JP2020010636A - Oligonucleotide probes for detecting toxin b gene (tcdb) - Google Patents

Abstract

To provide simpler and more sensitive methods for detecting toxin B gene (tcdB) of Clostridium difficile over the prior art.SOLUTION: Disclosed herein is an oligonucleotide probe having the following features (A) and (B): (A) a base sequence of at least consecutive 15 bases of a base sequence represented by any of SEQ ID:NOs 1 to 19 or complementary base sequences thereto; (B) at least one of the terminal bases is labelled with a fluorescent dye that quenches on the interaction with guanine.SELECTED DRAWING: Figure 3

Description

  The present invention relates to an oligonucleotide probe for detecting a toxin B gene (tcdB) of Clostridium difficile contained in a sample. Furthermore, the present invention relates to a method for detecting tcdB contained in a sample using the oligonucleotide probe, and a reagent kit for use in the method.

  Clostridium difficile (also called Clostridium difficile) (hereinafter also referred to as C. difficile) is one of the causative microorganisms of hospital-acquired infection, and it is important in clinical diagnosis to detect it easily, quickly, and with high sensitivity. .

  Specifically, C.I. difficile is known to be a major causative bacterium of diarrhea and enteritis associated with administration of antibacterial drugs. For example, if the normal intestinal flora is disrupted by antimicrobial treatment, C.I. Toxin-producing bacteria, which are toxic strains of C. difficile, produce toxins such as toxin A, toxin B, and binary toxin, which cause diarrhea and enteritis. Therefore, detection of the toxin produced by the present bacterium is performed by C. It is important for examination and diagnosis of difficile infection. Conventionally, toxin detection has been performed using an immunochromatography method, but this method has a problem of low sensitivity. Thus, a method for detecting genes involved in the production of these toxins by using a highly sensitive nucleic acid amplification method has been developed (Patent Document 1).

  However, C.I. It is desired to develop a simpler and more sensitive method for detecting genes involved in the production of C. difficile toxin, particularly the toxin B gene (tcdB), which is generally considered to be more toxic than toxin A.

JP-T-2015-529090

  The present invention has been made in the background of the problems of the related art. That is, an object of the present invention is to provide C.I. difficile tcdB is to be detected.

  As a result of earnest studies, the present inventors have found that tcdB contained in a sample can be detected more easily and with high sensitivity by using a specific oligonucleotide probe, and have reached the present invention. That is, the outline of the present invention is as follows.

[Item 1] An oligonucleotide probe having the following characteristics (A) and (B) used for detecting a toxin B gene (tcdB) of Clostridium difficile contained in a sample.
(A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 or at least 15 contiguous to a nucleotide sequence complementary thereto Includes base sequences of bases or more.
(B) At least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine.
[Item 2]
Item 1. The item (A) according to Item 1, which comprises the nucleotide sequence represented by any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 or a nucleotide sequence complementary thereto. Oligonucleotide probe.
[Item 3] Item 1 or 2, wherein the fluorescent quenching dye of (B) is at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, BODIPY and its derivatives. Oligonucleotide probe.
[Item 4] The fluorescence quenching dye of (B) is 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), From the group consisting of carboxy rhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue) Item 4. The oligonucleotide probe according to any one of Items 1 to 3, which is at least one fluorescent quenching dye selected.
[Item 5] The oligonucleotide probe according to any one of Items 1 to 4, wherein in (B), at least one terminal base labeled with a fluorescent quenching dye is cytosine.
[Item 6] A method for detecting a toxin B gene (tcdB) of Clostridium difficile contained in a sample, wherein a reaction solution containing at least the oligonucleotide probe according to any one of Items 1 to 5 is used. A tcdB detection method characterized by performing the following steps (1) to (3).
(1) Providing a sample that can contain tcdB.
(2) A step of performing a nucleic acid amplification reaction using the reaction solution.
(3) a step of hybridizing the oligonucleotide probe with the amplification product obtained in step (2) and measuring the fluorescence intensity of the reaction solution;
[Item 7] The method according to item 6, wherein the step (3) includes at least one of the following steps (3a) to (3c).
(3a) a step of monitoring the progress of the nucleic acid amplification reaction in real time by hybridizing the oligonucleotide probe to the obtained amplification product and measuring the fluorescence intensity of the reaction solution.
(3b) After completion of the step (2), the progress of the nucleic acid amplification reaction is monitored at an end point by hybridizing the oligonucleotide probe to the obtained amplification product and measuring the fluorescence intensity of the reaction solution. Process.
(3c) a step of, after the completion of the step (2), hybridizing the oligonucleotide probe to the obtained amplification product and measuring the temperature dependence of the fluorescence intensity of the reaction solution to detect tcdB.
[Item 8] The method according to Item 6 or 7, wherein the nucleic acid amplification reaction in the step (2) is PCR.
[Item 9] The method according to Item 8, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification reaction in the step (2) is a DNA polymerase belonging to Family B.
[Item 10] The method according to Item 9, wherein the DNA polymerase belonging to Family B is an archaebacterial DNA polymerase or a mutant thereof.
[Item 11] The method according to Item 10, wherein the archaebacterial DNA polymerase is KOD DNA polymerase or a mutant thereof.
[Item 12] A reagent for performing the detection method according to any one of Items 6 to 11.
[Item 13] A kit comprising a reagent for performing the detection method according to any one of Items 6 to 11.

