Primers for detecting the toxin B gene (tcdB)

Abstract

To provide simpler and more sensitive methods for detecting toxin B gene (tcdB) of Clostridioides difficile over the prior art.SOLUTION: According to the invention, toxin B gene (tcdB) of Clostridioides difficile in a sample is detected by using primers comprising a base sequence represented by any of SEQ ID NOs: 1 to 14 or at least 15 base sequences contiguous in complementary base sequences thereto.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a primer for detecting the toxin B gene (tcdB) of Clostridioides difficile contained in a sample. The present invention further relates to a method for detecting tcdB contained in a sample using the primer, and a reagent / kit for use in the method. [Background technology]

[0002] Clostridioides difficile (also known as Clostridium difficile) (hereafter referred to as C. difficile) is one of the microorganisms that cause hospital-acquired infections, and simple, rapid, and highly sensitive detection of the bacteria is important for clinical diagnosis.

[0003] Specifically, C. difficile is known to be a major causative bacterium of diarrhea and enteritis associated with the administration of antibacterial drugs. For example, when normal intestinal flora is disturbed by antibacterial drug treatment, toxin-producing bacteria, which are toxic strains of C. difficile, produce toxins such as toxin A, toxin B, and binary toxin, which cause diarrhea, enteritis, and the like. Therefore, it is important to detect the toxins produced by this bacterium in the examination and diagnosis of C. difficile infection. Conventionally, toxin detection has been performed using an immunochromatography method, but this method has a problem of low sensitivity. Therefore, a method has been developed to detect genes involved in the production of these toxins by using a highly sensitive nucleic acid amplification method (Patent Document 1).

[0004] However, there is a need for a simpler and more sensitive method for detecting genes involved in toxin production by C. difficile, especially the toxin B gene (tcdB), which is generally considered to be more toxic than toxin A. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2015-529090 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made against the background of the problems of the conventional techniques. That is, an object of the present invention is to provide a method for detecting tcdB of C. difficile contained in a sample in a simple and highly sensitive manner. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors discovered that tcdB contained in a sample can be detected with high sensitivity by using specific primers, and arrived at the present invention. That is, the outline of the present invention is as follows.

[0008] [Item 1] A primer used to detect the toxin B gene (tcdB) of Clostridioides difficile that may be contained in a sample, the primer comprising a base sequence of at least 15 consecutive bases in any of the base sequences shown in 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, or SEQ ID NO:14, or a base sequence complementary thereto. [Item 2] The primer according to Item 1, which satisfies any one or more of the following (1) or (2): (1) A primer consisting of a nucleotide sequence shown in any one of 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, or SEQ ID NO:8, or a nucleotide sequence complementary thereto. (2) A primer consisting of a nucleotide sequence shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, or a nucleotide sequence complementary thereto. [Item 3] A primer set for detecting tcdB, comprising the primer described in (1) above as a forward primer and the primer described in (2) above as a reverse primer. [Item 4] A method for detecting tcdB, comprising a step of carrying out a nucleic acid amplification reaction using the primer according to Item 1 or 2, or the primer set according to Item 3. [Item 5] The method for detecting tcdB according to Item 4, wherein the step of carrying out the nucleic acid amplification reaction involves carrying out a PCR method, a LAMP method, an LCR method, a TMA method, an SDA method, or an RT-PCR method. [Item 6] The method for detecting tcdB described in Item 4 or 5, wherein a PCR method is performed in the step of carrying out the nucleic acid amplification reaction. [Item 7] A method for detecting tcdB according to any one of Items 4 to 6, comprising a step of detecting by a fluorescent probe method or a nucleic acid chromatography method. [Item 8] A method for detecting tcdB described in any one of Items 4 to 7, comprising a step of hybridizing and detecting a nucleic acid probe consisting of a base sequence shown in either SEQ ID NO: 15 or 16, or a base sequence complementary thereto. [Item 9] The method for detecting tcdB described in Item 8, wherein the nucleic acid probe is a nucleic acid probe in which at least one of the terminal cytosines is labeled with a fluorescent dye. [Item 10] A method for detecting tcdB described in any one of Items 4 to 9, which uses at least one DNA polymerase selected from the group consisting of Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. [Item 11] A probe used to detect the toxin B gene (tcdB) of Clostridioides difficile that may be contained in a sample, the tcdB detection probe consisting of a base sequence shown in either SEQ ID NO: 15 or SEQ ID NO: 16 or a base sequence complementary thereto. [Item 12] A probe for detecting tcdB according to Item 11, in which at least one of the terminal cytosines is labeled with a fluorescent dye. [Item 13] A kit used for detecting tcdB, comprising the primer according to Item 1 or 2 or the primer set according to Item 3. [Item 14] The kit according to Item 13, further comprising the tcdB detection probe according to Item 11 or 12. [Item 13] The kit described in Item 13 or 14, further comprising at least one DNA polymerase selected from the group consisting of Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. Effect of the Invention

