JP2013208115A - Oligonucleotide for detection of acid-fast bacterium and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rapid, reliable and simple method for detection or species identification of an acid-fast bacterium which is important on clinical diagnosis.SOLUTION: A melting curve analysis is carried out by using a probe whose target nucleic acid is a region having a high bacteria species specificity in dnaJ1 gene in an acid-fast bacterium, whereby Mycobacterium kansasii is specifically detected and identified.

Description

The present invention relates to an oligonucleotide for rapidly, reliably and simply detecting Mycobacterium kansasii for clinical diagnosis, a method for detecting Mycobacterium, which uses the oligonucleotide, and reagents thereof.

Mycobacteria are Gram-positive rods with acid-fast properties, and there are many pathogens that cause serious lesions such as tuberculosis and leprosy. In general, mycobacteria have a slow growth and require a considerable amount of time for cultivation.

Of these, 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.

Traditionally, tubercle bacilli have been tested by culture methods. In general, M. tuberculosis is isolated and cultured on Ogawa's medium, properties on the medium (growth rate / temperature, colony shape, pigment production, etc.), niacin test, nitrate reduction test, heat-resistant catalase test, Tween 80 hydrolysis test, etc. Species are identified from the results. However, Mycobacterium tuberculosis is slow to grow, and it takes about 3 to 4 weeks for isolation culture alone, and about 2 to 3 weeks for various subsequent tests. Therefore, the detection or identification test for Mycobacterium tuberculosis takes about one month or more, regardless of its clinical importance and urgency, and has been a major limitation for flexible diagnosis and medication. Methods for detecting proteins produced by M. tuberculosis by antigen-antibody reaction have been developed, but sufficient bacterial species identification has not yet been achieved, and because there is a problem with sensitivity, separation culture is not possible. What was needed was unchanged.

In addition to Mycobacterium tuberculosis, mycobacterium tuberculosis, Mycobacterium intracellularis, Mycobacterium, Mycobacterium tuberculosis Atypical mycobacteria such as Mycobacterium fortuitum are known to be pathogenic to humans. Symptoms are lighter and less infectious than tuberculosis, but they are resistant to anti-tuberculosis drugs and there are no effective drugs. Like M. tuberculosis, these affect human lungs, lymph nodes, skin, etc., and there are many lung infections in particular.

In general, differentiation of pulmonary tuberculosis is extremely difficult from the clinical symptom and histopathological findings, so it must be done by identifying bacteria. The pathogenicity of atypical mycobacteria is large in the order of Mycobacterium kansasii> Mycobacterium avium, Mycobacterium intracellulare> Other in particular in order is important.

Conventionally, these atypical mycobacteria were isolated and cultured on Ogawa's medium, like M. tuberculosis, and properties on the medium (growth rate, temperature, colony shape, pigment production, etc.), niacin test, and nitrate reduction test The bacterial species are identified from the results of the heat-resistant catalase test, Tween 80 hydrolysis test, photoluminescence test and the like. However, these also generally have slow growth and often require about one month or more to identify the bacterial species.

Identification of Mycobacterium tuberculosis and atypical mycobacteria is extremely important clinically. Although mycobacterium tuberculosis has a serious medical condition, administration of anti-tuberculosis drugs such as streptomycin, rifampicin, ethambutol, and isonicotinic acid hydrazide is effective, and it is necessary to start treatment early. On the other hand, atypical mycobacteria are generally resistant to these drugs. The identification of the bacterial species greatly affects the subsequent treatment policy. However, as described above, these bacteria grow slowly, and it takes several weeks to identify the species. Similar non-pathogenic bacteria may also be detected, such as Mycobacterium smegmatis. This is why rapid and reliable detection and species identification methods are desired.

Recently, a method for detecting a pathogen with high sensitivity has been developed by artificially amplifying DNA or RNA and detecting the amplified nucleic acid. 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 is performed by repeatedly increasing and decreasing the temperature in the presence of a target nucleic acid, four types of deoxyribonucleoside triphosphates, a pair of oligonucleotide primers, and a thermostable DNA polymerase. A specific region of the target nucleic acid sandwiched between can be exponentially amplified.

As shown in Patent Document 3, a PCR method is usually performed 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) denaturation step, b) a primer-template hybrid is formed at a temperature in the range of 35-65 ° C. (preferably 37-60 ° C.) for 0.5-5 minutes (preferably 1-3 minutes). Annealing) step, and c) forming a primer extension product (extension) at a temperature in the range of 40-80 ° C (preferably 50-75 ° C) for 0.5-40 minutes (preferably 1-3 minutes). An amplification cycle consisting of steps is used.

Nucleic acid fragments 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, in the detection by electrophoresis, it is necessary to handle the amplification product of the target nucleic acid amplified exponentially, and there is a high risk of contamination. Further, since the operation is complicated and it takes 1 hour or more to complete the detection, it is difficult to apply the detection method to clinical diagnosis.

As a detection method other than electrophoresis, a method using a DNA insertion dye (intercalator) that exhibits the property of enhancing fluorescence when bound to a double-stranded nucleic acid is generally used. The increase in fluorescence with increasing DNA concentration during nucleic acid amplification can be used to measure the progress of the reaction and to determine the copy number of the target nucleic acid molecule. Furthermore, by monitoring the change in fluorescence accompanying a precisely controlled temperature change, a DNA melting curve can be generated, for example, at the end of the PCR cycle reaction. In such a melting curve analysis, the nature of the double-stranded nucleic acid can be distinguished to some extent, and when the melting temperature in the melting curve analysis is extremely low, a nucleic acid fragment shorter than the target nucleic acid fragment such as a primer dimer was amplified. If the main peak is not one, it indicates that there is a non-specific amplified nucleic acid fragment that is not the target nucleic acid fragment. As described above, according to the method for measuring fluorescence, it is possible to prevent the risk of contamination due to scattering of amplified nucleic acid fragments and the like, and a correct determination result cannot be obtained, and the human error that can occur from complicated operations. It is suitable for clinical diagnosis. However, since the general intercalator method generates fluorescence specifically for double-stranded nucleic acid, it is non-specific to the nucleic acid sequence when detection specific to the target nucleic acid sequence is required especially for clinical diagnosis. It is not very effective because it may detect a typical double-stranded nucleic acid.

