JP2009148245A - Quick detection method of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable quick detection of a target nucleic acid while keeping specificity and high sensitivity by solving the problems, of a conventional method using a nucleic acid probe or a nucleic acid primer whose fluorescent light quenches in hybridization with the target nucleic acid, that the nucleic acid probe interferes with a nucleic acid amplification process, the elongation process in particular, causing the sensitivity to drop when shortening the time and preventing the specific detection of the target nucleic acid by non-specific amplification of the nucleic acid primer. <P>SOLUTION: A relation between a Tm value of a first primer and a second primer and a Tm value of a marker probe and further a relation between a concentration of the primer and a concentration of the marker probe are controlled and optimized to quickly detect the target nucleic acid while keeping specificity and high sensitivity. <P>COPYRIGHT: (C)2009,JPO&INPIT

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 consisting of 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 as 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.

Patent Document 7 provides a method using a nucleic acid probe whose fluorescence is quenched during hybridization. A method in which a nucleic acid probe labeled with a fluorescent dye is hybridized to a target nucleic acid, and the amount of decrease in light emission of the fluorescent dye before and after hybridization is measured, or a nucleic acid primer labeled with a fluorescent dye is hybridized to the target nucleic acid and then extended. And a method of measuring the decrease in the amount of fluorescence during the annealing reaction and the extension reaction with respect to the amount of fluorescence during denaturation of the nucleic acid. Furthermore, a real-time quantitative PCR method using the method, and a data analysis method having a process of correcting the fluorescence intensity value during the annealing reaction with that during the denaturation reaction when analyzing the data obtained by the PCR method are described Has been. However, only the description of Patent Document 7 cannot easily imagine a method for rapidly detecting a nucleic acid while maintaining specificity and high sensitivity. In other words, when a nucleic acid probe whose fluorescence is quenched during hybridization is used, the nucleic acid probe inhibits the nucleic acid amplification process, particularly the extension process. When a quenching nucleic acid primer is used, extension starts from non-specific hybridization of the nucleic acid primer, resulting in a non-specific amplification product that detects a decrease in fluorescence and is difficult to detect specifically. It has been difficult to detect nucleic acids quickly while maintaining specificity and high sensitivity using the technique disclosed in Document 7.
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 Japanese Patent No. 3437816 Nucleic acid sequence-based amplification method; Nature Vol. 350, 91 (1991) Strand Displacement Amplification: Nucleic acid research Vol. 20, p. 1691 (1992) Transcription mediated amplification method; Clin. Microbiol. Volume 31, Page 3270 (1993) loop-mediated thermal amplification method: J Clin Microbiol. 2004 Volume 42: 1,956 isometric and chimeric primer-initiated amplification of nucleic acids: Kekaku. 2003 Volume 78, Page 533

As described above, in the method using the nucleic acid probe or nucleic acid primer whose fluorescence is quenched upon hybridization with the target nucleic acid, the nucleic acid probe inhibits the nucleic acid amplification process, particularly the extension process, and therefore the sensitivity is reduced when shortening the time. There is a problem that the target nucleic acid-specific detection is hindered by non-specific amplification of the nucleic acid primer. An object of the present invention is to overcome these problems and to rapidly detect a target nucleic acid while maintaining specificity and high sensitivity.

As a result of intensive studies in view of the above points, the present inventors have determined 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 the sample whose target nucleic acid is to be confirmed or at least the following (a) and (b), light of the excitation wavelength of the fluorescent substance of the labeled probe is given continuously or intermittently and the fluorescence of the labeled substance is measured A detection method comprising performing high-speed PCR and examining the presence or amount of the target nucleic acid.
(A) a labeled probe having the following properties (i) to (iv): (i) an oligo that can form a double-stranded nucleic acid structure with a sequence complementary to the target nucleic acid and has a length of 14 to 30 bases It has a nucleotide (ii) The end of the oligonucleotide is modified with a fluorescent substance that has a property of quenching when the double-stranded nucleic acid structure is formed. (Iii) It is modified with a fluorescent substance when the double-stranded nucleic acid structure is formed. At least one pair of G (guanine) and C (cytosine) is present at the terminal part. (Iv) The concentration of the labeled probe is 10 times or less with respect to the concentration of the second primer. (B) Next The first primer and the second primer having the properties (i) to (iii) of (i) (i) The extension product of the first primer has a sequence 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 serves as 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. (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. The first primer and the second primer of (b) are 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 (A), when the distance from the region where the labeled probe forms a double-stranded nucleic acid structure to the 3 ′ end of the extension product is (B), the value of (A / B) is 0.1 or more and 3 0.0 or less,
(1) A detection method according to (1).
3. (V) introducing a target site and 3 or less non-complementary bases into the labeled probe of (a);
1 or 2 detection method characterized by these.
4). 3. Three detection methods, wherein a non-complementary base is introduced into the center of the labeled probe.
5. 5. The detection method according to any one of 1 to 4, wherein the fluorescent substance modified with the labeled probe of (a) is modified at the end of the labeled probe, and the base at the end is G or C.
6). The detection method according to any one of 1 to 5, wherein the concentration of the first primer is 4 times or more the concentration of the second primer.
7). The detection method according to any one of 1 to 6, wherein the high-speed PCR repeats the increase and decrease in temperature at a rate of 10 ° C / second or more.
8). further,
(C) in the presence of a DNA polymerase having a DNA synthesis rate of 100 bases / second or more;
8. The detection method according to any one of 1 to 7, wherein high-speed PCR is performed.
9. 8. The detection method according to 8, wherein the DNA polymerase further does not have 5 ′ exonuclease activity.
10. 10. The detection method according to 8 or 9, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a variant thereof.
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 (a) is a continuous base sequence of 14 to 30 bases selected from SEQ ID NO: 2,
The first primer of (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
(B) the second primer is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The present invention is 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 fluorescent substance of the labeled probe and measuring the fluorescence of the fluorescent substance. The detection method according to any one of 1 to 10.
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 (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The second primer of (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
The present invention is 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 fluorescent substance of the labeled probe and measuring the fluorescence of the fluorescent substance. The detection method according to any one of 1 to 10.
Subsequent to any of the methods of 13.1 to 12, while giving a light having an excitation wavelength of the fluorescent substance of the labeled probe in a sealed state and measuring the fluorescence of the fluorescent substance, 0.1 ° C./second or more A detection method comprising performing melting curve analysis with continuous 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 at position 2142 or 2143 of the 23S rRNA gene of H. pylori, and the presence of a mutation that discriminates antibiotic resistance is examined.
15. 13. The detection method according to 13 or 14, wherein the mutation site is located in the center of the oligonucleotide sequence of the labeled probe.
16. A kit for detecting the presence of H. pylori and / or the presence of H. pylori antibiotic resistance, comprising at least the following (a) and (b):
(A) a labeled probe having the following properties (i) to (ii) (i) having a oligonucleotide having a continuous base sequence of 14 to 30 bases selected from SEQ ID NO: 1 or SEQ ID NO: 2 (ii) A fluorescent substance having a property of quenching when the oligonucleotide forms a double-stranded nucleic acid structure with a sequence complementary to the oligonucleotide is labeled at the end of the oligonucleotide (b) Tm value is 70 And a primer containing a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3 and a Tm value of 70 to 90 ° C and 20 bases to 35 selected from SEQ ID NO: 4 Primer containing a continuous base sequence of no more than bases 17. 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 (a) is 4 times or more than the concentration of the other primer.
18. further,
(C) 16 or 17 kits comprising a DNA polymerase having a DNA synthesis rate of 100 bases / second or more.
20. 18 kits characterized in that the DNA polymerase further has no 5 ′ exonuclease activity.
20. 18 or 19 kit, wherein the DNA polymerase is KOD DNA polymerase (from Thermococcus kodakaraensis KOD1) or a variant thereof.
21. The kit of 16-20, 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.

