JP6379871B2 - Method for detecting Rifampicillin-resistant Mycobacterium tuberculosis - Google Patents

Description

The present invention relates to a method for detecting a rifampicillin resistance gene mutation in M. tuberculosis rapidly, reliably and simply.

Mycobacteria are Gram-positive rods with acid-fast properties, and there are many pathogens that cause serious lesions such as tuberculosis and leprosy. Among them, Mycobacterium tuberculosis is a pathogen that causes tuberculosis in humans, and its examination is extremely important clinically. Pulmonary tuberculosis is predominant in lesions caused by Mycobacterium tuberculosis, but tuberculosis lesions occur in almost all organs, including tuberculous pleurisy, tuberculous meningitis, caries, intestinal tuberculosis, renal tuberculosis, joint tuberculosis And so on. The source of infection is the patient, who is infected via the respiratory tract due to droplet infection.

Treatment of Mycobacterium tuberculosis is centered on chemotherapy, and most can be cured with appropriate chemotherapy. Examples of therapeutic agents used include isodidianide (INH), rifampicillin (RFP), rifabutin (RBT), pyrazinamide (PZA), ethambutol (EB), and streptomycin (SP) (Non-patent Document 1).

On the other hand, there are strains that have acquired drug resistance among Mycobacterium tuberculosis. As a test for drug-resistant bacteria, a drug susceptibility test is generally performed in which a therapeutic agent is contained in a culture medium and cultured. However, Mycobacterium tuberculosis is a bacterium that requires several weeks for culturing, and during that time, there is a problem that the treatment policy for tuberculosis patients cannot be determined.

It has been reported that drug resistance is acquired by mutation of a specific gene. Therefore, a method for estimating drug resistance by examining a gene mutation that causes drug resistance has been established.

Among these, it has been known for some time that resistance to rifampicillin is acquired when a mutation occurs in the rpoB gene (Non-Patent Documents 2 to 4). Therefore, it is possible to confirm rifampicillin resistance by examining the rpoB gene, and a drug resistance test kit by genetic testing is also commercially available (Non-patent Document 2). The kit amplifies a partial sequence of the rpoB gene by nested-PCR, and then detects a mutation in the base sequence corresponding to the codons 511 to 533 with a probe fixed to the test piece.

  Among the mutations in the rpoB gene, it is said that mutations occurring at codons 509 to 533 account for 95% of the total (Non-patent Documents 2 and 5). Therefore, most of the RFP-resistant Mycobacterium tuberculosis can be detected by detecting a mutation in the codon region of the Mycobacterium tuberculosis rpoB gene.

  Most mutations in the codon region are base substitution mutations. In addition, it has been reported that base insertion mutation can occur at codon 514, and base deletion mutation can occur at some codons (Non-patent Document 5).

  In genetic testing, detection is usually performed after the target nucleic acid is amplified by a nucleic acid amplification method such as PCR.In this case, contamination with a nucleic acid amplification product in the detection system and false positives is a problem. Become. One of the causes of contamination is opening the reaction vessel after the nucleic acid amplification reaction. Since the kit also detects a gene mutation by opening the reaction vessel after nested-PCR and mixing the nucleic acid amplification product with a detection reagent, the possibility of contamination cannot be excluded.

  Further, the above kit requires 2 hours 30 minutes or more for PCR and 105 minutes or more for detection after PCR, and a more rapid rpoB mutation detection method has been desired.

Tuberculosis Practice Guidelines, 2009, Japanese Society for Tuberculosis, edited by Nanedo Inagaki et al., Tuberculosis, 2010, 85, p. 703-709 Telenti et al. Lancet. 1993, 341, p. 647-650 Kapur et al. Pathol Lab Med. 1995, 119, p. 131-138 James M.M. Musser, Clin Micro Rev. 1995, p. 496-514

The problem to be solved by the present invention is the construction of an rpoB gene test system using a closed system in which no contamination occurs and the time reduction of the rpoB gene test.

As a result of intensive studies in view of the above problems, the present inventors have found that rpoB gene detection in a closed system is possible by using a specific probe including a quenching probe. Moreover, it was confirmed that the gene detection system can be tested in a shorter time than a conventional test kit, and the present invention was completed.

[1]
A method for detecting Rifampicillin resistance of Mycobacterium tuberculosis, comprising the following steps (1) to (3):
(1) Step of hybridizing a nucleic acid derived from Mycobacterium tuberculosis in a sample and the nucleic acid probe described in (A) and (B) below to form a complex (A) represented by SEQ ID NOS: 1 and 2 Two fluorescently labeled nucleic acid probes (B), one unlabeled nucleic acid probe represented by SEQ ID NO: 3
(2) A step of detecting the complex obtained in step (1) (3) A step of discriminating rifampicillin resistance of Mycobacterium tuberculosis by analyzing the data measured or detected in step (2) [2]
A method for detecting Rifampicillin resistance of Mycobacterium tuberculosis, comprising the following steps (1) to (3):
(1) Step of hybridizing a nucleic acid derived from Mycobacterium tuberculosis in a sample and the nucleic acid probe described in (A) and (B) below to form a complex (A) represented by SEQ ID NOs: 4 and 5 Two fluorescently labeled nucleic acid probes (B) one unlabeled nucleic acid probe represented by any one of SEQ ID NOs: 6 to 9
(2) The step of detecting the complex obtained in step (1) (3) The step of determining the rifampicillin resistance of Mycobacterium tuberculosis by analyzing the data measured or detected in step (2) [3]
The method for detecting rifampicillin-resistant Mycobacterium tuberculosis according to [1] or [2], wherein the detection target is codons 509, 510, 511, 512, 513, 514, 515, 516, 520, 521, 522, 523, The method according to [1] or [2], which is a base mutation occurring in any one or more of the group consisting of 526, 527, 529, 531 and 533.

According to the present invention, rifampicillin-resistant Mycobacterium tuberculosis can be detected in a closed system using a total of three nucleic acid probes.

It is a model figure which shows a part of detection system of this invention. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 4. It is a figure which shows the result of Example 4. It is a figure which shows the result of Example 4.

  The present invention relates to an oligonucleotide for detecting Rifampicillin tuberculosis and a method for detecting Rifampicillin-resistant tuberculosis using the oligonucleotide.

In the method of the present invention, Rifampicillin resistance of M. tuberculosis is performed by analyzing mutations in the rpoB gene. The rpoB gene in the present invention means the rpoB gene of Mycobacterium tuberculosis unless otherwise specified. The base sequence of the rpoB gene is shown as SEQ ID NO: 18 in this specification.

[Nucleic acid probe used in the present invention]
The nucleic acid probe used in the present invention is a nucleic acid probe for detecting rifampicillin resistance of Mycobacterium tuberculosis by detecting a mutation in the rpoB gene. The nucleic acid probe is not particularly limited as long as it can hybridize with part or all of the rpoB gene to form a complex. More preferably, it is a nucleic acid probe that specifically forms a complex only with the rpoB gene and does not form a complex with a nucleic acid having any other nucleotide sequence, more preferably a part or all of the nucleotide sequence of the rpoB gene A nucleic acid probe having the same or complementary base sequence.
The target with which the nucleic acid probe forms a complex may be the rpoB gene in the Mycobacterium tuberculosis genome present in the sample, or a nucleic acid amplification product obtained by nucleic acid amplification of a part or all of the gene. More preferably, it is a nucleic acid amplification product.