The nucleic acid sequence specific probe method utilizes an additional nucleic acid reaction component (nucleic acid probe) to monitor the progress of the amplification reaction. This method often utilizes fluorescence energy transfer (FET) as the basis for detection. One or more nucleic acid probes are labeled with a fluorescent dye so that the fluorescent dye labeled on one probe can act as an energy donor molecule and the other fluorescent dye can act as an energy acceptor molecule. These are sometimes known as reporter molecules and quencher molecules, respectively. The energy donor molecule is excited with light of a specific wavelength in the excitation spectrum range and subsequently emits light in the range of the fluorescence emission wavelength. The energy acceptor molecule is also excited at this wavelength by receiving energy from the donor molecule by various distance-dependent energy transfer mechanisms. A special example of fluorescence energy transfer is fluorescence resonance energy transfer (FRET). In general, when an energy acceptor molecule and an energy donor molecule are in close proximity, for example when they are on the same or adjacent molecules, the acceptor molecule can receive the emitted energy of the donor molecule. Generally, there are two types of FET or FRET probes, one that uses nucleic acid probe hydrolysis to separate the energy donor from the energy acceptor, and one that changes the spatial relationship between the donor and acceptor molecules. For this purpose, those using hybridization are used.

Probes using hydrolysis are commercially available as TaqMan® probes. This probe consists of an oligonucleotide labeled with an energy donor and energy acceptor molecule and is designed to hybridize to a specific region of one strand of the amplified nucleic acid fragment. For example, in PCR, after a primer anneals to this strand, Taq polymerase extends the DNA by polymerase activity from 5 'to 3'. At this time, the TaqMan (registered trademark) probe is protected by phosphorylation at the 3 'end in order to prevent initiation of extension by Taq. If the TaqMan® probe is hybridized to the product strand, the growing Taq molecule hydrolyzes the probe, releasing the energy acceptor and energy donor, generating a detectable signal. This signal is cumulative and the concentration of free energy donor and acceptor molecules increases with each cycle of the amplification reaction. However, this method has a time penalty because the generation of a fluorescent signal depends on the occurrence of a probe hydrolysis reaction. In addition, when a large number of amplification cycles such as 50 cycles or more are required, a problem that hydrolysis can occur nonspecifically has also been found.

Many types of probes using hybridization are available. For example, molecular beacons are oligonucleotides with complementary 5 ′ and 3 ′ sequences that form hairpin loops, and fluorescent dyes labeled at their ends cause FRET due to the formation of hairpin structures It is in such a close position. Detection is performed by utilizing the fact that FRET does not occur when the energy acceptor and the energy donor are separated along with the hybridization of the molecular beacon to the target nucleic acid sequence.

Paired labeled probes can also be used. The probe labeled with the energy donor molecule and the probe labeled with the energy acceptor molecule hybridize closely on the strand of the amplified nucleic acid fragment to generate FRET, and the fluorescence of the wavelength generated by this FRET is detected. This type of variant involves the use of a labeled amplification primer and a single adjacent labeled probe. However, the use of two labeled oligonucleotides or a molecular beacon containing two labeled molecules is generally problematic because of the high cost of detection. Furthermore, for example, when it is intended to identify bacterial species for a plurality of pathogens in clinical diagnosis, there are a plurality of energy acceptor and energy donor pairs and a device for detecting fluorescent signals of a plurality of wavelengths emitted from them. The problem is that it is necessary and requires very expensive systems and complex reagent designs. As mentioned above, identification of Mycobacterium tuberculosis and atypical mycobacteria is important for clinical diagnosis, and rapid and reliable detection and species identification methods are desired. I can't.

In Patent Document 7, a nucleic acid probe including a DNA double-strand binding agent and a reactive molecule that can absorb or give fluorescence energy from the DNA double-strand binding agent is used to perform amplification reaction of a target nucleic acid. Then, a detection method for monitoring fluorescence is disclosed. This detection method is advantageous in terms of cost because only one labeled oligonucleotide is required. However, even if the technique disclosed in Patent Document 7 is used, the bacterial species of a plurality of pathogens in clinical diagnosis as described above can be obtained. When identifying, multiple energy acceptor and energy donor pairs and devices that detect the fluorescence signals of multiple wavelengths emitted from them are required, a very expensive system and complex reagent design The problem that is forced to be solved is not solved.

Patent Document 8 discloses a detection method using a nucleic acid probe whose fluorescence is quenched upon 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 fluorescence emitted by the fluorescent dye before and after the hybridization is measured, or a nucleic acid primer labeled with a fluorescent dye is hybridized to the target nucleic acid And a method for measuring the decrease in the amount of fluorescence during the annealing reaction and the extension reaction with respect to the amount of fluorescence upon denaturation of nucleic acid. Further, a real-time quantitative PCR method using the method and a data analysis method having a process of correcting the fluorescence intensity value at the annealing reaction with the one at the denaturation reaction when analyzing the data obtained by the PCR method are described. ing. In this detection method, a single labeled oligonucleotide is sufficient, and no intercalator is used, which is advantageous in terms of cost. However, even if the technique disclosed in Patent Document 8 is used, a plurality of pathogens in clinical diagnosis can be obtained. Species identification requires multiple fluorescent dyes and devices that detect multiple wavelength fluorescent signals emitted from them, such as the very expensive system and complex reagent design described above The problem that is forced to be solved is not solved.

US Pat. No. 4,683,195 U.S. Pat.No. 4,683,202 US Pat. No. 4,965,188 International Publication No.89 / 12696 JP-A-2-2934 International Publication No. 90/1069 Special table 2003-500001 Japanese Patent No. 3437816

Nucleic acidsequence-based amplification method; Nature vol. 350, p. 91 (1991) Strand Displacement Amplification: Nucleic acid research Vol. 20, page 1691 (1992) Transcription mediated amplification method; Clin. Microbiol. Volume 31, Page 3270 (1993) Loop-mediated isometric amplification method: J Clin Microbiol. 2004 Volume 42: 1,956 Isothermal and chimeric primer-initiated amplification of nuclear acids: Kekaku. 2003 Volume 78, Page 533

As described above, since detection by electrophoresis handles the amplification product of the target nucleic acid amplified exponentially, there is a risk that contamination will occur and correct determination results may not be obtained, which is suitable for clinical diagnosis. Absent.

