JP5593582B2 - Rapid detection method of nucleic acid - Google Patents

Description

    The present invention relates to methods for rapidly detecting specific target nucleic acid molecules present in a sample, and probes and kits utilized by these methods.

In general, the amount of nucleic acid held by an organism is extremely small, and most of them involve a nucleic acid amplification step when they are detected. As known nucleic acid amplification methods, PCR (Patent Literature 1, Patent Literature 2, Patent Literature 3), NASBA (Non Patent Literature 1), LCR (Patent Literature 4, Patent Literature 5), SDA (Non Patent Literature 2) , RCR (patent document 6), TMA (non-patent document 3) LAMP (non-patent document 4), ICAN (non-patent document 5), and the like.

  In particular, the PCR method uses the above-mentioned pair of oligonucleotide primers by repeatedly increasing and decreasing the temperature in the presence of the target nucleic acid, four types of deoxynucleoside triphosphates, a pair of oligonucleotide primers and a heat-resistant DNA polymerase. A specific region of the target nucleic acid to be sandwiched can be amplified exponentially.

Usually, as shown in Patent Document 3, the PCR method uses an amplification cycle comprising the following a) to c).
a) a step of denaturing at a temperature in the range of 90 to 105 ° C. (preferably 90 to 100 ° C.) for 0.5 to 5 minutes (preferably 0.5 to 3 minutes); b) in the range of 35 to 65 ° C. At a temperature of (preferably 37 to 60 ° C.) for 0.5 to 5 minutes (preferably 1 to 3 minutes) to form a primer-template hybrid (annealing), and c) within the range of 40 to 80 ° C. A step of forming a primer extension product (extension) at temperature (preferably 50 to 75 ° C.) for 0.5 to 40 minutes (preferably 1 to 3 minutes).

  Replication of the target nucleic acid amplified by repeating the cycle of the PCR method can be detected using various detection methods. A typical detection method currently used is agarose gel electrophoresis. However, the detection by electrophoresis has to deal with exponentially amplified replication of the target nucleic acid and has a high risk of contamination. In addition, the operation is complicated, and the time until the end of the detection is one hour or more.

  As a detection method other than electrophoresis, a method using a DNA insertion dye (intercalator) that exhibits enhanced fluorescence when bound to a double-stranded nucleic acid is generally used. Fluorescence enhancement by increasing DNA concentration during amplification can be used to measure the progress of the reaction and to determine the copy number of the target molecule. Furthermore, by monitoring the fluorescence accompanying a controlled temperature change, a DNA melting curve can be created, for example, at the end of the PCR cycle reaction.

When a general nucleic acid detection method is used to monitor the increase in the nucleic acid concentration, the above-described method using an intercalator can detect a nucleic acid in a relatively short time. At the same point in each reaction, a single fluorescent signal value is acquired. Melting curve analysis at the final time point can identify to some extent the nature of the double-stranded nucleic acid. When the melting temperature in the melting curve analysis is extremely low, it is considered to be a primer dimer, and when there is not one main peak, it indicates that there is a non-specific amplification product that is not the target PCR product.

However, the general intercalator method generates fluorescence specifically for double-stranded nucleic acids. Therefore, when detection of a specific nucleic acid sequence is required, there is a possibility of detecting a non-specific double-stranded nucleic acid, which is not so effective.

Nucleic acid sequence specific probing methods utilize additional nucleic acid reaction components to monitor the progress of the amplification reaction. These methods often utilize fluorescence energy transfer (FET) as the basis for detection. One or more nucleic acid probes are labeled with a fluorescent molecule, one of which can act as an energy donor and the other is an energy acceptor molecule. These are sometimes known as reporter molecules and quencher molecules, respectively. The donor molecule is excited at a specific wavelength of light in the excitation spectrum range and subsequently emits light in the range of fluorescence emission wavelengths. The acceptor molecule is also excited at this wavelength by receiving energy from the donor molecule by various distance-dependent energy transfer mechanisms. A particular example of fluorescence energy transfer that can occur is fluorescence resonance energy transfer (FRET). In general, when an acceptor molecule and a donor molecule are in close proximity (eg, on the same or adjacent molecules), the acceptor molecule receives the emitted energy of the donor molecule. The basis of FET detection is to monitor changes in donor and acceptor emission wavelengths.

There are two commonly used types of FET or FRET probes, one that uses nucleic acid probe hydrolysis to separate the donor from the acceptor, and one that changes the spatial relationship between the donor and acceptor molecules. Hybridization is used.

Hydrolysis probes are commercially available as TaqMan® probes. These consist of DNA oligonucleotides labeled with donor and acceptor molecules. This probe is designed to bind to a specific region of one strand of the PCR amplification product. After the PCR primer anneals to this strand, the Taq enzyme extends the DNA by polymerase activity from 5 'to 3'. The TaqMan® probe is phosphorylated at the 3 'end to prevent initiation of Taq extension. If the TaqMan® probe is hybridized to the product strand, the extending Taq molecule hydrolyzes the probe, releasing the donor from the acceptor as a basis for detection. The signal in this case is cumulative and the concentration of free donor and acceptor molecules increases with each cycle of the amplification reaction.

The fact that the generation of a fluorescent signal is dependent on the occurrence of a probe hydrolysis reaction means that there is a time penalty associated with this method. In addition, the presence of the probe could interfere with the primer extension process in the PCR process. It has also been found that hydrolysis can be non-specific when multiple amplification cycles, such as 50 cycles or more, are required.

Many types of hybridization probes are available. Molecular beacons are oligonucleotides with complementary 5 'and 3' sequences that form hairpin loops. The terminal fluorescent label is in close proximity to FRET due to the formation of the hairpin structure. Following hybridization of the molecular beacon to the complementary sequence, the fluorescent label is separated, so no FRET occurs, which is the basis for detection.

Labeled probe pairing is also available. FRET can be generated by close hybridization of the probe labeled donor molecule and the probe labeled acceptor molecule on the strand of the PCR product. The fluorescence of the wavelength generated by FRET at this time is the basis of detection. This type of variant involves the use of a labeled amplification primer and a single adjacent labeled probe.

The use of a molecular beacon type that includes two probes or two labeled molecules increases the costs associated with the process. In addition, this method requires the existence of a reasonably long known sequence in order to know two probes that are long enough to bind specifically in close proximity. This is a problem in certain diagnostic applications, and it may be difficult to design an effective probe when the length of the conserved sequence is relatively short, such as for example, HIV virus.

In Patent Document 7, a nucleic acid probe containing a DNA double-strand binding agent and a reactive molecule that can absorb or give fluorescence energy from the DNA double-strand binding agent is used to carry out amplification reaction of a target nucleic acid. After that, a detection method for monitoring fluorescence is clearly shown. However, the description in Patent Document 7 cannot easily achieve rapid and highly sensitive detection of nucleic acids. In other words, the DNA double-strand binding agent (that is, the intercalating dye) and the nucleic acid probe inhibit the nucleic acid amplification process, particularly the extension process, and thus there is a problem that the sensitivity is remarkably lowered when trying to shorten the detection method. .
US Pat. No. 4,683,195 U.S. Pat.No. 4,683,202 US Pat. No. 4,965,188 International Publication No.89 / 12696 JP-A-2-2934 International Publication No. 90/1069 Special table 2003-500001 Nucleic acid sequence-based amplification method; Nature Vol. 350, 91 (1991) Strand Displacement Amplification: Nucleic acid research Volume 20, 1691 (1992) Transcription mediated amplification method; J. Clin. Microbiol. Vol. 31, p. 3270 (1993) loop-mediated isothermal amplification method: J Clin Microbiol. 2004 Volume 42: 1,956 isothermal and chimeric primer-initiated amplification of nucleic acids: Kekkaku. 2003 78, 533

  As described above, in the method using FRET, a DNA double-strand binding agent (sometimes referred to as “insertion dye” or “intercalator” in this specification) and a nucleic acid probe are used in a nucleic acid amplification step, particularly an extension step. In order to inhibit this, there is a problem that the sensitivity is remarkably reduced when the detection method is shortened. An object of the present invention is to overcome these problems and to detect a target nucleic acid more quickly and with higher sensitivity.

  In view of the above points, the present inventors have conducted extensive studies, and as a result, the relationship between the Tm value of the first primer and the second primer and the Tm value of the labeled probe, and the relationship between the concentration of the primer and the concentration of the labeled probe. The inventors have found that the target nucleic acid can be detected more rapidly and more sensitively by adjusting and optimizing, and the present invention has been achieved. That is, the present invention has the following configuration.