  The nucleic acid probe is any one selected from the group consisting of codons 509, 510, 511, 512, 513, 514, 515, 516, 520, 521, 522, 523, 526, 527, 529, 531 and 533 of the rpoB gene. Examples include nucleic acid probes for detecting mutations that can occur in one or more. Hereinafter, the mutation that can occur in the rpoB gene is referred to as an rpoB gene mutation group (when it is clear from the context, it is simply “mutation group”). The nucleic acid probe has a single base sequence and can detect one or more mutations in the codon.

The nucleic acid probe may have a property of “detecting a wild-type rpoB gene and distinguishing whether the codon has a mutation” or “the codon has a mutation. It may have the property of “detecting the rpoB gene only in the case. More preferably, it is a nucleic acid probe having “a property of detecting a wild-type rpoB gene and distinguishing whether or not there is a mutation in the codon”.

  The number of bases of the nucleic acid probe used in the present invention is not particularly limited. A preferred lower limit is 15 bases, more preferably 16 bases, and even more preferably 17 bases. The upper limit is preferably 33 bases, more preferably 31 bases, and even more preferably 29 bases.

  Examples of the nucleotide sequence of such a nucleic acid probe include SEQ ID NOs: 1-9.

As the nucleic acid probe used in the present invention, one having a sequence complementary to SEQ ID NOs: 1 to 9 may be used. Further, in SEQ ID NOs: 1 to 9, substitution of several bases may be included. The term “several bases” as used herein refers to 10 bases or less, more preferably 5 bases or less, more preferably 4 bases or less, more preferably 3 bases or less, more preferably 2 bases or less, more preferably 1 base or less.

Moreover, at least one of the nucleic acid probes used in the present invention may be composed of a part of the base sequence (or their complementary sequence) described in any one of SEQ ID NOs: 1 to 9. For example, each sequence number may be composed of an oligonucleotide consisting of 15 bases or more, more preferably 18 bases or more.
In other words, the longest nucleotide sequence of SEQ ID NOS: 1 to 9 is 29 bases, and the shortest one is 19 bases. Most preferably, it has the full length of the sequence shown in the sequence. As long as the “part of the base sequence” is within a range in which the total length is not less than 15 bases, at least one of the ends may be missing on the premise of maintaining the continuity of the sequence. In that case, the length of the base sequence is preferably longer. For example, in the case of SEQ ID NO: 1, it may be 15 bases or more, more preferably 16 bases or more, more preferably 17 bases or more, more preferably 18 bases or more, more preferably 19 bases or more, more preferably 20 bases or more. More preferably 21 bases or more, more preferably 22 bases or more, more preferably 23 bases or more, more preferably 24 bases or more, more preferably 25 bases or more, more preferably 26 bases or more, more preferably 27 bases or more, More preferably, it is 28 bases or more, and most preferably 29 bases. For example, in the case of SEQ ID NO: 9, it may be 15 bases or more, more preferably 16 bases or more, more preferably 17 bases or more, more preferably 18 bases or more, and most preferably 19 bases.

Furthermore, the nucleic acid probe may have several bases added to either end, as long as it includes the base sequence described in any of the above. By adding a base to either end, the total length of the probe is preferably 18 bases or more, more preferably 20 bases or more, and more preferably 23 bases or more. In this case, the upper limit of the total probe length is preferably 35 bases, more preferably 32 bases, and even more preferably 30 bases.

  In the present invention, the rpoB gene mutation group is detected by a plurality of nucleic acid probes. The nucleic acid probe used for detection of the mutation group is both a fluorescently labeled nucleic acid probe and an unlabeled nucleic acid probe. The number of these nucleic acid probes is not particularly limited, but the preferred lower limit is 2 and more preferably 3. The upper limit is preferably 6, more preferably 5, more preferably 4, and even more preferably 3. The number of unlabeled nucleic acid probes is preferably equal to or less than the number of fluorescently labeled nucleic acid probes.

  The position and number of nucleic acids to be labeled in the fluorescently labeled nucleic acid probe are not limited, but preferably the nucleic acid at the end of the nucleic acid probe is labeled, and more preferably one of the ends is labeled. is there.

  The fluorescent substance of the fluorescently labeled nucleic acid probe is not particularly limited, but is preferably a fluorescent substance that exhibits fluorescence alone and quenches when it forms a hybrid with the target nucleic acid. Examples of the fluorescent substance include, but are not limited to, fluorescein, phosphor, rhodamine, polymethine dye derivatives, and the like. Examples of commercially available fluorescent dyes include BODIPY FL (trademark, manufactured by Molecular Probes), FluorePrime (trademark, Amersham). Pharmacia), Fluoredite (trademark, manufactured by Millipore), FAM (ABI), Cy3 and Cy5 (Amersham Pharmacia), TAMRA (Molecular Probes), carboxyrhodamine 6G (CR6G), and the like.

  An unlabeled nucleic acid probe refers to a nucleic acid probe in which no fluorescent substance or biotin is labeled on any nucleic acid in the nucleic acid probe. However, it is not included in the label that either or both ends of the nucleic acid probe are phosphorylated. For this reason, even a non-labeled nucleic acid probe does not prevent the terminal nucleic acid from being phosphorylated.

[Nucleic acid primer set]
In the detection of rifampicillin-resistant Mycobacterium tuberculosis according to the present invention, the detection target is preferably a nucleic acid amplification product obtained by nucleic acid amplification of the rpoB gene in the genome of Mycobacterium tuberculosis contained in the sample. When using a nucleic acid amplification product as a detection control, a nucleic acid primer set for specifically amplifying a part of the rpoB gene may be used together with the nucleic acid probe described in the present invention.

  The nucleic acid primer set is used for nucleic acid amplification of a part of the rpoB gene, and the nucleic acid primer set is composed of one forward primer and one reverse primer. Furthermore, it is preferable that both the forward primer and the reverse primer have a Tm value equal to or higher than the Tm value of the nucleic acid probe.

  A known calculation method can be used for calculating the Tm value of the nucleic acid primer or nucleic acid probe in the present invention, and any method may be used. Specific calculation methods include closest base pair method, Wallace method, and GC% method.

  The number of bases of the nucleic acid primer constituting the nucleic acid primer set is not particularly limited. A preferred lower limit is 24 bases, more preferably 25 bases, and even more preferably 26 bases. A preferred upper limit is 35 bases, more preferably 34 bases, and even more preferably 33 bases.

  Examples of such a nucleic acid primer set include a combination of a base sequence represented by any of SEQ ID NOs: 10 to 13 as a forward primer and a base sequence represented by any of SEQ ID NOs: 14 to 17 as a reverse primer. .

  The primer set may be selected from a pair of oligonucleotides having a base sequence complementary to SEQ ID NOs: 10 to 17. In addition, substitution of several bases may be included in SEQ ID NOs: 10 to 17. The term “several bases” as used herein refers to 10 bases or less, more preferably 5 bases or less, more preferably 4 bases or less, more preferably 3 bases or less, more preferably 2 bases or less, more preferably 1 base or less.

