JP2009039106A - Oligonucleotide for species identification of acid-fast bacterium and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a rapid, reliable and simple method for the detection or the species identification of an acid-fast bacterium which is important on clinical diagnosis. <P>SOLUTION: This method comprises carrying out a melting curve analysis with a probe whose target nucleic acid is a region having high bacteria species specificity in dnaJ1 gene in an acid-fast bacterium. The method comprises hybridizing a primer or an elongation product of the primer with an oligonucleotide probe used for detection and containing a nucleic acid sequence comprising at least ten continuous bases among specific nucleic acid sequences to form a complex, and then discriminating the formed complex. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention provides an oligonucleotide for detecting or identifying a species of a mycobacteria genus such as Mycobacterium tuberculosis in a clinical diagnosis rapidly, surely and simply, The present invention relates to a method for detecting or identifying a species of Mycobacterium belonging to the genus Mycobacterium and a reagent 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. And so on. The source of infection is the patient, who is infected via the respiratory tract due to droplet infection.

  Traditionally, tuberculosis bacteria have been tested by culture methods. In general, M. tuberculosis is isolated and cultured on Ogawa medium, properties on 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 has a slow growth, 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 avium, Mycobacterium intracellularis, Mycobacterium bacterium, Mycobacterium bacterium It is known that atypical mycobacteria such as Scrofulceum and Mycobacterium fortuitum are 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 higher in the order of Mycobacterium kansasii> Mycobacterium avium, Mycobacterium intracellulare> Other species in order Is particularly 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. In addition, similar non-pathogenic bacteria such as Mycobacterium smegmatis may be detected. 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 further, 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 acid sequence-based amplification method; Nature Vol. 350, 91 (1991) Strand Displacement Amplification: Nucleic acid research Vol. 20, p. 1691 (1992) Transcription mediated amplification method; Clin. Microbiol. Volume 31, Page 3270 (1993) Loop-mediated isometric amplification method: J Clin Microbiol. 2004 Volume 42: 1,956 Isothermal and chimeric primer-initiated amplification of nucleic 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 detect, or quickly, reliably and easily detect acid-fast bacilli such as Mycobacterium tuberculosis important in clinical diagnosis. An object of the present invention is to provide an oligonucleotide for species identification, a method for detection or species identification of Mycobacteria using the oligonucleotide, and reagents thereof.

  As a result of intensive studies in view of the above problems, the present inventors have 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. By using a probe, it has been found that detection or species identification of a plurality of acid-fast bacterium belonging to the art can be achieved with a single type of fluorescent dye, which has been difficult with the prior art, and the present invention has been completed. That is, the present invention has the following configuration.

