JP2013000093A - Identification method of bacterium or virus belonging to same genus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quickly, highly sensitively and specifically identifying two or more kinds of bacteria or viruses belonging to the same genus.SOLUTION: There is provided a method for identifying n (n≥2) kinds of bacteria or viruses belonging to the same genus by using n kinds of oligonucleotide probes that can form double strands each complemental to a part of a sequence in a specific nucleic acid derived from n kinds of bacteria or viruses belonging to the same genus, wherein the sequence of the specific nucleic acid is a sequence having high homology between the bacteria or the viruses to be identified, the oligonucleotide probes are probes changing the fluorescence properties compared with those before formation when forming a complemental double strand to a part of the sequence in the specific nucleic acid, and the changing amount of the fluorescence properties with the oligonucleotide probes is different by bacteria or viruses to be identified with the oligonucleotide probes.

Description

  The present invention relates to a method for identifying two or more types of bacteria or viruses belonging to the same genus rapidly, sensitively and specifically.

  Mycobacterium is a gram-positive gonococcus having acid-fast properties and is also called acid-fast bacterium. There are many pathogenic bacteria that cause severe lesions in mycobacteria, and among them, Mycobacterium tuberculosis is a pathogen that causes tuberculosis in humans, and its examination is extremely important clinically. In addition to Mycobacterium tuberculosis, nontuberculous mycobacteria such as Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium kansasii are known to be pathogenic to humans.

  Nontuberculous mycobacteria, like M. tuberculosis, affect human lungs, lymph nodes, skin, etc., and in particular, many cases infect the lungs. Nontuberculous mycobacteria do not cause human-to-human infection like tuberculosis, but are resistant to drugs against M. tuberculosis and there are few effective drugs. Thus, since the treatment method for acid-fast bacilli varies depending on the species, it is indispensable to quickly identify the species in acid-fast bacilli.

Japanese Patent No. 2998546 JP 2000-014400 A JP 2005-218317 A US Pat. No. 5,925,517 US Pat. No. 6,103,476 US Patent 5210015 US Pat. No. 5,487,972 Japanese Patent No. 3437816 US Pat. No. 5,011,769 US Pat. No. 5,403,711

Ishiguro T. et al. , Anal. Biochem. , 314, 77-86 (2003)

  As described above, in the mycobacterial test, it is indispensable to quickly identify the bacterial species because the treatment method differs depending on the bacterial species of the infected acid-fast bacterium. In mycobacteria testing, it is needless to say that identification of Mycobacterium tuberculosis causing tuberculosis in humans is important, but among non-tuberculous mycobacteria, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium intracellulare, In particular, it is important to identify Mycobacterium avium and Mycobacterium intracellulare, which frequently appear.

  When identifying the species of infected acid-fast bacilli, it is extremely difficult to identify from the clinical symptoms. In general, mycobacteria grow slowly, and it takes a considerable amount of time to test using culture. Therefore, a method for amplifying and detecting a nucleic acid (specific nucleic acid) in a specific region derived from acid-fast bacteria is effective for rapidly identifying the species of acid-fast bacteria.

  The present applicant has previously amplified a nucleic acid (specific nucleic acid) in a specific region contained in a sample by PCR in the presence of a fluorescent dye whose fluorescent property changes by reacting with the nucleic acid. Has been invented a method for detecting nucleic acid in a sample by measuring (Patent Document 1). In addition, the present applicant has previously described a double-stranded DNA containing a promoter sequence by using a primer set (one of which contains a promoter sequence), reverse transcriptase and, if necessary, ribonuclease H (RNase H) against a target RNA. A method for amplifying RNA simply by a chain reaction using RNA polymerase to synthesize RNA containing a specific base sequence of RNA to be targeted and subsequently using the RNA as a template for double-stranded DNA synthesis containing a promoter sequence (Patent Document 2 and Non-Patent Document 1). Further, in Patent Document 2 and Non-Patent Document 1, when RNA amplified by the above-described method is detected and labeled with an intercalating fluorescent dye and forms a complementary double strand with a target RNA, the complementary two An oligonucleotide probe (hereinafter referred to as an INAF probe) designed so that the fluorescent dye is intercalated into the chain portion to change the fluorescence characteristics (fluorescence intensity increases) is used. The probe enables RNA amplification and detection in a simple, constant temperature and one step.