In addition, at least one of the primer sets may be composed of a part of the base sequence (or a complementary sequence thereof) described in any one of SEQ ID NOs: 10 to 17. For example, in one set of primers, the forward primer and / or the reverse primer may be composed of an oligonucleotide consisting of 15 bases or more, more preferably 18 bases or more of each SEQ ID NO.
In other words, the longest of the base sequences described in SEQ ID NOs: 10 to 17 is 32 bases, and the shortest is 27 bases. Most preferably, it has the full length of the sequence shown in the sequence. As long as the “part of the base sequence” is within a range in which the total length is not less than 15 bases, at least one of the ends may be missing on the premise of maintaining the continuity of the sequence. In that case, the length of the base sequence is preferably longer. For example, if it is SEQ ID NO: 10, it may be 15 bases or more, more preferably 16 bases or more, more preferably 17 bases or more, more preferably 18 bases or more, more preferably 19 bases or more, more preferably 20 bases or more. More preferably 21 bases or more, more preferably 22 bases or more, more preferably 23 bases or more, more preferably 24 bases or more, more preferably 25 bases or more, more preferably 26 bases or more, more preferably 27 bases or more, More preferably, it is 28 bases or more, more preferably 29 bases or more, more preferably 30 bases or more, more preferably 31 bases or more, and most preferably 32 bases. Further, for example, in the case of SEQ ID NO: 17, it may be 15 bases or more, more preferably 16 bases or more, more preferably 17 bases or more, more preferably 18 bases or more, more preferably 19 bases or more, more preferably 20 Bases or more, more preferably 21 bases or more, more preferably 22 bases or more, more preferably 23 bases or more, more preferably 24 bases or more, more preferably 25 bases or more, more preferably 26 bases or more, and most preferably 27 bases It is.

Furthermore, as long as at least one of the primer sets includes the base sequence described in any of the above, several bases may be added to any end. By adding a base to any terminal, the total length of the primer is preferably 18 bases or more, more preferably 20 bases or more, and more preferably 23 bases or more. In this case, the upper limit of the total probe length is preferably 35 bases, more preferably 32 bases, and even more preferably 30 bases.

[Method for detecting rifampicillin-resistant Mycobacterium tuberculosis]
The method for detecting Rifampicillin-resistant Mycobacterium tuberculosis according to the present invention is characterized by detecting a mutation in the rpoB gene of Mycobacterium tuberculosis present in a sample.

One embodiment of the present invention is a method for detecting rifampicillin resistance of Mycobacterium tuberculosis, comprising the steps shown in the following (1) to (3).
(1) Step of hybridizing a nucleic acid derived from Mycobacterium tuberculosis in a sample and the nucleic acid probe described in (A) and (B) below to form a complex (A) represented by SEQ ID NOS: 1 and 2 Two fluorescently labeled nucleic acid probes (B), one unlabeled nucleic acid probe represented by SEQ ID NO: 3
(2) A step of detecting the complex obtained in step (1) (3) A step of discriminating rifampicillin resistance of M. tuberculosis by analyzing data measured or detected in step (2)

One embodiment of the present invention is a method for detecting rifampicillin resistance of Mycobacterium tuberculosis, comprising the steps shown in the following (1) to (3).
(1) Step of hybridizing a nucleic acid derived from Mycobacterium tuberculosis in a sample and the nucleic acid probe described in (A) and (B) below to form a complex (A) represented by SEQ ID NOs: 4 and 5 Two fluorescently labeled nucleic acid probes (B) one unlabeled nucleic acid probe represented by any one of SEQ ID NOs: 6 to 9
(2) A step of detecting the complex obtained in step (1) (3) A step of discriminating rifampicillin resistance of M. tuberculosis by analyzing data measured or detected in step (2)

  In each of the above embodiments, two nucleic acid probes fluorescently labeled for rifampicillin resistance of Mycobacterium tuberculosis and one unlabeled nucleic acid probe are used.

  In the above embodiment, a nucleic acid probe having the nucleotide sequence represented by SEQ ID NO: 1 or 2 as two fluorescently labeled nucleic acid probes is a nucleic acid having a nucleotide sequence represented by SEQ ID NO: 3 as an unlabeled nucleic acid probe. Each probe can be exemplified. Alternatively, a nucleic acid probe having the nucleotide sequence represented by SEQ ID NOs: 4 and 5 as two fluorescently labeled nucleic acid probes, or a nucleotide sequence represented by any one of SEQ ID NOs: 6 to 9 as an unlabeled nucleic acid probe Each of the nucleic acid probes having can be exemplified. However, the nucleic acid probe used in the method of the present invention is not limited to SEQ ID NOs: 1 to 9, and any nucleic acid probe can be used as long as it can be used to detect Rifampicillin resistance of Mycobacterium tuberculosis using molecular biological techniques. Even base sequences and labeled forms can be used.

The detection target in the method of the present invention is any of the group consisting of codons 509, 510, 511, 512, 513, 514, 515, 516, 520, 521, 522, 523, 526, 527, 529, 531 and 533. Or base mutations occurring in one or more. In the detection method of the present invention, mutations that can occur in all of the codons may be detected, or only some of the codons may be detected. Further, all mutations that can occur in codons may be detected, or only some mutations may be detected.

In the method of the present invention, the nucleic acid derived from Mycobacterium tuberculosis in the sample may be a nucleic acid corresponding to the rpoB gene in the Mycobacterium tuberculosis genome present in the sample, and obtained by amplifying part or all of the nucleic acid. It may be a nucleic acid amplification product. More preferably, it is a nucleic acid amplification product.
That is, the method for detecting rifampicillin resistance of Mycobacterium tuberculosis according to the present invention may be a method including the following step (0).
(0) A step of amplifying a test nucleic acid with a reaction solution containing a composition comprising a nucleic acid primer set for nucleic acid amplification of rpoB gene. In this embodiment, step (0) is performed at the latest before the start of step (3). It is preferable to carry out. More preferably, it is performed before the step (2). Moreover, it is also possible to carry out before the said process (1).
In this embodiment, in step (2), the nucleic acid amplification product obtained in step (1) is hybridized with a nucleic acid probe capable of forming a complex with a part of the nucleic acid amplification product. Form the body. The nucleic acid primer sets and nucleic acid probes used in this embodiment can be those described above.

In the present invention, the method for discriminating rifampicillin resistance of Mycobacterium tuberculosis is not particularly limited. For example, it can be carried out by determining the presence or absence of a mutation in the rpoB gene of the test nucleic acid. If the rpoB gene can be detected, a new process may be added to the above process.

  When the method of the present invention involves a nucleic acid amplification step (the above step (0)), steps (0) to (3) can be carried out either with or without opening the reaction system, but without opening. It is preferable that all of (0) to (3) are performed. “Open” means that the nucleic acid amplification product is exposed by, for example, opening a reaction container lid. “Do not open” means that the reaction container is not opened and the nucleic acid amplification product is not exposed to the outside.

One embodiment of step (2) of the method includes the following step (2 ′).
(2 ′) The presence or absence of dissociation between the nucleic acid amplification product forming the complex and the nucleic acid probe by performing a melting curve analysis by gradually increasing the temperature of the reaction system containing the complex obtained in step (1) Detecting process

As one embodiment of the step (3) of the method, the following step (3 ′) may be mentioned.
(3 ′) a step of determining that the rpoB gene was present when dissociation could be detected in step (2 ′), and further determining whether the rpoB gene has a mutation from the difference in melting temperature of the complex.

  The time from the start of the steps (0), (1), (2), and (3), or the steps (0), (1), (2 ′), and (3 ′) to completion thereof is not particularly limited. It is preferable to finish within one hour. More preferably, it is within 55 minutes, is within 50 minutes, and is within 45 minutes. This time refers to the time from the start of nucleic acid amplification in step (0) to the completion of complex detection in step (3) or detection of the presence or absence of dissociation in (3 ′), and is used in step (0). The reaction solution preparation time is not included.