1. In the presence of at least the following (a) to (c) and the sample to be confirmed for the presence or amount of the target nucleic acid, light of the excitation wavelength of the intercalator is given continuously or intermittently, and the fluorescence from the labeled probe is measured, A detection method comprising performing high-speed PCR and examining the presence or amount of the target nucleic acid.
(A) Intercalator (b) Labeled probe having the following properties (i) to (iii)
(i) It can form a double-stranded nucleic acid structure with a sequence complementary to the target nucleic acid, and has an oligonucleotide having a length of 14 to 30 bases
(ii) The oligonucleotide is labeled with one or more fluorescent substances capable of absorbing fluorescence energy from the intercalator and emitting fluorescence itself.
(iii) The concentration of the labeled probe is 3 to 15 times the concentration of the second primer of (c) (c) the first primer having the following properties (i) to (iii) and Second primer
(i) The extension product of the first primer has a property complementary to the target nucleic acid and serves as a template for the second primer, and the extension product of the second primer has a sequence homologous to the target nucleic acid. And has the property of becoming a template for the first primer
(ii) The length of the amplification product of the first primer and the second primer is 50 bp or more and 500 bp or less
(iii) The Tm value of the first primer and the second primer is 70 ° C. or more and 90 ° C. or less, and the Tm value of the second primer is larger than the Tm value of the labeled probe of (b). 1. The detection method according to 1, wherein the concentration of the first primer is 4 times or more the concentration of the second primer.
3. The detection method according to 1 or 2, wherein the high-speed PCR repeats the temperature increase and decrease at a rate of 10 ° C./second or more.
4). further,
(D) in the presence of a DNA polymerase having a DNA synthesis rate of 100 bases / second or more;
The detection method according to any one of 1 to 3, wherein high-speed PCR is performed.
5. 4. The detection method according to 4, wherein the DNA polymerase further does not have 5 ′ exonuclease activity.
6). 6. The detection method according to 4 or 5, wherein the DNA polymerase is KOD DNA polymerase (from Thermococcus kodakaraensis KOD1) or a mutant thereof.
7). (C) the first primer and the second primer further
(iv) In the extension product of the first primer, the distance from the 5 ′ end of the extension product to the region where the labeled probe forms a double-stranded nucleic acid structure is (A), and the labeled probe is a double-stranded nucleic acid structure. When the distance from the region forming the 3 ′ end of the extension product is (B), the value of (A / B) is 0.1 or more and 3.0 or less,
Any one of the detection methods of 1-6 characterized by these.
8). The labeled probe of (b) is further
(iv) have no more than 3 non-complementary bases with the target site;
Any one of the detection methods of 1-7 characterized by these.
9. 8. The detection method according to 8, wherein a non-complementary base is located in the center of the labeled probe.
10. The intercalator is SYBR Green I ™, LC Green I ™, LC Green Plus ™, or [2- [N-[(3-dimethylaminopropyl) -N-propylamino] -4- [2,3 -dihydro-3-methyl- (benzo-1,3-thiazol-2-yl) -methylidene] -1-phenyl-qunolinium], and the fluorescent substance of the labeled probe is Texas Red (trademark). The detection method according to any one of 1 to 9, wherein:
11. The target nucleic acid is the sense strand of the 23S rRNA gene of H. pylori,
The oligonucleotide sequence of the labeled probe of (b) is a continuous base sequence of 14 to 30 bases selected from SEQ ID NO: 2,
The first primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
The second primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
1) characterized in that the presence or amount of the 23S rRNA gene of H. pylori is examined by performing high-speed PCR while continuously or intermittently applying light of the excitation wavelength of the intercalator and measuring fluorescence from the labeled probe. Any one of 10 detection methods.
12 The target nucleic acid is the antisense strand of the 23S rRNA gene of H. pylori,
The oligonucleotide sequence of the labeled probe of (a) is a continuous base sequence of 14 to 30 bases selected from SEQ ID NO: 1,
The first primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The second primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
1) characterized in that the presence or amount of the 23S rRNA gene of H. pylori is examined by performing high-speed PCR while continuously or intermittently applying light of the excitation wavelength of the intercalator and measuring fluorescence from the labeled probe. Any one of 10 detection methods.
Subsequent to any of the methods of 13.1 to 12, in a sealed state, while applying light at the excitation wavelength of the intercalator and measuring the fluorescence from the labeled probe, continuously at 0.1 ° C./second or more A detection method characterized by conducting a melting curve analysis accompanied by a temperature rise or fall and examining the presence of a mutation in a target nucleic acid.
14 13. The detection method according to 13, wherein the mutation is examined for the presence of a mutation at position 2142 or position 2143 of the 23S rRNA gene that discriminates antibiotic resistance of Helicobacter pylori.
15. The detection method according to 13 or 14, wherein the mutation site is located in the center of the labeled probe.
16. A kit for detecting the presence of H. pylori and / or the presence of antibiotic resistance of H. pylori, which comprises at least the following (a) to (c):
(A) Intercalator (b) having an oligonucleotide having a base sequence of 14 to 30 bases selected from SEQ ID NO: 1 or SEQ ID NO: 2, and the oligonucleotide absorbs fluorescence energy from the intercalator and itself A labeled probe (c) for detecting the presence of H. pylori labeled with one or more fluorescent substances capable of emitting fluorescence and / or the presence of antibiotic resistance of H. pylori (c) 20 bases or more and 35 bases or less 18. A first primer containing a continuous base sequence selected from SEQ ID NO: 3 and a second primer containing a continuous base sequence selected from SEQ ID NO: 4 and having a base sequence of 20 to 35 bases 16 kits, wherein the concentration of the primer on the side that produces an extension product capable of forming a double-stranded nucleic acid structure with the labeled probe of (b) is at least 4 times the concentration of the other primer.
18. further,
(D) A kit of 16 or 17, comprising a DNA polymerase having a DNA synthesis rate of 100 bases / second or more.
19. 18 kits characterized in that the DNA polymerase further has no 5 ′ exonuclease activity.
20. 18. The kit according to 18 or 19, wherein the DNA polymerase is KOD DNA polymerase (from Thermococcus kodakaraensis KOD1) or a variant thereof.
21. The intercalator is SYBR Green I ™, LC Green I ™, LC Green Plus ™, or [2- [N-[(3-dimethylaminopropyl) -N-propylamino] -4- [2,3 -dihydro-3-methyl- (benzo-1,3-thiazol-2-yl) -methylidene] -1-phenyl-qunolinium], and the fluorescent substance of the labeled probe is Texas Red (trademark). The kit according to any one of 16 to 20, which is characterized by being.
22. The kit of 16-21, wherein a site corresponding to position 2142 or position 2143 of the 23S rRNA gene of H. pylori is located in the center of the labeled probe.
23. Existence of Helicobacter pylori labeled with two fluorescent substances and / or presence of antibiotic resistance of Helicobacter pylori having an oligonucleotide having a length of 14 to 30 bases selected from SEQ ID NO: 1 or SEQ ID NO: 2 Labeled probe for detecting.
24. 23 labeled probes, wherein the fluorescent substance is Texas Red (trademark).

According to the present invention, rapid and highly sensitive nucleic acid detection is possible. Specifically, a fluorescent signal is generated by hybridization of a labeled probe to a target nucleic acid or first primer extension product, and nucleic acid is amplified by extension of a second primer using the target nucleic acid or first primer extension product as a template. Can be achieved during the same cycle of PCR. Furthermore, the use of DNA polymerase, which has a high extension rate, shortened the amplification reaction time and realized a nucleic acid detection method that was faster and more sensitive than ever.
Furthermore, using the present invention, the presence or amount of the 23S rRNA gene of H. pylori is examined, the presence of a mutation in the 23S rRNA gene of H. pylori, the mutation at position 2142 or 2143 of the 23S rRNA gene The presence of H. pylori can be determined quickly and with high sensitivity.

  In the present invention, high-speed PCR is a nucleic acid amplification step, that is, a series of steps consisting of “denaturation”, “annealing”, “extension” as described later, or “denaturation” and “annealing” by performing annealing and extension simultaneously in this step. By shortening the time required for a series of steps called “and extension”, there is an advantage of high speed. FRET also has the advantage of high sensitivity and accuracy because it can clearly distinguish a specific amplified nucleic acid reflecting the presence or amount of the target nucleic acid from a non-specific amplified nucleic acid that does not depend on the target nucleic acid. Yes. However, even if these are simply combined, nucleic acid detection cannot be performed quickly and with high sensitivity. This is because in high-speed PCR, the target nucleic acid is amplified by annealing the primer to the target nucleic acid, extending the primer using the target nucleic acid as a template (extension reaction), and dissociating the target nucleic acid and the primer extension product (denaturation reaction). In FRET, a fluorescent signal is generated by hybridization of the labeled probe to the target nucleic acid. Therefore, nucleic acid amplification starting from primer annealing to the target nucleic acid and target nucleic acid This is because it is difficult to generate a fluorescent signal due to hybridization of the labeled probe in the same cycle of PCR. That is, even if high-speed PCR and FRET are simply combined, competition for the target nucleic acid occurs between the annealing of the primer for nucleic acid amplification and the hybridization of the labeled probe for generating a fluorescent signal, and the target nucleic acid is amplified. However, no fluorescent signal is generated, or a fluorescent signal is generated but the target nucleic acid is not amplified, both of which quickly and sensitively determine the presence or amount of the target nucleic acid and the presence of the mutation in the target nucleic acid. I can't. That is, in the present invention, various conditions are examined, and both the advantage of high speed of high-speed PCR and the advantage of high sensitivity and accuracy of FRET are compatible, and the presence or amount of the target nucleic acid and the mutation in the target nucleic acid It has established a nucleic acid detection method for quickly and highly sensitively detecting the presence.

  The target nucleic acid of the present invention refers to either the base sequence of a nucleic acid held by an organism (including bacteria) or a complementary base sequence thereof.