[0009] According to the present invention, it is possible to easily and sensitively detect tcdB contained in a sample. By using the primer of the present invention, and the detection method, reagent, and kit using the primer, it is possible to easily and sensitively detect tcdB, which can greatly contribute to the field of clinical diagnosis. [Brief description of the drawings]

[0010] [Figure 1] 1 shows the results of Example 1. It shows the presence or absence of non-specific products when different nucleic acid primers were used. [Diagram 2] 1 shows the results of Example 2. FIG. 1 shows the detection results when the tcdB gene was used. [Diagram 3] 1 shows the results of Example 3. It shows the detection results of the tcdB gene when genomic DNA was used. [Figure 4] Fig. 1 shows the results of Example 4. Fig. 1 shows the results of evaluating the minimum detection sensitivity of the tcdB gene when genomic DNA was used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in further detail below with reference to embodiments of the present invention, but the present invention is not limited thereto.

[0012] [Primers for detecting Clostridioides difficile toxin B gene (tcdB)] One embodiment of the present invention is a primer used for detecting tcdB of C. difficile that may be contained in a sample, characterized in that it contains a base sequence of at least 15 consecutive bases in any of the base sequences shown in 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 or SEQ ID NO:14, or a base sequence complementary thereto.

[0013] [Clostridioides difficile toxin B gene (tcdB)] Clostridioides difficile is one of the microorganisms that cause hospital-acquired infections, and toxin-producing bacteria cause antibiotic-associated diarrhea and enteritis by producing toxins. The main toxins are toxin A and toxin B, but other toxins such as binary toxin can also be produced.

[0014] Conventionally, immunochromatography and the like have been used to detect these toxins, but they 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).

[0015] The genes involved in toxin production by C. difficile include tcdA, tcdB, tcdC mutation, cdtA, and cdtB, but it is particularly important to detect tcdB, which encodes toxin B, which is considered to be highly toxic.

[0016] The present inventors conducted extensive research and developed the primers of the present invention in order to detect tcdB more simply than with conventional techniques.

[0017] The nucleic acid primers of the present invention have the following characteristics: It contains a base sequence of at least 15 consecutive bases in any of the base sequences shown in 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 or SEQ ID NO:14, or a base sequence complementary thereto.

[0018] The primer having the above characteristics is not particularly limited as long as it consists of a base sequence of at least 15 or more consecutive bases in the base sequence shown in any one of 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, or SEQ ID NO:14, or a base sequence complementary thereto. However, a base sequence of at least 17 or more consecutive bases in the base sequence is preferred, a base sequence of at least 18 or more consecutive bases in the base sequence is more preferred, a base sequence of at least 19 or more consecutive bases in the base sequence is even more preferred, and a full-length sequence of the base sequence is particularly preferred.

[0019] In certain embodiments, a primer corresponding to any one or more of the following (1) or (2) is used as the tcdB primer: (1) A primer consisting of a nucleotide sequence shown in any one of 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, or SEQ ID NO:8, or a nucleotide sequence complementary thereto. (2) A primer consisting of a nucleotide sequence shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, or a nucleotide sequence complementary thereto. In a specific preferred embodiment, at least one primer consisting of a base sequence shown in any one of SEQ ID NOs: 1 to 8 described in (1) above is used, and at least one primer consisting of a base sequence shown in any one of SEQ ID NOs: 9 to 14 described in (2) above is used. In particular, it is preferable to use a primer set for detecting tcdB in which the primer described in (1) above is used as a forward primer and the primer described in (2) above is used as a reverse primer. In these cases, one type of primer may be selected and used for each of the primers described in (1) or (2) above, or two or more types of primers may be used in combination as the primer described in (1), or two or more types of primers may be used in combination as the primer described in (2).