  According to the present invention, tcdB contained in a sample can be easily detected with high sensitivity. By using the oligonucleotide probe of the present invention and the detection method, reagent, and kit using the probe, simple and highly sensitive detection of tcdB can be performed, which greatly contributes to the field of clinical diagnosis.

FIG. 9 is a diagram showing the results of Example 2. Solid lines, dotted lines, and broken lines indicate changes in fluorescence of different oligonucleotide probes. FIG. 9 is a view showing a result of Example 3. FIG. 9 is a view showing a result of Example 4. The solid line, the dotted line, and the broken line show the results of the melting curve analysis when the colony suspensions having different template concentrations were measured.

  Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.

[Oligonucleotide for detecting Clostridium difficile toxin B gene (tcdB)]
One of the embodiments of the present invention is a method for preparing C.I. An oligonucleotide probe having the following features (A) and (B) used for detecting tddB of C. difficile.
(A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 or at least 15 contiguous to a nucleotide sequence complementary thereto Includes base sequences of bases or more.
(B) At least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine.

[Clostridium difficile toxin B gene (tcdB)]
Clostridium difficile (also known as Clostriidioides difficile) is one of the causative microorganisms of hospital-acquired infection, and toxin-producing bacteria cause diarrhea and enteritis associated with antibacterial drugs by the toxin. The major toxins are toxin A and toxin B, but may also produce binary toxin and the like as toxins.

  Conventionally, immunochromatography and the like have been used as methods for detecting these toxins, but have a problem of low sensitivity. Therefore, a method for detecting genes involved in the production of these toxins by using a highly sensitive nucleic acid amplification method has been developed (Patent Document 1).

  C. The genes involved in the production of C. difficile toxin include tcdA, tcdB, tcdC mutation, cdtA, cdtB, and the like. In particular, detection of tcdB, a gene encoding toxin B, is important.

  The present inventor has developed the oligonucleotide probe of the present invention as a result of intensive studies in order to detect tcdB more easily than in the prior art.

[Oligonucleotide probe]
The oligonucleotide probe of the present invention has the following features (A) and (B).
(A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 or at least 15 contiguous to a nucleotide sequence complementary thereto Includes base sequences of bases or more.
(B) At least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine.

  The above-mentioned features (A) include (A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 Those consisting of a base sequence of at least 15 bases or more continuous in a base sequence complementary thereto are more preferable, those consisting of a base sequence of at least 17 bases or more in the base sequence are more preferable, More preferably, the base sequence has a base sequence of 18 bases or more, and preferably a base sequence of at least 19 bases or more in the base sequence. More preferably made, made from the full-length sequence of the base sequence is more preferred.

  Nucleic acid testing methods using oligonucleotide probes have been known in the art, and have already been established in the art (for example, JP-T-2015-529090, JP-A-5354216). As an oligonucleotide probe used for such a nucleic acid test method, a TaqMan hydrolysis probe, a molecular beacon, a FRET hybridization probe, a QProbe, and the like are known. In the present invention, any labeled probe known in the art can be used as long as it has the feature that (B) at least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine. However, it is preferable to use QProbe from the viewpoint that a good result is more easily obtained.

  QProbe is a fluorescent probe (fluorescence quenching probe) developed by KURATA et al. (Japanese Patent No. 5354216). This probe is a hybridization probe in which at least one terminal base is labeled with a fluorescent quenching dye that is quenched by interaction with guanine.