In addition, although the possibility of contamination is reduced by the method using an intercalator, since double-stranded nucleic acid-specific fluorescence occurs, double-stranded nucleic acid that is non-specific for the target nucleic acid sequence is also detected and the correct determination is made. It is not suitable for clinical diagnosis because there is a risk that results cannot be obtained.

In the method using a nucleic acid sequence-specific probe, the above-mentioned problems related to the accuracy of the determination result have been improved to some extent, but it is important for clinical diagnosis, for example, identification of Mycobacterium tuberculosis and atypical mycobacteria In order to identify bacterial species for multiple pathogens, multiple fluorescent dyes and devices for detecting fluorescent signals of multiple wavelengths emitted from them are required, which is very expensive and complicated. It is not suitable for clinical diagnosis because it requires forced reagent design.

The present invention has been made in view of the above-mentioned conventional problems, and its purpose is to rapidly and reliably and easily detect or identify the species of mycobacteria that are important in clinical diagnosis, An object of the present invention is to provide a method for detecting an acid-fast bacterium using the oligonucleotide and a reagent thereof.

As a result of intensive studies in view of the above problems, the present inventors have a high species-specificity of a specific region in the dnaJ1 gene of Mycobacterium, including Mycobacterium tuberculosis, and the gene region is used as a target nucleic acid. It has been found that by using a probe, Mycobacterium kansasii (Mycobacterium kansasii) can be detected or identified with high accuracy, and the present invention has been completed. That is, the present invention has the following configuration.

[Claim 1]
A combination of oligonucleotides represented by the following (A) or (B).
(A) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 29 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 23.
(B) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 11 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 25.
[Section 2]
The detection method of Mycobacterium kansasii (Mycobacterium kansasii) shown by the following (A) or (B).
(A) A method comprising at least the following steps (1) to (3).
(1) a first step of performing nucleic acid amplification using a combination of the oligonucleotide shown in SEQ ID NO: 10 and the oligonucleotide shown in SEQ ID NO: 29 as a primer pair;
(2) a second step in which the amplification product obtainable in the first step and the detection oligonucleotide probe (Ck) shown in SEQ ID NO: 23 are hybridized to form a complex (Pk);
(3) A third step of detecting the complex (Pk) obtainable in the second step.
(B) A method comprising at least the following steps (1) to (3).
(1) a first step of performing nucleic acid amplification using a combination of the oligonucleotide shown in SEQ ID NO: 10 and the oligonucleotide shown in SEQ ID NO: 11 as a primer pair;
(2) a second step in which the amplification product obtainable in the first step and the detection oligonucleotide probe (Ck) shown in SEQ ID NO: 25 are hybridized to form a complex (Pk);
(3) A third step of detecting the complex (Pk) obtainable in the second step.
[Section 3]
Item 3. The detection method according to Item 2, wherein the steps (1) to (3) are performed in a sealed state.
[Claim 4]
Item 4. The detection method according to Item 2 or 3, wherein the detection oligonucleotide probe (Ck) is labeled with a fluorescent dye.
[Section 5]
Item 5. The detection method according to any one of Items 2 to 4, wherein an IFP method is used to detect the complex (Pk).
[Claim 6]
Item 5. The detection method according to any one of Items 2 to 4, wherein a Q-Probe method is used for detection of the complex (Pk).
[Claim 7]
A detection kit for Mycobacterium kansasi, comprising at least the combination of oligonucleotides represented by the following (A) or (B):
(A) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 29 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 23.
(B) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 11 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 25.

According to the present invention, as described above, it is possible to quickly and reliably and easily detect or identify the species of mycobacteria belonging to clinical diagnosis. Specifically, by performing a melting curve analysis using a probe whose target nucleic acid is a region having extremely high species specificity in the dnaJ1 gene of mycobacterial bacteria such as Mycobacterium tuberculosis, -It has the effect of being able to detect or identify the species of kansasii (Mycobacterium kansasii) very accurately.

It is a figure which shows the area | region with high microbial species specificity in dnaJ1 gene of the acid-fast bacterium belonging to this invention. It is a figure which shows the principle of a melting curve analysis typically. It is a figure which shows an evolution phylogenetic tree about the dnaJ1 gene region and 16S rRNA gene of this invention. It is a figure which shows the result detected using the oligo 12 of Example 1. It is a figure which shows the result detected using the oligo 23 of Example 2. FIG. It is a figure which shows the result detected using the oligo 24 of the comparative example 1. It is a figure which shows the result detected using the oligo 12 of Example 4. It is a figure which shows the result detected using the oligo 27 of Example 5.

In the present specification, the “region having high species-specificity in the dnaJ1 gene of Mycobacteria” refers to two oligonucleotides having the following sequences using the PCR method described below,
5′-CTACAAAGGAGCTGGGCGTCTCTCTCTG-3 ′ (SEQ ID NO: 3)
And 5'-CCCGAGGGAGCGCCCGCGACACCCGGCCCTCGCCCT-3 '(SEQ ID NO: 4)
Is used as a primer and is included in the gene region of the acid-fast bacterium that is amplified. Regarding the amplified region, Mycobacterium avium is in SEQ ID NO: 13, Mycobacterium intracellulare is in SEQ ID NO: 14, Mycobacterium kansasi is in SEQ ID NO: 15, Mycobacterium tuberculosis describes the specific nucleic acid sequence in SEQ ID NO: 16, respectively.
In addition, the above-mentioned “region with high bacterial species specificity in the dnaJ1 gene of mycobacterial bacteria” will be described later as “region A and region B”, but a partial sequence of the conventionally known dnaJ gene (Japanese Patent No. 2966478). , Japanese Patent No. 3134940, LClin. Microbio 1. Vol. 31, p. 446), and is not used for detection and / or identification of mycobacteria in the prior art.