  The high-speed PCR in the present invention is shown below. To speed up the PCR method, it is possible to shorten the time required to change the temperature to reach each temperature of `` denaturation '', `` annealing '', and `` extension '', shorten the time to maintain the temperature of each process, reduce the number of cycles, etc. It is done. Particularly important are shortening the time required for temperature change and shortening the temperature holding time of each process. The shortening of the time required for the temperature change specifically indicates that the temperature is increased and decreased at a rate of 10 ° C./second or more. There is no particular limitation on the means for achieving a temperature increase / decrease of 10 ° C./second or more, but an example is the Light Cycler (registered trademark) system manufactured by Roche Diagnostics. In this system, the temperature is controlled by using air to increase and decrease the temperature, and the temperature of the glass capillary containing the reaction solution is directly changed by hot and cold air. As a result, a temperature increase / decrease rate of about 20 ° C./second is achieved.

  Another means for achieving high-speed nucleic acid amplification is to reduce the time required for the extension step. There are several ways to shorten the elongation process time. The most effective strategy is to minimize the amplification region by PCR. Then, naturally, the time required for expansion can be shortened. Even in such a case, the minimum amplification region including the first primer and the second primer is about 60 base pairs. If a labeled probe is set for the purpose of detection, it is necessary to secure that portion, and amplification of about 100 base pairs is required. The length of the amplified product is preferably a short product of 50 bp to 500 bp, and preferably 50 bp to 300 bp for high speed amplification. More preferably 80 bp to 200 bp.

  Furthermore, the length of this amplification product is limited by the extension rate of the DNA polymerase. Taq polymerase as a currently known DNA polymerase (single or in combination), EX-Taq, LA-Taq, Expand series, Platinum series, Tbr, Tfl, Tru, Tth, T1i, Tac, Tne, Tma, Tih , Tfi (above PolI type enzyme), Pfu, Pfutubo, Pyrobest (registered trademark), Pwo, KOD, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT, DEEPVENT (above are α type enzymes) Among them, KOD is mentioned as an enzyme having a high elongation rate. KOD DNA polymerase has a DNA synthesis rate of 100 to 140 bases / second or more, which is twice the most common Taq polymerase. Moreover, it is thought that it is one of the factors with a high expansion | extension speed | velocity that processability etc. are also excellent. As the KOD DNA polymerase, for example, those manufactured by Toyobo (product code KOD-101, etc.) can be easily obtained. The amino acid sequence of the natural polymerase may be one (mutant) in which one or several amino acid sequences are deleted, substituted or added by known means. Alternatively, the enzyme (natural type, mutant) may be further modified by means such as chemical modification.

  In recent years, the above-mentioned DNA polymerase has been further improved by mutation, modification, etc. to achieve a deoxyribonucleic acid synthesis rate of 100 bases / second or more, or a combination that has achieved this performance (the combination includes KOD DNA) Can also be used as the DNA polymerase of the present invention.

    It is further preferred that the DNA polymerase does not have 5 'exonuclease activity. In the present invention, fluorescence from the labeled probe is used for detection. Therefore, when the labeled probe and the second primer are hybridized to the extension product of the first primer in the high-speed PCR process, the labeled probe is not used in a DNA polymerase having 5 ′ exonuclease activity, such as Taq polymerase. Decomposes with a decrease in detection signal. A DNA polymerase that does not have 5 'exonuclease activity, such as, but not limited to, KOD polymerase is compatible with the present invention.

  The insertion dye (intercalator) may be any substance that specifically binds to a double-stranded nucleic acid and emits fluorescence, such as SYBR Green I (trademark), LC Green I (trademark), LC Green Plus (trademark), ethidium bromide, Examples include, but are not limited to, acridine orange, thiazole orange, oxazole yellow, and rhodamine. [2- [N-[(3-dimethylaminopropyl) -N-propylamino] -4- [2,3-dihydro-3-methyl- (benzo-1,3-thiazol-2-yl) -methylidene] -1- phenyl-qunolinium] may be used.

  A duplex nucleic acid structure is formed by base pairing that occurs between a base in the target nucleic acid and a base in its complementary nucleic acid sequence. Base pairing occurs by hydrogen bonding that occurs between cytosine and guanine, adenine and thymine, or adenine and uracil. The intercalator binds this double-stranded nucleic acid structure to the target and produces fluorescence.

  The fluorescent substance to be labeled on the nucleic acid probe does not have to be decomposed or attenuated during the nucleic acid amplification process and may be detected by the fluorescence detection process. Fluorescent substances include FITC, 6-FAM, HEX, TET, TAMRA, Texas Red (trademark), Cy3, Cy5, rhodamine, etc., but are preferably fluorescent substances that produce FRET, and more preferably interact with an intercalator. As a particularly preferable fluorescent substance, Texas Red (trademark) can be mentioned, but it is not limited to this. If the FRET phenomenon is used, the fluorescence emitted only by the presence of the nucleic acid probe in the fluorescence detection step is suppressed, and the fluorescence emitted by the nucleic acid probe hybridized with the target nucleic acid can be specifically detected.

  Preferred intercalator and fluorescent substance labeling nucleic acid probes used in the present invention are (SYBR Green I (TM) and Texas Red (TM)), (LC Green I (TM) and Texas Red (TM)) , (LC Green Plus (TM) and Texas Red (TM)) or ([2- [N-[(3-dimethylaminopropyl) -N-propylamino] -4- [2,3-dihydro-3-methyl- (benzo -1,3-thiazol-2-yl) -methylidene] -1-phenyl-qunolinium] and Texas Red (trademark).

  As means for achieving the present invention, the Tm value of each primer and labeled probe is defined. The Tm value represents the temperature at which the primer anneals with the complementary base and dissociates. The Tm value can also be calculated. Various calculation methods have been devised for calculating the Tm value using the salt concentration in hybridization as a parameter. Commonly used calculation methods include nearest neighbor pairing (Schildkraut C., Lifson S. (1965) Dependence of the melting temperature of DNA on salt concentration-Biopolymers 3, 195-208, Breslauer KJ, Frank R ., Blocker H., Markey LA (1986) Predicting DNA duplex stability from the base sequence-Proc. Natl. Acad. Sci. USA 83, 3746-3750) and Wallance method (Wallace, RB; Shaffer, J .; Murphy RF Bonner, J .; Hirose, T .; Itakura, K .; (1979) Nucelic Acid Res. 6, 3543), GC% method (Dependence of the Melting Temperature of DNA on Salt Concentration, Schildkraut C., Lifson S. (1965) BIOPOLYMERS 3.195-208, Optimization of the annealing temperature for DNA amplification in vitro, W. Rychlik, Nucl. Acids Res. (1990) 18 (21) 6409-6412), etc. Or, depending on the detection conditions, etc., the difference in Tm value due to the calculation method will affect, but it will be experimentally conducted by a study that can be easily conceived by those skilled in the art Fixed differences of the Tm value can be set primers and labeled probes having the effect of the present invention.

  The relationship between the Tm value of the first primer and the second primer and the Tm value of the labeled probe is one of the important factors for obtaining the effects described in the present invention. Therefore, as an example for explaining the invention more specifically, in the present invention, Breslauer KJ, Frank R., Blocker H., Markey LA (1986) Predicting DNA duplex stability from the base sequence-Proc. Natl. Acad. The Tm value calculated by the method described in Sci. USA 83, 3746-3750 is used.

  As an example, according to the above calculation method, the Tm value of a continuous base sequence of 24 bases selected from SEQ ID NO: 3 (5′-GAGATGGGAGCTGTCTCAACCAGA-3 ′: SEQ ID NO: 5) is selected from 70.52 and SEQ ID NO: 4. The Tm value of a continuous base sequence of 25 bases (5′-TGCGCATGATATTCCCATTAGCAGT-3 ′: SEQ ID NO: 10) is 73.39.

  The Tm value of the primer is preferably 70 ° C. or higher. More preferably, it is 70 degreeC or more and 90 degrees C or less. Use of this primer enables stable annealing at a temperature of 65 ° C. or higher. When a primer of 91 ° C. or higher is used, nonspecific hybridization is likely to occur, resulting in a decrease in amplification efficiency of the target amplification product.

  In the present invention, a fluorescent signal is generated by hybridization of a labeled probe to an extension product of a target nucleic acid or a first primer, and nucleic acid is amplified by extension of a second primer using the extension product of the target nucleic acid or the first primer as a template. Balance is important. This relationship is optimized by the Tm values of the second primer and labeled probe, as well as the concentration of the second primer and labeled probe. In order to optimize this relationship, the Tm value should be designed so that the second primer is larger than the labeled probe, and the concentration should be higher for the labeled probe than the second primer, preferably 3 times or more, preferably It is 4 times or more, more preferably 5 times or more. Moreover, 15 times or less is good, Preferably it is 10 times or less. Preferably they are 3 times or more and 15 times or less, More preferably, they are 5 times or more and 10 times or less.

  By satisfying the relationship between the Tm value and the concentration, generation of a fluorescent signal by hybridization of the labeled probe to the target nucleic acid or the extension product of the first primer, which cannot be achieved by the conventional method, and the target nucleic acid or the first primer Nucleic acid amplification by extension of the second primer using the extension product as a template can be achieved during the same cycle of PCR.