[0020] Nucleic acid testing methods have been well known and have been established in the art. In such nucleic acid testing methods, it is important to design nucleic acid primers that specifically amplify target nucleic acids accurately in a short period of time.

[0021] In one embodiment, in the present invention, a primer (sometimes referred to as a "nucleic acid primer" in the present invention) or a primer set (sometimes referred to as a "nucleic acid primer pair" in the present specification) having the above characteristics is selected and used to carry out a nucleic acid amplification reaction. This allows the tcdB gene to be accurately and specifically amplified, enabling highly sensitive detection of tcdB.

[0022] [tcdB detection method] One embodiment of the present invention is a method for detecting the toxin B gene (tcdB) of Clostridioides difficile that may be contained in a sample, the method being characterized by carrying out the following steps (1) to (3) using a reaction solution containing at least any one of the primers or primer sets described above. (1) Providing a sample that may contain tcdB. (2) A step of carrying out a nucleic acid amplification reaction using the reaction solution. (3) A step of hybridizing a probe to the amplification product obtainable in the step (2) and measuring the fluorescence intensity of the reaction solution.

[0023] [sample] The sample that can be used in the present invention is not particularly limited as long as it may contain tcdB. Examples include biological samples, food, and environmental samples, as well as purified nucleic acids. The sample may be subjected to nucleic acid extraction or some pretreatment. Nucleic acid extraction and pretreatment of the sample are commonly performed in the art. Pretreatments include filtration, centrifugation, dilution, heat treatment, acid treatment, alkali treatment, organic solvent treatment, suspension treatment, crushing treatment, grinding treatment, etc., but are not limited to these in the present invention.

[0024] Examples of biological samples include, but are not limited to, animal and plant tissues, body fluids, excrement, cells, bacteria, viruses, etc. Further examples include blood, blood culture fluid, urine, pus, cerebrospinal fluid, pleural fluid, throat swabs, nasal swabs, sputum, tissue slices, skin, vomit, feces, isolated culture colonies, catheter washings, etc.

[0025] Examples of foods include water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, seafood, eggs, dairy products, raw meat, raw fish, prepared foods, etc. When food is used as a measurement sample, not only can a part or all of the food be used, but also the surface of the food can be wiped. Furthermore, materials used to wipe cooking utensils and doorknobs, or the cleaning fluid used to wash them, can also be used as samples.

[0026] Examples of environmental samples include water, ice, soil, air, aerosols, etc. Examples of water include tap water, seawater, and water collected from rivers, waterfalls, lakes, ponds, etc. In addition, wipes from walls, floors, equipment, fixtures, toilets, etc. of facilities, or cleaning fluids used to wash these can also be used as samples.

[0027] Any of the above-mentioned samples can be used in the present invention, but from the viewpoint that C. difficile is the main causative bacterium of diarrhea and enteritis, it is preferable to use a biological sample (e.g., animal or plant tissue, body fluid, excrement, tissue slice, skin, vomit, feces, or isolated culture colony), more preferably to use excrement, vomit, feces, or isolated culture colony, and even more preferably to use feces or isolated culture colony. According to the present invention, even when such a sample is used, the target sequence of tcdB can be specifically amplified, and tcdB can be detected with high sensitivity.

[0028] The method of collecting and preparing the sample is not particularly limited, and any known method can be used depending on the type and purpose of the sample.

[0029] [Nucleic acid amplification reaction] The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a level that allows visualization, i.e., hundreds of millions of copies or more, and is widely used not only in the field of life science research, but also in the fields of clinical diagnosis, food hygiene testing, environmental testing, etc. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, TMA, etc. These techniques have already been established in the technical field, and a method can be selected according to the purpose. From the viewpoint of more reliably obtaining the effects of the present invention, the nucleic acid amplification method used in the tcdB detection method of the present invention is preferably PCR, LAMP, LCR, TMA, SDA, or RT-PCR, and among them, PCR is preferable, but is not limited thereto.