  Among the RNA amplification detection methods disclosed in Patent Document 2 and Non-Patent Document 1, the INAF probe used for detection of amplified RNA has the same intercalator fluorescent dye to be labeled. The fluorescence characteristics (fluorescence intensity increase rate) are different (Patent Document 3). Therefore, when trying to identify bacteria or viruses using the RNA amplification detection methods disclosed in Patent Document 2 and Non-Patent Document 1, target RNAs derived from the bacteria or viruses to be identified are different from each other (for example, one is If rRNA and the other is mRNA), it can be identified from the difference in fluorescence characteristics of the probe. However, identification between genus Mycobacterium using ribosomal RNA (rRNA) or ribosomal DNA (rDNA), identification between Legionella genus using rRNA or rDNA, RNA of structural protein region Using a target RNA (or target DNA) having a very high base sequence homology between bacteria belonging to the same genus or between viruses, such as identification between the genotypes of Norovirus used, etc. In the case of identification, since the nucleotide sequences of oligonucleotide probes used for detection of target RNA (or target DNA) are also very close to each other, it is unclear whether bacteria or viruses can be identified due to differences in the fluorescence characteristics of the probes. It was.

  Accordingly, an object of the present invention is to provide a method capable of identifying a bacterium or virus even when the base sequence of a target nucleic acid used for the identification of the bacterium or virus has extremely high homology between the identified bacterium or virus. is there.

  As a result of intensive studies in view of the above object, the present inventors have completed the present invention.

That is, the first aspect of the present invention is:
Using n types of oligonucleotide probes capable of forming a complementary double strand with a part of the base sequence of specific nucleic acids derived from n types (n is 2 or more) of bacteria or viruses belonging to the same genus, the same genus A method for identifying n types of fungi or viruses belonging to
The base sequence of the specific nucleic acid is a sequence having high homology among the bacteria or viruses to be identified,
The oligonucleotide probe is a probe whose fluorescence characteristics change when it forms a complementary double strand with a part of the base sequence of the specific nucleic acid, compared to before formation,
The amount of change in fluorescence characteristics by the oligonucleotide probe varies depending on the bacteria or virus identified by the oligonucleotide probe.
Said method.

The second aspect of the present invention is as follows.
Using n types of oligonucleotide probes capable of forming a complementary double strand with a partial base sequence of a specific nucleic acid derived from n types (n is 2 or more) belonging to the genus Mycobacterium , A method for identifying n types of fungi belonging to the genus Mycobacterium,
The specific nucleic acid is ribosomal RNA or ribosomal DNA,
The oligonucleotide probe is a probe whose fluorescence characteristics change when it forms a complementary double strand with a part of the base sequence of the ribosomal RNA or ribosomal DNA, compared to before formation,
The amount of change in fluorescence characteristics due to the oligonucleotide probe varies depending on the bacteria identified by the oligonucleotide probe.
Said method.

  In the third aspect of the present invention, n types of bacteria belonging to the genus Mycobacterium are Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. The method according to the second aspect, which is n types of bacteria selected from intracellulare).

  The fourth aspect of the present invention is formed when n kinds of oligonucleotide probes are labeled with a common intercalating fluorescent dye and form a complementary double strand with a part of the base sequence of a specific nucleic acid. 4. The method according to any one of the first to third aspects, wherein the fluorescent dye is a probe whose fluorescence characteristics change as compared to before formation by intercalating the fluorescent dye into a complementary double-stranded portion.

In the fifth aspect of the present invention, the specific nucleic acid is RNA,
(1) a step of synthesizing cDNA complementary to a base sequence of a specific nucleic acid by an enzyme having RNA-dependent DNA polymerase activity using RNA as a template;
(2) a step of degrading RNA-DNA double-stranded RNA by an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA),
(3) having a promoter sequence capable of transcribing a base sequence of a specific nucleic acid or a sequence complementary to the base sequence of the specific nucleic acid by an enzyme having DNA-dependent DNA polymerase activity using a single-stranded DNA as a template Generating double-stranded DNA;
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity; and
(5) a step of generating an RNA transcript in a chain by using the RNA transcript as a template for cDNA synthesis in the step of generating the RNA-DNA duplex;
5. The method according to any one of the first to fourth aspects, which is RNA amplified by the above.

  Hereinafter, the present invention will be described in detail.

  The n types of bacteria or viruses identified by the identification method of the present invention only need to belong to the same genus. As an example, n types of bacteria belonging to the genus Mycobacterium, Legionella ( N types of bacteria belonging to the genus Legionella, n types of viruses belonging to the genus Norovirus / nenotypes having different genotypes, n types of viruses belonging to the genus Sapovirus having different genotypes, rota Examples include n types of viruses having different genotypes belonging to the genus Rotavirus and n types of viruses having different genotypes belonging to the genus Papillomavirus.