  The important disclosure of the present invention will be described in more detail with reference to FIG. In the conventional method, the target nucleic acid is amplified by annealing and extension of the second primer, but no fluorescence signal is generated because the labeled probe cannot hybridize to the target nucleic acid or the extension product of the first primer (FIG. 1 (1)-). A and FIG. 1 (2) -A), or the hybridization of the labeled probe to the target nucleic acid or the extension product of the first primer generates a fluorescent signal, but the hybridization of the labeled probe inhibits the extension of the primer, and the target When the nucleic acid is not amplified (FIGS. 1 (1) -B and 1 (2) -B), the rate of occurrence of either one is large, and in either case, the presence or amount of the target nucleic acid and the variation in the target nucleic acid The presence could not be examined quickly and with high sensitivity. On the other hand, according to the method of the present invention, the relationship between the Tm value of the first primer and the second primer and the Tm value of the labeled probe, and the relationship between the concentration of the primer and the concentration of the labeled probe are defined as described above. The labeled primer hybridizes to the target nucleic acid or the extension product of the first primer while the temperature decreases from the denaturation temperature to the annealing / extension temperature in the PCR process, and the second primer extends to the region that forms the double-stranded nucleic acid structure. To do. Subsequently, during the temperature increase from the annealing / extension temperature to the denaturation temperature of the next cycle, the labeled probe is dissociated from the target nucleic acid or the extension product of the first primer to extend to the region where the double-stranded nucleic acid structure is formed. Since this does not inhibit further extension of the second primer, an extension product of the second primer is formed to serve as a template for the first primer (FIGS. 1-C and 1 (2) -C). Thus, generation of a fluorescent signal by hybridization of the labeled probe to the target nucleic acid or first primer extension product, and amplification of the nucleic acid by extension of the second primer using the target nucleic acid or first primer extension product as a template, Is achieved during the same cycle of PCR, thereby combining the advantages of high-speed PCR with high speed and the advantages of FRET with high sensitivity and accuracy, and the presence or amount of target nucleic acid, as well as mutations in target nucleic acid. It was possible to establish a nucleic acid detection method for examining the presence quickly and with high sensitivity.

  Furthermore, by using the DNA polymerase having a high extension rate as described above, the extension of the second primer can reach the base sequence complementary to the first primer, and the template of the first primer can be more efficiently obtained. An extension product of the second primer such that

  As another important disclosure of the present invention, in order to efficiently generate a fluorescent signal and amplify a nucleic acid as described above, the labeled probe forms a double-stranded nucleic acid structure in the extension product of the first primer. The position to do is also important. As described above, in the method of the present invention, the fluorescent signal is generated by hybridization of the labeled probe to the target nucleic acid or the extension product of the first primer, and the second primer using the extension product of the target nucleic acid or the first primer as a template. It is important to achieve nucleic acid amplification by extension during the same cycle of PCR, and further, during the temperature decrease from the denaturation temperature to the annealing / extension temperature in the high-speed PCR step, The second primer is extended from the hybridization of the primer to the extension product to the region where the double-stranded nucleic acid structure is formed, and the duplex is subsequently raised during the temperature increase from the annealing / extension temperature to the denaturation temperature of the next cycle. When the second primer extended to the region where the strand nucleic acid structure was formed, further extension It is important that the extension product of the second primer as a template for the first primer are formed. That is, the distance from the 5 ′ end of the extension product of the first primer to the region where the labeled probe forms a double-stranded nucleic acid structure (A), and from the region where the labeled probe forms a double-stranded nucleic acid structure to the first primer When the distance to the 3 ′ end of the extension product of (B) is (B) (FIG. 2), the balance between (A) and (B) is important. More specifically, (A) is from the 5 ′ end of the extension product of the first primer to the 3 ′ end of the labeled probe forming the double-stranded nucleic acid structure, and (B) is the double-stranded nucleic acid structure. The distance from the 5 ′ end of the labeled probe to the 3 ′ end of the extension product of the first primer. In particular, if (A) is too larger than (B), the extension of the second primer does not reach the first primer, and the extension product of the second primer that becomes the template of the first primer cannot be formed. Fluorescence signal generation by hybridization of labeled probe to nucleic acid or first primer extension product and nucleic acid amplification by extension of second primer using target nucleic acid or first primer extension product as template It cannot be achieved during the cycle. As a balance between (A) and (B) for efficiently performing the generation of a fluorescent signal and the amplification of nucleic acid, specifically, the value of (A / B) is 0.1 or more and 3.0 or less. The extension product of the first primer and the second primer is more preferably 80 bp to 300 bp. More preferably, the value of (A / B) is 0.3 or more and 1.5 or less, and the extension products of the first primer and the second primer are 100 bp to 250 bp.

  A method of preferentially amplifying a single-stranded nucleic acid in a specific region of a target nucleic acid by making a difference between the concentrations of the first primer and the second primer used in the nucleic acid amplification step can also be applied to the present invention. For example, in designing the first primer, the second primer, and the nucleic acid probe as shown in FIG. 3, by setting the concentration of the first primer higher than the concentration of the second primer, the extension product by the first primer is preferentially generated. It can also be made. Since the extension product of the second primer and the labeled probe act competitively with the extension product of the first primer, thus intentionally amplifying the single-stranded nucleic acid containing the target nucleic acid in this way in the fluorescence detection step The hybridization efficiency of the labeled probe can be increased and more fluorescent signals can be generated. The concentration ratio of the first primer to the second primer is not particularly limited, but is preferably 4 times or more, more preferably 5 times or more, and further preferably 10 times or more. Moreover, 50 times or less is good, More preferably, 30 times or less is good. Preferably they are 4 times or more and 50 times or less, More preferably, they are 10 times or more and 30 times or less. The concentration of the second primer of the present invention is not particularly limited, but is preferably 25 nM to 500 nM. More preferably, it is 50 nM to 250 nM.

  In the fluorescence detection, for example, the nucleic acid probe may be hybridized with the extension product of the target nucleic acid or the first primer, and a decrease in fluorescence caused by dissociation of the nucleic acid probe and the target nucleic acid due to an increase in temperature may be detected. The rate of temperature rise is preferably 0.1 ° C./second or more, 0.1 ° C./second or more and 5 ° C./second or less, more preferably 0.5 ° C./second or more and 2 ° C./second or less. When it exceeds 5 ° C./second, the dissociation temperature between the nucleic acid probe and the target nucleic acid cannot be accurately captured.

  The definition of non-complementary base is a base in a labeled probe that does not base pair with a base in the target nucleic acid when the labeled probe hybridizes. When the labeled probe that matches the wild-type base sequence and the wild-type labeled nucleic acid are hybridized, there is no non-complementary base because they match completely. However, when a labeled probe that matches the wild-type base sequence and a target nucleic acid having a single base mutation relative to the wild-type are hybridized, the labeled probe has a non-complementary base. The meaning of the mutation of the present invention is to have a different base with respect to the wild type and is an event in the target nucleic acid. The non-complementary base in the labeled probe base sequence generates a fluorescent signal by hybridization of the labeled probe to the target nucleic acid or first primer extension product, and uses the target nucleic acid or first primer extension product as a template. Advantageously, nucleic acid amplification by extension of the second primer is achieved during the same cycle of PCR. This is because a labeled probe containing a non-complementary base is likely to dissociate when the temperature rises, so that the extension of the second primer can be achieved more smoothly. In this case, if 4 or more non-complementary bases are present in one labeled probe, hybridization is unlikely to occur within the range of 14 to 30 bases of the aforementioned labeled probe. The number of non-complementary bases is preferably 3 or less. The introduction position of the non-complementary base in the labeled probe is not particularly limited, but it is preferably introduced at a position excluding the mutated base to be detected in the target nucleic acid, and preferably at least 2 bases away from the end of the labeled probe. . Further, when guanine or cytosine having a relatively strong base pair binding force is present in the labeled probe for 3 or more bases, it is preferably introduced into a continuous region of guanine or cytosine.

  The positional relationship between the oligonucleotide sequence of the labeled probe and the mutation detects, for example, the decrease in fluorescence caused by the hybridization between the nucleic acid probe and the target nucleic acid or extension product of the first primer and the dissociation of the nucleic acid probe and the target nucleic acid due to an increase in temperature. When using this method, there is no particular limitation as long as the difference in dissociation temperature between the completely identical sequence and the sequence having a single nucleotide mutation is known. Preferably, it should be 3 bases or more away from both ends of the oligonucleotide sequence, more preferably 5 bases or more away from both ends. Particularly preferably, the mutation site may be located at the center of the labeled probe. In this case, the central portion is within 3 bases from the center of the oligonucleotide sequence toward both ends. The center in the case where the number of the oligonucleotide sequence is an even number means a base obtained by counting the number of the oligonucleotide sequences divided by 2 including the both ends and counting inward from the bases at both ends. In the case of an odd number of oligonucleotide sequences, the center is the number of bases counted inward from the bases at both ends by counting the number of oligonucleotide sequences plus 1 and dividing by 2 by including both ends. Say.

  By labeling both ends of the probe, the fluorescence intensity is higher than when labeling one end, and stable detection is possible. For example, in the FRET phenomenon that occurs between two different fluorescent dyes, the fluorescence intensity emitted by the acceptor fluorescent dye after energy transfer is generally weak. When an intercalator is used as in the present invention, the acceptor fluorescent dye is emitted because the number of molecules of the fluorescent dye that can be a donor is larger than in other methods, for example, when a fluorescent dye labeled with a probe is used as a donor. Although the fluorescence intensity becomes relatively strong, it is still more preferable to label both ends of the probe from the viewpoint of the intensity and reproducibility of the fluorescence signal. When using a probe with one end labeled, the 3 'end is labeled with a fluorescent dye, or phosphorylation or other treatment is added to the 3' end to extend during high-speed PCR and increase the Tm value of the labeled probe. It is preferable to devise not to do so. However, as a fluorescence detection method, for example, when using a method in which the labeled probe and the target nucleic acid or the extension product of the first primer are hybridized and the decrease in fluorescence caused by dissociation of the labeled probe and the target nucleic acid due to temperature rise is detected. If the probe is extended during high-speed PCR, the dissociation temperature between the nucleic acid probe and the target nucleic acid cannot be measured accurately.