[Section 1]
An oligonucleotide comprising a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 6 or a nucleic acid sequence complementary to the sequence.
[Section 2]
A method for identifying the species of a mycobacterial bacterium comprising at least the following steps (1) to (3):
(1) SEQ ID NOs: 1 to 6 using at least one type of oligonucleotide primer (A) and at least one type of oligonucleotide primer (B) complementary to a part of the extension product of the primer (A) A first step of amplifying a nucleic acid fragment comprising a nucleic acid sequence shown in any of the above or a nucleic acid sequence that is 90% or more identical to a nucleic acid sequence complementary to the sequence
(2) From the extension product of primer (A) or (B) that can be obtained in the first step, and at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to the sequence And at least one kind of detection oligonucleotide probe (Ct) containing a nucleic acid sequence to form a complex (Pt),
At least one kind comprising an extension product of the primer (A) or (B) and a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 3 or 4 or a nucleic acid sequence complementary to the sequence The oligonucleotide probe for detection (Cm) is hybridized to form a complex (Pm),
An extension product of the primer (A) or (B), and at least one kind containing a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 5 or 6 or a nucleic acid sequence complementary to the sequence A second step of hybridizing the detection oligonucleotide probe (Ck) with a complex (Pk),
(3) A third step of determining which of the complexes (Pt), (Pm), or (Pk) that can be obtained in the second step is formed.
[Claim 2-1]
A method for identifying the species of a mycobacterial bacterium comprising at least the following steps (1) to (3):
(1) SEQ ID NOs: 1 to 6 using at least one type of oligonucleotide primer (A) and at least one type of oligonucleotide primer (B) complementary to a part of the extension product of the primer (A) A first step of amplifying a nucleic acid fragment comprising a nucleic acid sequence shown in any of the above or a nucleic acid sequence that is 90% or more identical to a nucleic acid sequence complementary to the sequence
(2) a nucleic acid sequence comprising at least 10 consecutive bases of the extension product of primer (B) obtained in the first step and the nucleic acid sequence shown in SEQ ID NO: 1 or 2, or a nucleic acid sequence complementary to the sequence At least one kind of oligonucleotide probe for detection (Ct) contained therein is hybridized to form a complex (Pt),
An extension product of the primer (B) and at least one kind of detection oligo containing a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 3 or 4 or a nucleic acid sequence complementary to the sequence A nucleotide probe (Cm) is hybridized to form a complex (Pm),
An extension product of the primer (B) and at least one kind of detection oligo containing a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 5 or 6 or a nucleic acid sequence complementary to the sequence A second step of hybridizing a nucleotide probe (Ck) to form a complex (Pk);
(3) A third step of determining which of the complexes (Pt), (Pm), or (Pk) that can be obtained in the second step is formed.
[Section 3]
Item 3. The bacterial species identification method according to Item 2, wherein the Tm values of the probes (Ct), (Cm), and (Ck) differ from each other by 3 ° C. or more.
[Section 4]
Item 4. The bacterial species identification method according to Item 2 or 3, wherein the lengths of the probes (Ct), (Cm), and (Ck) differ from each other by one or more bases.
[Section 5]
Item 5. The bacterial species identification method according to any one of Items 2 to 4, wherein the steps (1) to (3) are performed in a sealed state.
[Section 6]
Item 6. The bacterial species identification method according to any one of Items 2 to 5, wherein the detection oligonucleotide probes (Ct), (Cm), and (Ck) are labeled with a fluorescent dye.
[Section 7]
While measuring the fluorescence emitted from the complex (Pt), (Pm), or (Pk), a melting curve analysis with continuous temperature increase or decrease is performed, and the complex (Pt), (Pm), or Item 7. The bacterial species identification method according to any one of Items 2 to 6, wherein which complex is formed among (Pk).
[Section 8]
Item 8. The bacterial species identification method according to Item 7, wherein the IFP method is used to detect a complex (Pt), (Pm), or (Pk) when performing a melting curve analysis.
[Section 9]
Item 8. The bacterial species identification method according to Item 7, wherein the Q-Probe method is used to detect a complex (Pt), (Pm), or (Pk) when performing a melting curve analysis.
[Section 10]
A method for identifying the species of a mycobacterial bacterium comprising at least the following (1) to (4):
(1) at least one kind of detection oligonucleotide probe (Ct) containing a nucleic acid sequence represented by SEQ ID NO: 1 or 2 or a nucleic acid sequence consisting of at least 10 consecutive bases of a nucleic acid sequence complementary to the sequence;
(2) at least one type of oligonucleotide probe for detection (Cm) containing a nucleic acid sequence represented by SEQ ID NO: 3 or 4 or a nucleic acid sequence consisting of at least 10 consecutive bases among nucleic acid sequences complementary to the sequence;
(3) at least one type of oligonucleotide probe for detection (Ck) containing a nucleic acid sequence represented by SEQ ID NO: 5 or 6 or a nucleic acid sequence consisting of at least 10 consecutive bases of a nucleic acid sequence complementary to the sequence;
(4) An oligonucleotide primer capable of amplifying a nucleic acid fragment containing a nucleic acid sequence of 90% or more of the nucleic acid sequence of probes (Ct), (Cm), and (Ck) or a complementary nucleic acid sequence to the sequence (A) and oligonucleotide primer (B).

  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 is difficult to detect or identify a plurality of mycobacterial bacteria using a single type of fluorescent dye. Therefore, an inexpensive system that detects only a single type of fluorescence has the effect of being able to detect or identify mycobacterial bacteria without risking misjudgment due to contamination.

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'-CCCGAGGGAGCGCCCGCGCAACCCGGCTCCGCCCT-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: 18, Mycobacterium intracellulare is in SEQ ID NO: 19, and Mycobacterium kansasi is in SEQ ID NO: 20. , Mycobacterium tuberculosis describes the specific nucleic acid sequence in SEQ ID NO: 21, respectively.
In addition, the above-mentioned “region having a high bacterial species specificity in the dnaJ1 gene of mycobacterial bacteria” will be described later as “region A and region B”. , Japanese Patent No. 3134940, L Clin. Microbio1, Vol. 31, p. 446), and is not used for detection and / or identification of mycobacteria in the prior art. .