The method for detecting an acid-fast bacterium of the present invention includes a detection step of detecting a region having a high species-specificity in the dnaJ1 gene of the acid-fast bacterium as described above.

The detection step preferably includes an amplification step of amplifying a nucleic acid sequence encoding at least a part of a region having a high species-specificity in the dnaJ1 gene of an acid-fast bacterium. In the amplification step, only the nucleic acid sequence (target nucleic acid) that is desired to be amplified out of all nucleic acid sequences in the sample is amplified using a primer that is complementary to the unique sequence. Specifically, in the amplification step, four types of deoxynucleoside triphosphates (dNTPs), DNA polymerase and two or more types of oligonucleotides (primers) are added to the chromosome of a mycobacterial bacterium or a fragment thereof denatured to a single strand. ) And the like are used to amplify the sequence between the primers.

The specific nucleic acid amplification method used in the amplification step is not particularly limited, and a known method can be used as appropriate. For example, as the nucleic acid amplification method, PCR, NASBA (Nucleicacid sequence-based amplification method; Nature, Vol. 350, page 91, 1991), LCR (see International Publication No. 89/12696, JP-A-2-2934) , SDA (Strand Displacement Amplification: Nucleic Acids Res. 20, pp. 1691, 1992), RCA (see International Publication No. 90/1069), TMA (Transcribion mediated amplification method, Vol. 31, J. ro. 3270, 1993), LAMP (Loop-mediated isoth). rmalamplification method: J. Clin Microbiol.Volume 42: see 1,956, 2004), ICAN (see the isothermative primer-initiated amplification of nucleic acid: vol.3, year 78). Can be, but is not limited to. By using the nucleic acid amplification method, it is possible to detect even a very small amount of sample nucleic acid (which may be one molecule), which could not be detected in the past, and can be suitably used for the amplification step. .

In addition, when the amount of nucleic acid in the sample is small, prior to the amplification step, it is possible to amplify the mycobacterial bacterium chromosome or a fragment thereof by the same method as in the amplification step.

Among the above methods, the PCR method is preferably used as the nucleic acid amplification method used in the amplification step. The PCR method is a reaction cycle comprising three steps of a denaturation step, an annealing step, and an extension reaction in the presence of a sample nucleic acid, a pair of primers (A and B), four types of deoxynucleoside triphosphates, and a heat-resistant DNA polymerase. By repeating the above, the region in the sample nucleic acid sandwiched between the pair of primers (A and B) can be amplified exponentially. That is, the sample nucleic acid is denatured in the denaturation step, each primer and a region in the sample nucleic acid complementary to the primer are hybridized in the subsequent annealing step, and each primer is used as the starting point in the subsequent extension step. To extend a complementary DNA strand. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers (A and B) is theoretically amplified to 2 n times.

The pair of primers (A and B) used in the amplification step is not particularly limited as long as the amplified nucleic acid fragment contains a region having a high species-specificity in the dnaJ1 gene of the above acid-fast bacterium. It is preferable to design in a region where the GC content is 65% or less. More preferably, it can be designed in the nucleic acid sequence of SEQ ID NO: 17 (region C) or SEQ ID NO: 18 (region D) or SEQ ID NO: 19 (region E) or in a nucleic acid sequence complementary to each sequence. The nucleic acid sequence region amplified when SEQ ID NO: 3 and SEQ ID NO: 4 are used as primers has a high GC content (about 70%). Among the regions, the regions C, D, and E are regions having a relatively low GC content of 58.62%, 58.14%, and 50.00%, respectively. Therefore, nonspecific reaction of the primer due to the high GC content hardly occurs, and the target nucleic acid in the sample can be specifically amplified.
For example,
As a primer (A),
5′-ACGGCGCTGAGGTTCAACCTACACG-3 ′ (SEQ ID NO: 5)
5′-CCAAGCGCAAGGAGTACGACGAA-3 ′ (SEQ ID NO: 6)
As a primer (B),
5′-GAACAAGCCACCGAACAAAGTCACCGAT-3 ′ (SEQ ID NO: 7)
5′-CGTCGAACATTTCGTTGAGGTTGAACT-3 ′ (SEQ ID NO: 8)
5′-CAAGCCACCGAACAGGTCGCCGAT-3 ′ (SEQ ID NO: 9)
Oligonucleotides having the following sequences can be used.

The nucleic acid fragment amplified using at least the primer (A) and the primer (B) and the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to the nucleic acid sequence "match 90% or more" The coincidence rate is shown when there is a mutation in the nucleic acid fragment between Mycobacterium kansasii strains. This shows that kansasii can be specifically detected by distinguishing it from mycobacteria other than kansasii even when there is a mutation between Mycobacterium kansasii strains. The coincidence rate is preferably 90% or more. More preferably, it is 97% or more. Table 1 shows the coincidence rate between the nucleic acid sequences possessed by 35 types of clinically isolated M. tuberculosis and 35 non-tuberculous mycobacteria and the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a complementary nucleic acid sequence. Show. The species most closely matching SEQ ID NO: 1 is Mycobacterium marinum, with a match rate of 83%, and the species most closely matching SEQ ID NO: 2 is Mycobacterium gordonae. The coincidence rate is 83%. Even in the case of mycobacteria Kansasii and the same genus mycobacteria, the concordance rate is less than 90%. Further, the coincidence ratio between the nucleic acid sequence possessed by the 7 strains of Mycobacterium kansasii and the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to the sequence is 90% or more for all 7 strains.

The detection oligonucleotide probe used in the detection step in the method for detecting an acid-fast bacterium of the present invention will be described. The region having high species-specificity in the dnaJ1 gene of the acid-fast bacterium belonging to the present invention is shown in FIG. 1 with partial sequences of the dnaJ1 gene for four types of mycobacteria belonging to, for example, Mycobacterium tuberculosis. As shown and used, there are two regions, region A and region B, and the effect of the present invention can be obtained by using either region. In FIG. 1, Ma is a nucleic acid sequence derived from Mycobacterium avium described in SEQ ID NO: 13, and Mi is derived from Mycobacterium intracellulare described in SEQ ID NO: 14. Mk is a nucleic acid sequence derived from Mycobacterium kansasii described in SEQ ID NO: 15, and Mt is a nucleic acid sequence derived from Mycobacterium tuberculosis described in SEQ ID NO: 16. It is.