  For example, the fluorescent quenching dye used in Qprobe is not particularly limited, but fluorescein or a derivative thereof (eg, fluorescein isothiocyanate), rhodamine or a derivative thereof (eg, tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate, carboxy rhodamine, x- Rhodamine, sulforhodamine 101 acid chloride), BODIPY or a derivative thereof (for example, BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665) and the like. That. The details of the fluorescent quenching dye are described in Japanese Patent No. 581263 and the like, and the present invention can also refer to the technique.

  In particular, in the present invention, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) and carboxyrhodamine 6G are used as fluorescent quenching dyes. , TAMRA, Rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). It is preferable to use at least one.

Further, an oligonucleotide probe in which at least one terminal base labeled with a fluorescent quenching dye (that is, at least one terminal base when both ends are labeled) is cytosine is more preferable.
When such an oligonucleotide probe is hybridized to an amplification product, it can be quenched by forming a base pair with a guanine base in the amplification product and interacting with the guanine base. Can be measured.

  In addition, when the probe hybridizes, even if the cytosine base of the probe and the guanine base in the amplification product do not form a base pair, the fluorescence can be quenched if the distance between the bases is short. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to the technology. That is, when the probe hybridizes, it can be quenched if the guanine base in the amplification product is within, for example, 1 to 3 bases with respect to the cytosine base of the probe (forming a base pair with the cytosine base). Base is 1).

  In a more particular embodiment, the oligonucleotide probe has a non-cytosine at least one terminal base (ie, at least one terminal base, if both ends are labeled) labeled with a fluorescent quenching dye. The change in the fluorescence intensity of the liquid can be measured. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to the technology. For example, when the probe hybridizes, it can be quenched if the guanine base in the amplification product is in the range of, for example, 1 to 3 bases with respect to at least one terminal base labeled with the fluorescent quenching dye ( The base forming a base pair with the terminal base is defined as 1).

  According to the purpose, one or more labeling substances can be further added to the oligonucleotide probe in addition to the fluorescent quenching dye quenched with guanine defined in the above feature (B). Such additional labeling substances include fluorescent substances, chemiluminescent substances, radioactive substances, biotin, alkaline phosphatase, digoxigenin, peroxidase and the like. In the present invention, the fluorescent quenching dye quenched with guanine defined in the above feature (B) A labeling substance other than the above may be further added. The labeling substance is not particularly limited except that it comprises a fluorescent quenching dye that quenches with guanine defined in the above feature (B), and an oligonucleotide probe labeled with a known labeling substance according to the test method can be used. . Further, a linker, a spacer, or the like may be added for labeling the oligonucleotide probe.

  In a more specific embodiment, the bases that make up the oligonucleotide probe may include nucleotide analogs such as inosine, peptide nucleic acid (PNA), LNA. The use of nucleotide analogs can be expected to improve specificity.

  The oligonucleotide probe of the present invention comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 or a nucleotide sequence complementary thereto And characterized in that it contains a continuous nucleotide sequence of at least 15 bases or more. It is clear from the present invention that tcdB can be detected more easily and with higher sensitivity than conventional techniques by using an oligonucleotide probe containing such a base sequence.

  Above all, a base sequence represented by any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 or a base sequence complementary to them, having at least 15 or more bases, Oligonucleotide probes are preferable, and at least 17 or more bases continuous in the base sequence represented by any of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 or a base sequence complementary thereto Oligonucleotide probes comprising the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 or a nucleotide sequence complementary thereto are continuous. An oligonucleotide probe containing a base sequence of at least 19 bases or more And an oligonucleotide probe comprising a nucleotide sequence represented by any of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 or a nucleotide sequence complementary thereto, is even more preferred. An oligonucleotide probe consisting of the nucleotide sequence represented by any of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 or a nucleotide sequence complementary thereto is particularly preferable.