According to the present invention, rapid nucleic acid detection is possible while maintaining specificity and high sensitivity. Specifically, the fluorescence signal decreases due to hybridization of the labeled probe to the target nucleic acid or the extension product of the first primer, and the extension of the second primer using the extension product of the target nucleic acid or the first primer as a template. Nucleic acid amplification can now be achieved during the same cycle of PCR. Furthermore, the use of a DNA polymerase with a high elongation rate shortened the amplification reaction time, and a nucleic acid detection method that was faster and more sensitive than ever was realized.
Furthermore, using the present invention, the presence or amount of the 23S rRNA gene of H. pylori is examined, the presence of a mutation of 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. In addition, by using a labeled probe that quenches fluorescence during hybridization apart from the primer for nucleic acid amplification, a specific amplified nucleic acid reflecting the presence or amount of the target nucleic acid can be made non-specific independent of the target nucleic acid. Can be clearly distinguished from typical amplified nucleic acids, and thus has the advantage of high sensitivity and accuracy. However, even if these are simply combined, rapid nucleic acid detection cannot be performed while maintaining specificity and 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). Is repeated in a short time, and the fluorescence probe quenches the fluorescence signal for the first time by hybridization to the target nucleic acid, so nucleic acid amplification starting from primer annealing to the target nucleic acid, This is because it is difficult to cause a decrease in the fluorescence signal due to hybridization of the labeled probe to the target nucleic acid during the same cycle of PCR. That is, even if a labeled probe that causes a decrease in fluorescence due to hybridization with a target nucleic acid is added to a high-speed PCR solution, annealing of the primer for nucleic acid amplification and a labeled probe that causes a decrease in fluorescence signal Competition with the target nucleic acid occurs during hybridization, and the target nucleic acid is amplified but does not decrease the fluorescent signal, or the hybridization of the labeled probe causes a decrease in the fluorescent signal but not the target nucleic acid. Neither can the presence or amount of the target nucleic acid, as well as the presence of mutations in the target nucleic acid, be examined rapidly while maintaining specificity and sensitivity. 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 a labeled probe that causes a decrease in fluorescence by hybridization are achieved. A nucleic acid detection method has been established in which the amount and the presence of a mutation in a target nucleic acid are rapidly examined while maintaining specificity and high sensitivity.

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. A method for speeding up the PCR method is disclosed in, for example, Japanese Patent Publication No. 2005-328709. Examples include shortening the time required for temperature change to reach each temperature of “denaturation”, “annealing”, and “extension”, shortening the time for maintaining the temperature of each step, and reducing the number of cycles. 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.

Further, as another means for achieving high-speed nucleic acid amplification, it is conceivable to shorten 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 100 bp to 300 bp in order to perform high-speed amplification and detection with a labeled probe.

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) Etc. 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. For example, 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. KOD DNA polymerase can be easily obtained from Toyobo (product code KOD-101, etc.).

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. For example, in addition to the above KOD DNA polymerase, as a DNA polymerase having a deoxyribonucleic acid synthesis rate of 100 bases / second or more, KOD Dash DNA polymerase (manufactured by Toyobo), PrimeSTAR MAX DNA polymerase (manufactured by TAKARA), Platinum Pfx DNA polymerase (manufactured by Invitrogen) ) Etc. are also commercially available.

It is further preferred that the DNA polymerase does not have 5 'exonuclease activity. In the present invention, the amount of decrease in fluorescence from the labeled probe is used for detection of the target nucleic acid. 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 for a DNA polymerase having 5 ′ exonuclease activity, such as Taq polymerase. Decomposes with increased non-specific signal. In the present invention, since the decrease in fluorescence that occurs upon hybridization of the labeled probe to the target nucleic acid is used for detection, an increase in non-specific signal increases the background, and accurate quantification or detection cannot be performed. The DNA polymerase having no 5 'exonuclease activity is not particularly limited, but for example, KOD polymerase is compatible with the present invention.

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. When the target nucleic acid and the labeled probe are hybridized, a double-stranded nucleic acid structure is formed, and the fluorescence of the labeled probe is reduced.

The “terminal” in the present invention refers to the base present at the extreme end on the 5 ′ side of the labeled probe, or the base present at the extreme end on the 3 ′ side in the base sequence of the labeled probe. The “terminal portion” indicates a base of 5 bases from the 5 ′ terminal base to the 3 ′ terminal or 5 bases from the 3 ′ terminal base to the 5 ′ terminal.

The fluorescent substance to be labeled on the labeled probe may be any fluorescent substance that does not have to be decomposed or attenuated in fluorescence during the nucleic acid amplification step, and may be a fluorescent substance that causes quenching during hybridization. In particular, a fluorescent substance that causes quenching of fluorescence upon base pairing of guanine and cytosine at the end of the labeled probe is preferable. Specifically, fluorescein or a derivative thereof (for example, fluorescein isothiocyanate (FITC)), BODIPY (trademark) series, rhodamine or a derivative thereof (for example, 5-carboxyrhodamine 6G (CR6G) or tetramethylrhodamine (TAMRA)), etc. However, the use of the BODIPY series or CR6G is particularly preferable. The method of binding the fluorescent dye to the oligonucleotide can be performed according to a usual method. By using quenching of fluorescent dyes, one fluorescent substance can be labeled without using dyes inserted into other double-stranded nucleic acid structures such as intercalators and without using two kinds of probes that cause the FRET phenomenon. The target nucleic acid sequence can be detected simply and specifically using the prepared probe. Although the labeling position of the fluorescent substance in the base sequence of the labeled probe is not particularly limited, it is preferably labeled at the end, and more preferably labeled at the end.

The selection of the base sequence of the labeled probe and the base sequence of the target nucleic acid complementary thereto is important in order to cause fluorescence quenching during hybridization. The base sequence of the labeled probe may be any base sequence that causes quenching upon hybridization with the target nucleic acid, and G (guanine) and C ( It may be designed so that at least one pair of cytosine) exists. If the end of the labeled probe is labeled, the terminal base is G or C, or G is present 1 or 3 bases away from the target nucleic acid base that forms a base pair with the end of the labeled probe. More preferably, the base sequence of the labeled probe is designed. It is further preferable that the end of the labeled probe is labeled and designed so that the terminal base is C.