  The method for detecting or identifying the species of 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 bacterial 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 the 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 (see Nucleic acid 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 (Transcribing medium amplification. Mic. 31, p. 3270, 1993), LAMP (Loop-mediate) I. (Thermal amplification method of J. Clin Microbiol. Vol. 42: see 1,956, 2004), ICAN (Isothermal and chimerized primer of nuclei. ) And the like, but is not limited thereto. 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 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 region C, region D or region E shown in FIG. 5 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 these regions, region C, region D, and region E shown in FIG. 5 are regions having a relatively low GC content of 65% or less in all mycobacteria. 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: 9)
5′-CCAAGCGCAAGGAGTACGACGAA-3 ′ (SEQ ID NO: 10)
As a primer (B),
5′-GAACAAGCCACCGAACAAAGTCACCGAT-3 ′ (SEQ ID NO: 11)
5′-CGTCGAACATTTCGTTGAGGTTGAACT-3 ′ (SEQ ID NO: 12)
5′-CAAGCCACCGAACAGGTCGCCGAT-3 ′ (SEQ ID NO: 13)
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 NOs: 1 to 6 or a nucleic acid sequence complementary to the nucleic acid sequence "match 90% or more" For example, when targeting Mycobacterium tuberculosis, the agreement rate is shown when the nucleic acid fragment is mutated between strains of Mycobacterium tuberculosis. This shows that even when there is a mutation between M. tuberculosis strains, M. tuberculosis can be specifically detected by distinguishing it from non-tuberculous mycobacteria. The coincidence rate is preferably 90% or more. Table 1 shows, as an example, the coincidence ratio between the nucleic acid sequences possessed by 35 types of clinically isolated nontuberculous mycobacteria and the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to the nucleic acid sequence. The bacterial species most consistent with SEQ ID NO: 1 is Mycobacterium malmoens with a match rate of 88%, and the bacterial species most consistent with SEQ ID NO: 2 is Mycobacterium chitae. ), The coincidence rate is 88%. Even in the case of nontuberculous mycobacteria belonging to the same genus as M. tuberculosis, the concordance rate is less than 90%. Further, the coincidence ratio between the nucleic acid sequence possessed by 20 strains of Mycobacterium tuberculosis 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 20 strains. Similar results are obtained in Mycobacterium avium and Mycobacterium intracellulare of SEQ ID NO: 3 or 4 and Mycobacterium kansasii of SEQ ID NO: 5 or 6.

  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: 18, and Mi is a Mycobacterium intracellulare described in SEQ ID NO: 19. Mk is a nucleic acid sequence derived from Mycobacterium kansasii described in SEQ ID NO: 20, and Mt is derived from Mycobacterium tuberculosis described in SEQ ID NO: 21 Nucleic acid sequence.

  As a detection oligonucleotide probe (Ct) for specifically detecting Mycobacterium tuberculosis, the nucleic acid sequence shown in SEQ ID NO: 1 (region A) or SEQ ID NO: 2 (region B) or the sequence The nucleic acid sequence is not particularly limited as long as it has a nucleic acid sequence consisting of at least 10 consecutive bases. When the nucleic acid sequence consists of 9 or fewer bases, the nucleic acid sequence of the oligonucleotide probe (Ct) matches the nucleic acid sequence of mycobacteria other than Mycobacterium tuberculosis and bacteria of other genera, and is specific for Mycobacterium tuberculosis. Detection is not possible. Preferably, a nucleic acid sequence comprising at least 5′-GGTTTCGGGG-3 ′ (SEQ ID NO: 22) in SEQ ID NO: 1 (region A) or a nucleic acid sequence complementary to the sequence can be used. Preferably, a nucleic acid sequence comprising at least 5′-AACCGGCGGT-3 ′ (SEQ ID NO: 23) in SEQ ID NO: 2 (region B) or a nucleic acid sequence complementary to the sequence can be used. Since the nucleic acid sequences of SEQ ID NO: 22 and SEQ ID NO: 23 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 clinical diagnosis, Mycobacterium avium and Mycobacterium intracellulare are often treated in the same way as Mycobacterium avium complex (MAC). As a detection type oligonucleotide probe (Cm) for specifically detecting M. tuberculosis or Mycobacterium intracellulare, that is, MAC, SEQ ID NO: 3 (region A) or SEQ ID NO: 4 (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 of the nucleic acid sequence shown or a complementary nucleic acid sequence. When the nucleic acid sequence has 9 or fewer consecutive bases, the nucleic acid sequence of the oligonucleotide probe (Cm) matches the nucleic acid sequence of acid-fast bacteria other than MAC or bacteria of other genera, and detection specific to MAC becomes impossible. Preferably, a nucleic acid sequence comprising at least 5′-TCAACGTCGG-3 ′ (SEQ ID NO: 24) in SEQ ID NO: 3 (region A) or a nucleic acid sequence complementary to the sequence can be used. Preferably, a nucleic acid sequence comprising at least 5′-AGCGGGCGCA-3 ′ (SEQ ID NO: 25) in SEQ ID NO: 4 (region B) or a nucleic acid sequence complementary to the sequence can be used. Since the nucleic acid sequences of SEQ ID NO: 24 and SEQ ID NO: 25 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.