[0030] [PCR] PCR is a reaction catalyzed mainly by DNA polymerase, and the target nucleic acid is amplified by repeating three steps, namely (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using DNA polymerase, as one cycle. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and their variants. In view of enabling simpler and more specific nucleic acid amplification, it is preferable to use a DNA polymerase belonging to family B in the present invention.

[0031] In this specification, the DNA polymerase mutant refers to a mutant having, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and especially preferably 99% or more sequence identity with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived, and having the activity of amplifying DNA similar to the wild-type DNA polymerase. Here, the method of calculating the identity of the amino acid sequence can be performed by any means known in the art. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). For example, it is possible to calculate the identity of the amino acid sequence by using the default (initial setting) parameters in the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) http: / / www.ncbi.nlm.nih.gov / BLAST / . Furthermore, the mutant that can be used in the present invention may be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted and / or added (hereinafter, these are also collectively referred to as "mutation") in the amino acid sequence of the wild-type DNA polymerase from which it is derived, and may have DNA amplifying activity similar to that of the wild-type DNA polymerase. Here, "one or several" may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, and even more preferably 1 to 5, but is not particularly limited thereto.

[0032] [Family B DNA polymerase] The DNA polymerase used in the present invention is preferably, but not limited to, a DNA polymerase belonging to Family B. The DNA polymerase belonging to Family B is not particularly limited, but is preferably a DNA polymerase derived from Archea.

[0033] [Archaemic DNA polymerase] Examples of DNA polymerases derived from archaea belonging to family B include DNA polymerases isolated from bacteria of the genus Pyrococcus and Thermococcus. The present invention also includes mutants of DNA polymerases derived from archaea belonging to family B that do not lose their activity. Examples of DNA polymerase mutants include, but are not limited to, mutants that are intended to enhance polymerase activity, to lack exonuclease activity, to adjust substrate specificity, and the like. Examples of DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as mutants derived therefrom that have not lost their DNA polymerase activity. Examples of DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9°N-7), and Thermococcus siculi, and mutants thereof that have not lost their DNA polymerase activity. PCR enzymes using these DNA polymerases are commercially available, and examples include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[0034] Among these, KOD DNA polymerase and its mutants (eg, KOD DNA polymerase lacking 3'→5' exonuclease activity) having excellent extensibility and thermostability are preferred.

[0035] KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and crude sample resistance compared to Taq DNA polymerase, which is a DNA polymerase belonging to family A. In the present invention, the use of such KOD DNA polymerase enables simple yet specific amplification of tcdB, and enables highly sensitive detection of tcdB, as shown in the Examples described later.

[0036] In the tcdB detection method of the present invention, a nucleic acid probe (preferably a nucleic acid probe in which at least one of the terminal cytosines is labeled with a fluorescent dye) designed to be capable of forming a complex with a part of the amplified product obtained by the nucleic acid amplification step performed in a reaction solution containing any of the above primers or primer sets is hybridized to the amplified product, and the fluorescence intensity of the reaction solution is measured to detect tcdB. In addition, tcdB may be detected by a nucleic acid chromatography method instead of the fluorescent probe method. The nucleic acid chromatography method can be performed by any method known in the art, and for example, the amplified product can be detected by hybridizing the amplified product obtained as described above with a labeled probe capable of forming a complex with a nucleic acid oligonucleotide immobilized on a membrane strip capable of forming a complex with the amplified product.

[0037] The conditions of the nucleic acid amplification process including any of the above-mentioned nucleic acid primers (e.g., temperature, pH, cation concentration, the presence of an organic solvent in the solution, etc.) may be optimized in accordance with the hybridization conditions of the nucleic acid probe, etc., and can be appropriately set by a person skilled in the art.

[0038] When the nucleic acid amplification step is carried out by PCR, the thermal cycle conditions are not particularly limited and can be appropriately set by those skilled in the art. As an example, it is preferable that the nucleic acid amplification reaction is carried out such that the first thermal deformation step is carried out at 80 to 100°C for 10 seconds to 15 minutes, the repeated thermal deformation steps are carried out at 80 to 100°C for 0.5 to 300 seconds, the annealing step is carried out at 40 to 80°C for 1 to 300 seconds, and the extension reaction step is carried out at 60 to 85°C for about 1 to 300 seconds, and this is repeated 30 to 70 times.