If the terminal part cannot be designed to G or C from the base sequence of the target nucleic acid, the labeled probe is hybridized by intentionally introducing G or C into the base sequence of the first primer or the second primer, and the label It is possible to create a complementary base sequence that can quench the fluorescence of the probe. In this case, it is preferable to introduce G or C into the first primer in order to achieve a decrease in the fluorescence signal and amplification of the nucleic acid by extension of the second primer during the same PCR cycle. In addition, in order to avoid non-specific hybridization of the labeled probe to the first primer, the intentional introduction position of G or C is 4 to 8 bases from the 3 ′ end of the first primer to the 5 ′ end including the end base. It is preferable to design away. When designed at a position of 3 bases or less, the primer cannot be extended, and when designed at a distance of 9 bases or more, non-specific hybridization between the labeled probe and the primer is not likely to occur due to amplification.

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 50% dissociates. The Tm value can also be calculated by calculation. Various calculation methods have been devised for calculating the Tm value using the salt concentration in hybridization as a parameter. As a commonly used calculation method, the closest base pair method (Schildkraut C., Lifeson S. (1965) Dependence of the melting temperature of DNA on salt concentration-Biopolymers 3, B. 195-208, 195-208). , Frank R., Blocker H., Markey LA (1986) Predicting DNA duplex stability from the base sequence-Proc. Natl. Acad. Sci. Shaffer, J .; M Bonner, J .; Hirose, T .; Itakura, K .; (1979) Nucleic Acid Res. 6, 3543), GC method (Dependence of the Censor Temporation). (1965) BIOPOLYMERS 3.195-208, Optimization of the annealing for DNA amplification in vitro, W. Rychlik, Nucl. Acids Res. (1960) 18 The target nucleus The difference in Tm value due to the calculation method is affected depending on the sequence of the DNA or the detection conditions, etc., but the primer having the effect of the present invention is experimentally corrected by a study that can be easily conceived by those skilled in the art. And labeled probes can be set.

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 K. et al. J. et al. , Frank R. et al. Blocker H. , Markey L. A. (1986) Predicting DNA duplex stability from the base sequence-Proc. Natl. Acad. Sci. The Tm value calculated by the method described in USA 83, 3746-3750 is used.

Specifically, according to the above calculation method, the Tm value of a continuous base sequence of 24 bases selected from SEQ ID NO: 3 (5′-GAGATGGGAGCTGTCTCAACCCAGA-3 ′: SEQ ID NO: 5) is selected from 70.52, SEQ ID NO: 4. The Tm value of a continuous base sequence of 25 bases (5′-TGCGCATGAATTCCCATTAGCAGT-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 decrease in fluorescence signal by hybridization of a labeled probe to a target nucleic acid or first primer extension product, and amplification of a nucleic acid by extension of a second primer using the target nucleic acid or first primer extension product as a template. Balance is important. This balance is optimized by the Tm values of the second primer and the labeled probe, as well as the concentration of the second primer and the labeled probe. In order to optimize the balance, it is necessary to design the Tm value so that the second primer is larger than the labeled probe. Since this detection method uses the fact that the fluorescence emitted from the labeled probe is reduced by hybridization, the background fluorescence signal is high when the labeled probe concentration is high. The concentration of the labeled probe is preferably a concentration at which the background fluorescence signal decreases with respect to the fluorescence signal with decreased fluorescence upon hybridization. Specifically, it is preferably 10 times or less of the second primer. More preferably, it is 5 times or less. When it exceeds 10 times, it becomes impossible to detect the difference in fluorescence intensity when the fluorescence decreases due to the influence of the background fluorescence signal. The lower limit of the labeled probe concentration is not particularly limited, but a concentration that does not extremely reduce the hybridization efficiency between the labeled probe and the target nucleic acid or the extension product of the first primer is preferable. In order to balance the decrease in the fluorescence signal and the amplification of the nucleic acid, the concentration of the labeled probe is preferably higher than the concentration of the second primer. Specifically, the concentration of the labeled probe is preferably 10 nM or more. If it is less than 10 nM, it is difficult to detect the fluorescence signal with high sensitivity.

By satisfying the relationship between the Tm value and the concentration, the fluorescence signal is reduced 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 in the same cycle of high-speed 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. However, when the labeled probe cannot hybridize to the target nucleic acid or the extension product of the first primer, the decrease in the fluorescence signal does not occur (FIG. 1 (1). ) -A and FIG. 1 (2) -A), or hybridization of the labeled probe to the target nucleic acid or extension product of the first primer causes a decrease in fluorescence signal, but hybridization of the labeled probe inhibits primer extension In the case where the target nucleic acid is not amplified (FIG. 1 (1) -B and FIG. 1 (2) -B), the rate of occurrence of either one is large. The presence of this mutation 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. During the temperature decrease from the denaturation temperature to the annealing / extension temperature in the PCR process, the labeled primer hybridizes to the target nucleic acid or the extension product of the first primer, 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. In order not to inhibit the further extension of the second primer, the extension product of the second primer is formed as a template for the first primer (FIGS. 1 (1) -C and 1 (2) -C). Thus, the fluorescence signal is reduced by hybridization of the labeled probe to the extension product of the target nucleic acid or the first primer, and the nucleic acid is amplified by extension of the second primer using the extension product of the target nucleic acid or the first primer as a template. Is achieved during the same cycle of high-speed PCR, thereby combining the advantages of high-speed PCR with high speed and the advantages of labeled probe with high sensitivity and accuracy, and the presence or amount of target nucleic acid, as well as in target nucleic acid. A nucleic acid detection method for rapidly and highly sensitively detecting the presence of mutations could be established.

Furthermore, by using the DNA polymerase having a high extension rate 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 reduce the fluorescence signal and amplify the 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 fluorescence signal is reduced 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. The reduction of the fluorescence signal by hybridization of the labeled probe to the nucleic acid or the extension product of the first primer and the amplification of the nucleic acid by extension of the second primer using the extension product of the target nucleic acid or the first primer as a template are the same in PCR. It cannot be achieved during the cycle. As a balance between (A) and (B) for efficiently reducing the fluorescence signal and amplifying the 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 0.4 or more and 2.0 or less, and the extension products of the first primer and the second primer are 100 bp to 300 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, when designing the first primer, the second primer, and the labeled probe as shown in FIG. 3, the extension product by the first primer is preferentially generated by setting the concentration of the first primer higher than the concentration of the second primer. 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 still more 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-250 nM.

In fluorescence detection, for example, a double-stranded nucleic acid structure is formed by hybridization of the labeled probe with the target nucleic acid or the extension product of the first primer, and then the amount of increase in fluorescence caused by dissociation of the labeled probe and the target nucleic acid due to temperature rise is detected. It 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 mutation of the present invention is to have a different base relative to the wild type and is an event in the target nucleic acid. The presence of a non-complementary base with the target site in the labeled probe base sequence results in a decrease in fluorescence signal due to hybridization of the labeled probe to the extension product of the target nucleic acid or first primer, and the target nucleic acid or first primer. Advantageously, amplification of the nucleic acid by extension of the second primer using the extension product as a template 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 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 mutant base to be detected in the target nucleic acid, and two or more bases including the end are separated from the end of the labeled probe. Preferably it is. Further, when guanine or cytosine having a relatively strong base pair binding force is present in the labeled probe for 3 or more consecutive bases, it is preferably introduced into a continuous region of guanine or cytosine.