[TcdB detection method]
One embodiment of the present invention is a method for detecting a toxin B gene (tcdB) of Clostridium difficile contained in a sample, comprising at least the oligonucleotide probe according to any of the above. A tcdB detection method characterized by performing the following steps (1) to (3) using a reaction solution.
(1) Providing a sample that can contain tcdB.
(2) A step of performing a nucleic acid amplification reaction using the reaction solution.
(3) a step of hybridizing the oligonucleotide probe with the amplification product obtained in step (2) and measuring the fluorescence intensity of the reaction solution;

[sample]
The sample that can be used in the present invention is not particularly limited as long as it may contain tcdB. For example, not only biological samples, foods, and environmental samples, but also purified nucleic acids and the like can be mentioned. The sample may be subjected to nucleic acid extraction and some pretreatment. Nucleic acid extraction and pretreatment of a sample are generally performed in the art. Examples of the pretreatment include filtration, centrifugation, dilution, heat treatment, acid treatment, alkali treatment, organic solvent treatment, suspension treatment, crushing treatment, and grinding treatment, but the present invention is not limited thereto.

  Examples of biological samples include, but are not limited to, animal and plant tissues, body fluids, excrement, cells, bacteria, viruses, and the like. Further examples include blood, blood culture solution, urine, pus, cerebrospinal fluid, pleural effusion, pharyngeal swab, nasal swab, sputum, tissue section, skin, vomit, feces, separated culture colony, catheter washing solution and the like.

  Examples of foods include water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, marine products, eggs, dairy products, raw meat, raw fish, prepared dishes, and the like. When a food is used as the measurement sample, not only a part or all of the food but also a food whose surface is wiped can be used. Further, a material obtained by wiping a cooking utensil or a door knob or a cleaning solution for cleaning the same can also be used as a sample.

  Examples of environmental samples include water, ice, soil, air and aerosol. The water mentioned here includes, for example, tap water, seawater, water collected from rivers, waterfalls, lakes, ponds, and the like. In addition, a sample obtained by wiping a wall surface, a floor surface, equipment and fixtures, a toilet bowl, and the like of a facility, or a cleaning solution obtained by cleaning them can also be used.

  In the present invention, any of the samples described above can be used. In view of the fact that D. difficile is a major causative bacterium of diarrhea and enteritis, it is preferable to use biological samples (eg, animal and plant tissues, body fluids, excrement, tissue sections, skin, vomit, feces, and isolated culture colonies). It is more preferable to use excreta, feces and fecal culture colonies, and it is even more preferable to use feces and isolated culture colonies. According to the present invention, tcdB can be detected with high sensitivity even when such a sample is used.

  The method for collecting and preparing the sample is not particularly limited, and a known method can be used depending on the type and purpose of the sample.

[Nucleic acid amplification reaction]
Nucleic acid amplification is a technology that amplifies several copies of a target nucleic acid to a level that can be visualized, that is, more than several hundred million copies. Used. Examples of such a nucleic acid amplification method include a PCR method, a LAMP method, an LCR method, a TMA method, an SDA method, an RT-PCR method, an RT-LAMP method, a NASBA method, a TRC method, a TMA method, and the like. These techniques are already established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method used in the tcdB detection method of the present invention is preferably a PCR method, but is not limited thereto.

[PCR]
The PCR reaction is a reaction mainly catalyzed by a DNA polymerase, (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), and (2) primers to template single-stranded DNA. The three steps of annealing and (3) extension of the primer using DNA polymerase are defined as one cycle, and the cycle is repeated to amplify the target nucleic acid. Examples of the DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and variants thereof. In the present invention, it is preferable to use a DNA polymerase belonging to Family B from the viewpoint that tcdB detection can be performed more easily and with high sensitivity.

[DNA polymerase belonging to family B]
The DNA polymerase used in the present invention is preferably a DNA polymerase belonging to Family B, but is not limited thereto. The DNA polymerase belonging to the family B is not particularly limited, but is preferably a DNA polymerase derived from archaea (Archea).

[A DNA polymerase derived from archaebacteria]
Examples of DNA polymerases derived from archaea belonging to the family B include DNA polymerases isolated from bacteria belonging to the genus Pyrococcus and the genus Thermococcus. The present invention also includes a mutant thereof that has not lost DNA polymerase activity derived from an archaea belonging to Family B. Variants of DNA polymerase include, but are not limited to, variants for the purpose of enhancing polymerase activity, deficient exonuclease activity, adjusting substrate specificity, and the like.
Pyrococcus DNA polymerases include Pyrococcus furiosus and Pyrococcus sp. Examples include, but are not limited to, DNA polymerases isolated from GB-D, Pyrococcus woesii, Pyrococcus abyssi, Pyrococcus horikoshii, and variants thereof that have not lost DNA polymerase activity derived therefrom.
Examples of DNA polymerases derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, and Thermococcus sp. JDF-3, Thermococcus sp. 9degreases North-7 (Thermococcus sp. 9 ° N-7), DNA polymerases isolated from Thermococcus siculi, and variants thereof that have not lost the DNA polymerase activity derived therefrom.
PCR enzymes using these DNA polymerases are commercially available and include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), and Deep Vent (New England). , Tgo (Roche), Pwo (Roche) and the like, any of which can be used in the present invention.