  As a detection oligonucleotide probe (Ck) for specifically detecting Mycobacterium kansasii, the nucleic acid sequence shown in SEQ ID NO: 5 (region A) or SEQ ID NO: 6 (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 (Ck) matches the nucleic acid sequence of mycobacteria other than Mycobacterium kansasii and bacteria of other genera 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: 26) or 5′-CTACGGCTCC-3 ′ (SEQ ID NO: 27) in SEQ ID NO: 5 (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: 28) in SEQ ID NO: 6 (region B) or a nucleic acid sequence complementary to the sequence can be used. Since the nucleic acid sequence of SEQ ID NOs: 26 to 28 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.

  The probes (Ct), (Cm), and (Ck) can be arbitrarily mixed and used for detection because of their high bacterial species specificity. The combinations described in the examples can be used.

  In the method for detecting Mycobacterium belonging to the genus Mycobacterium or the method for identifying the bacterial species, if a method using a fluorescent dye such as IFP method or Q-Probe method is used for detection, the detection or detection can be performed without opening the reaction vessel after nucleic acid amplification. So-called homogeneous detection that identifies bacterial species is possible, and the risk of erroneous determination due to fatal contamination in clinical diagnosis can be suppressed.

  Further, in the method for identifying species of the acid-fast bacterium belonging to the present invention, as described above, when the probes (Ct), (Cm), and (Ck) are mixed and used for detection, The method for discriminating which complex is formed among the complexes (Pt), (Pm), and (Pk) hybridized with the target nucleic acid or amplified nucleic acid fragment is not particularly limited, and various conventionally known various types are known. Although the method can be used, it is preferred to use melting curve analysis. For detection of a signal in the melting curve analysis, an IFP method, a Q-Probe method, or the like can be used.

  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. 2 (a), 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, such as SYBR Green I (trademark), LC Green I (trademark), LC Green Plus (trademark), ethidium bromide, acridine orange, thiazole. There are orange, oxazole yellow, rhodamine and the like, but this is not the case. [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, Texas Red (trademark), Cy3, Cy5, rhodamine, etc., but are preferably fluorescent substances that produce FRET, more preferably interact with an intercalator. As a particularly preferable fluorescent substance, Texas Red (trademark) can be mentioned, but it is not limited to this. If the FRET phenomenon is used, the fluorescence emitted only by the presence of the nucleic acid probe in the fluorescence detection step is suppressed, and the fluorescence emitted by the nucleic acid probe hybridized with the target nucleic acid can be specifically detected.

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

  In the method for identifying species of Mycobacteria of the present invention, when the melting curve analysis is performed as described above, the probes (Ct), (Cm), and (Ck) each hybridize with a target nucleic acid or an amplified nucleic acid fragment. The melting temperature of the composites (Pt), (Pm), and (Pk), that is, the temperature at which the double-stranded nucleic acid dissociates into the single-stranded nucleic acid in the composite is important.

  In general, the melting temperature in melting curve analysis using an oligonucleotide probe depends on the Tm value of the probe. The Tm value represents the temperature at which the primer anneals with the complementary base and dissociates. The Tm value can also be calculated by calculation. Various calculation methods have been devised for calculating the Tm value using the salt concentration in hybridization as a parameter. As a commonly used calculation method, the closest base pair method (Schildkraut C., Lifeson S. (1965) Dependence of the melting temperature of DNA on salt concentration-Biopolymers 3, B. 195-208, 195-208). , Frank R., Blocker H., Markey LA (1986) Predicting DNA duplex stability from the base sequence-Proc. Natl. Acad. Sci. Shaffer, J .; M Bonner, J .; Hirose, T .; Itakura, K .; (1979) Nucleic Acid Res. 6, 3543), GC method (Dependence of the Censor Temporation). (1965) BIOPOLYMERS 3.195-208, Optimization of the annealing for DNA amplification in vitro, W. Rychlik, Nucl. Acids Res. (1960) 18 The target nucleus The difference in Tm value due to the calculation method is affected depending on the sequence of the detection or the detection condition, etc., but the probe having the effect of the present invention is experimentally corrected by the study that can be easily conceived by those skilled in the art. Can be set. Breslauer K.M. J. et al. , Frank R. et al. Blocker H. , Markey L. A. (1986) Predicting DNA duplex stability from the base sequence-Proc. Natl. Acad. Sci. It is preferable to use the Tm value calculated by the method described in USA 83, 3746-3750.