The positional relationship between the nucleotide sequence of the labeled probe and the mutation detects, for example, a decrease in fluorescence caused by hybridization between the nucleic acid probe and the target nucleic acid or the extension product of the first primer, and dissociation of the nucleic acid probe and the target nucleic acid due to an increase in temperature. In the case of using the 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 base mutation is known. Preferably, it should be 3 bases or more away from both ends of the base sequence of the labeled probe, 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. The central part refers to 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. The center in the case of an odd number of oligonucleotide sequences means the number of bases counted inward from the bases at both ends by counting the number of the oligonucleotide sequences plus 1 and dividing by 2 by including both ends. Say.

It is preferable to devise measures such that the Tm value of the labeled probe does not increase by labeling the 3 'end with a fluorescent dye or by adding treatment such as phosphorylation to the 3' end so as to extend during high-speed PCR. 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 increase in fluorescence caused by the dissociation of the labeled probe and the target nucleic acid due to temperature rise is detected. If the labeled probe is extended during high-speed PCR, the dissociation temperature between the labeled probe and the target nucleic acid cannot be accurately measured, and thus the above-described device is particularly necessary.

The present invention also includes the following.
A sample for which the presence or amount of the target nucleic acid is to be confirmed and at least in the presence of the following (a) and (b), light of an excitation wavelength of a fluorescent substance labeled on the labeled probe is applied continuously or intermittently and from the labeled probe Measure the presence or amount of the target nucleic acid while measuring the fluorescence of the target nucleic acid, and then, in the sealed state, give the light of the excitation wavelength of the fluorescent substance labeled on the labeled probe and from the labeled probe A detection method characterized by conducting a melting curve analysis with a continuous temperature rise or fall of 0.1 ° C./second or more while measuring the fluorescence of and measuring the presence of mutations in the target nucleic acid. In this case,
(A) a labeled probe having the following properties (i) to (iv): (i) an oligo that can form a double-stranded nucleic acid structure with a sequence complementary to the target nucleic acid and has a length of 14 to 30 bases (Ii) The end of the oligonucleotide is modified with a fluorescent substance that has a property of quenching when the double-stranded nucleic acid structure is formed. (Iii) The fluorescent substance is modified when the double-stranded nucleic acid structure is formed. There are at least one pair of G (guanine) and C (cytosine) within 3 bases from the terminal nucleotides. (Iv) The concentration of the labeled probe is 10 times the concentration of the second primer. (B) a first primer and a second primer having the following properties (i) to (iii): (i) the extension product of the first primer has a sequence complementary to the target nucleic acid and the second primer (Ii) the first primer and the second primer, and the extension product of the second primer has a sequence homologous to the target nucleic acid and serves as a template for the first primer. The length of the amplification product by the 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 the label. It is larger than the Tm value of the 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 is 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 triple-drug combination therapy of a proton pump inhibitor, amoxicillin and clarithromycin is effective, and has been widely practiced in Japan since 2000 AD as insurance medical treatment. 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, thereby achieving higher sensitivity. Detection of Helicobacter pylori is possible.

In recent years, the emergence of clarithromycin-resistant H. pylori (hereinafter referred to as CAM-resistant bacteria) has been reported, and detection of the CAM-resistant gene of H. pylori (Helicobacter pylori) has attracted attention. CAM-resistant Helicobacter pylori has been cited as one of the major factors for the reduction in the success rate of Helicobacter pylori eradication. CAM exerts its action by binding to the peptidyltransferase loop of H. pylori 23S rRNA and inhibiting its protein synthesis. However, it has been clarified that Helicobacter pylori acquires CAM resistance by a point mutation of 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 positions 2142 or 2143 in the above region (A2142G or A2143G), and 7% of CAM-resistant bacteria have cytosine at position 2142. Mutant (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-base mutations) A labeled probe that perfectly matches the 23S rRNA gene of the CAM resistant H. pylori species is used simultaneously. The fluorescence signal is detected in the “extension” step or the “annealing / extension” step in the PCR step. In this detection, when a labeled probe that completely matches the 23S rRNA gene of wild-type H. pylori is used, the wild-type H. pylori can be specifically detected. When a labeled probe corresponding to the above is used, specific detection of CAM-resistant H. pylori is possible. Furthermore, it is also included in the category of the present invention that the detection of wild-type H. pylori and CAM-resistant H. pylori can be performed simultaneously with one labeled probe by performing melting curve analysis. As for CAM-resistant H. pylori, as described above, the majority of the single nucleotide mutations are adenine at position 2142 or 2143 mutated to guanine (A2142G or A2143G) or mutated to cytosine at position 2142 (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, non-complementary bases of 3 bases or less indicate that when the labeled probe is the same and the target nucleic acid is different, the base nucleic acid is 3 bases or less including a single base mutation in the target nucleic acid.

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

Furthermore, the following detection method may be implemented using the present invention.
Using the first primer and the second primer of (b) while applying the excitation wavelength light of the fluorescent substance labeled on the labeled probe continuously or intermittently and measuring the fluorescence from the labeled probe of (a) High-speed PCR to examine the presence or amount of the 23S rRNA gene of Helicobacter pylori, and then, in a sealed state, give light at the excitation wavelength of the fluorescent substance labeled on the labeled probe and emit fluorescence from the labeled probe. A detection method comprising performing melting curve analysis with continuous temperature increase or decrease of 0.1 ° C./second or more while measuring, and examining the presence of a mutation in the 23S rRNA gene of H. pylori.

Furthermore, the following detection method may be implemented using the present invention.
Using the first primer and the second primer of (b) while applying the excitation wavelength light of the fluorescent substance labeled on the labeled probe continuously or intermittently and measuring the fluorescence from the labeled probe of (a) And the presence or amount of the 23S rRNA gene of Helicobacter pylori is examined, and subsequently, light in the excitation wavelength of the fluorescent substance labeled on the labeled probe is applied in a sealed state, and the labeled probe of (a) Melting curve analysis with continuous temperature increase or decrease of 0.1 ° C./sec or more while measuring fluorescence from the urine and examining the presence of mutations at positions 2142 or 2143 of the 23S rRNA gene. A detection method characterized by distinguishing antibiotic resistance.

A method for examining the presence or amount of the 23S rRNA gene of H. pylori, a method for examining the presence of a mutation in the 23S rRNA gene of H. pylori, and the presence of a mutation at positions 2142 or 2143 of the 23S rRNA gene. In the method for determining antibiotic resistance, when the target nucleic acid is the sense 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: 2,
The first primer of (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
(B) the second primer is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
When 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 (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The second primer of (b) may be a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3.

Moreover, although not necessarily limited, this invention can be utilized also for the detection of a mycobacteria. Culture methods are used to identify acid-fast bacteria, but acid-fast bacteria have a slow growth rate. In culture using Ogawa medium or the like, identification may take about one month. In recent years, genetic testing has been introduced for testing mycobacteria, and even if it is shortened, it takes 1 to 2 days. If the method of this invention is applied, a test | inspection can be completed within 30 minutes, and it becomes possible to detect an acid-fast bacterium in an extremely short time.