[0039] In one embodiment, an example of a method for detecting tcdB includes the following steps (1) to (5). (1) Prepare at least one forward primer specific to a region of the tcdB gene of Clostridioides difficile. (2) At least one reverse primer specific to a region of the tcdB gene of Clostridioides difficile is prepared. (3) A reaction solution containing a test nucleic acid and the set of nucleic acid primers is prepared, and the test nucleic acid is amplified by using the reaction solution. (4) A step of hybridizing the nucleic acid amplification product obtained in the step (3) with a nucleic acid probe designed to form a complex with a part of the nucleic acid amplification product to form a complex. (5) detecting the complex obtained in step (4).

[0040] Of the above primers described in (1) and (2), in order to suppress non-specific amplification and detect the tcdB gene with high sensitivity compared to conventional methods, it is desirable to use as the primer (1) a primer consisting of any one of the nucleotide sequences shown in 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, or SEQ ID NO: 8, or a nucleotide sequence complementary thereto, and it is desirable to use as the primer (2) a primer consisting of any one of the nucleotide sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a nucleotide sequence complementary thereto.

[0041] In a specific embodiment, in the above method, it is preferable to use a nucleic acid primer consisting of a base sequence of 20 to 26 consecutive bases in the base sequence shown in any one of SEQ ID NOs: 4 to 8 in (1) above, preferably SEQ ID NO: 8, as a forward primer, and to use a nucleic acid primer consisting of a base sequence of 20 to 26 consecutive bases in the base sequence shown in any one of SEQ ID NOs: 11 to 14 in (2) above, preferably SEQ ID NO: 13 and / or SEQ ID NO: 14, as a reverse primer.

[0042] The above nucleic acid primer pair is not particularly limited as long as it satisfies the above (1) and (2), but must be a combination of a sequence derived from the sense strand and a sequence derived from the antisense strand.

[0043] The amplification product obtained using the primer or primer set of the present invention can be detected by any analysis method, for example, electrophoresis, fluorescence melting curve analysis, various probe methods (Q probe, TaqMan probe, molecular beacon probe, FRET hybridization probe, Scorpion probe, etc.), intercalator method (for example, a method using a commercially available intercalator dye such as SYBRGREEN or EvaGreen), or any combination of these methods, etc. From the viewpoint of enabling more sensitive detection, it is preferable to detect by melting curve analysis using a Q probe.

[0044] The Q probe (also called the "guanine quenching probe") is a fluorescent probe (fluorescence quenching probe) developed by KURATA et al. (Patent Publication No. 5354216). This probe is a hybridization probe in which at least one terminal base is labeled with a fluorescence quenching dye that quenches by interaction with guanine.

[0045] For example, the fluorescent quenching dye used in the Q probe is not particularly limited, but may be fluorescein or a derivative thereof (e.g., fluorescein isothiocyanate), rhodamine or a derivative thereof (e.g., tetramethylrhodamine, tetramethylrhodamine isothiocyanate, carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), BODIPY or a derivative thereof (e.g., 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), etc. Details of the fluorescent quenching dye are described in Japanese Patent No. 5813263, etc., and the present invention may also refer to this technology.

[0046] In the present invention, a probe having a base sequence in which at least one terminal base is cytosine and the terminal base cytosine is labeled with a fluorescent quenching dye is preferred. When such a probe hybridizes to an amplification product, it can be quenched by forming a base pair with a guanine base in the amplification product and interacting with it, so that the change in the fluorescence intensity of the reaction solution can be measured very easily.

[0047] In addition, when the probe is hybridized, even if the cytosine base of the probe and the guanine base in the amplified product do not form a base pair, the fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. That is, when the probe is hybridized, the fluorescence can be quenched if the guanine base in the amplified product is present within a range of, for example, 1 to 3 bases relative to the cytosine base of the probe (a base that forms a base pair with the cytosine base is considered to be 1).