As a detection oligonucleotide probe (Ck) for specifically detecting Mycobacterium kansasii, the nucleic acid sequence shown in SEQ ID NO: 1 (region A) or SEQ ID NO: 2 (region B), The nucleic acid sequence is not particularly limited as long as it has a nucleic acid sequence consisting of at least 10 consecutive bases among nucleic acid sequences complementary to the sequence. Nucleotide sequence of oligonucleotide probe (Ct) matches the nucleic acid sequence of mycobacteria other than Mycobacterium kansasii and other genus bacteria when it becomes a continuous nucleic acid sequence of 9 bases or less, and is specific to Mycobacterium kansasii Cannot be detected. Preferably, a nucleic acid sequence comprising at least 5′-GGAGGCGGCT-3 ′ (SEQ ID NO: 20) or 5′-CTACGGCTCC-3 ′ (SEQ ID NO: 21) in SEQ ID NO: 1 (region A) or complementary to the sequence Nucleic acid sequences can be used. Preferably, a nucleic acid sequence comprising at least 5′-TTCCGGCGGA-3 ′ (SEQ ID NO: 22) in SEQ ID NO: 2 (region B) or a nucleic acid sequence complementary to the sequence can be used. Since the nucleic acid sequence of SEQ ID NOs: 20 to 22 or a nucleic acid sequence complementary to the sequence is a region where there are particularly many mutations among species of the mycobacteria genus, more specific detection is possible. More preferably, the oligonucleotides described in the examples of the present specification can be used.

In the method for detecting Mycobacterium belonging to the present invention, if a method using a fluorescent dye such as IFP method or Q-Probe method is used for detection, detection or species identification can be performed without opening the reaction vessel lid after nucleic acid amplification. The so-called homogeneous detection can be performed, and the risk of misjudgment due to fatal contamination in clinical diagnosis can be suppressed.

Here, the principle of the melting curve analysis will be described with reference to FIG. FIG. 2 is a diagram schematically showing the principle of melting curve analysis, (a) showing the principle of melting curve analysis using the IFP method for detection, and (b) melting curve analysis using Q-Probe method for detection. (C) shows an example of the result of confirming the result of nucleic acid amplification by melting curve analysis.

As shown in FIG. 2A, in the IFP method, as described in Patent Document 7, when a compound that binds to a double-stranded nucleic acid (intercalator) and the compound binds to the double-stranded nucleic acid, , And a probe labeled with a reactive molecule whose emitted fluorescence changes. That is, the fluorescence emitted from the reaction molecule in a state where the double-stranded nucleic acid is dissociated into the single-stranded nucleic acid may be detected, or the fluorescence emitted from the reaction molecule in a state where it is not dissociated may be detected.

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

Double-stranded nucleic acid structures are formed by base pairing that occurs between a base in the target nucleic acid and a base in its complementary nucleic acid sequence. Base pair formation is caused by hydrogen bonds occurring between C (cytosine) and G (guanine), A (adenine) and T (thymine), or A (adenine) and U (uracil). The intercalator binds this double-stranded nucleic acid structure to the target and produces fluorescence.

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

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

As shown in FIG. 2 (b), in the Q-Probe method, as described in Patent Document 8, it has C (cytosine) at the 3 ′ end, and C (cytosine) and G (guanine) are hydrogen bonded. A probe labeled with a fluorescent dye that fluoresces when used is used. When the temperature is gradually raised after the nucleic acid amplification and the double-stranded nucleic acid is dissociated into the single-stranded nucleic acid, the quenched fluorescent dye emits light again. By detecting this fluorescence, nucleic acid amplification can be confirmed.

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)), BODIRY (trademark) series, rhodamine or a derivative thereof (for example, 5-carboxyrhodamine 6G (GR6G) 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, probes labeled with one type of fluorescent substance without using dyes inserted into double-stranded nucleic acid structures such as intercalators and without using two types of probes that cause the FRET phenomenon Can be used to detect a target nucleic acid sequence simply and specifically. The labeling position of the fluorescent substance in the base sequence of the labeled probe is not particularly limited, but 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 (guanine) or C (cytosine), or 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 so that G (guanine) is present. It is more preferable to design such that the end of the labeled probe is labeled and the terminal base is C (cytosine).

As shown in FIG. 2C, the result of the melting curve analysis may be output according to the amount of change in temperature and fluorescence intensity. In FIG. 2C, the vertical axis represents the amount of change in fluorescence intensity. This shows the case where the above-described IFP method detects fluorescence emitted from the reaction molecule in a state where the double-stranded nucleic acid is dissociated into the single-stranded nucleic acid. Moreover, it is also a case where light emission of the fluorescent dye is detected by the Q-Probe method. In the case of detecting fluorescence emitted from the above-mentioned reactive molecules in the dissociated state by the IFP method, the change in the amount of change in fluorescence intensity is opposite to that in the case of detecting fluorescence emitted in the dissociated state. That is, the shape of the obtained table is a shape obtained by inverting the shape of each line in the table shown in FIG. In other words, if the fluorescence emitted from the reactive molecule in the non-dissociated state is detected and the vertical axis is negative of the amount of change in fluorescence intensity, the shape of each line in the table shown in FIG. become.

FIG. 2C shows the result of nucleic acid amplification performed on three types of samples. That is, since the dissociation temperature varies depending on the sequence of the double-stranded nucleic acid formed, the temperature at which the peak of each line varies indicates that the nucleic acid sequence contained in the sample is different. The samples showing the results of the solid line and the broken line indicate that one type of target nucleic acid having a different base sequence was included. The sample showing the result of the alternate long and short dash line shows that both of the target nucleic acids contained in the sample showing the result of the solid line and the broken line were included. By performing the melting curve analysis in this way, it is possible to discriminate amplification by nucleic acid amplification and the base sequence of the target nucleic acid contained in the sample, that is, the type of mycobacteria.