In addition, it can be applied to infectious disease items that need to be detected more quickly. For example, it can be used for detection of sexually transmitted diseases such as Chlamydia and Neisseria gonorrhoeae, hepatitis C virus, hepatitis B virus, multi-drug resistant Staphylococcus aureus, multi-drug resistant Pseudomonas aeruginosa, and septic bacteria. Furthermore, it can be applied to human genetic testing. For example, cancer gene testing can be detected within 30 minutes, so intraoperative diagnosis is possible.

“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.

  A method for determining clarithroma machine resistance on the 23S rRNA gene of H. pylori is described below.

Example 1
(Sample collection)
Genes were extracted from stomach 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 23S rRNA gene. All were designed from the known sequence of the 23S rRNA gene of H. pylori (GenBank accession number U27270).

PCR was performed using GeneAmp (registered trademark) 9700 (manufactured by Applied Biosystems) as a thermal cycler with 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. for 15 seconds, an annealing step of 60 ° C. for 30 seconds, and an extension step of 68 ° C. for 60 seconds was repeated 35 times. Other PCR conditions are the same as usual (see, for example, Gene Manipulation Technology Manual, Medical School, published in 1995). The obtained nucleic acid fragment was ligated to pUC18 which was cleaved with a blunt end 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 mutant of the 23S rRNA gene is 2142G, and the 2143 mutant 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 contractor (Nippon Bio Service, Sawaddy, GENSET KK, Sigma-Aldrich Japan, 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 primers, primers F1 to F4 each have 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 in which 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 with the reaction solution shown in Table 2 as a template. 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. Moreover, the composition of Table 2 is an example, and optimization of the reaction composition can be easily performed by those skilled in the art. The amount of plasmid used was 10 ⁴ copies and 10 ² copies. Primers F4 and R4 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. The measurement mode was 530 nm. Unless otherwise specified, all measurements shall be made with a light cycler (registered trademark). 4 to 6 are graphs showing the analysis results with the horizontal axis of the graph being the number of cycles n and the vertical axis being the fluorescence signal value. Since the fluorescence signal value emitted from the labeled probe A that appears as the background is calculated as 0 by the analysis of the light cycler software, the smaller the numerical value on the vertical axis, the greater the target nucleic acid for the labeled probe.

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 beyond the 30 cycles at 10 ⁴ copies, from the vicinity beyond the 38 cycles for the amplification products of labeled probe target nucleic acid 10 ² copies A decrease in the fluorescence signal value upon hybridization was confirmed. When a mutant plasmid in which a single base mutation exists is used, two non-complementary bases exist in the labeled probe A, and the hybridization ability of the labeled probe A to the target nucleic acid during the amplification reaction is reduced. No decrease 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. It was found that even in the plasmid of 10 ² copies can be detected with labeled probes specific and 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 a probe in which one deliberate non-complementary base is introduced into a 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, this 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 a decrease in the fluorescence signal value only when the 2142G mutant plasmid was used as a template. When NC, WT and 2143G templates were used, no decrease in fluorescence signal value occurred. According to the present invention, it can be seen that the target nucleic acid can be detected by a decrease in the fluorescence signal specific to the base sequence of the target nucleic acid.

(Example 5)
The examination which changed the combination with primer F1-F5 and primer R1-R5 was performed. Conditions other than the primer were in accordance with Example 3. Template was used the 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 emitted from the BODIPY-FL (trademark) labeled with the labeled probe A falls below a certain value (−0.1) during measurement in the light cycler real-time PCR ( Ct value). Combinations in which no decrease in fluorescence signal was observed indicated (-) instead of numerical values. The value of (A / B) is (distance from the base homologous to the 5 ′ end of the 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 the primer F in the target nucleic acid) The distance from the base to the base complementary to the 5 ′ end of the labeled probe).
The conditions used were that the labeled probe was 14 to 30 bases, the concentration of the labeled probe was 10 times or less the concentration of the second primer, and the Tm values of the first primer and the second primer were 70 ° C. or more and 90 ° C. The following conditions are satisfied and all the conditions that the Tm value of the second primer is larger than the Tm value of the labeled probe are satisfied.

From Table 4, when the value of (A / B) is 0.5 to 1.5 and the size of the amplification product is a combination of primers up to 300 bp, a result equivalent to the combination of primers F4 and R4 can be obtained. It was possible. When R5 having a large A value was used, the extension reaction with each primer F was not completed up to primer R5, and no amplification was observed. Further, even when B was extremely short (A / B numerical value of 0.3 or less), no fluorescence signal value was obtained. From this result, it can be seen that it is advantageous that the amplification product length is within 300 bp and the A / B value is close to 1.
Moreover, although all the results of Table 4 are performed under the conditions of high-speed PCR, for example, the combination of primer F5 and primer R1 has not led to detection. When the reaction solution using the primer F5 and the primer R1 was confirmed by agarose gel electrophoresis, an electrophoresis band could not be confirmed. It can be seen that the target nucleic acid cannot be detected rapidly only by using conventional high-speed PCR conditions.

The number of cycles when the fluorescence signal falls below (−0.1) is an indicator of amplification detection efficiency. In Table 4, when the number of cycles is divided into 34 or less, 34 to 35.5 or less, 35.5 to 38 or less, 38 to 42 or less, the amplification detection efficiency is 34 or less> 34 and> 35.5 or less> More than 35.5 and 38 or less> 38 and 42 or less. The result of 34 or less was obtained under the condition that the numerical value of (A / B) was 0.50 to 1.5 and the size of the amplification product was included in 100 to 300 bp. Below 34, the balance between primer and probe positions and amplification product length was the best. The result of 34 to 35.5 was shown under the condition that the numerical value (A / B) was 0.40 to 2.0 and the size of the amplification product was included in 100 to 300 bp. Next, the result of exceeding 35.5 and not more than 38 was a condition in which the numerical value of (A / B) was 0.30 to 3.0 and the size of the amplification product was included in 100 to 300 bp. The result exceeding 38 and not more than 42 was near the detection limit under the conditions of Example 5, and was a condition in which the numerical value of (A / B) was 0.30 to 3.0 and the size of the amplification product was included in ~ 400 bp. .
Although the results are not illustrated, if other gene sequences are targeted, the condition is (A / B) that is 0.10 to 3.0 and the size of the amplification product is within 50 bp to 500 bp. Rapid detection is possible by optimizing conditions other than the position of the primer / probe in the invention and the length of the amplified product. Using the information disclosed in the present invention, it is not difficult to optimize the conditions.

(Example 6)
(Examination of first primer concentration and second primer concentration ratio)
The concentration ratio of the first primer to the second primer was examined at 1 and 10 times. In 1-fold, primer F4 500 nM and primer R4 500 nM were used. For 10 times, primer F4 150 nM and primer R4 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). FIG. 6 (comparative example) shows the results with a concentration ratio of 1 and FIG. 5 shows the results with a concentration ratio of 10.

  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, the fluorescence signal decreased specifically only with the perfect match template. When the concentration ratio was 1 time, detection was not possible due to a decrease in the fluorescence signal even when a labeled probe whose template and base sequence were completely identical was used. When asymmetric PCR is performed, more target nucleic acid is amplified, and it can be seen that specific detection can be performed with higher sensitivity than symmetric PCR.