[0048] When the tcdB detection method of the present invention uses a nucleic acid probe, the base sequence of the nucleic acid probe is not particularly limited as long as it can form a complex with a part of the amplification product obtained when the primer or primer set of the present invention is used. In a specific preferred embodiment, the probe is preferably a probe containing a base sequence of at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 15 or 16 or a base sequence complementary thereto, and is preferably a probe containing a base sequence of at least 17 consecutive bases in the base sequence. A particularly preferred probe is a probe consisting of the base sequence shown in SEQ ID NO: 15 or 16 or a base sequence complementary thereto. By using such a probe, it is possible to detect tcdB with higher sensitivity.

[0049] [Reagents for detecting tcdB] Another embodiment of the present invention includes a reagent for detecting tcdB that may be contained in a sample. The reagent contains at least components necessary for nucleic acid amplification in addition to the nucleic acid primer having the above-mentioned characteristics. The necessary components differ depending on the nucleic acid amplification reaction to be performed, and a known method can be used for each. For example, when detecting tcdB contained in a sample using a PCR reaction, it is preferable to 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, but for example, the oligonucleotide probe is preferably 0.1 to 1 μM, more preferably 0.2 to 0.5 μM. The DNA polymerase is preferably 0.01 to 1 U / uL, more preferably 0.1 to 0.5 U / uL. The oligonucleotide primers are different, but are preferably 0.1 to 10 μM. The deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The magnesium salt is preferably at 0.1 to 6 mM, more preferably at 1 to 5 mM.

[0050] Furthermore, additives known in the art may be added for the purpose of suppressing non-specific amplification or promoting the reaction. Examples of additives for suppressing non-specific amplification include anti-DNA polymerase antibodies and phosphoric acid. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, 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), tetramethylammonium acetate (TMAA), polyethylene glycol, Triton, Tween 20, and Nonidet P40. In the present invention, one or more of these additives may be used in combination, but is not limited to these.

[0051] For example, a nucleic acid amplification product amplified with a set of nucleic acid primers corresponding to (1) and (2) above is hybridized with a nucleic acid probe designed to form a complex with a part of the nucleic acid amplification product to form a complex.

[0052] The timing of adding the nucleic acid probe to a sample containing a nucleic acid amplification product is not particularly limited, and the nucleic acid probe may be added to the reaction system of the amplification reaction, for example, before, during, or after the aforementioned nucleic acid amplification reaction. In particular, since the amplification reaction and the detection reaction can be carried out consecutively, it is preferable to add the nucleic acid probe before the amplification reaction. When the nucleic acid probe is added before the nucleic acid amplification reaction in this manner, it is preferable to add, for example, a fluorescent dye or a phosphate group to the 3' end of the nucleic acid probe.

[0053] The nucleic acid probe may be added to a liquid sample containing the nucleic acid amplification product, or may be mixed with the nucleic acid amplification product in a solvent. The solvent is not particularly limited, and examples of the solvent include conventionally known solvents such as a buffer solution such as Tris-HCl, a solvent containing KCl, MgCl2, MgSO4, glycerol, etc., and a PCR reaction solution.

[0054] [Kit for detecting tcdB] Further, as another embodiment of the present invention, there is a kit containing a reagent for detecting tcdB that may be contained in a sample. The configuration of the kit is not particularly limited as long as it contains the tcdB detection reagent containing the primer or primer set and is configured to detect tcdB. The kit may further contain a probe (preferably a probe in which at least one of the terminal cytosines is labeled with a fluorescent dye) capable of forming a complex with a part of the amplification product obtained using the primer or primer set. Furthermore, in order to perform efficient and highly specific nucleic acid amplification, the kit preferably further contains at least one DNA polymerase selected from the group consisting of Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. EXAMPLES

[0055] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0056] Example 1: Confirmation of non-specific amplification in tcdB gene amplification (1-1) Method In order to design nucleic acid primers capable of detecting tcdB with high sensitivity while suppressing non-specific amplification, the presence or absence of non-specific amplification was confirmed using a total of 10 nucleic acid primer pairs without adding a template. The combinations of nucleic acid primer pairs used in this test are shown in Table 1. Here, each primer was prepared by synthesizing nucleic acids consisting of the base sequences shown in SEQ ID NOs: 1 to 14 and 17 to 24 according to a conventional method. [Table 1]