The present invention also includes the following.
In the presence of at least the following (a) to (c) and the sample to be confirmed for the presence or amount of the target nucleic acid, light of the excitation wavelength of the intercalator is given continuously or intermittently, and the fluorescence from the labeled probe is measured, Perform high-speed PCR to determine the presence or amount of the target nucleic acid, and then in a sealed state, give light at the excitation wavelength of the intercalator and measure the fluorescence from the labeled probe at 0.1 ° C./second or higher A detection method characterized by conducting a melting curve analysis with continuous temperature rise or fall of and examining the presence of mutations in the target nucleic acid. In this case,
(A) Intercalator (b) Labeled probe having the following properties (i) to (iii)
(i) It can form a double-stranded nucleic acid structure with a sequence complementary to the target nucleic acid, and has an oligonucleotide having a length of 14 to 30 bases
(ii) The oligonucleotide is labeled with one or more fluorescent substances capable of absorbing fluorescence energy from the intercalator and emitting fluorescence itself.
(iii) The concentration of the labeled probe is 3 to 15 times the concentration of the second primer of (c) (c) the first primer having the following properties (i) to (iii) and Second primer
(i) The extension product of the first primer has a property complementary to the target nucleic acid and serves as a template for the second primer, and the extension product of the second primer has a sequence homologous to the target nucleic acid. And has the property of becoming a template for the first primer
(ii) The length of the amplification product of the first primer and the second primer is 50 bp or more and 500 bp or less
(iii) The Tm value of the first primer and the second primer is 70 ° C. or more and 90 ° C. or less, and the Tm value of the second primer is larger than the Tm value of the labeled probe.

  As an example of the target nucleic acid, a specific nucleic acid held by a bacterium causing infection can be considered. For example, detection of 23S rRNA gene of Helicobacter pylori can be mentioned. Helicobacter pylori is considered to be a causative agent of gastric / duodenal ulcer, and has recently been pointed out to be related to gastric cancer. If there is a suspicion of ulcer, if a breath test, blood or fecal Helicobacter pylori antibody test is performed, and there is a possibility of Helicobacter pylori infection, a gastric biopsy is performed in an endoscopy. Can be confirmed by rapid urease test or culture method. When the presence of Helicobacter pylori is confirmed by endoscopy, treatment such as sterilization therapy is performed. As a sterilization therapy for Helicobacter pylori, a combination therapy of a proton pump inhibitor, amoxicillin and clarithromycin is effective, and it has been widely practiced in Japan since 2000 AD as an insurance practice. In order to detect H. pylori according to the present invention, the first primer, the second primer, and the labeled probe were designed using the 23S rRNA gene sequence of H. pylori as a target. In the process of amplifying a specific region of the 23S rRNA gene sequence with the first primer and the second primer, using a labeled probe, the fluorescence signal is detected in the “extension” step or the “annealing / extension” step, resulting in higher sensitivity. Detection of Helicobacter pylori is possible.

  Recently, the appearance of clarithromycin-resistant H. pylori (hereinafter referred to as CAM-resistant bacterium) has been reported, and detection of the CAM-resistant gene of H. pylori has attracted attention. CAM-resistant Helicobacter pylori has been cited as one of the major factors in reducing the success rate of Helicobacter pylori eradication. CAM exerts its action by binding to the peptidyltransferase loop of 23S rRNA of H. pylori and inhibiting its protein synthesis. However, it has been shown that Helicobacter pylori acquires CAM resistance by a point mutation in the 23S rRNA gene (James versalovic et. Al. Antimicrobial Agents and Chemotherapy p477-480 1996). In this regard, 93% of CAM-resistant bacteria have mutated adenine at position 2142 or 2143 in the above region (A2142G or A2143G), and 7% of CAM-resistant bacteria have cytosine at position 2142. Mutation (A2142C) has been reported (Stone, GG et. Al. Antimicrobial Agents & Chemotherapy. 41 (3): 712-4, 1997).

  In order to determine the CAM resistance of Helicobacter pylori, for example, the following method can be considered. A specific region of the 23S rRNA gene of H. pylori is amplified by the first and second primers designed using a sequence common to wild-type H. pylori and CAM-resistant H. pylori. A labeled probe that completely matches the 23S rRNA gene of wild-type H. pylori using the products amplified in this step (two types of wild-type and amplified products with CAM-resistant single nucleotide mutations) or the above two A labeled probe that perfectly matches the 23S rRNA gene of a variety of CAM resistant H. pylori is used simultaneously. The fluorescence signal is detected in the “extension” step or “annealing / extension” step in the PCR process. In this detection, when a labeled probe that completely matches the 23SrRNA gene of wild-type H. pylori is used, detection of wild-type H. pylori is possible, and the two types of CAM-resistant H. pylori completely match the 23SrRNA gene. When a labeled probe is used, CAM resistant H. pylori can be detected. Furthermore, it is also included in the scope of the present invention that wild-type H. pylori and CAM-resistant H. pylori can be simultaneously detected with one labeled probe by performing melting curve analysis. Regarding CAM-resistant H. pylori, the single-base mutations are mostly due to H. pylori where the adenine at position 2142 or 2143 is mutated to guanine (A2142G or A2143G), or the 2142 position is mutated to cytosine (A2142C). This is not the case. The target nucleic acid in Helicobacter pylori includes a nucleic acid possessed by a wild type, and includes a nucleic acid possessed by a CAM resistant bacterium in which a single nucleotide mutation exists. In this case, the number of non-complementary bases is one base for a CAM-resistant H. pylori in a labeled probe having a completely identical base sequence to the wild type. That is, a non-complementary base of 3 bases or less means a mutation of 3 bases or less including a 1 base mutation in the target nucleic acid when the labeled probe is the same and the target nucleic acid is different.

That is, the following detection method may be implemented using the present invention.
High-speed PCR using the first primer and the second primer of (c) while continuously or intermittently giving light of the excitation wavelength of the intercalator of (a) and measuring the fluorescence from the labeled probe of (b) And detecting the presence or amount of the 23S rRNA gene of Helicobacter pylori.

Furthermore, the following detection method may be implemented using the present invention.
High-speed PCR using the first primer and the second primer of (c) while continuously or intermittently giving light of the excitation wavelength of the intercalator of (a) and measuring the fluorescence from the labeled probe of (b) To determine the presence or amount of the 23S rRNA gene of H. pylori, followed by applying light at the excitation wavelength of the intercalator in (a) and measuring the fluorescence from the labeled probe in the sealed state, while measuring 0. A detection method characterized by performing a melting curve analysis with continuous temperature increase or decrease of 1 ° C./second or more and examining the presence of a 23S rRNA gene mutation of H. pylori.

Furthermore, the following detection method may be implemented using the present invention.
High-speed PCR using the first primer and the second primer of (c) while continuously or intermittently giving light of the excitation wavelength of the intercalator of (a) and measuring the fluorescence from the labeled probe of (b) To determine the presence or amount of the 23S rRNA gene of H. pylori, and then, in the sealed state, give light of the excitation wavelength of the intercalator of (a) and measure the fluorescence from the labeled probe of (b). However, the melting curve analysis with continuous temperature rise or fall of 0.1 ℃ / second or more was performed, and the presence of mutation at position 2142 or 2143 of the 23S rRNA gene was examined to determine the antibiotic resistance of H. pylori A detection method characterized by:

The method of examining the presence or amount of the 23S rRNA gene of H. pylori, the method of examining the presence of the mutation of the 23S rRNA gene of H. pylori, the presence of the mutation at position 2142 or 2143 of the 23S rRNA gene In the method of determining antibiotic resistance, if the target nucleic acid is the sense strand of the 23S rRNA gene of H. pylori,
The oligonucleotide sequence of the labeled probe of (b) is a continuous base sequence of 14 to 30 bases selected from SEQ ID NO: 2,
The first primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
The second primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
If the target nucleic acid is the antisense strand of the 23S rRNA gene of H. pylori,
The oligonucleotide sequence of the labeled probe of (a) is a continuous base sequence of 14 to 30 bases selected from SEQ ID NO: 1,
The first primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The second primer of (c) may be a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3.

  “In one container” indicates that a solution containing a specimen is filled in a container made of plastic, glass, or the like, and there is no operation to transfer or remove the solution to another container from amplification to detection. The sealed state indicates that there is no entry of impurities from the outside and no evaporation of the solution from the inside. It is important to prevent contamination such as impurities, and to prevent differences in results due to changes in solution composition.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

  The following is a method for determining the clarithroma resistance on the 23S rRNA gene of H. pylori.

Example 1
(Sample collection)
Genes were extracted from gastric biopsy material using QIAamp (registered trademark) DNA micro Kit (manufactured by Qiagen). The extracted DNA was stored at -20 ° C.
(Preparation of template plasmid)
PCR was performed using 1 μl of the extracted DNA solution as a template. The primer pair used here was a primer capable of full-length amplification of the H. pylori 23SrRNA gene. All were designed from known sequences of the 23S rRNA gene (GenBank accession number U27270) of H. pylori.

