JPH05276996A - Oligonucleotide for detecting microorganism capable of producing vibrio cholerae toxin, method for detecting microorganism capable of producing vibrio cholerae toxin and reagent kit for detection - Google Patents

Abstract

PURPOSE:To obtain an oligonucleotide useful for directly, simply, rapidly and specifically detecting Vibrio cholerae capable of producing Vibrio cholerae toxin with high sensitivity. CONSTITUTION:The objective oligonucleotide for detecting Vibrio cholerae capable of producing Vibrio cholerae toxin has a nucleic acid sequence expressed by sequence Nos.1 to 6 in a sequence table or a sequence complementary thereto. The objective method for detecting the microorganism capable of producing the Vibrio cholerae toxin in a sample and the objective reagent kit for detection using a labeled oligonucleotide prepared by labeling the oligonucleotide and the oligonucleotide as a probe or a primer are obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cholera toxin-producing bacterium, and more particularly to a simple and rapid detection of a cholera toxin-producing bacterium in Vibrio cholerae.

[0002]

Cholera is a legal infectious disease-causing bacterium that causes cholera by oral infection through food and drink. In developing countries, there is a large epidemic, and vomiting and massive diarrhea often cause patients to become dehydrated and shocked, resulting in death. However, among the cholera bacteria classified by biochemical properties, there are cholera toxins that do not produce cholera toxin and do not show pathogenicity, and determination of cholera toxin productivity is important. Therefore, the detection and identification of the cholera toxin-producing cholera bacterium are important in the food industry, public health, and clinical testing fields, and there is a demand for rapid measurement in view of the fact that cholera is a legally transmitted disease. However, cholera toxin producing cholera and cholera toxin non-producing cholera cannot be distinguished serologically or biochemically at all. Therefore, it is necessary to see the productivity of cholera toxin itself. Conventional methods for detecting the productivity of cholera toxin include De test (a method of ligating the intestinal tract of a rabbit and inoculating a bacterial solution into it), and Duttatest (a method of orally administering a bacterial solution to a suckling rabbit. There is a biological detection method such as a method of causing symptoms, but the procedure is complicated and cannot be said to be a simple test. Vibrio cholerae is generally identified by its reactivity with antisera. This is a classification according to the sugar chain antigen on the surface of the bacterium called O antigen. In leaves-type antigen for cholera,
There are two types of antigens called brook-type antigens, and they can produce their own antisera. The antigens specific to this cholera bacterium are collectively referred to as the O1 antigen. Vibrio cholera
Bacteria that cause an immune reaction with this anti-O1 serum in rae) are classified as cholera. On the other hand, there are non-O-1 type cholera bacteria (Vibrio cholerae non O-1: sometimes called Nagvibrio) that have different serological properties and do not cause an immunoreaction with this anti-O1 serum. Some produce the same toxins as cholera toxin, and some cause food poisoning. Therefore, the determination of Nagvibrio that produces these toxins is also important. In addition, polymyxin B sensitivity and chicken hemagglutination are classified into Asian-type cholera and ertor cholera, and at present, ertor cholera is predominant. Furthermore, there are phage type classification, immunological classification, etc., but these are not directly related to cholera toxin productivity. In recent years, the identification of bacteria and the detection of toxin genes have been frequently performed by genetic tests including DNA probes. Although the gene sequence of cholera toxin has been revealed,
Cholera toxin is a heat-labile toxin of enterotoxigenic E. coli (Enteroxigen
ic Escherichia coli heat-labile enterotoxin: ETEC-
It is known that it has a high similarity with LT) and a high similarity at the gene level, and it is difficult to separate it with a DNA probe. Further, in this intestinal tract infection caused by ETEC-LT toxin, the symptoms are very similar to those of cholera and it is difficult to discriminate them from clinical symptoms, so an accurate test method is desired.

[0003]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide an oligonucleotide for use in the detection of cholera toxin-producing cholera bacteria which is direct, convenient, rapid, specific and highly sensitive.

[0004]

Means for Solving the Problems As a result of various studies on the gene of cholera toxin, the present inventor has found that ETEC-L
To obtain a highly specific, highly sensitive, simple, and rapidly diagnosable oligonucleotide that does not react with similar toxins such as T,
The detection method using it was established and the present invention was completed.