[0057] (1-2) Reaction solution A reaction solution containing the components shown below was prepared using SYBR qPCR Mix (Toyobo Co., Ltd.) The reaction solution was prepared according to the instruction manual for SYBR qPCR Mix (Toyobo Co., Ltd.). 1.5μM forward primer(*) 0.5μM reverse primer(*) (*) When two types of forward primers (or reverse primers) were used, the amount of each forward primer (or reverse primer) was halved so that the total amount was the specified amount above. (1-3) Reaction The reaction solution was reacted in the following temperature cycles using Rotor-Gene(TM) Q, and the fluorescence intensity in each cycle was measured. 94℃ 30 seconds, 98℃ 1 sec - 52℃ 10 sec - 63℃ 10 sec (50 cycles) (1-4) Results Figure 1 shows the fluorescence intensity obtained by the measurement plotted against the number of cycles. Even though no template was added, an increase in fluorescence intensity due to non-specific amplification was observed as the number of cycles increased depending on the combination of nucleic acid primers. On the other hand, it was shown that no non-specific amplification was observed in combinations No. 1 to No. 5.

[0058] Example 2: Confirmation of tcdB gene amplification (2-1) Method In order to design nucleic acid primers capable of detecting tcdB with high sensitivity while suppressing non-specific amplification, 10,000 copies of the tcdB gene were added as a template per reaction, and tcdB gene amplification was confirmed using a total of five nucleic acid primer pairs. The combinations of nucleic acid primer pairs are shown in Table 2. Here, each primer was prepared by synthesizing nucleic acids consisting of the base sequences shown in SEQ ID NOs: 1 to 14 according to a conventional method. [Table 2]

[0059] (2-2) Reaction solution A reaction solution containing the components shown below was prepared using SYBR qPCR Mix (Toyobo Co., Ltd.) The reaction solution was prepared according to the instruction manual for SYBR qPCR Mix (Toyobo Co., Ltd.). 1.0μM forward primer(*) 1.0μM reverse primer(*) (*) When two types of forward primers (or reverse primers) were used, the amount of each forward primer (or reverse primer) was halved so that the total amount was the specified amount above. (2-3) Reaction The reaction solution was reacted in the following temperature cycles using Rotor-Gene(TM) Q, and the fluorescence intensity in each cycle was measured. 98℃ 120 seconds, 98℃ 10 seconds - 50℃ 10 seconds - 68℃ 30 seconds (40 cycles) (2-4) Results Figure 2 is a plot of the fluorescence intensity obtained by the measurement against the cycle number. Amplification of the tcdB gene was confirmed with all nucleic acid primer pairs.

[0060] Example 3: Detection of tcdB using genomic DNA (3-1) Method The tcdB gene was detected using five nucleic acid primer pairs shown in Table 2 of Example 2 above (however, in nucleic acid primer pair combination No. 5, only the nucleic acid primer consisting of the base sequence of SEQ ID NO: 14 was used as the reverse primer) and a nucleic acid probe labeled with BODIPY-FL at the 3' end and designed to form a complex with the amplified product. The genomic DNA used for the measurement was Clostridioides difficile genomic DNA (ATCC 9689D-5), and 8,000 copies / test were measured. (3-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). 3μM forward primer 1.2 μM reverse primer (0.6 μM nucleic acid primer shown in SEQ ID NO: 14 (combination No. 5 only)) 0.4 μM Nucleic acid probe consisting of the base sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16 (3' end is labeled with BODIPY-FL) (3-3) Reaction The reaction solution was reacted in the following temperature cycle using GENECUBE (registered trademark) to carry out nucleic acid amplification. After the nucleic acid amplification reaction, a melting curve analysis was carried out under the following conditions. 97℃ 15 seconds, 97℃ 1 sec -54℃ 5 sec -63℃ 2 sec (60 cycles) 94℃ 30 seconds 39℃ 30 seconds 40-75℃ 0.09℃ / sec (3-4) Results The results of the melting curve analysis are shown in Figure 3. Figure 3 shows that tcdB contained in a sample can be detected by melting curve analysis when any of the nucleic acid primer pair combinations No. 1 to 5 is used. In particular, when the nucleic acid primer pair combinations No. 3 to 5 are used, a high peak is observed, indicating that detection is possible with particularly high sensitivity.