The 16S ribosomal RNA (rRNA) gene has been widely used for detection of mycobacteria. However, the 16S rRNA gene has a low bacterial species specificity compared to the dnaJ1 gene region of the present invention, and therefore, the probe bacterial species specificity is low, so that a non-specific signal is generated upon detection. For example, the 16S ribosomal RNA genes of Mycobacterium kansasii and Mycobacterium gastrii were exactly the same. Therefore, in the detection of Mycobacterium kansasii using the conventional 16S rRNA gene, Mycobacterium gastrii was detected as a false positive. The dnaJ1 gene targeted in the present invention has different nucleotide sequences between Mycobacterium kansasii and Mycobacterium gastrii. Since the bacterial species specificity of the dnaJ1 gene region of the present invention is high, the bacterial species specificity of the probe targeting the gene is also high, and the bacterial species can be accurately identified.

FIG. 3 shows an evolutionary tree for the dnaJ1 gene region and the 16S rRNA gene of the present invention. The evolutionary phylogenetic tree is an index indicating the probability that the gene sequence differs between the bacterial species, and indicates that the greater the numerical value, that is, the evolution distance, the greater the difference in gene sequence between the bacterial species. As shown in FIG. 3, the dnaJ1 gene region of the present invention is about 10 times higher in gene sequence difference depending on the bacterial species than the 16S rRNA gene, and is excellent as a gene for identifying the species of mycobacteria. I understand that. The gene evolutionary tree can be calculated from the gene sequence using, for example, gene analysis software GENETYX (manufactured by Genetics Co., Ltd.).

In addition, the evolution distance of the dnaJ1 gene region of the present invention is the known partial sequence of the dnaJ gene (Japanese Patent No. 2540023, Japanese Patent No. 2966478, Japanese Patent No. 3134940, Japanese Patent No. 3151415, Japanese Patent No. 3360737, Japanese Patent Laid-Open No. 2001-103986, J. Clin. Microbiol. This means that the bacterial species specificity of the dnaJ1 gene region of the present invention is higher than that of a conventionally known partial sequence of the dnaJ gene, and is excellent as a gene for detecting mycobacteria or identifying the bacterial species. To do.

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 Mycobacterium kansasii using Q-Probe method]
(1) Sample preparation Four types of mycobacterial bacteria, namely Mycobacterium tuberculosis, avian Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium kansasii, respectively, using the phenol-chloroform method The extracted DNA was used as a sample. As a negative control, water was used as a sample.

(2) Synthesis of Primer and Labeled Probe Oligonucleotide having the nucleic acid sequence shown in SEQ ID NOs: 10 to 12 (hereinafter referred to as oligos 10 to 12) by the phosphoramidite method using a DNA synthesizer type 392 manufactured by PerkinElmer ) Was synthesized. The synthesis was carried out according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, DNA commissioned companies (Nippon Bioservice, Sigma-Aldrich Japan, Operon Biotechnology, etc.) were requested. Oligo 10 is the primer (A), oligo 11 is the primer (B), and oligo 12 is the probe. Oligo 12 has 5 ′ end labeled with BODIPY-FL and 3 ′ end phosphorylated.

(3) Nucleic acid amplification and melting curve analysis The following reagents were added to each of the four types of mycobacterial bacteria DNA and negative control, and Mycobacterium kansasii was detected under the following conditions. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for nucleic acid amplification and melting curve analysis. The measurement mode was 530 nm, and the results were analyzed using light cycler software.

A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution oligo 10 250 nM,
Oligo 11 1500 nM,
Oligo 12 (5 ′ end labeled with BODIPY-FL) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO4 4 mM,
KOD plus DNA polymerase 0.2U,
Extracted DNA solution 20ng

Nucleic acid amplification and melting curve analysis conditions 94 ° C., 2 minutes, 98 ° C., 0 seconds,
65 ° C for 5 seconds (50 cycles)
94 ℃ for 1 minute,
40 ℃ for 1 minute,
40 ° C to 75 ° C (temperature increase at 0.5 ° C / second).

(4) Results FIG. 4 is a diagram showing the analysis results by analyzing the light cycler software, with the horizontal axis of the graph as the temperature and the vertical axis as the differential value of the fluorescence signal. As is clear from FIG. 4, a clear peak is observed only when Mycobacterium kansasii (MK) DNA is used as a sample, and M. tuberculosis (MT), M. tuberculosis (MA), mycobacteria When the DNA of each of um. Intracellulare (MI) was used as a sample, no peak was obtained as in the negative control (NC). This indicates that the specificity of the probe (oligo 12) is very high, and that Mycobacterium kansasii can be specifically detected by using the probe (oligo 12).

[Example 2: Detection 2 of Mycobacterium kansasii using Q-Probe method]
(1) Preparation of sample Four types of acid-fast bacterium DNAs prepared in the same manner as in Example 1 were used as samples. As a negative control, water was used as a sample.

(2) Primer As in Example 1, oligonucleotides having the nucleic acid sequences shown in SEQ ID NOs: 23 and 29 (hereinafter referred to as oligos 23 and 29) were synthesized. Oligo 29 is a primer (B). Oligo 23 is a probe, and the 3 ′ end is labeled with BODIPY-FL.

(3) Nucleic acid amplification and melting curve analysis The following reagents were added to each of the four types of mycobacterial bacteria DNA and negative control, and MK was detected under the following conditions. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for nucleic acid amplification and melting curve analysis. The measurement mode was 530 nm, and the results were analyzed using light cycler software.

A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution oligo 10 250 nM,
Oligo 29 1500 nM,
Oligo 23 (BODIPY-FL labeling) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO4 4 mM,
KOD plus DNA polymerase 0.2U,
Extracted DNA solution 20ng

Nucleic acid amplification and melting curve analysis conditions The same conditions as in Example 1 were used.