That is, the present invention is an oligonucleotide specific to cholera toxin-producing Vibrio cholerae, Sequence Listing, SEQ ID NOs: 1 to 6 (where A is adenine, C is cytosine, G is guanine and T is thymine). Further, T at an arbitrary position may be substituted with uracil (U).), Or an oligonucleotide for detecting cholera toxin-producing Vibrio cholerae having the sequence complementary to the nucleic acid sequence and the oligo. It is a labeled oligonucleotide in which nucleotides are labeled. The present invention is characterized by labeling the oligonucleotide for detecting cholera toxin-producing Vibrio cholerae, hybridizing the resulting labeled nucleic acid probe with DNA or RNA in the sample, and measuring the label of the hybridized conjugate. Method for detecting cholera toxin-producing cholera bacteria in a sample, or the oligonucleotide for detecting cholera toxin-producing cholera bacteria is directly used as a nucleic acid primer, or a labeled primer obtained by labeling is hybridized with DNA or RNA in a sample Then, the primer is extended, and the obtained primer extension product is measured, which is a method for detecting cholera toxin-producing cholera bacteria in a sample. Furthermore, the present invention is a reagent kit for detecting a cholera toxin-producing bacterium containing the oligonucleotide as a probe, and a reagent kit for detecting a cholera toxin-producing bacterium containing the oligonucleotide or a labeled oligonucleotide as a primer.

Since the oligonucleotide of the present invention can be prepared by chemical synthesis, it is possible to obtain an oligonucleotide of a certain quality easily, in a large amount and at a low cost, as compared with a cloned oligonucleotide or polynucleotide. The oligonucleotide of the present invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, it goes without saying that the thymidine residue (T) is replaced with the uridine residue (U). Alternatively, a DNA containing a uridine residue may be used, which is synthesized by changing T at any position to U during the synthesis. Similarly, U at any position can be T
It may be RNA containing the thymidine residue changed to. Further, the oligonucleotide may have a point mutation such as deletion, insertion or substitution, or a modified nucleotide.
A labeled oligonucleotide is obtained by introducing a labeling substance such as an antigen, a hapten, an enzyme, a fluorescent substance, a luminescent substance, an enzyme substrate, a radioactive substance, an insoluble carrier into the oligonucleotide. The label binding method follows conventional methods. For example, the enzyme can be labeled by substituting a nucleotide having a linker arm as a member of the oligonucleotide of SEQ ID NO: 1 in the sequence listing (Nucleic Asids Rese
arch, 14, 6115, 1986, etc.). As an example thereof, uridine having a linker arm at the 5-position can be chemically synthesized from deoxyuridine by the synthetic method described in JP-A-60-500717 and introduced into vapor nucleotides.
The labeling method may be end labeling or labeling in the middle of the sequence. It may also be a label on the sugar, phosphate group or base moiety.

When the cholera toxin-producing cholera bacteria in a sample are detected using the oligonucleotide of the present invention, (1)
A method in which an oligonucleotide is used as a probe to hybridize with DNA or RNA in a sample and a hybrid is detected, or (2) an oligonucleotide is used as a primer to hybridize with DNA or RNA in a sample, and an extension reaction is performed with a DNA polymerase or the like. There is a method of detecting a target nucleic acid from the obtained extension product. In these cases, detection can be easily performed by introducing a label such as an antigen, a hapten, an enzyme, a fluorescent substance, a luminescent substance, an enzyme substrate, a radioactive substance, an insoluble carrier into the oligonucleotide as described above. When the labeled oligonucleotide is used as a probe, the cholera toxin-producing cholera bacterium in the sample can be detected by detecting the above-mentioned label by an appropriate detection method after hybridization with the sample. When an oligonucleotide is used as a primer, only the cholera toxin gene can be specifically amplified by performing gene amplification (PCR; see JP-A-62-281) with DNA polymerase or the like. In PCR, the presence of the cholera toxin gene, that is, the cholera toxin-producing cholera bacterium, can be easily detected by incorporating a radiolabeled nucleotide during the reaction or by fractionating the amplified product by electrophoresis and detecting a specific band. be able to. Further, the amplification product can be directly detected by using the above-mentioned labeled oligonucleotide as a primer.

The oligonucleotide of the present invention can be bound to a solid phase carrier and used as a capture probe. In this case, a sandwich assay may be performed using a combination of a capture probe and a labeled probe, or there is a method of labeling and capturing a target nucleic acid. There is also a method in which an oligonucleotide is labeled with biotin, and after hybridization, it is captured with an avidin-binding carrier. In the sandwich assay, if the oligonucleotide of the present invention is used in either one of the two, it becomes possible to perform more specific measurement by the oligonucleotide, and there is no problem even if the other oligonucleotide has a slightly low specificity.