  As the nucleic acid primer set and the nucleic acid probe for amplifying and detecting the rpoB gene used in the method, those described above can be used. Furthermore, for example, for the purpose of amplifying and detecting a nucleic acid different from the rpoB gene, addition of a nucleic acid primer set or nucleic acid probe different from the nucleic acid primer set and nucleic acid probe is not particularly limited.

[Amplification of test nucleic acid]
The test nucleic acid in the present invention may be, for example, a single strand or a double strand. In the case of a double strand, for example, it is preferable to include a step of dissociating the double strand into a single strand by heating in order to hybridize a test nucleic acid and a probe to form a hybrid.

  The type of the test nucleic acid is not particularly limited, and examples thereof include DNA, RNA such as total RNA and mRNA, and the like. Examples of the test nucleic acid include nucleic acids contained in a sample such as a biological sample. This sample may be used as it is, or may be diluted with an appropriate solution. Suitable solutions include water, physiological saline, buffer solution, alkaline aqueous solution, acidic aqueous solution, nucleic acid extraction reagent, surfactant, organic solvent and the like. Alternatively, nucleic acids such as DNA and RNA may be prepared from the biological sample by an appropriate method. The preparation method is not particularly limited, and a conventionally known method such as a known nucleic acid purification kit, an automatic nucleic acid purification apparatus, or a phenol / chloroform method can be used.

  The biological sample is not particularly limited, and examples thereof include sputum, tissue pieces, stool, urine, gastric fluid, pleural effusion, pharyngeal wiping fluid, bronchial lavage fluid, and blood. Moreover, it is good also considering the tuberculosis microbe culture | cultivated by adding these biological samples to a culture medium or culture solution.

  Subsequently, the isolated genomic DNA is used as a template to amplify a sequence containing a base site to be detected by a nucleic acid amplification method such as PCR using the above-described nucleic acid primer set. The specific nucleic acid amplification method is not particularly limited, and a known method can be used as appropriate. For example, PCR (Polymerase Chain Reaction) method, NASBA (Nucleic acid sequence based amplification) method, TMA (Transscription-mediated amplification (LMP) method, SDA (Strand Displacement Amplification method), SDA (Strand Displacement Amplification method). It is preferable to use the PCR method. In each of these methods, the conditions for the amplification reaction are not particularly limited, and can be performed by a conventionally known method.

  In the PCR method, usually, a three-stage reaction of a denaturation reaction, an annealing reaction, and an extension reaction is repeated. However, the two-stage reaction may be repeated by setting the same reaction conditions for the annealing reaction and the extension reaction. In the present invention, the reaction of the PCR method may be two steps, three steps, or four steps or more, preferably three steps.

The temperature and time conditions for each reaction are not limited, but the temperature for the denaturation reaction is preferably 90 to 100 ° C. The time for the denaturation reaction is preferably 0 to 10 seconds. Here, 0 seconds means that the temperature is set to a predetermined temperature for a moment and immediately proceeds to the next stage reaction, and does not mean that the denaturation reaction is not performed. The annealing reaction temperature is preferably 50 to 70 ° C., and the annealing reaction time is preferably 0 to 15 seconds. The temperature of the extension reaction is preferably 58 to 75 ° C., and the extension reaction time is preferably 1 to 15 seconds.

  Performing a denaturation reaction, an annealing reaction, and an extension reaction once each is called a PCR cycle, and the number of PCR cycles performed in the entire nucleic acid amplification reaction by the PCR method is called a cycle number. The number of cycles that can be carried out in the present invention is not particularly limited, but is preferably in the range of 35 to 100 times. The lower limit of the number of cycles is preferably 40 times, more preferably 50 times. The upper limit of the number of cycles is preferably 80 times, more preferably 70 times.

[DNA polymerase]
When the PCR method is used for nucleic acid amplification, the DNA polymerase to be used is not particularly limited, but α-type DNA polymerase is preferably used. The reason will be described below.

When the rpoB gene is amplified in a reaction system containing the probe of the present invention, the nucleic acid probe can bind to the rpoB gene of the sample or their amplification product during the nucleic acid amplification step. The nucleic acid probe bound to the test nucleic acid during the nucleic acid amplification step inhibits the nucleic acid amplification reaction by the nucleic acid primer and DNA polymerase.

PolI type DNA polymerases such as Taq DNA Polymerase are known to have 5'-3 'exonuclease activity. Due to this activity, if there is a nucleic acid bound to the rpoB gene as a template during the nucleic acid amplification reaction, the bound nucleic acid is degraded by exonuclease activity. For this reason, the nucleic acid probe in the reaction system may be reduced, causing a problem in the nucleic acid detection process. Therefore, it is not preferable to carry out the present invention using PolI type DNA polymerase.

On the other hand, α-type DNA polymerases such as KOD DNA Polymerase (derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1) do not have 5'-3 'exonuclease activity but have 3'-5' exonuclease activity. Therefore, the use of α-type DNA polymerase not only solves the above problem, but also exhibits high accuracy in the nucleic acid amplification step due to the 3′-5 ′ exonuclease activity.

Usually, α-type DNA polymerase has a 3 ′ → 5 ′ exonuclease activity, and therefore the nucleic acid amplification rate tends to be lower than that of PolI-type enzyme. However, although KOD DNA Polymerase is an α-type DNA polymerase, it has high DNA synthesis activity, has a DNA synthesis rate of 100 bases / second or more, and has excellent elongation efficiency. Therefore, it is preferable to use KOD DNA Polymerase (trade name, manufactured by Toyobo Co., Ltd.) among the α-type DNA polymerases for carrying out the present invention.

In addition, a mutant type in which α-type DNA polymerase is mutated to achieve a deoxyribonucleic acid synthesis rate of 100 bases / second or more, or a DNA polymerase composition in which the performance is achieved by a combination of wild type and / or mutant type Can be used as a DNA polymerase suitable for the practice of the present invention.
For example, as a DNA polymerase having a deoxyribonucleic acid synthesis rate of 100 bases / second or more in addition to the above KOD DNA Polymerase, “KOD FX (Toyobo, Trademark)”, “KOD-Plus- (Toyobo, Trademark)”, “KOD” Dash (trade name, manufactured by Toyobo Co., Ltd.), PrimeSTAR HS DNA polymerase (trade name, manufactured by Takara Bio Inc.) and the like can also be used.
Among these, KOD-Plus- having both high accuracy and DNA synthesis activity is desirable.

  By using the polymerase, PCR can be performed in a shorter time than conventional. In ordinary Taq polymerase, the deoxyribonucleic acid synthesis rate is said to be about 60 bases / second, but KOD DNA Polymerase and the like have a deoxyribonucleic acid synthesis rate exceeding 100 bases / second, so the extension time is reduced to half of the usual time. Can be shortened.

  In addition, PCR is further accelerated by using a nucleic acid amplification apparatus that performs a nucleic acid amplification reaction using a thin capillary reaction vessel, such as LightCycler (Roche Diagnostics) or GENECUBE (Toyobo). It is possible. By using these techniques, PCR within 1 hour is possible as shown in the examples of the present invention.

[Complex formation of nucleic acid amplification product and probe]
In the rpoB gene detection method of the present invention, the nucleic acid amplification product obtained in step (1) is hybridized with a nucleic acid probe designed to form a complex with a part of the nucleic acid amplification product. Form.