(4) Results FIG. 5 is a diagram showing the results of detection using oligo 23 instead of oligo 12 in FIG. As is clear from FIG. 5, if the nucleic acid sequence shown in SEQ ID NO: 2 (region B) or a nucleic acid sequence complementary to the sequence has a nucleic acid sequence consisting of at least 10 consecutive bases, MK DNA A specific peak is observed when using as a sample. Further, as apparent from FIG. 4, if the nucleic acid sequence shown in SEQ ID NO: 1 (region A) or a nucleic acid sequence complementary to the sequence has a nucleic acid sequence consisting of at least 10 consecutive bases, MK A specific peak is observed when this DNA is used as a sample.

[Comparative Example 1: Detection 3 of Mycobacterium kansasii using Q-Probe method]
(1) Preparation of sample Four types of acid-fast bacterium DNAs prepared in the same manner as in Example 1 were used as samples. As a negative control, water was used as a sample.

(2) Primer As in Example 1, an oligonucleotide having the nucleic acid sequence shown in SEQ ID NO: 24 (hereinafter referred to as oligo 24) was synthesized. Oligo 24 is a probe. Oligo 24 is labeled at its 5 ′ end with BODIPY-FL.

(3) Nucleic acid amplification and melting curve analysis The following reagents were added to each of the four types of mycobacterial bacteria DNA and negative control, and MK was detected under the following conditions. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for nucleic acid amplification and melting curve analysis. The measurement mode was 530 nm, and the results were analyzed using light cycler software.

A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution oligo 10 250 nM,
Oligo 29 1500 nM,
Oligo 24 (BODIPY-FL labeling) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO4 4 mM,
KOD plus DNA polymerase 0.2U,
Extracted DNA solution 20ng

Nucleic acid amplification and melting curve analysis conditions The same conditions as in Example 1 were used.

(4) Results FIG. 6 is a diagram showing the results of detection using an oligo 24 instead of the oligo 12 of FIG. The oligo 24 is a probe having a nucleic acid sequence that does not contain 10 or more consecutive bases of region A or region B or their complementary nucleic acid sequences. As is clear from FIG. 6, when oligo 24 was used, a detection peak was obtained when MK was used as a template, but a similar detection peak was also obtained when MA was used as a template. That is, a detection peak specific for MK was not obtained.

[Example 3: Detection 4 of Mycobacterium kansasii using Q-Probe method]
(1) Preparation of samples 36 types of mycobacteria belonging to each of McFarland No. The suspension was suspended in TE buffer to a concentration of 1 and placed in a bead-filled tube for MORA-EXTRACT manufactured by AMR. The tube was heated at 95 ° C. for 10 minutes, and the cells were disrupted by vortexing for 5 minutes. Thereafter, the solution after the crushing treatment was diluted 100 times to obtain a cell disruption solution, which was used as a sample. As a negative control, water was used as a sample.

(2) Nucleic acid amplification and melting curve analysis MK was detected under the following conditions by adding the following reagents to 36 types of mycobacterial bacteria disruption solutions and negative controls, respectively. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for nucleic acid amplification and melting curve analysis. The measurement mode was 530 nm, and the results were analyzed using light cycler software.

A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution oligo 10 250 nM,
Oligo 11 1500 nM,
Oligo 12 (3 ′ end labeled with BODIPY-FL) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO4 4 mM,
KOD plus DNA polymerase 0.2U,
Cell disruption solution 1μL

Nucleic acid amplification and melting curve analysis conditions The same conditions as in Example 1 were used.

(3) Results Table 2 shows the results of detection of 36 types of mycobacteria. “+” In the detection column is a detectable bacterial species. "-" Indicates a bacterial species that is not detected. Detection was confirmed only when the MK cell disruption solution was used as a sample. Even in the case of mycobacteria, when 35 kinds of mycobacterial bacteria disruption solutions were used as samples, no peak was obtained as in the negative control (NC). This indicates that the specificity of the probe (oligo 12) is very high, and that Mycobacterium kansasii can be specifically detected by using the probe (oligo 12).

[Example 4: Discrimination between Mycobacterium kansasii and Mycobacterium gastrii using Q-Probe method]
(1) Preparation of Samples Mycobacterium kansasii (MK) and Mycobacterium gastrii (MG) were obtained from McFarland No. The suspension was suspended in TE buffer to a concentration of 1 and placed in a bead-filled tube for MORA-EXTRACT manufactured by AMR. The tube was heated at 95 ° C. for 10 minutes, and the cells were disrupted by vortexing for 5 minutes. Thereafter, the solution after the crushing treatment was diluted 100 times to obtain a cell disruption solution, which was used as a sample. As a negative control, water was used as a sample.

(2) Primer Similarly to Example 1, oligonucleotides having the nucleic acid sequences shown in SEQ ID NOs: 25 and 26 (hereinafter referred to as oligos 25 and 26) were synthesized. The oligos 25 and 26 are probes. Oligo 25 is labeled at the 3 ′ end with BODIPY-FL. Oligo 26 is labeled at its 5 ′ end with BODIPY-FL.

(3) Nucleic Acid Amplification and Melting Curve Analysis The following reagents were added to each MK and MG cell disruption solution and negative control, respectively, and MK was detected under the following conditions. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for nucleic acid amplification and melting curve analysis. The measurement mode was 530 nm, and the results were analyzed using light cycler software.

Reagent 1
A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution oligo 10 250 nM,
Oligo 11 1500 nM,
Oligo 12, 26 (BODIPY-FL label) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO4 4 mM,
KOD plus DNA polymerase 0.2U,
MK or MG cell disruption solution 1μL
Reagent 2
A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution oligo 10 1500 nM,
Oligo 11 250 nM,
Oligo 25 (BODIPY-FL labeling) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO4 4 mM,
KOD plus DNA polymerase 0.2U,
MK or MG cell disruption solution 1

Nucleic acid amplification and melting curve analysis conditions The same conditions as in Example 1 were used.