  In the identification method of the present invention, a specific nucleic acid to be detected (identified) using an oligonucleotide probe is characterized in that the homology of its base sequence is high among n types of bacteria or viruses to be identified. In the present invention, high homology means that the homology of the base sequence of a specific nucleic acid among n types of bacteria or viruses to be identified is 80% or more, preferably 85% or more, more preferably 90% or more. is there. In addition, when the homology of the base sequence of a specific nucleic acid among n types of bacteria or viruses to be identified is 100%, that is, when they completely match, the amount of change in fluorescence characteristics by the oligonucleotide probe is equivalent between the bacteria or viruses. Therefore, it is not preferable. As an example of a preferable specific nucleic acid in the case where the identification target is a bacterium belonging to the same genus, a ribosomal RNA (5SrRNA, 5.8SrRNA, 16SrRNA, 18SrRNA, 23SrRNA, 28SrRNA) or a gene thereof (ribosome) having high homology among the bacteria to be identified (DNA) all or a partial region. Since ribosomal RNA has a large number of expression copies per bacterium, it can be said to be preferable in terms of identifying the bacterium with high sensitivity. As an example of the case where the identification target is a virus belonging to the genus Norovirus, the entire region or partial region of the RNA polymerase region of ORF1, the entire region or partial region of the structural protein (capsid) region that is ORF2, the RNA polymerase region and the structural protein region The junction area is given. An example of the specific nucleic acid when the identification target is a virus belonging to the genus Sapovirus is the entire region or a partial region of the structural protein region. As an example of the specific nucleic acid when the identification target is a virus belonging to the genus Rotavirus, the entire region or a partial region of a P (protease-cleaved protein) region can be mentioned. An example of the specific nucleic acid when the identification target is a virus belonging to the genus Papillomavirus is the entire region or a partial region of the L1 gene region.

  The oligonucleotide probe used in the identification method of the present invention is a probe capable of forming a double strand complementary to a partial sequence of a specific nucleic acid derived from a bacterium or virus to be identified, and corresponds to the bacterium or virus to be identified. What is necessary is just to prepare a probe. The oligonucleotide probe used in the identification method of the present invention is preferably a probe capable of forming a complementary double strand under stringent conditions. Specific examples of stringent conditions include: 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at 42 ° C. (PH 6.5), 150 mM sodium chloride or 75 mM sodium citrate coexisting conditions. As specific examples of the oligonucleotide probe used in the identification method of the present invention, the bacteria to be identified are selected from Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare, which are the main bacteria belonging to the genus Mycobacterium (Mycobacterium). In the case of n types of bacteria (n is 2 or more), there are n types of probes selected from oligonucleotide probes consisting of the nucleotide sequences set forth in SEQ ID NOs: 1 to 7.

  The oligonucleotide probe used in the identification method of the present invention is a probe whose fluorescence characteristics change when it forms a complementary double strand with a partial sequence of a specific nucleic acid derived from the bacterium or virus to be identified. is there. As an example of a probe whose fluorescence characteristics change, a molecular beacon probe (Patent Documents 4 and 5), a TaqMan probe (Patent Documents 6 and 7), a Q-Probe (Patent Document 8), a cycling probe (Patent Document 9). And 10), and a probe labeled with an intercalating fluorescent dye (INAF probe, Patent Document 2 and Non-Patent Document 1). In particular, the INAF probe is preferable when a specific nucleic acid is amplified to identify a bacterium or a virus because identification can be performed in parallel with the amplification reaction of the specific nucleic acid.

  The intercalating fluorescent dye labeled with the INAF probe used in the preferred identification method of the present invention is not particularly limited, and examples thereof include oxazole yellow, thiazole orange, ethidium bromide, and derivatives thereof. As an example, oxazole yellow is a dye that significantly increases fluorescence at 510 nm (excitation wavelength: 470 nm) when intercalated into a double-stranded nucleic acid. The fluorescent dye is composed of 5 ′ terminal side, 3 ′ terminal side, phosphodiester part, base of oligonucleotide capable of forming a double strand complementary to a partial sequence of a specific nucleic acid derived from a fungus or virus to be identified. It may be introduced into any part via an appropriate linker. In addition, when performing identification with an INAF probe in parallel with the amplification reaction of a specific nucleic acid, it is preferable to prevent the extension from the 3′-terminal side hydroxyl group by modifying the 3′-terminal side hydroxyl group of the probe. . In addition, if an intercalator fluorescent dye introduced into n types of oligonucleotide probes is used in common, n types of bacteria or viruses can be simultaneously identified with the same excitation / fluorescence wavelength, and the configuration of the fluorescence detection apparatus can be simplified. preferable.

  The identification method of the present invention is a method for identifying a bacterium or a virus by detecting a difference in the amount of change in fluorescence characteristics by n kinds of oligonucleotide probes. Detection of the amount of change in fluorescence characteristics may be performed with the absolute value of the fluorescence intensity, or may be performed with a relative value (fluorescence intensity ratio) with respect to the fluorescence intensity value in a negative sample, or by amplifying a specific nucleic acid Or when identifying a virus, you may carry out by the relative value (fluorescence intensity ratio) with respect to the fluorescence intensity value in the sample before amplification of a specific nucleic acid.