  In the method for identifying the species of Mycobacterium belonging to the present invention, the Tm values of the probes (Ct), (Cm), and (Ck) are preferably different from each other by 3 ° C. or more. As an example, if (Ct)> (Cm)> (Ck) in the descending order of the Tm value of the probe, the difference between the Tm values of the probes (Ct) and (Cm) is 3 ° C. or more. The difference between the Tm values of (Cm) and (Ck) may be 3 ° C. or more. If the Tm values differ from each other by 3 ° C. or more, the type of complex (Pt), (Pm), or (Pk) in which each probe is hybridized with the target nucleic acid or amplified nucleic acid fragment using melting curve analysis or the like Can be clearly distinguished.

  Thus, in the method for identifying species of mycobacteria of the present invention, the reason why the type of the complex (Pt), (Pm), or (Pk) can be discriminated by the difference in Tm value of 3 ° C. or higher. It is mentioned that the species specificity of probes (Ct), (Cm), and (Ck) is high, that is, the species specificity of the dnaJ1 gene region discovered in the present invention is high.

  The 16S ribosomal RNA (rRNA) gene has been widely used for identifying bacterial species 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. It was difficult to mix and use the probes in the detection. In other words, in the method of identifying mycobacterial bacteria of the present invention, 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. It is possible to simultaneously discriminate between the bacterial species.

  FIG. 3 shows an evolutionary tree for the dnaJ1 gene region and the 16S rRNA gene of the present invention. Evolutionary phylogenetic tree is an index indicating the probability of gene sequence differences between species, and the larger the value, the greater the difference in gene sequence between 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, in the method for identifying the species of acid-fast bacterium of the present invention, in addition to the Tm value, the probe can be easily designed using the probe length as an index. The lengths of the probes (Ct), (Cm), and (Ck) are preferably different from each other by one or more bases. As an example, when (Ct)> (Cm)> (Ck) in the order of the longest probe, the difference in length between the probes (Ct) and (Cm) is one base or more, and the probe (Cm) The difference in length of (Ck) may be one base or more. If the lengths of the probes differ from each other by one or more bases, a complex (Pt), (Pm), or (Pk) in which each probe is hybridized with a target nucleic acid or an amplified nucleic acid fragment using melting curve analysis or the like Type can be clearly identified. Such a simple probe design method is also difficult with the prior art, and is derived from the high bacterial species specificity of the dnaJ1 gene region of the present invention.

  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: Identification of Mycobacterium tuberculosis and MAC 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 Using a DNA synthesizer type 392 manufactured by PerkinElmer, Inc., an oligonucleotide having the nucleic acid sequence shown in SEQ ID NOs: 14 to 17 (hereinafter referred to as oligos 14 to 17) is synthesized by the phosphoramidite method. did. The synthesis was performed according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C. with ammonia water. 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 14 is the primer (A), oligo 15 is the primer (B), oligo 16 is the probe (Ct), and oligo 17 is the probe (Cm). Oligo 16 is labeled at the 3 ′ end with BODIPY-FL, oligo 17 is labeled at the 5 ′ end with BODIPY-FL, and the 3 ′ end is 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 M. tuberculosis 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.

Reagents A 10 μl solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 14 250 nM,
Oligo 15 1500 nM,
Oligo 16 (3 ′ end labeled with BODIPY-FL) 200 nM,
Oligo 17 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 200 nM,
X10 buffer 1 μl,
dNTP 0.2 mM,
MgSO ₄ 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 being the temperature and the vertical axis being the differential value of the fluorescence signal. As is clear from FIG. 4, a clear peak is observed when a sample of Mycobacterium tuberculosis (MT) is used, and avian Mycobacterium tuberculosis (MA) and Mycobacterium intracellulare (MI), that is, MAC. When DNA is used as a sample, a clear peak is observed at a position different from that of M. tuberculosis (MT), and when Mycobacterium kansasii (MK) DNA is used as a sample, it is the same as the negative control (NC). No peak was obtained. This indicates that the specificity of the probe (Ct) and the probe (Cm) is very high. By using the probe (Ct) and the probe (Cm) in combination, It shows that the MAC can be identified.

