JP3141976B2 - Oligonucleotides for detection of Shigella and detection methods using them - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to quarantine, clinical examination,
In particular, it relates to testing for food poisoning or bacterial diarrhea, or detection of Shigella in food testing.

[0002]

2. Description of the Related Art Detection of Shigella is important for various medical and public health purposes, and conventionally, detection has been carried out by the following steps. First, isolation culture using a medium such as DHL agar or MacConkey agar is performed from the patient's feces and food as test materials, and then differential culture using a TSI agar, a LIM agar, or the like.

[0003]

However, in the conventional method, the time required for each culturing step is 18 to 24 hours, and the total required time is 3 to 4 days, which is poor in quickness. Reverse passive latex agglutination using a specific antibody against Shiga toxin of Shigella, and an EIA method using a specific antibody against a 140 megadalton plasmid product involved in the pathogenicity of Shigella and enteroinvasive E. coli (Kenichiro Ito) Et al., The Japanese Bacteriological Journal 41,414 (1986)) and the DNA probe method for detecting the ipaB gene, ipaC gene, or ipaD gene (US patent Applicatio).
n No. 888194), however, these test methods involve complicated and cumbersome preparation of reagents and specimens, and require much time. Therefore, an object of the present invention is to provide a simple, rapid and highly sensitive novel detection method for quarantine, clinical examination, and food inspection.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention detects Shiga toxin gene of Shigella by gene amplification technology in which an oligonucleotide functions as a primer for a nucleic acid synthesis reaction.

The oligonucleotide used in the present invention targets a nucleotide sequence encoding a Shiga toxin gene, is complementary to the nucleotide sequence, and is chemically synthesized so as to define both ends of the nucleotide sequence to be amplified. A pair of oligonucleotides, wherein the synthetic nucleotides are selected from the following sequences (a) and (b) and a sequence containing at least 10 or more consecutive bases of a complementary sequence corresponding thereto: is there. (5 ')-CAACACTGGATGATCTCAG- (3') (a; SEQ ID NO: 1) (5 ')-CCCCCTCAACTGCTAATA- (3') ... (b; SEQ ID NO: 2)

[0006] Gene amplification is carried out using the Polyme
It is carried out based on the rase chain reaction method (hereinafter abbreviated as PCR method; Science 230, 1350 (1985)).
This method is used for detecting a specific nucleotide sequence region (in the case of the present invention, Shiga toxin gene of Shigella).
One end of both ends of the region prepares an oligonucleotide that recognizes the + strand and the other strand recognizes the − strand, and prepares an oligonucleotide. By functioning as a primer for the polymerization reaction, the generated double-stranded nucleic acid
It is separated into main chains and the same reaction is caused again. By repeating this series of operations, the copy number of the region sandwiched between the two primers increases until it can be detected.

[0007] The specimen may be a clinical examination material such as feces, urine, blood, tissue homogenate, or a food material. In order to use these materials as a sample for PCR, an operation of releasing nucleic acid components from cells present in the materials is required as pretreatment. However, if the nucleic acid to which the primer can hybridize is present from several molecules to several tens of nucleons or more, the PCR will proceed. A sample solution having a nucleic acid amount 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 in consideration of selectivity, detection sensitivity and reproducibility. either will do. Further, the primer may not be particularly labeled for detection.

The amplification region in the nucleotide sequence of the Shigella gene specified by the primers is 50 to 2,000 bases, preferably 100 to 1,000 bases.
What is necessary is just to become 0 bases. A heat-resistant DNA polymerase is used in the template-dependent nucleotide polymerization reaction. The origin of this enzyme is 90 to 95 ° C., and the temperature of the annealing operation for hybridizing the primer is 37 to 65 ° C.
The polymerization is carried out at 50 to 75 ° C. at a temperature of 50 ° C. to 75 ° C. The PCR is carried out for 20 to 42 cycles in which the cycle is one cycle.

[0010] For detection, the presence of the amplified nucleotide fragment and its length can be confirmed by subjecting the reaction solution after PCR to agarose gel electrophoresis as it is. From the result, it can be determined whether or not a nucleotide having a sequence to be recognized by the primer exists in the sample. This determination directly determines the presence or absence of the Shiga toxin gene. Other electrophoresis and chromatography are also effective for detecting the amplified nucleotide fragment.

[0011]

EXAMPLES [Experimental Example 1] Preparation of Specimens The Shigella dysenteriae strains used were derived from patients and the like, and a total of 42 strains shown in Table 1 were used.
Each strain was inoculated into LB medium (1% tryptone, 0.5% yeast extract, and 1% sodium chloride,
Shaking culture was performed overnight at 37 ° C under aerobic conditions. The culture of each strain was treated with 10 mM Tris-HCl buffer pH 7.5.
(Hereinafter referred to as TE buffer), and the mixture was heated at 95 ° C. for 10 minutes, centrifuged, and the supernatant was used as a sample.