(Example 7)
(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 were examined: Taq polymerase (derived from Thermos aquaticus) and KOD polymerase (derived from Thermococcus kodakaraensis KOD1). Taq polymerase is Blend Taq-Plus- (registered trademark) manufactured by Toyobo Co., Ltd., and KOD polymerase is manufactured by Toyobo Co., Ltd. KOD-Plus-ver. 2 (registered trademark) was used. For Taq polymerase, the labeled probe A and primers F4 and R4 shown in Table 1 were used, and the reagent composition described in the protocol was referred to for other reagent compositions. The reaction conditions shown in Table 3 (nucleic acid amplification cycle) were used. For KOD polymerase, the same label probe A and primers F4 and R4 were used as in 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, it was possible to specifically detect H. pylori 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 can be adapted for high-speed amplification, but Taq polymerase cannot be adapted for high-speed amplification, and the desired nucleic acid amplification was not observed, so that H. pylori could not be detected. 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 8)
It was investigated whether a single labeled probe could detect a template when a single base mutation was present and a template when it was not present 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 F4 and R4 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 530 nm.

The results of fluorescence measurement when 10 ⁴ copies and 10 ² copies of plasmid were used as templates are shown in FIGS. 7 and 8, respectively. In the melting curve analysis using LightCycler (registered trademark), an increase in the amount of fluorescence when the labeled probe dissociates from the target nucleic acid is detected. In the figure, the vertical axis represents the value of the inverse sign of the first derivative of the fluorescence intensity (−dF / dt), and the horizontal axis represents the temperature (° C.). Two peaks were obtained from FIG. 7 and FIG. The peak observed at 61 ° C. is a WT peak, and the peak observed at 50 ° C. is a peak of two types of mutant H. pylori having a single base mutation relative to WT. From this result, it can be seen that WT and mutant types can be detected by separate peaks by performing melting curve analysis using one labeled probe.

Example 9
(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 which a sample was collected each 10 ⁹ cells, a heat treatment was carried out 95 ° C. 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 8 where the melting curve analysis was performed. The result is shown in FIG.

When wild type H. pylori and CAM resistant H. pylori were detected by melting curve analysis, both could be detected as a single peak by melting curve analysis. It can be performed within 30 minutes until the detection of microbial cells as a sample (CAM resistance determination).

(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 detection is performed by melting curve analysis, the temperature rise / fall rate is fast among the thermal cyclers. The Light Cycler (registered trademark), which has been used, requires more than 1 hour for amplification and detection.

  Under the reaction conditions of Example 3, the time required for detection of 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.

(Example 10)
(Examination of concentration ratio of second primer and labeled probe)
Using the labeled probe A, primer F4, and primer R4 shown in Table 1, the concentration ratio of the primer and the probe was examined. The conditions of Example 3 were used for conditions other than the primer and probe concentrations. Regarding the second primer (primer F4) and the probe concentration, the following four conditions were used.
Second primer 150 nM, probe 150 nM (probe / primer ratio = 1): FIG.
Second primer 100 nM, probe 500 nM (probe / primer ratio = 5): FIG.
Second primer 100 nM, probe 1000 nM (probe / primer ratio = 10): FIG.
Second primer 100 nM, probe 1100 nM (probe / primer ratio = 11)
Template with a plasmid 10 ⁴ H. pylori WT.

  4, 10, and 11, when the probe concentration / primer concentration is 1, MT can be detected without any problem. When the concentration ratio was 5, the amplification was limited due to the large number of probes. Therefore, although the Ct value was slightly slower than when the concentration ratio was 1, MT could be detected. At a concentration ratio of 10, amplification limitation became strong due to excessive probe, and the background signal increased due to an increase in the number of probes emitting fluorescence without hybridization to the probe that had been hybridized to the target nucleic acid and quenched. Thus, although the signal was weaker than the concentration ratio of 1 or 5, MT was detected. Although the data is not shown, if the concentration ratio is further increased to 11, the number of probes that have undergone amplification restriction and that have not hybridized at the signal detection temperature are extremely large, and the background signal causes a fluorescent signal. It could not be detected for two reasons that the difference in

(Example 11)
(Examination of probe base number)
The number of probe bases was examined using labeled probes A and C to E described in Table 1. The conditions of Example 3 were used for conditions other than the labeled probe. The labeled probe A has 19 bases (Tm = 62.20), the labeled probe C has 12 bases (Tm = 33.47), the labeled probe D has 16 bases (Tm = 54.34), and the labeled probe E has 32 bases (Tm = 80.65). The labeled probe C was detected by changing the annealing temperature to 50 ° C. Template with a plasmid 10 ⁴ H. pylori WT. The results detected using labeled probe A and labeled probe D are shown in FIGS. 4 and 12, respectively.

  From FIG. 4 and FIG. 12, it was confirmed that WT was detected with labeled probe A and labeled probe D containing 14 to 30 bases of the labeled probe. When the labeled probe C having 12 bases and the labeled probe E having 32 bases were used, no decrease in the fluorescence signal was observed and WT detection could not be confirmed. Particularly, the Tm value of the 32-base labeled probe E is 80.65, which is higher than the Tm value of 71.66 of the second primer, and the primer extension reaction is caused by hybridization of the labeled probe E to the target nucleic acid. It is thought that it was inhibited. This result also shows that rapid detection of the target nucleic acid cannot be realized simply by adding a fluorescently labeled nucleic acid probe to the conventional high-speed PCR method.

Example 12
(Examination of probe base number: melting curve analysis)
Detection by melting curve analysis was performed by examining the number of probe bases using the labeled probes A and C to E described in Table 1. Regarding the conditions other than the labeled probe, the conditions of Example 8 were used. Template by using the H. pylori WT, 2142G, three kinds of plasmids 2143G, amount of the plasmid using ¹⁰⁴ copies.

  From FIG. 7 and FIG. 13, the detection peaks of WT, 2142G, and 2143G could be confirmed in labeled probe A and labeled probe D, in which the number of labeled probe bases included in 14 to 30 bases. When the labeled probe C having 12 bases and the labeled probe E having 32 bases were used, respective detection peaks of WT, 2142G, and 2143G could not be confirmed. When the amplification product of the labeled probe E was confirmed by agarose gel electrophoresis, no electrophoresis band was observed at the target position, and amplification inhibition occurred.

  A method for detecting acid-fast bacteria (Mycobacterium spp.) Targeting the 16S rRNA gene is shown below.

(Example 13)
(Preparation of template)
Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium kansasii were each cultured in Ogawa's medium (manufactured by Nissui) for about 2 weeks. Using a solution obtained by suspending each cultured bacterial cell in a phosphate buffer, phenol / chloroform extraction was performed according to a conventional method to extract genomic DNA. The extracted genomic DNA was stored at -20 ° C. Mycobacterium tuberculosis is referred to as MT, avian M. tuberculosis is referred to as MA, and Mycobacterium kansasii is referred to as MK.

(Example 14)
(Synthesis of primer and labeled probe)
Primers and labeled probes used for detection were requested from a nucleic acid synthesis contractor (Nippon Bio Service, Sawaddy, GENSET KK, Sigma-Aldrich Japan, etc.).