PCR was performed using GeneAmp (registered trademark) 9700 (manufactured by Applied Biosystems) as a thermal cycler in a total volume of 26 μl. 1 μl of DNA solution and reagent mixture (KOD-plus 0.5 U, 10 × PCR buffer 2.5 μl, 200 μM dNTP, each 5 μM primer) were mixed. After initial denaturation of the target DNA at 94 ° C. for 2 minutes, a cycle of a denaturation step of 94 ° C./15 seconds, an annealing step of 60 ° C./30 seconds, and an extension step of 68 ° C./60 seconds was repeated 35 times. Other PCR conditions are as usual (see, for example, the Manual for Gene Manipulation Technology, Medical School, published in 1995). The obtained nucleic acid fragment was ligated to pUC18 that had been blunt-ended with a restriction enzyme, and a transformant was obtained using competent cell JM109. The transformant in which the target nucleic acid fragment was inserted was subjected to liquid culture and a plasmid was extracted.
The nucleic acid sequence of the obtained plasmid was analyzed by an autosequencer, and a plasmid having a wild-type base sequence, a plasmid having a base sequence in which the adenine at position 2142 or 2143 of H. pylori 23S rRNA gene was mutated to guanine A total of three types were obtained. Hereinafter, the wild type is WT, the 2142 mutation of the 23S rRNA gene is 2142G, and the 2143 mutation of the 23S rRNA gene is 2143G.

(Example 2)
(Synthesis of primer and labeled probe)
Primers and labeled probes used for detection were requested from a nucleic acid synthesis contract company (Japan Bioservice Co., Ltd., Sawaddy Co., Ltd., GENSET KK, Sigma Aldrich Japan Co., Ltd., etc.).

Table 1 shows the base sequences of the labeled probes and primers used. As the labeled probe, a base sequence of 12 to 22 bases selected from SEQ ID NO: 1 was used. As for the primers, primers F1 to F4 are each a base sequence composed of 24 bases, 29 bases, 29 bases and 22 bases selected from SEQ ID NO: 3. Primers R1 to R4 are each a base sequence composed of 25 bases, 31 bases, 24 bases and 26 bases selected from SEQ ID NO: 4. Primer F5 is a base sequence composed of 24 bases designed in a region in which the distance to the base sequence represented by SEQ ID NO: 1 is longer than the base sequence represented by SEQ ID NO: 3 in the base sequence of H. pylori 23S rRNA gene. Primer R5 is a base sequence composed of 22 bases designed in a region where the distance to the base sequence represented by SEQ ID NO: 2 is longer than the base sequence represented by SEQ ID NO: 4 in the base sequence of H. pylori 23S rRNA gene.

(Example 3)
(Detection of wild-type H. pylori)
Each plasmid DNA of WT, 2142G, and 2143G was mixed as a template in the reaction solution shown in Table 2. Hereinafter, based on the reaction composition in Table 2, when the final concentration of any of the solutions constituting the reaction solution was changed, it was adjusted with milli-Q water to maintain a total amount of 20 μL. Further, the composition shown in Table 2 is an example, and the reaction composition can be easily optimized by those skilled in the art. The amount of the plasmid was used ¹⁰⁴ copies and 10 ² copies. Primers F1 and R1 shown in Table 1 were used as primers. As the labeled probe, labeled probe A shown in Table 1 was used. The labeled probe A is a probe in which a deliberate non-complementary base is introduced into a labeled probe having a nucleotide sequence that completely matches that of the wild type. For example, in labeled probe A, the intentional non-complementary base is a non-complementary base in the labeled probe corresponding to the mutation site, that is, a non-complementary base is introduced into the base sequence of the labeled probe excluding positions 2142 and 2143 To do. Labeled probe A is a labeled probe in which two non-complementary bases are present for CAM-resistant H. pylori (mutant) 2142G and 2143G. The reaction conditions at the time of PCR amplification were those shown in Table 3 (nucleic acid amplification cycle). A light cycler (registered trademark) manufactured by Roche Diagnostics was used for the measurement of amplification and fluorescence. Measurement modes used were 530 nm and 640 nm. Unless otherwise specified, all measurements shall be made with a light cycler (registered trademark).

  The fluorescence signal value FL (n) in the nth cycle of amplification was calculated by the formula (I) and analyzed.

FL (n) = FL640 (n) − {k × FL530 (n)} − FLtb (n) (Formula (I))

FL640 (n): Fluorescence measurement value in light cycler measurement mode 640 nm in the nth cycle,
FL530 (n): Fluorescence measurement value in light cycler measurement mode 530 nm in the nth cycle,
k: a correction coefficient such that k × FL530 (n) is a background fluorescence signal value at a wavelength of 640 nm of SYBR Green I (trademark) in the nth cycle,
FLtb (n): Background fluorescence signal value at a wavelength of 640 nm of Texas Red ™ in the nth cycle.
The background fluorescence signal refers to a fluorescence signal generated irrespective of the hybridization of the labeled probe to the target nucleic acid or the extension product of the first primer.

4 to 8 are graphs showing the analysis results with the horizontal axis of the graph being the cycle number n and the vertical axis being the fluorescence signal value FL (n).

As a result of the analysis, each with the amount of plasmid ¹⁰⁴ copies and ¹⁰² copies of labeled probe A Figure 4, the results shown in FIG. 5 were obtained. 4 and 5, 2143G is the 2143-position mutant of the H. pylori 23S rRNA gene, 2142G is the 2142-position mutant of the H. pylori 23S rRNA gene, NC is a negative control using milli-Q water as a template, and WT is The wild type of H. pylori 23S rRNA gene is shown, and the same applies to FIGS. When the plasmid was introduced pylori 23S rRNA gene of the wild-type template, from the vicinity of the ¹⁰⁴ copies exceeding the 30 cycles, an increase in fluorescent signal values with labeled probe from the vicinity of over 40 cycles at 10 ² copies confirmed. When using a mutant plasmid in which a single nucleotide mutation exists, two non-complementary bases exist in labeled probe A, which reduces the hybridization ability of labeled probe A to the target nucleic acid during the amplification reaction. No increase in fluorescence signal value was observed. Thus, WT could be specifically detected by adjusting the number of non-complementary bases in the labeled probe. The intentional introduction of non-complementary bases into the labeled probe is effective as a technique for adjusting the hybridization ability of the labeled probe to the target nucleic acid and the ease of dissociation. In the plasmid of 10 ² copies it is possible to detect the labeled probe specifically and in a short time.

Example 4
(Detection of H. pylori CAM resistance gene mutation)
CAM-resistant H. pylori (mutant) was detected using the labeled probe B described in Table 1. Labeled probe B is one in which a deliberate non-complementary base 1 base is introduced at a position excluding positions 2142 and 2143 in the base sequence that completely matches the 2142G mutant. For the wild type, there are two non-complementary bases in total, one non-complementary base present at the mutation site and one intentional non-complementary base. For the 2143G mutant, it is a labeled probe having 3 non-complementary bases. The conditions of Example 3 were used except for the labeled probe.
In contrast to FIGS. 4 and 5, the labeled probe B was able to confirm an increase in fluorescence signal value only when the 2142G mutant plasmid was used as a template. When NC, WT and 2143G templates were used, no fluorescence signal value was obtained. According to the present invention, it can be seen that a fluorescent signal can be obtained specifically for the base sequence of the target nucleic acid.

(Example 5)
In Example 3, the primer F1 was changed to the primers F2 to F5 shown in Table 1, the primer R1 was changed to the primers R2 to R5 shown in Table 1, and the combination was changed between F1 to F5 and R1 to R5. . Other conditions were in accordance with Example 3. The template used was 10 ⁴ copy plasmid of WT. The results are shown in Table 4. The numerical values in Table 4 indicate the number of cycles when the fluorescence signal value of Texas Red (trademark) of the labeled probe obtained from the formula (I) exceeds a certain value (0.25). Combinations in which no increase in fluorescence signal was observed showed (−) instead of numerical values. The value of (A / B) is (distance from the base homologous to the 5 ′ end of primer R in the target nucleic acid to the base complementary to the 3 ′ end of the labeled probe / complementary to the 5 ′ end of primer F in the target nucleic acid. The distance from the base to the base complementary to the 5 ′ end of the labeled probe).

  From Table 4, it was possible to obtain the same result as the combination of the primers F1 and R1 when the combination of the primers (A / B) was 0.3 to 2.0 and the size of the amplification product was up to 250 bp. . When R5 having a large A value was used, the extension reaction with primer F was not completed up to primer R5, and no amplification was observed. When B was extremely short, the fluorescence signal value tended to be weak. From this result, it can be seen that it is advantageous that the length of the amplified product is short and the length of A is short.

(Example 6)
(Examination of Tm value of labeled probe)
The Tm value (length) of the labeled probe without intentional mutation is 39.64 (12 bases), 50.68 (14 bases), 60.07 (16 bases), 63.68 (18 bases), 69 The Tm value of the labeled probe affects the rapid nucleic acid detection method of the present invention using each of the six types of labeled probes C to H shown in Table 1 with .39 (20 bases) and 76.29 (22 bases). The impact was examined. Except for the labeled probe, the same conditions and composition as in Example 3 were used. Labeled probes C to H have base sequences represented by SEQ ID NOs: 17 to 22, respectively. As templates, WT, 2142G and 2143G plasmids (10 ⁴ copies of plasmid) were used. Each labeled probe is designed so that the mutated base site is located in the center. The results with the nucleic acid probe D (Tm value 60.07) are shown in FIG.