  Among them, KOD DNA polymerase and its mutants (e.g., KOD DNA polymerase lacking 3 'to 5' exonuclease activity) having excellent extensibility and thermostability are preferred.

  KOD DNA polymerase is superior to Taq DNA polymerase, a DNA polymerase belonging to family A, in accuracy, amplification efficiency, elongation, and crude sample resistance. In the present invention, by using such a KOD DNA polymerase, tcdB can be detected easily and with high sensitivity, as shown in Examples described later.

  In the tcdB detection method of the present invention, any of the above oligonucleotide probes is hybridized to the amplification product obtained by any of the above methods, and the fluorescence intensity of the reaction solution is measured to detect tcdB.

  Hybridization conditions are affected by the temperature, pH, cation concentration, presence of an organic solvent in the solution, and the like, and may be optimized according to the nucleic acid amplification reaction to be performed.

The mode of nucleic acid detection in the step (3) in the above-described tcdB detection method is not particularly limited. For example, it is preferable to use a method including at least one of the following steps (3a) to (3c).
(3a) a step of monitoring the progress of the nucleic acid amplification reaction in real time by hybridizing the oligonucleotide probe to the obtained amplification product and measuring the fluorescence intensity of the reaction solution.
(3b) After completion of the step (2), the progress of the nucleic acid amplification reaction is monitored at an end point by hybridizing the oligonucleotide probe to the obtained amplification product and measuring the fluorescence intensity of the reaction solution. Process.
(3c) After completion of the step (2), the oligonucleotide probe is hybridized to the obtained amplification product, and the temperature dependence of the fluorescence intensity of the reaction solution is measured to detect the norovirus G1 nucleic acid. Analyzing the nucleotide sequence polymorphism of the nucleic acid.

[(3a) Monitor progress of nucleic acid amplification reaction in real time]
By monitoring the progress of the nucleic acid amplification reaction in real time, the target nucleic acid contained in the sample can be quickly detected. Furthermore, it is possible to quantify the target nucleic acid contained in the sample based on the amplification rate.
For example, the progress of a nucleic acid amplification reaction is monitored in real time by measuring the fluorescence intensity of a reaction solution containing an oligonucleotide probe labeled with a fluorescence quenching dye. At any point in the nucleic acid amplification reaction, the fluorescence intensity of the reaction solution is measured. When tcdB DNA is contained in the sample, the oligonucleotide probe hybridizes to the amplified nucleic acid depending on the amount of the amplified nucleic acid, and the fluorescence intensity increases (or decreases). The progress of the nucleic acid amplification reaction can be monitored in real time by plotting the obtained fluorescence intensity against time (the number of cycles in a PCR reaction) (for example, Example 2).

[(3b) Monitor progress of nucleic acid amplification reaction at endpoint]
By monitoring the progress of the nucleic acid amplification reaction at the endpoint, the target nucleic acid contained in the sample can be rapidly detected. Further, the target nucleic acid can be quantified by comparing the fluorescence intensity or the like at the end point.
For example, the progress of a nucleic acid amplification reaction is monitored at an endpoint by measuring the fluorescence intensity of a reaction solution containing an oligonucleotide probe labeled with a fluorescence quenching dye (for example, Example 3). After completion of the nucleic acid amplification reaction, the fluorescence intensity of the reaction solution is measured. By comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of the reaction solution before the reaction, the presence or absence of amplification of the target nucleic acid can be confirmed. Alternatively, the presence or absence of the target nucleic acid contained in the sample can also be confirmed by comparing the reaction intensity of the reaction solution after the reaction with the reaction intensity of the control reaction solution. The control reaction solution is a reaction solution to which a sample known to be negative or a sample known to be positive is added instead of the sample to be measured.
In general, it is preferable to monitor the progress of the nucleic acid amplification reaction in real time, but it is preferable to monitor the progress at the end point for the purpose of simplifying the detection step.