The timing of adding a nucleic acid probe to a sample containing a nucleic acid amplification product is not particularly limited. For example, it may be added to the reaction system of the amplification reaction before, during or after the nucleic acid amplification reaction. May be.
Especially, since an amplification reaction and a detection reaction described later can be performed continuously, it is preferable to add them before the amplification reaction. Thus, when the probe is added before the nucleic acid amplification reaction, for example, as described later, it is preferable to add a fluorescent dye or a phosphate group to the 3 ′ end.

The probe may be added to a liquid sample containing a nucleic acid amplification product, or may be mixed with a nucleic acid amplification product in a solvent. The solvent is not particularly limited, and examples thereof include conventionally known solvents such as a buffer solution such as Tris-HCl, a solvent containing KCl, MgCl ₂ , MgSO ₄ , glycerol and the like, and a PCR reaction solution.

[Detection of complex between nucleic acid amplification product and probe]
In the step shown in (2) of the method, the method for detecting the obtained complex is not particularly limited. For example, a method by melting curve analysis as described in (2 ′) above can be mentioned.

In the case of melting curve analysis, for example, the following is performed.
When a solution containing double-stranded DNA is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both strands in double-stranded DNA are unwound by heating and dissociated into single-stranded DNA (DNA melting). When all the double-stranded DNAs are dissociated into single-stranded DNA, the absorbance is about 1.5 times the absorbance at the start of heating (absorbance of only double-stranded DNA), thereby melting. Can be determined to be completed.

  In the present invention, the measurement of the signal fluctuation accompanying the temperature change for performing the melting curve analysis can be performed by measuring the absorbance at 260 nm based on the principle as described above, but the signal of the label added to the probe of the present invention is measured. It is preferable to measure. For this reason, as a probe used for the rifampicillin-resistant tuberculosis detection method of this invention, it is preferable to use a labeled probe.

  Examples of the labeled probe include a labeled probe that shows a signal alone and does not show a signal by hybridization, or a labeled probe that does not show a signal alone and shows a signal by hybridization. In the former probe, no signal is shown when a hybrid (double strand) is formed with the detection target sequence, and a signal is shown when the probe is released by heating. In the case of the latter probe, a signal is shown by forming a hybrid (double strand) with the sequence to be detected, and the signal decreases (disappears) when the probe is released by heating. Therefore, by detecting the signal from the label under signal-specific conditions (absorbance and the like), the progress of melting can be grasped in the same manner as the absorbance measurement at 260 nm.

  As a specific example of the labeled probe, for example, a probe that is labeled with a fluorescent dye, exhibits fluorescence alone, and fluorescence decreases (for example, quenches) by hybridization is preferable. Such a phenomenon is generally called a fluorescence quenching phenomenon. As a probe using this fluorescence quenching phenomenon, a probe generally called a guanine quenching probe is preferable. Such a probe is known as a so-called QProbe (registered trademark). A guanine quenching probe is, for example, a fluorescent dye designed so that the base at the 3 ′ end or 5 ′ end of an oligonucleotide becomes cytosine, and light emission becomes weaker when the base cytosine at the end approaches a complementary base guanine. It is a probe labeled at the end. In the probe of the present invention, for example, a fluorescent dye exhibiting a fluorescence quenching phenomenon may be bound to cytosine at the 3 ′ end of the oligonucleotide, or the 5 ′ end of the oligonucleotide is designed to be cytosine. It may be combined.

  The fluorescently labeled nucleic acid probe used in the present invention has the same structure as the guanine quenching probe, but the fluorescently labeled base may be other than guanine. Specific examples include SEQ ID NOS: 1 and 5.

  Even if the nucleic acid probe binds to a nucleic acid amplification product which is a template nucleic acid, the fluorescence-labeled base does not bind to guanine, so the fluorescence is not quenched, and the complex between the nucleic acid amplification product and the nucleic acid probe cannot be detected. . However, when the nucleic acid probe forms a complex with the nucleic acid amplification product, it has a base sequence that binds to the nucleic acid amplification product so as to be adjacent to the nucleic acid probe, and is further adjacent to the fluorescently labeled nucleic acid probe. By simultaneously using an unlabeled nucleic acid probe whose base is guanine, guanine quenching occurs in the nucleic acid probe fluorescently labeled with guanine of the unlabeled nucleic acid probe, and a complex with the nucleic acid amplification product can be detected.

When the detection system is used, if a mutation is contained in the binding region of the rpoB gene, which is a test nucleic acid, with the fluorescently labeled nucleic acid probe, the nucleic acid probe and the nucleic acid amplification product are converted into the rpoB gene. Binds weaker than when wild-type. In that case, for example, when melting curve analysis is performed, the nucleic acid probe melts at a lower temperature than when the wild type of the rpoB gene is examined, and emits fluorescence away from the unlabeled nucleic acid probe.
Similarly, when a mutation is included in the binding region of the rpoB gene with the unlabeled nucleic acid probe, the unlabeled nucleic acid probe and the nucleic acid amplification product bind more weakly than when the rpoB gene is a wild type. In that case, for example, when a melting curve analysis is performed, the unlabeled nucleic acid probe melts at a lower temperature than when the wild type of the rpoB gene is examined, and is adjacent to the fluorescently labeled base of the fluorescently labeled nucleic acid probe. Since the guanine thus separated is separated, the nucleic acid probe emits fluorescence.
Therefore, even if a mutation is contained at a position where it binds to any nucleic acid probe, the detection system can detect the mutation.

  A model diagram of the detection system is shown in FIG. A state in which both the fluorescently labeled probe and the unlabeled probe are released without binding to the nucleic acid to be detected is shown in FIG. By placing the reaction system in the state A at a temperature of about 35 to 50 ° C., for example, both probes can be bound to the detection target as shown by B in FIG. When a mutation is included in the binding region with the nucleic acid probe that is fluorescently labeled in the nucleic acid to be detected, that is, the rpoB gene, then, for example, by performing a melting curve analysis, it is shown in FIG. The nucleic acid probe is released as shown. On the other hand, if a mutation is included in the binding region with the unlabeled nucleic acid probe in the rpoB gene, for example, by performing a melting curve analysis, the unlabeled nucleic acid probe is released as shown by C ′ in FIG. The fluorescently labeled nucleic acid probe emits fluorescence.

  In addition, the rpoB gene can be detected alone with a normal guanine quenching probe whose cytochrome-labeled end is cytosine. In addition, for example, when melting curve analysis is performed, the nucleic acid probe melts and emits fluorescence at a lower temperature than when the wild type of the rpoB gene is examined, so that the nucleic acid probe of the rpoB gene binds depending on the melting temperature. It is possible to detect a mutation in the region to be performed.

  As one embodiment of the present invention, Rifampicillin-resistant Mycobacterium tuberculosis is detected using the above-described normal guanine quenching probe and a set of a special quenching probe and a non-labeled nucleic acid probe that are not guanine at the end. As an example of this embodiment, there can be exemplified a combination in which a normal guanine quenching probe is SEQ ID NO: 2, a special quenching probe is SEQ ID NO: 1, and an unlabeled nucleic acid probe is SEQ ID NO: 3.

  Further, as a different example, there can be exemplified a combination in which a normal guanine quenching probe is SEQ ID NO: 4, a special quenching probe is SEQ ID NO: 5, and an unlabeled nucleic acid probe is any one of SEQ ID NOs: 6 to 9.

  In the embodiment described above, all three nucleic acid probes may be added to a single detection system, or a set of a normal guanine quenching probe, a special quenching probe, and a non-labeled nucleic acid probe may be used as separate detection systems. May be used. More preferred is an embodiment in which all three nucleic acid probes are used in one detection system.