Synthesis of primers The Shiga toxin gene of Shigella is enterohemorrhagic Escherichia coli (entero).
hemorrhagic Escherichia coli (EHEC) or Verocytotoxin-producing Escherichia
coli; VTEC), which are considered to be the same gene, differing only by a few bases from the VT1 gene. Therefore, literature (Takao, T etal., Microb. Pathog., 5: 357-36
9 (1988)), and the oligonucleotides having the same sequences were chemically synthesized. Chemical synthesis was performed by a β-cyanoethylphosphoramidite method using a cyclone plus DNA synthesizer (Milligen / BioResearch). Purification of the synthesized oligonucleotide was performed by high performance liquid chromatography using a C18 reverse phase column.

PCR Using 3 μl of the sample solution, add 17.05 sterile distilled water
μl, 3 μl of 10x reaction buffer, dNTP solution 4.8
μl, Primer (1) 1.0 μl, Primer (2) 1.
0 μl and 0.15 μl of thermostable DNA polymerase
Was added to prepare a reaction solution having a total volume of 30 μl. 50 ml of mineral oil (manufactured by SIGMA) is added to the container containing
μl was added and overlaid on the reaction solution. The contents of each used solution and the combinations of the primers (1) and (2) are as follows. 10x reaction buffer: 500mM KCl, 100mM Tris-HCl pH8.3, 15mM MgCl2, 0.1% (w / V) gelatin dNTP solution: dATP, dCTP, dGTP, dTTP mixed, final concentration of 1.25mM Primers (1) and (2): Aqueous solution of the above-mentioned chemically synthesized purified product (concentration 3.75 OD / ml) Primer combination: The above-mentioned chemically synthesized purified product was used in combination as follows. Primer (1) + Primer (2) (a) + (b) Thermostable DNA polymerase: Taq DNA polymerase (5 units / ml; Perkin Elmer cetus)

The reaction conditions are as follows. Thermal denaturation: 94 ° C., 1 minute Annealing: 55 ° C., 1 minute Polymerization reaction: 72 ° C., 1 minute The process from heat denaturation to annealing to the polymerization reaction is one cycle (required time: 5.7 minutes), and this is 35 cycles (Total time required: about 3 hours). These operations are
The above reaction conditions were programmed into an NA thermal cycler (Perkin Elmer Cetus) and performed.

Detection In order to detect the amplified nucleotide fragment from the reaction solution, agarose gel electrophoresis was performed as follows. The agarose gel used had a gel concentration of 3% (W / V) and contained ethidium bromide (0.5 μl / ml). The electrophoresis was performed at a constant voltage of 100 V for 30 minutes. The operation method and other conditions were performed by the technique described in Maniatis et al., Molecular Cloning, 2nd edition (1989). In addition to the reaction solution, electrophoresis of a molecular weight marker was performed at the same time, and the length of the nucleotide fragment was calculated by comparing the relative mobilities.

Reverse passive latex agglutination (Reversed Pas)
sive Latex Agglutination (RPLA) test A commercially available RPLA kit for detection of E. coli verotoxin (manufactured by Denka Seiki) was purchased, and a test was performed by preparing a sample according to the attached instruction manual.

Results As described above, the Shiga toxin gene of Shigella has already been determined in base sequence, and the size of the oligonucleotide of the present invention, ie, the nucleotide to be amplified by the primer by PCR, can be easily estimated. According to this, in the combination of the primers (a) and (b), a nucleotide having a length of 349 bases (or 349 base pairs) should be amplified. If these estimates match the length of the amplified nucleotides, then this combination of primers
The target region in the Shiga toxin gene was correctly amplified, and the strain was determined to have the Shiga toxin gene. Table 1 shows the results of the tests performed on 34 test strains.

The primer of the present invention can be used in the RPLA test.
Vero toxin, the DN of a strain determined to be positive for Shiga toxin
Only A was amplified and did not react at all with the Shiga toxin gene-negative strain DNA. In other words, it indicates that Shiga toxin gene is correctly amplified, and Shigella having Shiga toxin gene is accurately detected.

[Table 1]

[Experimental Example 2] In order to confirm whether or not the results obtained in Experimental Example 1 were selective for the Shiga toxin gene, diarrhea other than Shigella, etc., to be tested in clinical tests. It was examined whether or not the primer of the present invention reacts with the above gene.
The method is the same as that shown in Experimental Example 1 except for the method of preparing the specimen.