  With a labeled probe having a Tm value of 60 ° C. to 70 ° C. (length from 16 to 20 bases), specific detection of H. pylori wild type was possible. In the case of the labeled probe H in which the Tm value exceeds the primers F1 and R1, the hybridization to the target nucleic acid was excessively stabilized, so that the hybridization inhibited the primer extension reaction in PCR. As a result, the amount of amplified product decreased and the fluorescence signal decreased. When the Tm value was less than 50 ° C. as in labeled probe C, the labeled probe close to 100% was dissociated at 65 ° C., and no fluorescence signal could be detected. When the Tm value of the primer is 70.52 for the primer F1, 73.39 for the primer R1, and the “annealing / extension” temperature is 65 ° C., the optimum Tm value for the labeled probe is 60 ° C. to 70 ° C.

(Example 7)
(Examination of labeled probe concentration)
Detection was performed when the labeled probe concentration was 150 nM and 1000 nM. Using the labeled probe, primer, reagent composition and reaction conditions of Example 3 as they were, the labeled probe concentration was examined by changing the labeled probe concentration of 1000 nM to 150 nM. The relationship between the labeled probe concentration and the second primer concentration is 1.0 times and 6.7 times for the labeled probe with respect to the second primer concentration, respectively. Template was used WT, 2142G, each plasmid 2143G (the plasmid of 10 ² copies). The examination result of the labeled probe concentration of 1000 nM is shown in FIG.
WT H. pylori could be specifically detected at a labeled probe concentration of 1000 nM. At a concentration of 1000 nM, a sufficient amount of labeled probe was present for the amplified product that hybridized with the labeled probe, and a fluorescent signal sufficient for detection of H. pylori could be obtained. It can be seen that when the labeled probe concentration is 150 nM, the proportion of labeled probe hybridized with the amplification product decreases and it is difficult to detect H. pylori with high sensitivity.

As a comparative example of Example 7, a labeled probe concentration of 3000 nM (concentration ratio of labeled probe to second primer 20 times) was examined. As shown in FIG. 7, at a probe concentration of 3000 nM, primer extension was inhibited and a fluorescence signal value could not be obtained. It can be seen that detection of Helicobacter pylori is difficult even when the labeled probe concentration is 20 times as high as the second primer concentration.

(Example 8)
(Examination of first primer concentration and second primer concentration ratio)
The concentration ratio of the first primer to the second primer was examined 1 and 10 times. For 1-fold, primer F1 500 nM and primer R1 500 nM were used. For 10-fold, primer F1 150 nM and primer R1 1500 nM were used. The same conditions as in Example 3 were used except for the primer concentration. Template was used WT, 2142G, each plasmid 2143G (the plasmid of 10 ² copies). The result of the concentration ratio of 1 time is shown in FIG. 8 (comparative example), and the result of 10 times is shown in FIG.

  When the concentration ratio was 10 times, more single-stranded target nucleic acid was amplified with respect to the labeled probe, so that the relationship between primer extension and labeled probe hybridization was well maintained. Therefore, a fluorescent signal was obtained specifically only with the perfect match template. When the concentration ratio was 1 time, when a labeled probe whose template and base sequence were completely identical was used, the fluorescence signal obtained was less than that obtained when the primer concentration ratio was 10 times. It can be seen that specific detection can be performed with higher sensitivity than symmetric PCR because asymmetric PCR amplifies more target nucleic acid.

Example 9
(Selection of DNA polymerase for high-speed amplification)
DNA polymerases that can be applied to speed up the nucleic acid amplification process were investigated. Two types of DNA polymerases, Taq polymerase (derived from Thermus aquaticus) and KOD polymerase (derived from Thermococcus kodakaraensis KOD1), were examined. Taq polymerase was Toyobo Co., Ltd. Blend Taq-Plus- (registered trademark), and KOD polymerase was Toyobo Co., Ltd. KOD-Plus- ver.2 (registered trademark). For Taq polymerase, labeled probe A and primers F1 and R1 shown in Table 1 were used, and other reagent compositions were referred to the reagent composition described in the protocol. Reaction conditions shown in Table 3 (nucleic acid amplification cycle) were used. Regarding KOD polymerase, labeled probe A and primers F1, R1 were used in the same manner as Taq polymerase, and the basic reaction conditions were the same as in Example 3. Template was used WT, 2142G, each plasmid 2143G (the amount of plasmid ¹⁰⁴ copies). The results when KOD polymerase is used are as shown in FIG. In addition, Table 5 shows the results of confirming the amount of each amplified product by electrophoresis on 3% agarose gel and staining with ethidium bromide.

When Taq polymerase was used, the reaction conditions were fixed and various reagent compositions were examined in addition to the above reagent composition, but specific H. pylori bacteria could not be detected. On the other hand, when KOD polymerase was used, H. pylori could be specifically detected corresponding to high-speed amplification. From the results of the electrophoresis, amplification with Taq polymerase is slightly to the extent that the band is visible amplified in the case of rapid amplification WT, could not be detected in the electrophoretic band when WT plasmids (10 ² copies) to the template . KOD polymerase could be adapted for high-speed amplification, but Taq polymerase could not be adapted for high-speed amplification and could not detect H. pylori because the desired nucleic acid amplification was not observed. It can be seen that KOD polymerase having a high extension rate and no 5 ′ exonuclease activity is more suitable as the DNA polymerase of the present invention.

(Example 10)
We investigated whether single-label mutations present in the template could be detected with a single labeled probe by performing a melting curve analysis following the nucleic acid amplification step. Template by using the H. pylori WT, 2142G, three kinds of plasmids 2143G, amount of the plasmid using ¹⁰⁴ copies and 10 ² copies. The same reagent composition and reaction conditions as those in Example 3 were used as shown in (Nucleic acid amplification cycle) / (melting curve analysis) / (cool down) in Table 3. Primers F1 and R1 and nucleic acid probe A shown in Table 1 were used as primers and labeled probes, respectively. The obtained fluorescence signal value was not corrected, and the result of the melting curve analysis in the light cycler (registered trademark) was used as it was. The measurement range was 640 nm.

¹⁰⁴ copies and ¹⁰² copies of plasmid results of fluorescence measurements when used as a template, respectively 9, is shown in FIG. 10. Originally, an increase in the amount of fluorescence is detected by real-time fluorescence detection using LightCycler (registered trademark), but a decrease in the amount of fluorescence when the labeled probe dissociates from the target nucleic acid is detected in the melting curve analysis. The vertical axis in the figure is the value of the inverse sign of the first derivative of the fluorescence intensity (-dF / dt), and the horizontal axis is the temperature (° C). From FIG. 9 and FIG. 10, two peaks were obtained. The peak observed at 60 ° C. is the peak of WT, and the peak observed at 51 ° C. is the peak of two types of mutant H. pylori having a single base mutation relative to WT. From this result, it is understood that WT and mutant types can be detected by separate peaks by performing melting curve analysis using one labeled probe.

(Example 11)
(Detection by melting curve analysis using cells directly as template)
Two ATCC strains of H. pylori (wild type 26695 strain and 2143G mutant UA1182 strain) were obtained. Of these, 10 ⁹ cells were collected and heat-treated at 95 ° C. for 5 minutes. Thereafter, 10 ⁵ heat-treated cells were used as templates in Table 2. Other than that, H. pylori was detected in the same manner as in Example 10 where the melting curve analysis was performed. The result is shown in FIG.
When detection of H. pylori wild type and CAM-resistant H. pylori was performed by melting curve analysis, both could be detected as a single peak by melting curve analysis. It is possible to detect the microbial cells as a sample (CAM resistance determination) within 30 minutes.

(Comparative Example 1)
(Time to detection of H. pylori)
When PCR is performed at a commonly used PCR cycle of 94 ° C for 15 seconds, 65 ° C for 30 seconds, 68 ° C for 30 seconds and detected by melting curve analysis, the temperature rise / fall rate is fast even among thermal cyclers. Even the Light Cycler (registered trademark) that was used took more than 1 hour for amplification and detection.

  Under the reaction conditions of Example 3, the time required to detect the target nucleic acid using LightCycler (registered trademark) is within 15 minutes, and even using melting curve analysis, detection of H. pylori and presence of H. pylori CAM resistance gene The time required to detect was within 20 minutes. When the detection method of the present invention is used, it is possible to detect H. pylori in a very short time and with high sensitivity.

  According to the present invention, rapid and highly sensitive nucleic acid detection is possible. Specifically, the fluorescence signal can be detected while maintaining the amplification efficiency due to the nature and concentration of the labeled probe that does not inhibit the extension reaction of the second primer. In addition, the use of DNA polymerase, which has a high extension rate, shortens the amplification reaction time, realizes a faster and more sensitive nucleic acid detection method than ever before, and examines the presence or amount of the 23S rRNA gene of H. pylori. In addition, the presence of mutations in the 23S rRNA gene of Helicobacter pylori, the presence of mutations at positions 2142 or 2143 of the 23S rRNA gene, and the determination of antibiotic resistance in Helicobacter pylori can be quickly and highly sensitive. And contribute greatly to the industry.

