular Cloning by Iatis et al.
This was done using the technique described in (1982). In addition to the reaction solution, a molecular weight marker was also run at the same time, and the length of the nucleotide fragment was calculated by comparing the relative mobility.

Results As mentioned above, the nucleotide sequence of the LT gene has already been determined, and the size of the oligonucleotides of the present invention, ie, the nucleotides amplified by the primers by PCR, can be estimated. According to it, primer (a
) and (b), (c) and (d), and (e) and (
d), 589 and 55, respectively.
Nucleotides of length 0 and 264 bases should be amplified. When these estimated values and the length of the amplified nucleotides matched, it was determined that each primer was correctly amplifying the targeted region of the LT gene. Table 1 shows the results of examining 492 clinical isolates of E. coli using the above method and measuring the length of the amplified nucleotides.
- Shown in Table 13. Tables 1 to 13 Primers (prime
In the r) column, + indicates that the length of the amplified nucleotide matched the estimated value, or - indicates that no amplified nucleotide was detected.

[0026] As shown in the same table, when PCR was performed with each combination of primers, only strains having the LT gene (those with +, 2+, and w in the LTgene column in Tables 1 to 13) , amplifying nucleotides. Moreover, all of the amplified nucleotides are consistent with the predicted nucleotide length. Therefore, it is clear that the oligonucleotides, ie, the primers of the present invention, correctly amplify the targeted region of the LT gene of toxigenic E. coli.

(Experimental Example 2) The results obtained in Experimental Example 1 are as follows:
In order to confirm whether it is selective for LT-producing E. coli, we conducted a comparative study of bacterial species other than pathogenic E. coli that can be tested in clinical tests.

The method was the same as that shown in Experimental Example 1, except that Clostridium perfringen
s, Campylobacter jejuni,
Campylobacter coli, Bacte
roides flagilis, Bacteroides
es vulgatus, and Lactobacillus
For Illus acidophilus, culture was performed overnight at 37°C under anaerobic conditions, and PCR was performed.
Samples applicable to the method were prepared. The 50 bacterial strains shown in the vertical headings of Tables 14 and 15 were cultured in the preparation of the specimens. In addition, human placenta-derived DNA (Human p
lacenta DNA) was prepared at a concentration of 1 μg/ml, and PCR was also performed on this in the same manner.

The results are shown in Tables 14 and 15. PCR was performed using each combination of primers, but no matter which of the three sets of primers was used, DNA of various bacteria other than pathogenic E. coli was not amplified. Therefore, it can be concluded that the oligonucleotide, ie, the primer, of the present invention selectively reacts only with heat-labile toxin-producing E. coli.

On the other hand, in the present invention, CT-producing Vibr
io cholerae (in Table 15, V. cholerae
It can also be seen that no cross-reaction occurs with raeO1ctx+). As mentioned above, immunologically, CT and LT
They have common antigens and can be detected by immunological methods.
I couldn't tell them apart. However, since the present invention clearly detects only LT-producing E. coli,
It is considered that the reliability is higher than that of the conventional method.

Note that if the agarose electrophoresis used in the examples of the present invention is performed under the above-mentioned electrophoresis conditions, if the nucleotide is 100 base pairs or less, 5 to 10 base pairs, 10
For nucleotides ranging from 0 to 500 bases, 10
A difference in length of 20 base pairs of nucleotides can be distinguished. Furthermore, by using acrylamide or the like in the gel, the accuracy of measuring nucleotide length can be improved, and it is thought that the reliability in selective detection of the LT gene will further increase.

[0032]

[Table 1]

[0033]

[Table 2]

[0034]

[Table 3]

[0035]

[Table 4]

[0036]

[Table 5]

[0037]

[Table 6]

[0038]

[Table 7]

[0039]

[Table 8]

[0040]

[Table 9]

[0041]

[Table 10]

[0042]

[Table 11]

[0043]

[Table 12]

[0044]

[Table 13]

[0045]

[Table 14]

[0046]

[Table 15]

[0047] In the table, the LTgene column (+):
2+): Carrying the LT gene
(w): Same as possessing LT gene (-)
:Does not have LT gene Primer column (+): Length of amplified nucleotide matches estimated value
What I did
Same (w): The length of the amplified nucleotide matches the estimated value.
However, the degree of amplification is slightly weaker.
(−): No amplified nucleotides were observed.
Where to obtain each bacterial strain in Tables 1 to 13: Kyoto University School of Medicine Microbiology Department Where to obtain each bacterial strain in Tables 14 and 15 (No. 1 to 39): ATCC (American Type Cul
ture Collection) JCM (RIKEN Microbial System Collection Facility) IFO (Fermentation Research Institute) Sources of bacterial strains in Table 15 (No. 41 to 50): Kyoto University School of Medicine Microbiology Department Table 15 (No. 51) Source: Takara Shuzo Co., Ltd.

[0048]

[Effect] By using the PCR method in the present invention, in the detection of toxigenic E. coli, high detection sensitivity due to gene amplification effect and high selectivity due to the reaction being defined by two or more primers. can be obtained.

Furthermore, due to the high detection sensitivity, a large amount of specimen is not required, and pretreatment of the specimen is simple. Moreover, the reaction time is short, detection requires simple equipment, and operation is easy, so the time required for identification can be significantly shortened. For example, in Examples, the reaction time is approximately 3 hours, and the detection operation is 30 minutes.

[0050] Furthermore, by using agarose gel electrophoresis and nucleic acid staining with ethidium bromide for detection, detection can be performed without labeling primers, etc. Furthermore, since the length of the nucleic acid can be confirmed, the reliability of the results is increased. becomes high.

[0051] When identifying pathogenic Escherichia coli as a food poisoning bacterium, it is important to confirm whether it produces LT. Since no other species has the ability to produce LT, the nucleotide sequence targeted by the primer contains LT.
By using genes, selective detection of pathogenic E. coli becomes possible.

[0052]

[Sequence list]

Claims (2)

[Claims] Claim 1: Heat-labile enterotoxin produced by toxigenic Escherichia coli present in a specimen.
n, LT) An oligonucleotide that targets a nucleotide sequence encoding a gene and is chemically synthesized to be complementary to the nucleotide sequence, the synthetic nucleotides having the following sequence group: (5') d-CCCAGATGAAATAAAACGT
-(3')・・・(a) (5')d-CCTGAG
ATATATTGTGCTC-(3')...(b)
(5')d-ACAAACCGGCTTTGTCAG
ATAT-(3')・(c) (5')d-GTTAT
ATATGTCAAACCTCTGAC-(3')・(d
) (5')d-ACCGGTATTACAGAAAT
An oligonucleotide characterized by consisting of CTGA-(3')...(e) or a corresponding complementary sequence. 2. A method comprising selectively amplifying a target nucleotide sequence by causing an oligonucleotide having at least one of the sequences set forth in claim 1 to function as a primer for a chain length reaction. and (a
) A primer is hybridized to the single-stranded target nucleotide sequence in the sample to perform a chain length reaction by polymerization reaction of four types of nucleotides, and (b) the obtained double-stranded target nucleotide sequence is converted into a single-stranded one. If separated, the complementary strand serves as a template for a length reaction by the other primer, and (c) simultaneous length reactions by these two primers, separation of the primer length product from the template, and generation of a new By repeating hybridization with primers, a specific nucleotide sequence is amplified,
The method is characterized in that toxigenic E. coli is detected by detecting this amplified nucleotide fragment, and (d) determining whether or not the sequence to be recognized is present in the sample as a result. Method for detecting toxigenic E. coli.