  Table 6 shows the base sequences of the labeled probes and primers used. SEQ ID NOs: 34 to 38 are labeled probes, SEQ ID NOs: 34, 37, and 38 are MT, SEQ ID NO: 35 is a probe for detecting MA, and SEQ ID NO: 36 is a probe for detecting MK. SEQ ID NOs: 20 to 33 are common primers that can amplify a partial region of the acid-fast bacterium 16S rRNA gene.

(Example 15)
(MT, MA, MK detection)
20 ng of each genomic DNA of MT, MA, and MK was mixed with the reaction solution shown in Table 7 or Table 2 as a template. Hereinafter, based on the reaction composition of Table 7 or 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. Moreover, the composition of Table 7 or Table 2 is an example, and optimization of the reaction composition can be easily performed by those skilled in the art. Primers F6 and R6 shown in Table 6 were used as primers. Labeled probes F to H listed in Table 6 were used as the labeled probes. The labeled probe F is a labeled probe having a base sequence that completely matches the base sequence of MT. The labeled probe G is a labeled probe having a base sequence that completely matches the base sequence of MA. The labeled probe H is a labeled probe having a base sequence that completely matches the base sequence of MK. The composition shown in Table 7 was used when the labeled probe F was used, and the composition shown in Table 2 was used when the labeled probes G and H were used. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for the measurement of amplification and fluorescence. The conditions shown in Table 8 were used as PCR conditions. The measurement mode was 530 nm. Hereinafter, unless otherwise specified, all measurements shall be made with a light cycler (registered trademark). 14 to 18 are graphs showing analysis results with the horizontal axis of the graph being the cycle number n and the vertical axis being the fluorescence signal value. Since the fluorescence signal value emitted by the labeled probe that appears as the background is calculated as 0 by the analysis of the light cycler software, the smaller the numerical value on the vertical axis, the more target nucleic acid to the labeled probe.

  As a result of the analysis, MT using the labeled probe F, MA using the labeled probe G, and MK using the labeled probe H are shown in FIGS. 14-16, NC shows the negative control which uses milli Q water for a template. From FIG. 14 using the labeled probe F, the detection signal specific to MT is detected. From FIG. 15 using the labeled probe G, the detection signal specific to MA is displayed. From FIG. Each specific detection signal could be confirmed.

(Example 16)
(Examination of second primer and probe concentration ratio)
Using the labeled probe F, the primer F6, and the primer R6 described in Table 6, the concentration ratio of the primer and the probe was examined. The conditions of Example 12 were used for conditions other than the primer and probe concentrations. As a template, 20 ng of MT genomic DNA was used. Regarding the second primer (primer R6) and the probe concentration, the following three conditions were used.
Second primer 150 nM, probe 150 nM (probe / primer ratio = 1): FIG.
Second primer 150 nM, probe 450 nM (probe / primer ratio = 5): FIG.
Second primer 150 nM, probe 1650 nM (probe / primer ratio = 11): FIG.

  14, 17, and 18, MT can be detected without problems when the probe concentration / primer concentration is 1, whereas when the concentration ratio is 5, the Ct value is limited due to amplification limitation due to the large number of probes. It is late. Further, in FIG. 18 where the concentration ratio was as high as 11, the number of probes that were not hybridized at the signal detection temperature was extremely increased along with amplification limitation, and the difference in fluorescence signal was not observed due to the background signal. In the examination using labeled probe F, primer F6, and primer R6, detection was not possible at probe concentration / primer concentration = 11, but the fluorescence intensity of the labeled probe was low, and the Tm of the second primer was lower than the Tm of the probe concentration. In the case of extremely large values, there is a combination in which detection can be confirmed at probe concentration / primer concentration = 10.

(Example 17)
(Examination of Tm of primer and Tm of probe)
Using the labeled probes F, I, and J shown in Table 6, primers F6-8, and primers R6-8, it was examined whether a difference in MT detection would appear depending on the Tm of the primer and the probe. Primers F6 and R6, primers F7 and R7, and primers F8 and R8 were immobilized as primer sets, and primer sets having different Tm were obtained. A combination of 3 types of labeled probes and 3 types of primer sets was examined. Basically, the conditions of Example 15 were used for conditions other than the labeled probe and primer set. Only in the case of labeled probe I, the annealing / extension temperature of the PCR conditions in Table 8 was changed from 60 ° C to 57 ° C. The results are shown in Table 9. “◯” in Table 9 indicates that Ct value = less than 40, “Δ” indicates Ct value = 40 or more, and “x” is not detected. For example, the result of the combination of the labeled probe F and the primers F6 and R6 is shown in FIG. 14. In this case, the Ct value = 31.35 is “◯”. FIG. 14 is also obtained for other combinations, but only the results are shown here.

  From Table 9, a difference was observed in the Ct values detected by the Tm of the primer and the probe. For example, in the combination of primers F6 and R6 and labeled probe F, detection was possible without problems because probe Tm <primer Tm. On the other hand, the combination of primers F8 and R8 and labeled probe J could not be detected because probe Tm> primer Tm. When probe Tm> primer Tm in the high-speed detection, it is considered that primer extension was inhibited by the hybridization of the probe, and amplification was inhibited.

(Example 18)
(Examination of primer Tm)
Using the labeled probe F and the primers F6 and F8 to 12, and the primers R6 and R8 to 12 shown in Table 6, whether or not a difference in MT detection appears depending on the primer Tm value was examined. Detection was performed by combining six types of primer F and six types of primer R. The conditions of Example 15 were basically used for the conditions other than the primers. However, the annealing / extension temperature under PCR conditions was set in the range of 50 ° C. to 60 ° C. according to the Tm of each primer set. The results are shown in Table 10. The determination criteria were the same as in Example 17.

  From Table 10, if the Tm of the primer is 70 or more, it is detected without any problem, but if the primer Tm of either primer F or R is less than 70, the condition balance of high-speed detection is lost and the Ct value is delayed. I can see that In the present invention, since the balance of conditions such as the concentration of the primer and the labeled probe and Tm becomes important, the primer Tm is suitably 70 ° C. or higher for stable detection.

Example 19
(Detection by melting curve analysis of MT and MA)
It was investigated whether a single labeled probe could detect a template when a single base mutation was present and a template when it was not present by performing a melting curve analysis following the nucleic acid amplification step. The template used was 20 ng of MT or MA genome. The same reagent composition and reaction conditions as in Example 15 were used as shown in Table 8 (Nucleic acid amplification cycle) / (Melting curve analysis) / (Cool down). Primers F6 and R6 and nucleic acid probe F shown in Table 6 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 530 nm.

    FIG. 19 shows the result of fluorescence measurement when MT and MA genomes were used as templates. In the melting curve analysis using LightCycler (registered trademark), an increase in the amount of fluorescence when the labeled probe dissociates from the target nucleic acid is detected. In the figure, the vertical axis represents the value of the inverse sign of the first derivative of the fluorescence intensity (−dF / dt), and the horizontal axis represents temperature (° C.). In FIG. 19, two peaks were confirmed. The peak observed at 60 ° C. is an MT detection peak having a base sequence that completely matches the labeled probe F. The peak observed at 55 ° C. is a detection peak of MA having a base sequence in which a single base mutation exists with respect to the labeled probe F. From this result, it was found that MT and MA species, which are acid-fast bacteria, can be detected as separate peaks by performing melting curve analysis using one labeled probe.