[0009]

EXAMPLES Next, the present invention will be specifically described by way of examples. Example 1 Synthesis of Various Oligonucleotides Various oligonucleotides having the sequences shown in the sequence listing / SEQ ID NOS: 2 to 6 were synthesized by the phosphoamidite method using ABI DNA Synthesizer Model 391. Less than,
The various oligonucleotides shown in Sequence Listing and SEQ ID NOS: 2 to 6 are referred to as oligonucleotides (2) to (6), respectively.
The method was carried out on a 0.2 μmol scale according to the ABI company manual. Deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C overnight. Purified by Pharmacia F
Performed on a reverse phase column with PLC. If necessary, a ³² P-phosphate group was bound to the 5'end of the synthesized oligonucleotide by the following method. Reaction solution composition Oligonucleotide 5-20 pmoles 10 × T4 polynucleotide kinase buffer 10 μl 1 mM [γ- ³² PATP (10 mCi / ml)] 1 μl T4 polynucleotide kinase (manufactured by Toyobo) 10 Units Total amount of water 100 μl Above The reaction mixture prepared as above was reacted at 37 ° C. for 1 hour. Here, the 10 × T4 polynucleotide kinase buffer means 0.5 M Tris-HCl (pH 8.0), 0.1 M MgCl ₂ , and 0.1 M2-mercaptoethanol.

Example 2 (1) Reagent kit for amplifying nucleic acid derived from cholera toxin-producing Vibrio cholerae (a) Oligonucleotide of Example 1 (3) (b) Oligonucleotide of Example 1 (4) (c) Oligonucleotide of Example 1 (5) (d) Oligonucleotide of Example 1 (6) (e) Tth DNA Polymerase (Toyobo), dATP, dCT
P, dGTP, dTTP (2) Preparation of specimens Fecal samples were collected from cholera patients and suspended in PBS for 15,0
After centrifugation at 00 rpm for 10 minutes, 200 μl of water was added and the mixture was boiled to extract the nucleic acid. Vibrio Cholerae 569B strain was cultured as a standard strain of cholera toxin-producing Vibrio cholerae, treated in the same manner, and used as a sample. (3) To 90 μl of the PCR reaction solution, 10 μl of the above-mentioned nucleic acid solution and 5 μl of each of the oligonucleotides of Example 1 were added as primers to amplify the cholera toxin producing cholera bacterium-derived nucleic acid. The reaction solution composition is 1 mM dithiothreitol, 50 mM KCl, 10 mM Tris-
HCl (pH 8.3), 1.5 mM MgCl ₂ , 0.01% (wt / vol) gelatin, 0.2 mM dATP, dCTP, dGTP, dTTP and Tth DNA, respectively.
40 units / ml of polymerase. The reaction conditions are as follows. First, after denaturing treatment at 94 ° C for 5 minutes, thermal denaturation: 94 ° C, 1 minute, annealing: 58 ° C, 2 minutes, polymerization reaction: 75 ° C, 1.5 minutes were repeated 30 times. These operations were performed using a Perkin-Elmer / Cetus DNA thermal cycler.

(4) Detection 10 μl of the reaction solution was electrophoresed on a 2% agarose gel and stained with ethidium bromide, and then fluorescence from ultraviolet rays was detected. The electrical conditions for electrophoresis were a constant voltage of 100 V and a time of 30 minutes. The operating method and other conditions are described in Maniatis et al., Molecular.
The method described in Cloning (1982) was followed. In addition to the reaction solution, a molecular weight marker was also electrophoresed at the same time, and the length of the detected nucleotide fragment was calculated by comparing the relative mobilities. (5) Results The PCR product of the sample obtained from the patient is the primer (3) (4)
372 bases in the combination of, and the combination of primers (5) (6)
It showed 318 bases and had the same nucleotide length as the cholera toxin-producing Vibrio cholerae standard strain. This was in good agreement with the nucleotide length calculated from the nucleic acid sequences of domestic isolates (Table 1).

[0012]

[Table 1]

Reference Example 1 Confirmation of Specificity of Reagent Kit In order to confirm that the results of Example 2 are specific to the cholera toxin-producing cholera bacterium, the same operation as in Example 2 was performed for other bacteria. .. As other bacteria, the bacteria shown in Table 2 below were used. As a result, as shown in Table 2, no amplified band was observed except for V. cholerae producing Vibrio cholerae and N. vibrio and Vibrio mimicus, which were confirmed to produce cholera toxin.