[Method of melting curve analysis]
The dissociation of the nucleic acid amplification product obtained in step (1) and the hybridization between the single-stranded DNA obtained by the dissociation and the labeled probe can be performed, for example, by changing the temperature of the reaction solution.

  The heating temperature in the dissociation step is not particularly limited as long as the amplification product can be dissociated, and is, for example, 85 to 98 ° C. The heating time is not particularly limited, but is usually 1 second to 10 minutes, preferably 1 second to 5 minutes.

  The dissociated single-stranded DNA and the labeled probe can be hybridized, for example, by lowering the heating temperature in the dissociation step after the dissociation step. As temperature conditions, it is 35-50 degreeC, for example.

  The volume and concentration of each composition in the reaction system (reaction system) of the hybridization process are not particularly limited. As a specific example, in the reaction system, the concentration of DNA is, for example, 0.01-100 μmol / L, preferably 0.1-10 μmol / L, and the concentration of the labeled probe is, for example, relative to the DNA A range satisfying the addition ratio is preferable, for example, 0.01 to 100 μmol / L, and preferably 0.01 to 10 μmol / L.

  Then, the temperature of the reaction solution is changed, and a signal value indicating the melting state of the hybrid formed of the amplification product and the labeled probe is measured. Specifically, for example, the reaction solution (hybridized body of the single-stranded DNA and the labeled probe) is heated, and a change in signal value accompanying a temperature rise is measured. As described above, when a probe labeled with a terminal C base (guanine quenching probe) is used, fluorescence is reduced (or quenched) in a hybridized state with single-stranded DNA, and in a dissociated state, Fluoresce. Therefore, for example, the hybrid formed body in which the fluorescence is decreased (or quenched) may be gradually heated, and the increase in the fluorescence intensity accompanying the temperature increase may be measured.

  The temperature range for measuring the fluctuation of the fluorescence intensity is not particularly limited. For example, the start temperature is room temperature to 85 ° C, preferably 25 to 70 ° C, and the end temperature is 40 to 105 ° C, for example. is there. Moreover, the rate of temperature rise is not particularly limited, but is, for example, 0.05 to 20 ° C./second, and preferably 0.08 to 5 ° C./second.

  The presence / absence of the test nucleic acid can be determined, for example, by measuring signal fluctuations during hybridization. That is, when a hybrid is formed by lowering the temperature of the reaction solution containing the probe, the signal fluctuation accompanying the temperature drop is measured.

  As a specific example, when a labeled probe (for example, a guanine quenching probe) that shows a signal alone and does not show a signal by hybridization, it emits fluorescence when the single-stranded DNA and the probe are dissociated. However, when a hybrid is formed due to a decrease in temperature, the fluorescence is reduced (or quenched). Therefore, for example, the temperature of the reaction solution may be gradually decreased to measure the decrease in fluorescence intensity accompanying the temperature decrease. On the other hand, when a labeled probe that does not show a signal alone and shows a signal by hybridization, it does not emit fluorescence when the single-stranded DNA and the probe are dissociated, but the hybrid is not released due to a decrease in temperature. Once formed, it will fluoresce. Therefore, for example, the temperature of the reaction solution may be gradually lowered and the increase in fluorescence intensity accompanying the temperature drop may be measured.

  In the present invention, in order to determine the genotype at the target base site, the signal variation may be analyzed and determined as a Tm (melting temperature) value.

The method for measuring the activity of the DNA synthase used in the present specification is described below.
[DNA synthesis activity]
In the present invention, DNA synthesis activity refers to deoxyribonucleoside bound to deoxyribonucleic acid by covalently binding deoxyribonucleoside 5′-triphosphate α-phosphate to the 3′-hydroxyl group of the oligonucleotide or polynucleotide annealed to the template DNA. It refers to the activity of catalyzing the reaction of introducing 5′-monophosphate in a template-dependent manner.

When the enzyme activity is high, the activity is measured by diluting the sample with a storage buffer. In the present invention, 25 μl of the following A solution, 5 μl of each of B solution and C solution, and 10 μl of sterilized water are added to an Eppendorf tube and mixed with stirring. Then, 5 μl of the enzyme solution is added and reacted at 75 ° C. for 10 minutes. Thereafter, the mixture is ice-cooled, and 50 μl of E solution and 100 μl of D solution are added. This solution is filtered through a glass filter (Whatman GF / C filter), thoroughly washed with solution D and ethanol, and the radioactivity of the filter is measured with a liquid scintillation counter (manufactured by Packard) to incorporate nucleotides into the template DNA. taking measurement. One unit of enzyme activity is the amount of enzyme that incorporates 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction under these conditions.
A: 40 mM Tris-HCl (pH 7.5)
16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA
B: 2 μg / μl activated calf thymus DNA
C: 1.5 mM dNTP (250 cpm / pmol ^[3H] dTTP)
D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
E: 1 μg / μl carrier DNA

[3′-5 ′ exonuclease activity]
In the present invention, the 3′-5 ′ exonuclease activity refers to the activity of excising the 3 ′ terminal region of DNA and releasing 5′-mononucleotide.
The activity was measured using 50 μl of a reaction solution (120 mM Tris-HCl (pH 8.8 at 25 ° C.), 10 mM KCl, 6 mM ammonium sulfate, 1 mM MgCl ₂ , 0.1% Triton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) is dispensed into a 1.5 ml Eppendorf tube and DNA polymerase is added. After reacting at 75 ° C. for 10 minutes, the reaction was stopped by cooling with ice, and then 50 μl of 0.1% BSA was added as a carrier, and then 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution were added and mixed. To do. After leaving on ice for 15 minutes, the precipitate is separated by centrifugation at 12,000 rpm for 10 minutes. The radioactivity of 100 μl of the supernatant is measured with a liquid scintillation counter (manufactured by Packard), and the amount of nucleotide released in the acid-soluble fraction is measured.

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

[Example 1: Detection of rpoB gene mutation using two nucleic acid probes of the method of the present invention]
An experiment was conducted to show that the rpoB gene can be detected and a wild-type rpoB gene and a mutant rpoB gene can be distinguished by using a fluorescently labeled nucleic acid probe and an unlabeled nucleic acid probe.
(1) Preparation of sample A nucleotide sequence including a region corresponding to codon 509 to codon 533 in the rpoB gene was amplified by PCR using the forward primer shown in SEQ ID NO: 19 and the reverse primer shown in SEQ ID NO: 20 . Next, the nucleic acid amplification product was incorporated into the cloning vector pUC19 to obtain a wild type rpoB positive sample. Furthermore, by preparing a linear DNA into which mutation has been introduced from the wild-type rpoB positive sample by using site-directed mutagenesis by inverse PCR, and ligating the linear DNA, a predetermined mutant rpoB gene is obtained. Circular DNA containing a partial sequence was prepared. The circular DNA shown on the left was used as a mutant rpoB positive sample.
For nucleic acid amplification of rpoB gene, KOD-Plus-ver. 2 (manufactured by Toyobo) was used. For site-directed mutagenesis, KOD-Plus-Mutageness Kit (Toyobo) was used. A list of the prepared positive samples is shown in Table 1.

In Table 1, the partial sequence refers to a partial sequence of the rpoB gene that was nucleic acid amplified using nucleic acid primers consisting of SEQ ID NOs: 19 and 20.