[Example 2: Identification 1 of Mycobacterium tuberculosis, MAC and 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 primers Oligonucleotides having the nucleic acid sequences shown in SEQ ID NOs: 29 and 30 (hereinafter referred to as oligos 29 and 30) were synthesized in the same manner as in Example 1. The oligo 29 is a probe (Ck) and the oligo 30 is a probe (Ct). Oligo 29 has a 5 ′ end labeled with BODIPY-FL and a 3 ′ end phosphorylated. Oligo 30 is labeled with BODIPY-FL at the 3 ′ end.

(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 M. tuberculosis 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.

Reagents A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 14 250 nM,
Oligo 15 1500 nM,
Oligo 30 (3 ′ end labeled with BODIPY-FL) 100 nM,
Oligo 17 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 80 nM,
Oligo 29 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 300 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO ₄ 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. 6 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. The oligos 30, 17, and 29, which are labeled probes, hybridize to the extension product of oligo 15 used as the primer (B). That is, the three types of labeled probes used for detection all hybridize with the extension product extended using the same primer. As is clear from FIG. 6, when a tuberculosis tuberculosis (MT) sample was used, a clear peak was observed at 68 ° C., and avian tuberculosis (MA) and Mycobacterium intracellulare (MI), That is, when MAC DNA was used as a sample, a clear peak was observed at 63 ° C., and when Mycobacterium kansasii (MK) DNA was used as a sample, a peak was observed at 59 ° C. Specific detection peaks for MT, MAC, and MK were observed. Negative control (NC) did not obtain a peak. This indicates that the specificity of the probe (Ct), the probe (Cm) and the probe (Ck) is very high, and the three types of probes, ie, the probe (Ct), the probe (Cm) and the probe (Ck) By using in combination, it is shown that M. tuberculosis, MAC, and Mycobacterium kansasii that are particularly important in clinical diagnosis among acid-fast bacteria can be identified at one time by one measurement.

[Example 3: Identification 2 of Mycobacterium tuberculosis, MAC and 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) Primer Synthesis An oligonucleotide having the nucleic acid sequence represented by SEQ ID NO: 31 (hereinafter referred to as oligo 31) was synthesized in the same manner as in Example 1. The oligo 31 is a probe (Ct). Oligo 31 is labeled at its 3 ′ 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 M. tuberculosis 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.

Reagents A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 14 500 nM,
Oligo 15 500 nM,
Oligo 31 (3 ′ end labeled with BODIPY-FL) 100 nM,
Oligo 17 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 80 nM,
Oligo 29 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 300 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO ₄ 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. 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. The oligo 31 that is the labeled probe hybridizes to the extension product of the oligo 14 used as the primer (A), and the oligo 17 that is the labeled probe corresponds to the extension product of the oligo 15 used as the primer (B). 29 hybridizes. That is, among the three types of labeled probes used for detection, the probe (Ct) hybridizes with the “extension products extended using different primers” from the probes (Cm) and (Ck). As is clear from FIG. 7, when a tuberculosis tuberculosis (MT) sample is used, a clear peak is observed at 66 ° C., and avian tuberculosis (MA) and Mycobacterium intracellulare (MI), That is, when MAC DNA was used as a sample, a clear peak was observed at 63 ° C., and when Mycobacterium kansasii (MK) DNA was used as a sample, a peak was observed at 59 ° C. Specific detection peaks for MT, MAC, and MK were observed. Negative control (NC) did not obtain a peak. This indicates that the specificity of the probe (Ct), the probe (Cm) and the probe (Ck) is very high, and the three types of probes, ie, the probe (Ct), the probe (Cm) and the probe (Ck) By using in combination, it is shown that M. tuberculosis, MAC, and Mycobacterium kansasii that are particularly important in clinical diagnosis among acid-fast bacteria can be identified at one time by one measurement.

[Example 4: Identification of Mycobacterium tuberculosis, MAC and Mycobacterium kansasii using Q-Probe method 3]
(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 primers Oligonucleotides having the nucleic acid sequences shown in SEQ ID NOs: 32 and 33 (hereinafter referred to as oligos 32 and 33) were synthesized in the same manner as in Example 1. The oligo 32 is a probe (Cm), and the oligo 33 is a probe (Ck). Oligo 32 is labeled at its 3 ′ end with BODIPY-FL. Oligo 33 has a 5 ′ end labeled with BODIPY-FL and a 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 M. tuberculosis 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.