[0014]

[Table 2]

Example 3 As a probe for detecting cholera toxin-producing Vibrio cholerae nucleic acid, 5 '
Example 1 containing a nucleic acid having a ³² P-phosphate group at the end
Oligonucleotide (2) was prepared.

(1) Synthesis of Oligonucleotide An oligonucleotide for detecting cholera toxin-producing cholera having a linker arm was synthesized by a phosphoamidite method using a DNA synthesizer 380A type (Applied Biosystems). The base sequence is as follows. 5'-AATAGGGGCTACAGAGAXAGATATTACAGT-3 'In the sequence, X represents uridine having a linker arm at the 5-position. This sequence is obtained by substituting a part of the T portion of the sequence of SEQ ID NO: 1 with X. This uridine having a linker arm at the 5-position was prepared by chemical synthesis from deoxyuridine by the synthetic method described in JP-A-60-500717 and introduced into an oligonucleotide. The synthesized oligonucleotide was treated with 27% ammonia water at 55 ° C, 4
After the time deprotection treatment, the product was purified using anion exchange high performance liquid chromatography Mono-Q FPLC (Pharmacia). About 0.2 μmol scale synthesis
1.5A ₂₆₀ (absolute amount obtained from absorbance at 260nm)
Was obtained.

(2) Synthesis of Labeled Oligonucleotide The above linker oligonucleotide was bound to alkaline phosphatase via the linker arm according to the literature (Nucleic Acids Research, 14, 6115, 1986). Linker oligonucleotide 1.0 A ₂₆₀ to 0.2
It was dissolved in 60 μl of M NaHCO ₃ , and 1.25 mg of disuccinimidyl suberate (DSS) was added thereto and reacted at room temperature for 2 minutes. Sepha equilibrated with 1 mM CH ₃ COONa (pH 5.0)
Excess gel filtration using a dex G-25 column (1cmφx30cm)
The DSS was removed. A linker oligonucleotide in which the terminal amino group was activated was further added with an alkaline phosphatase (Boehringer Mannheim) (10
Alkaline phosphatase-labeled nucleic acid probe was obtained by reacting with 0 mM NaHCO ₃ , dissolved in 3M NaCl) at room temperature for 16 hours. The resulting labeled probe was purified using anion exchange high performance liquid chromatography Mono-Q FPLC (Pharmacia). Fractions containing the labeled probe were collected and concentrated by an ultrafiltration method using Centricon 30K (Amicon).

(3) Examination of alkaline phosphatase-labeled nucleic acid probe In this examination, the reverse passive hemagglutination reaction (RPLA) has already been performed.
Cholera strains 109 strains (88 clinical isolates, 21 environmental isolates) whose toxin-producing properties were identified by ELISA and ELISA were used. Inoculate each strain into alkaline peptone water (pH 8.4),
Rotation culture was performed overnight at 37 ° C. 2 μl of each bacterial solution was added dropwise to the nylon membrane, lysis was performed under alkaline conditions, and the nucleic acid was immobilized on the nylon membrane. After neutralizing this membrane, transfer the membrane to a hybridization bag (BRL), and add 25 μl of hybridization buffer containing alkaline phosphatase-labeled nucleic acid probe solution (5xSSC, 0.5% bovine serum albumin, 0.5% polyvinylpyrrolidone, 1% dodecyl sulfate). Sodium) 5m
l was added, and the mixture was sealed with a policyr and pre-hybridized at 50 ° C for 15 minutes. Remove the membrane from the polybag and wash solution-1 (1xSSC, 1% sodium dodecyl sulfate)
It was washed by shaking at 50 ° C. for 5 minutes twice. Further cleaning liquid-2
Washing was performed with (1xSSC, 1% Triton X-100) at 50 ° C for 5 minutes twice and at room temperature for 5 minutes twice with shaking. Finally, wash solution-3 (1xSS
Washing with C) was carried out twice with shaking at room temperature for 5 minutes. Transfer the membrane to a new hybridization bag and add substrate solution (0.1M Tris-H
Cl, 0.1 M NaCl, 0.1 M MgCl ₂ , 0.3 mg / ml, nitroblue tetrazolium, 0.3 mg / ml bromchloroindoferryl phosphate, pH 7.5) was added, and the mixture was sealed with a policyr and incubated at 37 ° C. for 1 hour. The spot of purple dye generated by alkaline phosphatase was visually determined.