The principle of a rapid and highly sensitive detection method that is made possible by the relationship between the primer and probe concentrations and the respective Tm values is shown. The positional relationships A and B between the target nucleic acid or the extension product of the first primer and the labeled probe are schematically shown. The positional relationship among the first primer, the second primer and the labeled probe is shown for the asymmetric PCR method in the present invention. The results of real-time detection of Helicobacter pylori using ⁴ copies of plasmid 104 as a template are shown. Plasmid ¹⁰² copies shows real-time detection results of H. pylori as the template. The result of real-time detection of Helicobacter pylori using a labeled probe having a 16-base sequence is shown. The real-time detection result of Helicobacter pylori when the labeled probe concentration is used 20 times with respect to the second primer concentration is shown. The real-time detection result of Helicobacter pylori when the concentration ratio of the first primer and the second primer is 1: 1 is shown. Plasmid ¹⁰⁴ copies shows the results of simultaneous detection of wild-type and mutant forms of H. pylori due to melting curve analysis as a template. The result of simultaneous detection of wild type and mutant of H. pylori by melting curve analysis using ² copies of plasmid as a template is shown. The result of having detected the Helicobacter pylori using what processed the microbial cell simply was used as a template is shown.

Claims (18)

In the presence of at least the following (a) to (c) and the sample to be confirmed for the presence or amount of the target nucleic acid, light of the excitation wavelength of the intercalator is given continuously or intermittently, and the fluorescence from the labeled probe is measured, 5 'no exonuclease activity, the presence of a DNA polymerase having a DNA synthesis rate of more than 100 bases / second, performs P CR, detection method characterized by determining the presence or amount of the target nucleic acid.
(A) Intercalator (b) Labeled probe having the following properties (i) to (iii) (i) A double-stranded nucleic acid structure can be formed with a sequence homologous to the target nucleic acid, and has 14 to 30 bases An oligonucleotide having a length (ii) the oligonucleotide is labeled with one or more fluorescent substances capable of absorbing fluorescence energy from the intercalator and emitting fluorescence itself (iii) of the labeled probe (C) The first primer and the second primer (i) having the following properties (i) to (iv) having a concentration of 3 to 15 times the concentration of the second primer (c) The extension product of one primer has a sequence homologous to the target nucleic acid and serves as a template for the second primer, and the extension product of the second primer has a sequence complementary to the target nucleic acid. And the length of the first primer having the property as a template (ii) said first primer and said second primer by amplification product is 80 bp or more 250 bp or less (iii) said first primer and said second The Tm value of the primer is 70 ° C. or more and 90 ° C. or less, and the Tm value of the second primer is larger than the Tm value of the labeled probe of (b) (iv) In the extension product of the first primer, The distance from the 5 ′ end to the region where the labeled probe forms a double-stranded nucleic acid structure (A), and the distance from the region where the labeled probe forms a double-stranded nucleic acid structure to the 3 ′ end of the extension product In the case of (B), the value of (A / B) is 0.3 or more and 2.0 or less. The detection method according to claim 1, wherein the concentration of the first primer is 4 times or more the concentration of the second primer. P CR is detecting method according to claim 1 or 2, characterized in that repeated rising and lowering of the temperature at 10 ° C. / sec or faster. The detection method according to claim 1 or 2, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a mutant thereof. (B) the labeled probe further has (iv) a target site and 3 or less non-complementary bases;
The detection method according to claim 1, wherein: 6. The detection method according to claim 5, wherein a non-complementary base is located at the center of the labeled probe. The intercalator is SYBR Green I (TM), LC Green I (TM), LC Green Plus (TM), or [2- [N-[(3-dimethylaminopropyl) -N-propylamino] -4- [2,3 -Dihydro-3-methyl- (benzo-1,3-thiazol-2-yl) -methylidene] -1-phenyl-quinolinium], and the fluorescent substance of the labeled probe is Texas Red (trademark) It exists, The detection method in any one of Claims 1-6 characterized by the above-mentioned. The target nucleic acid is the sense strand of the 23S rRNA gene of H. pylori,
(B) the oligonucleotide sequence of the labeled probe is a continuous base sequence of 14 to 26 bases selected from SEQ ID NO: 2,
The first primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
The second primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
While measuring the fluorescence from continuously or intermittently fed with light having an excitation wavelength of intercalator and labeled probe, performs P CR, and wherein the determining the presence or amount of 23S rRNA gene of H. pylori claims The detection method according to any one of 1 to 7. The target nucleic acid is the antisense strand of the 23S rRNA gene of H. pylori,
The oligonucleotide sequence of the labeled probe of (b) is a continuous base sequence of 14 to 26 bases selected from SEQ ID NO: 1,
The first primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The second primer of (c) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
While measuring the fluorescence from continuously or intermittently fed with light having an excitation wavelength of intercalator and labeled probe, performs P CR, and wherein the determining the presence or amount of 23S rRNA gene of H. pylori claims The detection method according to any one of 1 to 7. The target nucleic acid is the antisense strand of the 23S rRNA gene of H. pylori,
The oligonucleotide sequence of the labeled probe of (b) is any one of the nucleotide sequences of SEQ ID NOS: 15-22,
The first primer of (c) is any one of the nucleotide sequences of SEQ ID NOS: 10-14,
(C) the second primer is any one of the nucleotide sequences of SEQ ID NOS: 5-9,
While measuring the fluorescence from continuously or intermittently fed with light having an excitation wavelength of intercalator and labeled probe, performs P CR, and wherein the determining the presence or amount of 23S rRNA gene of H. pylori claims 10. The detection method according to 9. Subsequent to the method according to any one of claims 1 to 10, while being sealed, applying light of an excitation wavelength of an intercalator and measuring fluorescence from a labeled probe continuously at 0.1 ° C / second or more. A detection method comprising analyzing a melting curve accompanied with a temperature rise or fall and examining the presence of a mutation in a target nucleic acid. The detection method according to claim 11, wherein the mutation is examined for the presence of a mutation at position 2142 or position 2143 of the 23S rRNA gene of H. pylori. The detection method according to claim 11 or 12, wherein the mutation site is located in the center of the labeled probe. A kit for detecting the presence of H. pylori and / or the presence of a mutation at position 2142 or position 2143 of the 23S rRNA gene of H. pylori, comprising at least the following (a) to (d):
(A) Intercalator (b) Labeled probe for detecting the presence of H. pylori and / or the presence of antibiotic resistance of H. pylori having the following properties (i) to (iii) (I) An oligonucleotide having a continuous base sequence of 14 to 26 bases selected from SEQ ID NO: 1 or (II) an oligonucleotide having a continuous base sequence of 14 to 26 bases selected from SEQ ID NO: 2 (ii) The oligonucleotide is labeled with one or more fluorescent substances capable of absorbing fluorescence energy from the intercalator and emitting fluorescence itself. (Iii) The concentration of the labeled probe is the concentration of the second primer of (c) 3 times more than 1
5 times or less (c) Tm value is 70 ° C. or higher and 90 ° C. or lower, and SEQ ID NO: 3 or 4 (the labeled probe has the properties (I), (ii) and (iii) of (i)) And when the labeled probe has the property of (II), (ii) and (iii) of (i), it is 20 bases or more and 35 bases or less selected from SEQ ID NO: 3) . A first primer comprising a continuous base sequence and a Tm value of 70 ° C. or higher and 90 ° C. or lower and SEQ ID NO: 3 or 4 (the first base is a continuous base sequence of 20 bases to 35 bases selected from SEQ ID NO: 3) SEQ ID NO: 4 in the case of those containing, 20 base wherein the first primer may be intended to include contiguous nucleotide sequence of more than 20 bases or more 35 base selected from SEQ ID NO: 4 is selected from SEQ ID NO: 3) A combination of a first primer and a second primer having the following properties (i) to (iii) (i) The length of the amplification product of one primer and the second primer is 80 bp or more and 250 bp or less (ii) The Tm value of the second primer is larger than the Tm value of the labeled probe of (b) (iii) The length of the first primer In the extension product, the distance from the 5 ′ end of the extension product to the region where the labeled probe forms a double-stranded nucleic acid structure (A), and the extension product from the region where the labeled probe forms a double-stranded nucleic acid structure When the distance to the 3 ′ end of (B) is (A /
B) a value of 0.3 or more and 2.0 or less (d) a DNA polymerase not having 5 ′ exonuclease activity and having a DNA synthesis rate of 100 bases / second or more 15. The concentration of the primer on the side that generates an extension product capable of forming a double-stranded nucleic acid structure with the labeled probe of (b) is at least 4 times the concentration of the other primer. The described kit. The kit according to claim 14 or 15, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a variant thereof. The intercalator is SYBR Green I (TM), LC Green I (TM), LC Green Plus (TM), or [2- [N-[(3-dimethylaminopropyl) -N-propylamino] -4- [2,3 -Dihydro-3-methyl- (benzo-1,3-thiazol-2-yl) -methylidene] -1-phenyl-quinolinium], and the fluorescent substance of the labeled probe is Texas Red (trademark) The kit according to any one of claims 14 to 16, wherein the kit is provided. The kit according to any one of claims 14 to 17, wherein a site corresponding to position 2142 or position 2143 of the 23S rRNA gene of H. pylori is located in the center of the labeled probe.

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