(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the above samples, respectively, and nucleic acid amplification and melting curve analysis were performed under the following conditions to detect a complex of a nucleic acid probe and a nucleic acid amplification product. For analysis, GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used.

[Reagent for nucleic acid amplification]
A solution containing the following reagents was prepared. A sample prepared by adjusting the concentration of the wild type or mutant rpoB positive sample to 25 copies / μl was used.
0.25 μl of 100 μM forward primer (SEQ ID NO: 10)
100 μM reverse primer (SEQ ID NO: 14) 0.05 μl
100 μM nucleic acid probe (SEQ ID NO: 1, 5 ′ end BODIPY-FL label, 3 ′ end phosphorylation) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 2, 5 ′ end BODIPY-FL labeling, 3 ′ end phosphorylation) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 3, 3 ′ end phosphorylation) 0.06 μl
KOD Mix (Gene Cube (R) Test Basic, manufactured by Toyobo) 4 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 4 μl
DMSO 0.32 μl
Purified water 0.46μl
Sample 4μl
(WT (wild type), 511GTG, 512GGC, 516GTC, 516CAC, 531TTG, 531TGG, 533CCG were used as samples. In this specification, each mutant-positive sample is named “base sequence after codon number mutation”. For example, 511GTG is a sample in which the base of codon 511 is substituted with GTG.)

[Nucleic acid amplification and melting curve analysis]
94 ° C, 30 seconds (more than one cycle)
97 ° C · 1 second 58 ° C · 3 seconds 63 ° C · 5 seconds (over 60 cycles)
94 ° C, 30 seconds 39 ° C, 30 seconds 40 ° C to 80 ° C (temperature rise at 0.09 ° C / second)

[result]
FIG. 2 shows a nucleic acid primer set composed of a forward primer having a base sequence consisting of SEQ ID NO: 10 and a reverse primer having a base sequence consisting of SEQ ID NO: 14, and nucleic acids each having a base sequence consisting of SEQ ID NOs: 1-3. Using a probe, nucleic acid amplification was performed using WT and 511GTG as samples, and the change in fluorescence intensity as the temperature increased thereafter was expressed with the horizontal axis of the graph representing temperature and the vertical axis representing the differential value of the fluorescence signal. FIG. The nucleic acid probe used in this example is a quenching probe, which binds to and quenches the amplified target nucleic acid by placing it at 39 ° C. together with the nucleic acid amplification product. Thereafter, by gradually raising the temperature, the nucleic acid probe is released from the target nucleic acid and emits fluorescence. Therefore, if the amount of change in fluorescence changes positively in the process of gradually raising the temperature, this indicates that the nucleic acid probe bound to the target nucleic acid has been released.
FIG. 2 shows that when WT was used as a sample, a large change in fluorescence occurred at about 70 ° C., and a detection peak was obtained. On the other hand, with 511GTG, a large change in fluorescence occurs at both about 65 ° C. and about 70 ° C., and a detection peak is obtained. Since the detection peak shape was different between WT and 511GTG, it was shown that the wild-type rpoB gene and the rpoB gene in which codon 511 was mutated can be distinguished by the method of the present invention.
Similarly, FIGS. 3 to 8 show that 512 GGC, 516 GTC, 516 CAC, 531 TTG, 531 TGG, and 533 CCG have different detection peaks from WT, and based on this result, either codon 511, 512, 516, 531, 533 It became clear that rpoB genes with mutations can be distinguished from wild-type rpoB genes.

[Example 2: Detection of rpoB gene mutation using two nucleic acid probes of the method of the present invention]
An experiment was conducted to show that the rpoB gene can be detected and a wild-type rpoB gene and a mutant rpoB gene can be distinguished by using a fluorescently labeled nucleic acid probe and an unlabeled nucleic acid probe.
(1) Preparation of sample A nucleotide sequence including a region corresponding to codon 509 to codon 533 in the rpoB gene was amplified by PCR using the forward primer shown in SEQ ID NO: 19 and the reverse primer shown in SEQ ID NO: 20 . Next, the nucleic acid amplification product was incorporated into the cloning vector pUC19 to obtain a wild type rpoB positive sample. Furthermore, by preparing a linear DNA into which mutation has been introduced from the wild-type rpoB positive sample by using site-directed mutagenesis by inverse PCR, and ligating the linear DNA, a predetermined mutant rpoB gene is obtained. Circular DNA containing a partial sequence was prepared. The circular DNA shown on the left was used as a mutant rpoB positive sample.
For nucleic acid amplification of rpoB gene, KOD-Plus-ver. 2 (manufactured by Toyobo) was used. For site-directed mutagenesis, KOD-Plus-Mutageness Kit (Toyobo) was used. A list of the prepared positive samples is shown in Table 2.
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the above samples, respectively, and nucleic acid amplification and melting curve analysis were performed under the following conditions to detect a complex of a nucleic acid probe and a nucleic acid amplification product. For analysis, GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used.

[Reagent for nucleic acid amplification]
A solution containing the following reagents was prepared. A sample prepared by adjusting the concentration of the wild type or mutant rpoB positive sample to 25 copies / μl was used.
0.25 μl of 100 μM forward primer (SEQ ID NO: 10)
100 μM reverse primer (SEQ ID NO: 14) 0.05 μl
100 μM nucleic acid probe (SEQ ID NO: 1, 5 ′ end BODIPY-FL label, 3 ′ end phosphorylation) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 2, 5 ′ end BODIPY-FL labeling, 3 ′ end phosphorylation) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 3, 3 ′ end phosphorylation) 0.06 μl
KOD Mix (Gene Cube (R) Test Basic, manufactured by Toyobo) 4 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 4 μl
DMSO 0.32 μl
Purified water 0.46μl
Sample 4μl
(As samples, WT, 510_AG, 510CAT, 513TAA, 513AAT, 513CCA, 514TTC +, 515GTG, 523CGG, 526CAA, 529AAA were used. 510_AG indicates that the original base was deleted. Also, 514TTC + + Indicates an insertion mutation.)

[Nucleic acid amplification and melting curve analysis]
94 ° C, 30 seconds (more than one cycle)
97 ° C · 1 second 58 ° C · 3 seconds 63 ° C · 5 seconds (over 60 cycles)
94 ° C, 30 seconds 39 ° C, 30 seconds 40 ° C to 80 ° C (temperature rise at 0.09 ° C / second)

[result]
FIG. 9 shows a nucleic acid primer set composed of a forward primer having a base sequence consisting of SEQ ID NO: 10 and a reverse primer having a base sequence consisting of SEQ ID NO: 14, and a nucleic acid having a base sequence consisting of SEQ ID NOs: 1 to 3, respectively. Using the probe, nucleic acid amplification was performed using WT and 510_AG as samples, and the change in fluorescence intensity as the temperature increased thereafter was expressed with the horizontal axis of the graph representing temperature and the vertical axis representing the differential value of the fluorescence signal. FIG. FIG. 9 shows that when WT was used as a sample, a large change in fluorescence occurred at about 70 ° C., and a detection peak was obtained. On the other hand, with 510_AG, a change in fluorescence occurs at about 65 ° C. in addition to about 70 ° C., and a detection peak is obtained. Since the detection peak shape was different between WT and 510_AG, it was shown that the wild-type rpoB gene and the rpoB gene in which codon 511 was mutated can be distinguished by the method of the present invention.
Similarly, FIGS. 10 to 18 show that 510CAT, 513TAA, 513AAT, 513CCA, 514TTC +, 515GTG, 523CGG, 526CAA, and 529AAA have different detection peaks from WT. It was revealed that the rpoB gene having a mutation in any of 523, 526, and 529 can be distinguished from the wild-type rpoB gene.