[(3c) Measurement of temperature dependence of fluorescence intensity]
Measuring the temperature dependence of the fluorescence intensity means, specifically, measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from a low temperature to a high temperature (for example, Example 4). By first-order differentiation of the obtained fluorescence intensity with respect to temperature, a melting temperature (Tm value) specific to the oligonucleotide probe used can be obtained. The fluorescence intensity may be converted into a fluorescence quenching rate or the like according to the purpose.
Detection and analysis of the target nucleic acid using the Tm value are called melting curve analysis. Generally, the Tm value refers to the temperature at which the ratio of the oligonucleotide forming a double strand with its complementary strand is equal to the rate of forming a single strand without forming a double strand. Since the Tm value is a value specific to the nucleotide sequence, the melting curve analysis can be used as a technique for analyzing the nucleotide sequence polymorphism of the target nucleic acid. The term “base sequence polymorphism” used herein includes single nucleotide polymorphism, base substitution, base deletion, base insertion, and the like.

As an example, the melting curve analysis is also applied to SNP analysis and the like.
When there is a mutation in the base sequence of the target nucleic acid with respect to the oligonucleotide probe, the Tm value is generally low because the base is mismatched when the probe hybridizes. Therefore, single nucleotide polymorphism analysis (SNP analysis) can be performed by comparing the magnitudes of the Tm values.

[Reagent for detecting tcdB]
Another embodiment of the present invention includes a reagent for detecting tcdB contained in a sample. The reagent contains at least components necessary for nucleic acid amplification, in addition to the oligonucleotide probe having the above-mentioned characteristics (A) and (B). The necessary components vary depending on the nucleic acid amplification reaction to be performed, and each can be performed by a known method. For example, when detecting tcdB contained in a sample using a PCR reaction, it is preferable that the probe contain at least an oligonucleotide probe, a DNA polymerase, an oligonucleotide primer, deoxyribonucleoside triphosphates (dNTPs), and a magnesium salt. The concentration of each component can be appropriately adjusted depending on the intended experiment. For example, the oligonucleotide probe is preferably 0.1 to 1 μM, more preferably 0.2 to 0.5 μM. DNA polymerase is preferably 0.01 to 1 U / uL, more preferably 0.1 to 0.5 U / uL. Oligonucleotide primers are different, but preferably 0.1 to 10 μM. The amount of deoxyribonucleoside triphosphates (dNTPs) is preferably from 0.02 to 1 mM, more preferably from 0.1 to 0.5 mM. The magnesium salt is preferably from 0.1 to 6 mM, more preferably from 1 to 5 mM.

  Furthermore, additives or the like known in the art may be added for the purpose of suppressing non-specific amplification and promoting the reaction. Additives for the purpose of suppressing non-specific amplification include anti-DNA polymerase antibodies and phosphoric acid (Patent Document 4). As additives for promoting the reaction, bovine serum albumin (BSA), protease inhibitor, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, compounds containing sulfur or acetate, dimethyl sulfoxide (DMSO), glycerol, Ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethyl acetate Ammonium (TMAA), polyethylene glycol, Triton, Tween 20, Nonidet P40 and the like. That. In the present invention, one or more of these additives may be used in combination, but the invention is not limited thereto.

  An oligonucleotide primer is an oligonucleotide used as a starting point of nucleic acid amplification in a nucleic acid amplification reaction, and is generally used in the art (for example, Patent Document 1). A plurality of types of oligonucleotide primers may be used depending on the nucleic acid amplification reaction to be performed. Although it depends on the purpose of the experiment, preferably, a set of a forward primer and a reverse primer that can amplify about 50 to 500 bp, preferably about 50 to 200 bp including the target sequence to which the oligonucleotide probe of the present invention hybridizes, is used. Is good. It is important for the oligonucleotide primer with high specificity that the 3 'end is complementary to the target sequence. In other words, if the 3' end is complementary, it is effective even if the 5 'end is not complementary Primers. For this reason, it is preferable that the oligonucleotide primer used in the present invention is also designed so that the 3 'end has a sequence complementary to the genomic sequence. Furthermore, when the target nucleic acid has a base sequence polymorphism depending on the strain, a degenerate primer may be used.

[Kit for detecting tcdB]
Further, another embodiment of the present invention includes a kit including a reagent for detecting tcdB contained in a sample. The configuration of the kit is not particularly limited as long as it includes the tcdB detection reagent containing the oligonucleotide probe and is configured to detect tcdB.

  Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to Examples.

Example 1 Alignment of tcdB and Design of Oligonucleotide Probe (1-1) Method In order to design an oligonucleotide probe that can detect tcdB more easily and more sensitively than conventional techniques, tcdB alignment was performed. The nucleotide sequence of tcdB was obtained from the NCBI (National Center for Biotechnology Information) database, and their alignment was performed (total of 40 sequences).
(1-2) Results Based on the results of the alignment, a base sequence considered to satisfy the following characteristics (a) and (b) was selected.
(A) Can hybridize without difference between strains (b) Does not form non-specific products such as dimers (c) Tm value is 40 ° C. or higher As a result, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, Sequence SEQ ID NO: 17, SEQ ID NO: 18, or a part of the nucleotide sequence represented by any of the nucleotide sequences complementary to the nucleotide sequence represented by SEQ ID NO: 19, a nucleotide sequence of at least 15 consecutive nucleotides or more could be selected. .

Example 2: Detection of tcdB that can be monitored in real time (2-1) Method The oligonucleotide probe whose base sequence is complementary to SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 and whose 3 ′ end is labeled with BODIPY-FL Using either one, C.I. The PCR reaction was performed using the difficile (tcdB positive) colony suspension as a sample. In addition, the oligonucleotide primer used the base sequence of sequence number 20 and sequence number 21.
(2-2) Reaction solution A reaction solution containing the following components was prepared using KOD FX Neo (Toyobo Co., Ltd.). The preparation of the reaction solution, etc., followed the instruction manual of KOD FX Neo (Toyobo).
1.5 μM Primer represented by SEQ ID NO: 20 0.5 μM Primer represented by SEQ ID NO: 21 0.3 μM Oligonucleotide probe (base sequence complementary to any of SEQ ID NOs: 9 to 11)
(2-3) Reaction Using the Rotor-Gene (TM) Q, the reaction solution was reacted in the following temperature cycle, and the fluorescence intensity in each cycle was measured.
94 ° C for 30 seconds,
98 ° C 1 second -52 ° C 10 seconds -63 ° C 10 seconds (60 cycles)
(2-4) Results FIG. 1 is a diagram in which the fluorescence intensity (converted to the extinction ratio) obtained by the measurement is plotted on the number of cycles. The solid line, the dotted line, and the broken line are plots of the fluorescence intensity when using the oligonucleotide probe represented by the nucleotide sequence complementary to SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. Therefore, it was shown that the progress of the nucleic acid amplification reaction can be monitored in real time using the oligonucleotide probe of the present invention.

Example 3 Detection of tcdB Monitorable at Endpoint (3-1) Method Using an oligonucleotide probe having a nucleotide sequence complementary to SEQ ID NO: 11 and labeled at the 3 ′ end with BODIPY-FL, C.I. The PCR reaction was performed using the difficile (tcdB positive) colony suspension as a sample. In addition, the oligonucleotide primer used the base sequence of sequence number 20 and sequence number 21. The template concentrations used were 0, 100, and 1000 CFU / test colony suspensions, respectively.
(3-2) Reaction Solution A reaction solution containing the following components was prepared using GeneCube (registered trademark) Test Basic (Toyobo Co., Ltd.). Preparation of the reaction solution, etc., followed the instruction manual of GeneCube (registered trademark) Test Basic.
1.5 μM Primer represented by SEQ ID NO: 20 0.5 μM Primer represented by SEQ ID NO: 21 0.3 μM Oligonucleotide probe (3-3) Reaction Using GENECUBE (registered trademark), the reaction solution was subjected to the following temperature cycle. The reaction was performed, and the fluorescence intensity in each cycle was measured.
94 ° C for 30 seconds,
98 ° C 1 second -52 ° C 10 seconds -63 ° C 10 seconds (60 cycles)
(3-4) Results FIG. 2 shows the fluorescence intensity and the fluorescence change rate. The fluorescence change rate (representing the extinction rate in this example) was determined by the formula (fluorescence intensity before amplification−fluorescence intensity after amplification) / (fluorescence intensity before amplification). FIG. 2 shows that the fluorescence intensity changes according to the amount of the template. difficile (tcdB positive) was also possible. Therefore, it was shown that the progress of the nucleic acid amplification reaction can be monitored at the endpoint using the oligonucleotide probe of the present invention. Similar results were obtained when oligonucleotide probes having a sequence represented by a nucleotide sequence complementary to SEQ ID NOS: 9 and 10 were used.