[0061] Example 4: Confirmation of minimum detection sensitivity using genomic DNA (4-1) Method The tcdB gene was detected using the nucleic acid primer pair of combination No. 5 and a nucleic acid probe labeled at the 3' end with BODIPY-FL, which was designed to form a complex with the amplification product. The genomic DNA used for the measurement was Clostridioides difficile genomic DNA (ATCC 9689D-5), and 3 copies / test and 1 copy / test were measured. (4-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). 3 μM nucleic acid primer represented by SEQ ID NO:8 1.2 μM nucleic acid primer represented by SEQ ID NO: 13 0.6 μM nucleic acid primer represented by SEQ ID NO:14 0.4 μM of the nucleic acid probe shown in SEQ ID NO: 15 (labeled with BODIPY-FL at the 3' end) (4-3) Reaction The reaction solution was reacted in the following temperature cycle using GENECUBE (registered trademark) to carry out nucleic acid amplification. After the nucleic acid amplification reaction, a melting curve analysis was carried out under the following conditions. 97℃ 15 seconds, 97℃ 1 sec -54℃ 5 sec -63℃ 2 sec (60 cycles) 94℃ 30 seconds 39℃ 30 seconds 40-75℃ 0.09℃ / sec (4-4) Results The results of the melting curve analysis are shown in Figure 4. Figure 4 shows that tcdB contained in a sample can be detected with high sensitivity by melting curve analysis using the nucleic acid primer set of the present invention. This test result shows that, although detection is possible even with 1 copy / test by using the nucleic acid primer pair of the present invention, the minimum detection sensitivity that enables more accurate measurement is 3 copies / test. [Industrial Applicability]

[0062] By using a method, reagent, or kit using the primer or primer set described in the present invention, it has become possible to detect tcdB contained in a sample simply and with high sensitivity. Therefore, the present invention is not only useful for research purposes, but also makes it possible to quickly, simply, and highly sensitively detect tcdB, which is the toxin gene of the causative bacterium, in cases where there is a risk of hospital infection, etc., and can greatly contribute to clinical diagnosis, environmental testing, etc.

Claims

1. A primer set for detecting tcdB, which is used for detecting the toxin B gene (tcdB) of Clostridioides difficile that may be contained in a sample, comprising a primer described in (1) below as a forward primer and a primer described in (2) below as a reverse primer. (1) A primer consisting of the base sequence shown in SEQ ID NO:

8. (2) A primer consisting of the base sequence shown in SEQ ID NO: 13 or SEQ ID NO:

14.

2. A method for detecting tcdB, comprising a step of performing a nucleic acid amplification reaction using the primer set described in claim 1.

3. The method for detecting tcdB according to claim 2, wherein the step of carrying out the nucleic acid amplification reaction comprises carrying out a PCR method, a LAMP method, an LCR method, a TMA method, an SDA method, or an RT-PCR method.

4. The method for detecting tcdB according to claim 2 or 3, wherein a PCR method is carried out in the step of carrying out the nucleic acid amplification reaction.

5. The method for detecting tcdB according to any one of claims 2 to 4, comprising a step of detection by a fluorescent probe method or a nucleic acid chromatography method.

6. 6. A method for detecting tcdB according to any one of claims 2 to 5, comprising a step of hybridizing a nucleic acid probe consisting of a base sequence shown in any one of SEQ ID NOs: 15 and 16, or a base sequence complementary thereto, to detect tcdB.

7. The method for detecting tcdB according to claim 6 , wherein the nucleic acid probe is a nucleic acid probe in which at least one of the terminal cytosines is labeled with a fluorescent dye.

8. The method for detecting tcdB according to any one of claims 2 to 7, wherein at least one DNA polymerase selected from the group consisting of Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, and DEEPVENT is used.

9. A kit used for detecting tcdB, comprising the primer set described in claim 1.

10. The kit described in claim 9, further comprising a nucleic acid probe consisting of a base sequence shown in either SEQ ID NO: 15 or 16 or a base sequence complementary thereto.

11. The kit of claim 9 or 10, further comprising at least one DNA polymerase selected from the group consisting of Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, and DEEPVENT.

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