Reagents A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 14 500 nM,
Oligo 15 500 nM,
Oligo 30 (3 ′ end labeled with BODIPY-FL) 100 nM,
Oligo 32 (3 ′ end labeled with BODIPY-FL) 160 nM,
Oligo 33 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 300 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO ₄ 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. 8 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. The oligo 32, which is a labeled probe, hybridizes to the extension product of the oligo 14 used as the primer (A), and the oligo 30, which is the labeled probe, extends to the extension product of the oligo 15 used as the primer (B), 33 hybridizes. That is, of the three types of labeled probes used for detection, the probe (Cm) hybridizes with the “extension products extended using different primers” from the probes (Ct) and (Ck). As is clear from FIG. 8, when a sample of Mycobacterium tuberculosis (MT) is used, a clear peak is observed at 68 ° C., and avian Mycobacterium tuberculosis (MA) and Mycobacterium intracellulare (MI), That is, when MAC DNA was used as a sample, a clear peak was observed at 60 ° C., and when Mycobacterium kansasii (MK) DNA was used as a sample, a peak was observed at 63 ° C. Specific detection peaks for MT, MAC, and MK were observed. Negative control (NC) did not obtain a peak. This indicates that the specificity of the probe (Ct), the probe (Cm) and the probe (Ck) is very high, and the three types of probes, ie, the probe (Ct), the probe (Cm) and the probe (Ck) By using in combination, it is shown that M. tuberculosis, MAC, and Mycobacterium kansasii that are particularly important in clinical diagnosis among acid-fast bacteria can be identified at one time by one measurement.

[Example 5: Identification 4 of Mycobacterium tuberculosis, MAC and 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) 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 M. tuberculosis 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.

Reagents A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 14 500 nM,
Oligo 15 500 nM,
Oligo 31 (3 ′ end labeled with BODIPY-FL) 100 nM,
Oligo 32 (3 ′ end labeled with BODIPY-FL) 160 nM,
Oligo 33 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 300 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO ₄ 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).

(3) Results FIG. 9 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. The oligos 31 and 32, which are labeled probes, hybridize to the extension product of the oligo 14 used as the primer (A), and the oligos which are labeled probes to the extension product of the oligo 15 used as the primer (B). 33 hybridizes. That is, of the three types of labeled probes used for detection, the probe (Ck) hybridizes with the “extension products extended using different primers” from the probes (Ct) and (Cm). As is clear from FIG. 9, when a tuberculosis tuberculosis (MT) sample was used, a clear peak was observed at 66 ° C., and avian tuberculosis (MA) and Mycobacterium intracellulare (MI), That is, when MAC DNA was used as a sample, a clear peak was observed at 60 ° C., and when Mycobacterium kansasii (MK) DNA was used as a sample, a peak was observed at 63 ° C. Specific detection peaks for MT, MAC, and MK were observed. Negative control (NC) did not obtain a peak. This indicates that the specificity of the probe (Ct), the probe (Cm) and the probe (Ck) is very high, and the three types of probes, ie, the probe (Ct), the probe (Cm) and the probe (Ck) By using in combination, it is shown that M. tuberculosis, MAC, and Mycobacterium kansasii that are particularly important in clinical diagnosis among acid-fast bacteria can be identified at one time by one measurement.

[Example 6: Identification of Mycobacterium tuberculosis, MAC and Mycobacterium kansasii from Mycobacterium using the Q-Probe method]
(1) Preparation of Samples 36 types of mycobacteria belonging to each of McFarland 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 The following reagents were added to 36 types of mycobacterial bacteria lysate and negative control, respectively, and M. tuberculosis 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.

Reagents A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 14 250 nM,
Oligo 15 1500 nM,
Oligo 30 (3 ′ end labeled with BODIPY-FL) 100 nM,
Oligo 17 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 80 nM,
Oligo 29 (5 ′ end labeled with BODIPY-FL, 3 ′ end phosphorylated) 300 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO ₄ 4 mM,
KOD plus DNA polymerase 0.2U,
Extracted DNA solution 20ng

Nucleic acid amplification amplification and melting curve analysis conditions 94 ° C, 2 minutes 98 ° C, 0 seconds,
62 ° 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).

(3) Results Table 2 shows the results of detection of 36 types of mycobacteria. The description of the numerical value in the detection column indicates that the species is detectable. For example, “68” indicates that a detection peak is observed at 68 ° C. Species whose peak cannot be detected at 55 ° C. or higher are described as “−”. The bacterial species that can be detected by the combination of oligo 30 (Ct), oligo 17 (Cm), and oligo 29 (Ck) are MT, MAC, and MK for which specific detection peaks are obtained in FIG. Similarly to the results of Example 2, when the MT cell disruption solution was used as a sample, 68 ° C., and when the MAC cell disruption solution was used as a sample, 63 ° C., and the MK cell disruption solution was used as a sample. In some cases, a detection peak at 59 ° C. was observed. However, no peak was obtained in the same manner as in the negative control (NC) when 32 types of mycobacterial cells other than MT, MAC, and MK were used as samples. This is because the combination of Oligo 30 (Ct), Oligo 17 (Cm) and Oligo 29 (Ck) can specifically detect three types of clinically important MT, MAC, and MK. Shows that it can be clearly identified.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to detect or to identify a bacterial species quickly, surely and simply about a mycobacteria genus important for 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 difficult to detect or identify a plurality of mycobacterial bacteria using a single type of fluorescent dye. Therefore, an inexpensive system that detects only one type of fluorescence has the effect of being able to detect or identify mycobacterial bacteria without risking misjudgment due to contamination. It is expected to make a significant contribution to industry.