Example 4: Detection of tcdB by melting curve analysis (4-1) Method Using the same reaction solution as in Example 3 (including an oligonucleotide probe having a nucleotide sequence complementary to SEQ ID NO: 11) and reaction conditions, . The PCR reaction was performed using the difficile (tcdB positive) colony suspension as a sample. After the nucleic acid amplification reaction, melting curve analysis was performed under the following conditions. The template concentrations used were 0, 100, and 1000 CFU / test colony suspensions, respectively.
94 ° C for 30 seconds,
39 ° C for 30 seconds,
40-75 ° C 0.09 ° C / sec (4-2) Results FIG. 3 shows the results of the melting curve analysis. Solid lines, dotted lines, and broken lines show the results of melting curve analysis when the 1000, 100, and 0 CFU / test colony suspensions were measured, respectively. FIG. 3 shows that the melting curve analysis can detect tcdB contained in the sample. Similar results were obtained when oligonucleotide probes having a sequence represented by a nucleotide sequence complementary to SEQ ID NOS: 9 and 10 were used.

  By using the oligonucleotide probe according to the present invention and a method, reagent or kit using the probe, tcdB contained in a sample can be detected easily and with high sensitivity. Therefore, the present invention enables not only research use, but also the possibility of detecting tcdB, which is a toxin gene of the causative bacterium, quickly, simply, and with high sensitivity when there is a risk of hospital-acquired infection. And so on.

Claims (13)

An oligonucleotide probe having the following features (A) and (B) used for detecting a toxin B gene (tcdB) of Clostridium difficile contained in a sample.
(A) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 or at least 15 contiguous to a nucleotide sequence complementary thereto Includes base sequences of bases or more.
(B) At least one terminal base is labeled with a fluorescent quenching dye that quenches by interaction with guanine.   The said (A), The base sequence shown by any one of sequence number 5, sequence number 6, sequence number 9, sequence number 10, or sequence number 11, or the base sequence complementary thereto is included. Oligonucleotide probes.   The oligonucleotide probe according to claim 1 or 2, wherein the fluorescent quenching dye (B) is at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, BODIPY and its derivatives. .   The fluorescence quenching dye (B) is 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, At least one selected from the group consisting of TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue) The oligonucleotide probe according to any one of claims 1 to 3, which is one fluorescent quenching dye.   The oligonucleotide probe according to any one of claims 1 to 4, wherein in (B), at least one terminal base labeled with a fluorescence quenching dye is cytosine. A method for detecting a toxin B gene (tcdB) of Clostridium difficile contained in a sample, using a reaction solution containing at least the oligonucleotide probe according to any one of claims 1 to 5, A tcdB detection method comprising performing the following steps (1) to (3).
(1) Providing a sample that can contain tcdB.
(2) A step of performing a nucleic acid amplification reaction using the reaction solution.
(3) a step of hybridizing the oligonucleotide probe with the amplification product obtained in step (2) and measuring the fluorescence intensity of the reaction solution; The method according to claim 6, wherein the step (3) includes at least one of the following steps (3a) to (3c).
(3a) a step of monitoring the progress of the nucleic acid amplification reaction in real time by hybridizing the oligonucleotide probe to the obtained amplification product and measuring the fluorescence intensity of the reaction solution.
(3b) After completion of the step (2), the progress of the nucleic acid amplification reaction is monitored at an end point by hybridizing the oligonucleotide probe to the obtained amplification product and measuring the fluorescence intensity of the reaction solution. Process.
(3c) a step of detecting the tcdB by, after the completion of the step (2), hybridizing the oligonucleotide probe to the obtained amplification product and measuring the temperature dependence of the fluorescence intensity of the reaction solution.   The method according to claim 6 or 7, wherein the nucleic acid amplification reaction in the step (2) is PCR.   The method according to claim 8, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification reaction in the step (2) is a DNA polymerase belonging to Family B.   The method according to claim 9, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from archaea or a mutant thereof.   The method according to claim 10, wherein the archaebacterial DNA polymerase is KOD DNA polymerase or a mutant thereof.   A reagent for performing the detection method according to claim 6.   A kit comprising a reagent for performing the detection method according to claim 6.