[Example 3: Application of the method of the present invention to diffusion amplification products obtained using various nucleic acid primer sets]
The detection target is a nucleic acid amplification product obtained by nucleic acid amplification using a nucleic acid primer set different from those in Examples 1 and 2, and a rpoB gene is detected by using a fluorescently labeled nucleic acid probe and an unlabeled nucleic acid probe, In addition, an experiment was conducted to show that the wild-type rpoB gene and the mutant rpoB gene can be differentiated.
(1) Preparation of sample WT used in Examples 1 and 2 was used. The concentration was 25 copies / μl, and the concentration was adjusted with 10 mM Tris-HCl (pH 7.5).
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the above samples, respectively, and nucleic acid amplification and melting curve analysis were performed under the following conditions to detect a complex of a nucleic acid probe and a nucleic acid amplification product. For analysis, GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used.

[Reagent for nucleic acid amplification]
A solution containing the following reagents was prepared. Nucleic acid primer combinations are listed in Table 3.
0.25 μl of 100 μM forward primer (any one of SEQ ID NOs: 10, 11, 12, and 13)
100 μM reverse primer (any one of SEQ ID NOs: 14, 15, 16, and 17) 0.05 μl
100 μM nucleic acid probe (SEQ ID NO: 1, 5 ′ end BODIPY-FL label, 3 ′ end phosphorylation) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 2, 5 ′ end BODIPY-FL labeling, 3 ′ end phosphorylation) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 3, 3 ′ end phosphorylation) 0.06 μl
KOD Mix (Gene Cube (R) Test Basic, manufactured by Toyobo) 4 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 4 μl
DMSO 0.32 μl
Purified water 0.46μl
Sample 4μl
(WT was used as a sample)

[Nucleic acid amplification and melting curve analysis]
Same as Example 2

[result]
FIG. 19 shows a nucleic acid primer set composed of a forward primer having a base sequence consisting of SEQ ID NO: 10 and a reverse primer having a base sequence consisting of SEQ ID NO: 14, and a nucleic acid having a base sequence consisting of SEQ ID NOs: 1 to 3, respectively. Using the probe, nucleic acid amplification was performed using WT as a sample, and the change in fluorescence intensity as the temperature increased thereafter was shown in the graph with the horizontal axis of the graph representing temperature and the vertical axis representing the differential value of the fluorescence signal. is there.
Similarly, FIGS. 20 to 29 show the results of similar experiments with the nucleic acid primer combinations described in Table 3. From this example, the detection method of the present invention and the nucleic acid probe set used in the present invention are not intended for nucleic acid amplification products by nucleic acid primers having a specific base sequence, but using nucleic acid primer sets having various base sequences. It was shown that detection of the obtained nucleic acid amplification product was possible.

[Example 4: rpoB gene detection by the method of the present invention using nucleic acid probes having different base sequences]
Using a nucleic acid probe having a nucleotide sequence different from that in Examples 1 and 2, an rpoB gene was detected, and an experiment showing that a wild-type rpoB gene and a mutant rpoB gene can be distinguished.
(1) Preparation of sample WT, 511GTG, 512GGC, and 531TTG were used among the samples used in Examples 1 and 2. The concentration was 25 copies / μl, and the concentration was adjusted with 10 mM Tris-HCl (pH 7.5).
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the above samples, respectively, and nucleic acid amplification and melting curve analysis were performed under the following conditions to detect a complex of a nucleic acid probe and a nucleic acid amplification product. For analysis, GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used.

[Reagent for nucleic acid amplification]
A solution containing the following reagents was prepared. A sample prepared by adjusting the concentration of the wild type or mutant rpoB positive sample to 25 copies / μl was used. The combinations of nucleic acid primers are shown in Table 2.
0.25 μl of 100 μM forward primer (any one of SEQ ID NOs: 10, 11, 12, and 13)
100 μM reverse primer (any one of SEQ ID NOs: 14, 15, 16, and 17) 0.05 μl
100 μM nucleic acid probe (SEQ ID NO: 4, 3 ′ end BODIPY-FL label) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 5, 3 ′ end BODIPY-FL label) 0.03 μl
100 μM nucleic acid probe (SEQ ID NO: 6, 3 ′ end phosphorylation) 0.06 μl
KOD Mix (Gene Cube (R) Test Basic, manufactured by Toyobo) 4 μl
PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 4 μl
DMSO 0.32 μl
Purified water 0.46μl
Sample 4μl

[Nucleic acid amplification and melting curve analysis]
Same as Example 2

[result]
30 shows a nucleic acid primer set composed of a forward primer having a base sequence consisting of SEQ ID NO: 10 and a reverse primer having a base sequence consisting of SEQ ID NO: 14, and nucleic acids each having a base sequence consisting of SEQ ID NOs: 4-6. Using a probe, nucleic acid amplification was performed using WT and 511GTG as samples, and the change in fluorescence intensity as the temperature increased thereafter was expressed with the horizontal axis of the graph representing temperature and the vertical axis representing the differential value of the fluorescence signal. FIG.
Similarly, FIGS. 31 and 32 show the results of a similar experiment using 512 GGC and 531 TTG as samples instead of 511 GTG. From this example, it was clarified that the nucleic acid probe used in the present invention is not limited to SEQ ID NOs: 1 to 3, and a nucleic acid probe having a base sequence different from that shown on the left can also be used.

According to the present invention, rpoB gene can be detected in a closed system. In addition, it was confirmed that the gene detection system can be tested in a shorter time than a conventional test kit. This has made it possible to quickly, reliably and easily detect the rifampicillin resistance gene mutation of Mycobacterium tuberculosis.

Claims (3)

A method for detecting Rifampicillin resistance of Mycobacterium tuberculosis, comprising the following steps (1) to (3):
(1) Step of hybridizing a nucleic acid derived from Mycobacterium tuberculosis in a sample and the nucleic acid probe described in (A) and (B) below to form a complex (A) represented by SEQ ID NOS: 1 and 2 Two nucleic acid probes labeled with a fluorescent dye that quenches when close to guanine (B) One unlabeled nucleic acid probe represented by SEQ ID NO: 3 (2) Complex obtained in step (1) Step (3) for detecting Rifampicillin resistance of M. tuberculosis by analyzing data measured or detected in step (2) A method for detecting Rifampicillin resistance of Mycobacterium tuberculosis, comprising the following steps (1) to (3):
(1) Step of hybridizing a nucleic acid derived from Mycobacterium tuberculosis in a sample and the nucleic acid probe described in (A) and (B) below to form a complex (A) represented by SEQ ID NOs: 4 and 5 Two nucleic acid probes labeled with a fluorescent dye that quenches when close to guanine (B) One unlabeled nucleic acid probe represented by any one of SEQ ID NOs: 6 to 9 (2) Step (1) The step of detecting the complex obtained in step (3) The step of discriminating rifampicillin resistance of Mycobacterium tuberculosis by analyzing the data measured or detected in step (2) The method for detecting rifampicillin-resistant Mycobacterium tuberculosis according to claim 1 or 2, wherein the detection target is codons 509, 510 , 511 , 512 , 513 , 514 , 515 , 516 , 520 , 521 , 522 , 523 in the rpoB gene. The method according to claim 1 or 2, wherein the base mutation occurs in any one or more of the group consisting of 526, 527, 529, 531 and 533.