(Comparative Example 2)
(Time to MT detection)
The time required to detect a positive control using the Amplicomycobacterium tuberculosis kit of Roche Diagnostics, which is generally used for genetic testing of MT, is about 6 hours at the shortest until PCR and detection are possible.

  Under the reaction conditions of Example 15, the time required to detect the target nucleic acid using LightCycler (registered trademark) is about 20 minutes, and the time required to detect MT using melting curve analysis is within 30 minutes. Met. By using the detection method of the present invention, MT detection can be performed in a very short time.

  According to the present invention, rapid and highly sensitive nucleic acid detection is possible. Specifically, by measuring the amount of decrease in the required fluorescence signal, the unnecessary background fluorescence signal value is reduced as much as possible while maintaining the amplification efficiency, depending on the nature and concentration of the labeled probe that does not inhibit the extension reaction of the second primer. Is possible. Furthermore, by using a DNA polymerase with a high extension rate, the amplification reaction time can be shortened, and a nucleic acid detection method that is faster and more sensitive than ever can be realized. Further, the presence or amount of the 23S rRNA gene of H. pylori is examined. 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 the antibiotic resistance of 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 will be described. 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 between the first primer, the second primer and the labeled probe is shown for the asymmetric PCR method in the present invention. Plasmid ¹⁰⁴ copies shows real-time detection results of H. pylori as the template. Plasmid ¹⁰² copies shows real-time detection results of H. pylori as the template. 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. Plasmid ¹⁰² 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 having detected the Helicobacter pylori using what processed the microbial cell simply was used as a template is shown. The real-time detection result of Helicobacter pylori under the condition of probe / primer ratio = 5 is shown. The real-time detection result of Helicobacter pylori under the condition of probe / primer ratio = 10 is shown. The real-time detection of H. pylori using labeled probe D is shown. The result of simultaneous detection of wild type and mutant type of H. pylori by melting curve analysis when labeled probe D is used is shown. The real-time detection result of MT when targeting 16S rRNA gene is shown. The real-time detection result of MA when targeting 16S rRNA gene is shown. The real-time detection result of MK when targeting 16S rRNA gene is shown. The real-time detection result of MT in the condition of probe / primer ratio = 5 is shown. The real-time detection result of MT in the condition of probe / primer ratio = 11 is shown. The results of discriminating MT and MA by peak Tm by melting curve analysis using MT and MA genomes as templates are shown.

Claims (21)

In the presence of the sample whose target nucleic acid is to be confirmed or at least the following (a) and (b), light of the excitation wavelength of the fluorescent substance of the labeled probe is given continuously or intermittently and the fluorescence of the labeled substance is measured A detection method comprising performing high-speed PCR and examining the presence or amount of the target nucleic acid.
(A) a labeled probe having the following properties (i) to (iv): (i) an oligo that can form a double-stranded nucleic acid structure with a sequence complementary to the target nucleic acid and has a length of 14 to 30 bases It has a nucleotide (ii) The end of the oligonucleotide is modified with a fluorescent substance that has a property of quenching when the double-stranded nucleic acid structure is formed. (Iii) It is modified with a fluorescent substance when the double-stranded nucleic acid structure is formed. At least one pair of G (guanine) and C (cytosine) is present at the terminal part. (Iv) The concentration of the labeled probe is 10 times or less with respect to the concentration of the second primer. (B) Next The first primer and the second primer having the properties (i) to (iii) of (i) (i) The extension product of the first primer has a sequence 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 serves as 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. (Iii) The Tm value of the first primer and the second primer is 70 ° C. or higher and 90 ° C. or lower, and the Tm value of the second primer is larger than the Tm value of the labeled probe. The first primer and the second primer of (b) are 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 (A), when the distance from the region where the labeled probe forms a double-stranded nucleic acid structure to the 3 ′ end of the extension product is (B), the value of (A / B) is 0.1 or more and 3 0.0 or less,
The detection method according to claim 1. (V) introducing a target site and 3 or less non-complementary bases into the labeled probe of (a);
The detection method according to claim 1 or 2. The detection method according to claim 3, wherein a non-complementary base is introduced into the center of the labeled probe. 5. The detection according to claim 1, wherein the fluorescent substance modified with the labeled probe of (a) is modified at the end of the labeled probe, and the base at the end is G or C. Method. 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. The detection method according to any one of claims 1 to 6, wherein the high-speed PCR repeatedly increases and decreases in temperature at a rate of 10 ° C / second or more. further,
(C) in the presence of a DNA polymerase having a DNA synthesis rate of 100 bases / second or more;
8. The detection method according to claim 1, wherein high-speed PCR is performed. The detection method according to claim 8, wherein the DNA polymerase further does not have 5 'exonuclease activity. 10. The detection method according to claim 8 or 9, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a variant thereof. The target nucleic acid is the sense 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: 2,
The first primer of (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
(B) the second primer is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The present invention is 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 fluorescent substance of the labeled probe and measuring the fluorescence of the fluorescent substance. The detection method according to claim 1. 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 (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 4,
The second primer of (b) is a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3,
The present invention is 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 fluorescent substance of the labeled probe and measuring the fluorescence of the fluorescent substance. The detection method according to claim 1. Subsequent to the method according to any one of claims 1 to 12, while maintaining a sealed state, while applying light of an excitation wavelength of the fluorescent substance of the labeled probe and measuring fluorescence of the fluorescent substance, 0.1 ° C / second A detection method characterized by performing a melting curve analysis with the above continuous temperature rise or fall and examining the presence of a mutation in a target nucleic acid. The detection method according to claim 13, wherein the mutation is at position 2142 or 2143 of the 23S rRNA gene of Helicobacter pylori, and the presence of the mutation that determines antibiotic resistance is examined. The detection method according to claim 13 or 14, wherein the mutation site is located in the center of the oligonucleotide sequence of the labeled probe. A kit for detecting the presence of H. pylori and / or the presence of H. pylori antibiotic resistance, comprising at least the following (a) and (b):
(A) a labeled probe having the following properties (i) to (ii) (i) having an oligonucleotide having a continuous base sequence of 14 to 30 bases selected from SEQ ID NO: 1 or SEQ ID NO: 2 (ii) A fluorescent substance having a property of quenching when the oligonucleotide forms a double-stranded nucleic acid structure with a sequence complementary to the oligonucleotide is labeled at the end of the oligonucleotide (b) Tm value is 70 And a primer containing a continuous base sequence of 20 to 35 bases selected from SEQ ID NO: 3 and a Tm value of 70 to 90 ° C and 20 bases to 35 selected from SEQ ID NO: 4 Primer containing a continuous base sequence of less than the base 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 (a) is at least 4 times the concentration of the other primer. The described kit. further,
(C) The kit according to claim 16 or 17, comprising a DNA polymerase having a DNA synthesis rate of 100 bases / second or more. The kit according to claim 18, wherein the DNA polymerase further does not have 5 'exonuclease activity. The kit according to claim 18 or 19, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a variant thereof. The kit according to any one of claims 16 to 20, 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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