Preparation of Specimens Each of the strains shown in Table 2 was inoculated into an appropriate enrichment medium, and cultured overnight at 37 ° C. under aerobic or anaerobic conditions (of which anaerobic conditions were used). The strain cultured in
tridium perfringens, Campylobacter jejuni, Campyl
Bacteria coli, Bacteroides flagilis, Bacteroides vu
lgatus, Lactobacillus acidophilus, Bifidobacteriu
m adolescentis). The cells were collected from 0.5 ml of each culture by centrifugation, and the cells were collected with TE buffer.
Washed twice. Add 50 mM phosphate buffer pH 7.
5 was dissolved in an N-acetylmuraminidase solution and an achromopeptidase solution at a final concentration of 50 μg / m 2.
l, and 1 mg / ml.
Treated for 0 minutes and lysed. A mixture of phenol and chloroform (1: 1 mixing ratio) saturated with TE buffer was added to the lysate, and the mixture was stirred well. After centrifugation, the upper layer solution was collected and treated with ethanol to precipitate nucleic acid components. The precipitate was dissolved in 1 ml of a TE buffer to obtain a sample. In addition, human placenta-derived DNA (Human placen
ta DNA) was prepared at a concentration of 1 μg / ml and subjected to PCR in the same manner.

Results Table 2 shows the test results for the primer combinations. The combination of the primers of the present invention does not amplify various DNAs including diarrhea bacterium DNA except for DNAs of Shiga toxin-producing Shigella and Vero toxin type 1-producing Escherichia coli. Was. Therefore, it can be asserted that the oligonucleotide of the present invention, that is, the primer, selectively reacts only with the bacterium having the Shiga toxin gene.

When the agarose gel electrophoresis method used in the examples of the present invention is performed under the above-described conditions, if the nucleotide has a length of 100 bases (or 100 base pairs) or less, 5 to 10 bases (bases) Pair), and 100 to 5
For nucleotides in the range of 00 bases (base pairs), 1
Differences in nucleotide length from 0 to 20 bases (base pairs) can be distinguished. Furthermore, by using acrylamide or the like for the gel material, it is possible to improve the measurement accuracy of nucleotide length, and it is considered that the reliability in the selective detection of the Shiga toxin gene is further increased.

[Table 2]

[0023]

According to the present invention, the use of the PCR method and the use of a primer targeting the gene of Shiga toxin, which is one of the pathogenic factors of Shigella, enable the detection of bacteria having the Shiga toxin gene. High detection sensitivity due to the gene amplification effect and high selectivity due to the reaction being defined by two or more primers are obtained. Further, since the detection sensitivity is high, a large amount of the sample is not required, and the pretreatment of the sample can be simplified. In the example of the present invention, the reaction time was 3 hours, and the operation required for the detection was 30 minutes. Furthermore, by using agarose gel electrophoresis and nucleic acid staining with ethidium bromide for detection, detection can be performed without labeling primers or the like. Moreover, since the length of the amplified nucleotide can be confirmed, the reliability of the test result is high.

[0024] The test for Shigella requires accurate results without delay in order to promptly and appropriately treat the detected patients and take preventive measures. Further, the present invention selectively detects a gene of Shiga toxin, which is one of the virulence factors of Shigella. Therefore, according to the present invention, it is possible to accurately detect Shigella as the causative bacterium.

[0025]

[Sequence List] SEQ ID NO: 1 Sequence length 19 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin Shigella dysenteriae type 1 Sequence characteristics Method of determining characteristics S sequence CAACACTGGATGATCTCA
G

Sequence number (SEQ ID NO); 2 Sequence length 18 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin Shigella dysenteriae type 1 Sequence Features Method of Determining Features S Sequence CCCCCTCAACTGCTAATA

 ──────────────────────────────────────────────────続 き Continuing from the front page Referee, Hisashina Tanaka, Judge Masamichi Tokuhiro, Judge Akira Terumura, Judge (56) Reference Microbiological Pathology (1988) Vol. 5, p. 357-369

Claims (2)

(57) [Claims] 1. A serotype 1 subtype of Shigella A present in a sample.
To target the nucleotide sequence encoding the toxin gene (Shiga toxin gene) produced by the strain (Shigella dysenteriae type 1), to complement the nucleotide sequence and to define both ends of the nucleotide sequence to be amplified A chemically synthesized oligonucleotide, wherein the synthetic nucleotide is selected from the following sequences (a) and (b) and a sequence containing at least 10 consecutive bases or more of the complementary sequence corresponding thereto. A pair of oligonucleotides. (5 ')-CAACACTGGATGATCTCAG- (3') (a) (5 ')-CCCCCTCAACTGCTAATA- (3') ... (b) 2. The combination of oligonucleotides according to claim 1 defines both ends of a nucleotide sequence to be amplified and functions as a primer for chain length reaction to selectively amplify a target nucleotide sequence. A method for detecting Shigella, which is characterized in that: (A) the primer is hybridized to a single-stranded target nucleotide sequence in a sample, and a chain length reaction is carried out by a polymerization reaction of four nucleotides; (b) the resulting double-stranded nucleotide sequence is When separated into main strands, the complementary strand functions as a template for further chain length reaction, and (c) performs the chain length reaction with the primer, separation of the chain length product from the template, and hybridization with the further primer. (D) a method for detecting Shigella by determining whether or not a nucleic acid having a nucleotide sequence to be recognized is present in the sample, by repeating the process;