(4) Results FIG. 7 is a diagram showing the analysis results by analyzing the light cycler software with the horizontal axis of the graph as the temperature and the vertical axis as the differential value of the fluorescence signal. FIG. 7 shows the result of detecting MK and MG using oligo 12. As can be seen from FIG. 7, when Oligo 12 is used, a detection peak is obtained at around 60 ° C. when an MK cell disruption solution is used as a sample, and no detection peak is obtained when an MG cell disruption solution is used as a sample. It was. By using the target nucleic acid detection method of the present invention, it was possible to discriminate between MK and MG that could not be discriminated by the genetic testing method targeting the 16S rRNA gene. Even when Oligo 25 was used as a probe, a detection peak specific to MK was obtained, and MG was not detected. Even when Oligo 25 was used, the same results as when Oligo 12 was used as shown in FIG. 7 were obtained (however, the detection peak was around 65 ° C.). Oligo 12 and oligo 25 are base sequences including SEQ ID NO: 21 and SEQ ID NO: 20, respectively. Although data is not shown, when Oligo 26 was used, it was not possible to distinguish between MK and MG. Since the base sequence of oligo 26 is included in region A, MK can be detected. However, since it does not contain SEQ ID NO: 20 or 21, MK and MG could not be discriminated. As can be seen from this result, it can be seen that MK can be detected more specifically if a probe further comprising SEQ ID NOs: 20 to 22 is designed in region A or region B.

[Example 5: Detection of Mycobacterium kansasii using IFP method]
(1) Preparation of sample Four types of acid-fast bacterium DNAs prepared in the same manner as in Example 1 were used as samples. As a negative control, water was used as a sample.

(2) Synthesis of Primer and Labeled Probe As in Example 1, oligonucleotides having the nucleic acid sequences shown in SEQ ID NOs: 27 and 28 (hereinafter referred to as oligos 27 and 28) were synthesized. Oligos 27 and 28 are probes. Oligos 27 and 28 are labeled with Texas Red at the 5 ′ and 3 ′ ends.

(3) Nucleic acid amplification and melting curve analysis The following reagents were added to each of the four types of mycobacterial bacteria DNA and negative control, and MK was detected under the following conditions. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for nucleic acid amplification and melting curve analysis. The measurement mode was 640 nm, and the results were analyzed using light cycler software.

Reagent 1
A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution oligo 10 1500 nM,
Oligo 11 150 nM,
Oligo 27, 28 (Texas Red label) 1000 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO4 4 mM,
SYBR Green I (trademark) (Invitrogen) 1/20000
KOD plus DNA polymerase 0.2U,
Extracted DNA solution 20ng

Nucleic acid amplification and melting curve analysis conditions 94 ° C, 2 minutes 98 ° C, 0 seconds,
65 ° C for 5 seconds (50 cycles)
94 ℃ for 1 minute,
40 ℃ for 1 minute,
40 ° C to 75 ° C (temperature increase at 0.5 ° C / second).

(4) Results FIG. 8 shows the results of detection using the oligo 27, and is a diagram showing the analysis results by analyzing the light cycler software with the horizontal axis of the graph as the temperature and the vertical axis as the differential value of the fluorescence signal. . As shown in FIG. 8, clear peaks are observed only when MK DNA is used as a sample, and M. tuberculosis (MA), Mycobacterium intracellulare (MI), M. tuberculosis (MT) When each DNA was used as a sample, no peak was obtained as in the negative control (NC). This indicates that the specificity of the probe (oligo 27) is very high, and it is possible to specifically detect Mycobacterium tuberculosis by using the probe (oligo 27). Moreover, as a result of detecting using the oligo 28 instead of the oligo 27 of FIG. 8, the same peak was detected by MK and MI. The oligo 28 is a probe having a nucleic acid sequence that does not contain 10 or more consecutive bases of the region A or the region B or their complementary nucleic acid sequences. In the case of using Oligo 28, a detection peak is obtained when MK is used as a template, but since a similar detection peak is obtained when MI is used as a template, detection specific to MK cannot be performed. It was.

ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to detect or identify a bacterial species quickly, surely and simply about an acid-fast bacterium belonging to clinical diagnosis. Specifically, by performing a melting curve analysis using a probe whose target nucleic acid is a region having extremely high species specificity in the dnaJ1 gene of mycobacterial bacteria such as Mycobacterium tuberculosis, -It is expected to greatly contribute to the industry because it has the effect of being able to detect or identify the species of mycobacterium kansasii very accurately.

Claims (7)

A combination of oligonucleotides represented by the following (A) or (B).
(A) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 29 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 23.
(B) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 11 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 25. The detection method of Mycobacterium kansasii (Mycobacterium kansasii) shown by the following (A) or (B).
(A) A method comprising at least the following steps (1) to (3).
(1) a first step of performing nucleic acid amplification using a combination of the oligonucleotide shown in SEQ ID NO: 10 and the oligonucleotide shown in SEQ ID NO: 29 as a primer pair;
(2) a second step in which the amplification product obtainable in the first step and the detection oligonucleotide probe (Ck) shown in SEQ ID NO: 23 are hybridized to form a complex (Pk);
(3) A third step of detecting the complex (Pk) obtainable in the second step.
(B) A method comprising at least the following steps (1) to (3).
(1) a first step of performing nucleic acid amplification using a combination of the oligonucleotide shown in SEQ ID NO: 10 and the oligonucleotide shown in SEQ ID NO: 11 as a primer pair;
(2) a second step in which the amplification product obtainable in the first step and the detection oligonucleotide probe (Ck) shown in SEQ ID NO: 25 are hybridized to form a complex (Pk);
(3) A third step of detecting the complex (Pk) obtainable in the second step. The detection method according to claim 2, wherein the steps (1) to (3) are performed in a sealed state. The detection method according to claim 2 or 3, wherein the detection oligonucleotide probe (Ck) is labeled with a fluorescent dye. The detection method according to any one of claims 2 to 4, wherein an IFP method is used for detection of the complex (Pk). The detection method according to claim 2, wherein a Q-Probe method is used for detection of the complex (Pk). A detection kit for Mycobacterium kansasi, comprising at least the combination of oligonucleotides represented by the following (A) or (B):
(A) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 29 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 23.
(B) A combination of a primer set consisting of two oligonucleotides shown in SEQ ID NO: 10 and SEQ ID NO: 11 and a probe consisting of an oligonucleotide shown in SEQ ID NO: 25.