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 of Example 1. It is a figure which shows the area | region C, the area | region D, and the area | region E in the dnaJ1 gene of the mycobacteria genus bacteria of this invention. 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 4. It is a figure which shows the result of Example 5.

Claims (10)

  An oligonucleotide comprising a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 6 or a nucleic acid sequence complementary to the sequence. A method for identifying the species of a mycobacterial bacterium comprising at least the following steps (1) to (3):
(1) SEQ ID NOs: 1 to 6 using at least one type of oligonucleotide primer (A) and at least one type of oligonucleotide primer (B) complementary to a part of the extension product of the primer (A) A first step of amplifying a nucleic acid fragment comprising a nucleic acid sequence shown in any of the above or a nucleic acid sequence that is 90% or more identical to a nucleic acid sequence complementary to the sequence;
(2) From the extension product of primer (A) or (B) that can be obtained in the first step, and at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to the sequence And at least one kind of detection oligonucleotide probe (Ct) containing a nucleic acid sequence to form a complex (Pt),
At least one kind comprising an extension product of the primer (A) or (B) and a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 3 or 4 or a nucleic acid sequence complementary to the sequence The oligonucleotide probe for detection (Cm) is hybridized to form a complex (Pm),
An extension product of the primer (A) or (B), and at least one kind containing a nucleic acid sequence consisting of at least 10 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 5 or 6 or a nucleic acid sequence complementary to the sequence A second step of hybridizing the detection oligonucleotide probe (Ck) with a complex (Pk),
(3) A third step of determining which of the complexes (Pt), (Pm), or (Pk) that can be obtained in the second step is formed.   The method according to claim 2, wherein Tm values of the probes (Ct), (Cm), and (Ck) differ from each other by 3 ° C or more.   4. The method according to claim 2, wherein the lengths of the probes (Ct), (Cm), and (Ck) differ from each other by one or more bases.   The method according to any one of claims 2 to 4, wherein the steps (1) to (3) are performed in a sealed state.   6. The bacterial species identification method according to claim 2, wherein the detection oligonucleotide probes (Ct), (Cm), and (Ck) are labeled with a fluorescent dye.   While measuring the fluorescence emitted from the complex (Pt), (Pm), or (Pk), a melting curve analysis with continuous temperature increase or decrease is performed, and the complex (Pt), (Pm), or It distinguishes which complex formed among (Pk), The bacterial species identification method in any one of Claims 2-6 characterized by the above-mentioned.   The bacterial species identification method according to claim 7, wherein the IFP method is used to detect the complex (Pt), (Pm), or (Pk) when performing a melting curve analysis.   The bacterial species identification method according to claim 7, wherein the Q-Probe method is used for detection of a complex (Pt), (Pm), or (Pk) when performing a melting curve analysis. A method for identifying the species of a mycobacterial bacterium comprising at least the following (1) to (4):
(1) at least one kind of detection oligonucleotide probe (Ct) containing a nucleic acid sequence represented by SEQ ID NO: 1 or 2 or a nucleic acid sequence consisting of at least 10 consecutive bases of a nucleic acid sequence complementary to the sequence;
(2) at least one type of oligonucleotide probe for detection (Cm) containing a nucleic acid sequence represented by SEQ ID NO: 3 or 4 or a nucleic acid sequence consisting of at least 10 consecutive bases among nucleic acid sequences complementary to the sequence;
(3) at least one type of oligonucleotide probe for detection (Ck) containing a nucleic acid sequence represented by SEQ ID NO: 5 or 6 or a nucleic acid sequence consisting of at least 10 consecutive bases of a nucleic acid sequence complementary to the sequence;
(4) An oligonucleotide primer capable of amplifying a nucleic acid fragment containing a nucleic acid sequence of 90% or more of the nucleic acid sequence of probes (Ct), (Cm), and (Ck) or a complementary nucleic acid sequence to the sequence (A) and oligonucleotide primer (B).