The results are shown in the table below.

[0020]

[Table 3] Good results were obtained with an overall sensitivity of 98.6% and specificity of 94.7%.

The specificity of the above-mentioned alkaline phosphatase-labeled nucleic acid probe was examined for the following bacteria. The presence or absence of cross-hybridization was examined using Vibrio spp., Which is the same genus as Vibrio cholerae, and bacteria derived from human placenta, which can cause diarrhea causing bacteria like Vibrio cholerae. All bacteria were clinical isolates, grown in an appropriate medium, and measured in the same manner as in Example 8. As a result, only Nagvibrio and Vibrio mimicus, which were confirmed to produce Vibrio cholerae and cholera toxin-like toxin, were positive. The results are shown below.

[0022]

[Table 4]

[0023]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to measure cholera toxin-producing cholera bacteria rapidly, simply and specifically with high sensitivity. The oligonucleotide of the present invention can be used as a primer for amplification reaction or as a probe for direct detection. Especially in the amplification reaction, the cholera toxin-producing cholera bacterium can be detected from a small amount of sample because of high detection sensitivity, and its clinical significance is great.

[0024]

[Sequence list]

SEQ ID NO: 1 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..30 Method for determining features : S Other features: It has a sequence complementary to the sequence of cholera toxin-producing Vibrio cholerae. Array AATAGGGGCT ACAGAGATAGA TATTACAGT 30

SEQ ID NO: 2 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..30 Determined method: S Other characteristics: having a sequence complementary to the sequence of cholera toxin-producing Vibrio cholerae. Sequence TATCTTACTG AAGCTAAAGT CGAAAAGTTA 30

SEQ ID NO: 3 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Determined method: S Other characteristics: having a sequence complementary to the sequence of cholera toxin-producing Vibrio cholerae. Sequence GGTCAAACTA TATTGTCTGG TC 22

SEQ ID NO: 4 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..21 Features Determined method: S Other characteristics: having a sequence complementary to the sequence of cholera toxin-producing Vibrio cholerae. Sequence ACTCATCGAT GATCTTGGAG C 21

SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..21 Features Determined method: S Other characteristics: having a sequence complementary to the sequence of cholera toxin-producing Vibrio cholerae. Sequence GCATATGCAC ATGGAACACC T 21

SEQ ID NO: 6 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..21 Features Determined method: S Other characteristics: having a sequence complementary to the sequence of cholera toxin-producing Vibrio cholerae. Sequence CATACTAATT GCGGCAATCG C 21

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location // (C12Q 1/04 C12R 1:63) (72) Inventor Shuji Shibata Nii Katata, Otsu City, Shiga Prefecture 1-1-1 Toyobo Co., Ltd.

Claims (6)

[Claims] 1. SEQ ID NO: 1 to 6 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is replaced with uracil (U). An oligonucleotide for detecting a cholera toxin-producing bacterium, which has a nucleic acid sequence shown in (1) or a complementary sequence thereof. 2. A labeled oligonucleotide for detecting a cholera toxin-producing bacterium, which is obtained by labeling the oligonucleotide for detecting a cholera toxin-producing bacterium according to claim 1. 3. Labeling the cholera toxin-producing bacterium detecting oligonucleotide according to claim 1, crossing the obtained labeled nucleic acid probe with DNA or RNA in a sample, and measuring the label of the hybridized conjugate. A method for detecting a cholera toxin-producing bacterium in a sample, which comprises: 4. A labeled nucleic acid primer obtained by using the oligonucleotide for detecting cholera toxin-producing bacteria of claim 1 as a nucleic acid primer as it is or by labeling it with a DNA or RNA in a sample to extend the primer,
A method for detecting a cholera toxin-producing bacterium in a sample, which comprises measuring the obtained primer extension product. 5. A reagent kit for detecting cholera toxin-producing bacteria in a sample, which comprises a labeled nucleic acid probe labeled with the oligonucleotide for detecting cholera toxin-producing bacteria of claim 1. 6. Amplification of a cholera toxin-producing bacterium in a sample, which comprises a labeled nucleic acid probe obtained by using the oligonucleotide for detecting a cholera toxin-producing bacterium of claim 1 as a primer as it is or by labeling. And / or a reagent kit for detection.