  In the identification method of the present invention, biological samples such as sputum, gastric juice, blood, urine, feces, body cavity fluid, tissue, bronchial lavage fluid, bronchoalveolar lavage fluid may be used for identification as a sample containing a specific nucleic acid as it is. A sample obtained by extracting a nucleic acid from a sample derived from the living body using a known method or a known reagent may be used for identification as a sample containing a specific nucleic acid. Regardless of which sample is used, identification of bacteria and viruses can be performed with higher sensitivity by amplifying a specific nucleic acid by an appropriate method or detecting it using an oligonucleotide probe after amplification. Therefore, it is preferable. As a method for amplifying a specific nucleic acid, a PCR method, a LAMP (Loop-mediated isometric AMP qualification) method, a TRC method (Transcribation Reverse transcription transcribed reaction reaction, Patent Document 2), a NASBA (Nucleic Acetic Acid Method). Although known methods such as the Mediated Amplification method can be exemplified, the TRC method, the NASBA method, and the TMA method that can amplify a specific nucleic acid easily and quickly at a constant temperature (relatively low temperature) are preferable. Further, when amplifying a specific nucleic acid, it is preferable to remove or inactivate a substance that interferes with the activity of the enzyme for amplification in advance.

The preferred specific nucleic acid amplification method described above is a method of amplifying RNA, and the specific nucleic acid (RNA) is amplified by the steps shown in the following (1) to (5).
(1) a step of synthesizing cDNA complementary to a base sequence of a specific nucleic acid by an enzyme having RNA-dependent DNA polymerase activity using RNA as a template;
(2) a step of degrading RNA-DNA double-stranded RNA by an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA),
(3) having a promoter sequence capable of transcribing a base sequence of a specific nucleic acid or a sequence complementary to the base sequence of the specific nucleic acid by an enzyme having DNA-dependent DNA polymerase activity using a single-stranded DNA as a template Generating double-stranded DNA;
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity; and
(5) A step of generating RNA transcripts in a chain by using the RNA transcript as a template for cDNA synthesis in the step of generating the RNA-DNA duplex.

  In the amplification, an enzyme having RNA-dependent DNA polymerase activity using RNA as a template (reverse transcriptase), an enzyme having RNase H activity, an enzyme having DNA-dependent DNA polymerase activity using single-stranded DNA as a template, and RNA It proceeds by an enzyme having polymerase activity. As these enzymes, enzymes having several activities may be used, or a plurality of enzymes having respective activities may be used. As an example, a reverse transcriptase having three activities of RNA-dependent DNA polymerase activity using RNA as a template, RNase H activity and DNA-dependent DNA polymerase activity using single-stranded DNA as a template, and an enzyme having RNA polymerase activity And can be used in combination. However, an enzyme having RNase H activity may be further added to the enzyme having reverse transcriptase and RNA polymerase activity as necessary. Examples of the reverse transcriptase include AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase and derivatives thereof, among which AMV reverse transcriptase and derivatives thereof are preferable. Examples of enzymes having RNA polymerase activity include bacteriophage-derived T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and derivatives thereof widely used in molecular biology experiments and the like.

  In the amplification, in addition to the sample and the enzyme, a buffer, a magnesium salt, a potassium salt, a nucleoside triphosphate (NTP) and a deoxyribonucleoside triphosphate (dNTP) are added to adjust the reaction efficiency. Accordingly, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), bovine serum albumin (BSA), sugar and the like are added, and the reaction with the enzyme proceeds under an appropriate temperature condition. As an example of appropriate temperature conditions, when AMV reverse transcriptase and T7 RNA polymerase are used, the temperature may be in the range of 35 ° C. to 65 ° C., and preferably in the range of 40 ° C. to 50 ° C.

  In the amplification, a first primer having a sequence homologous to a part of the base sequence of the specific nucleic acid and a second primer having a sequence complementary to the part of the base sequence of the specific nucleic acid are used (here, Either one of the first and second primers has an RNA polymerase promoter sequence added to its 5 ′ end). When a promoter sequence is added to the first primer, an RNA transcript homologous to the base sequence of the specific nucleic acid is generated, and when a promoter sequence is added to the second primer, the base sequence of the specific nucleic acid A complementary RNA transcript is produced. The promoter sequence added to the primer may be a known promoter sequence corresponding to the enzyme having RNA polymerase activity used in the amplification. When the enzyme having RNA polymerase activity is T7 RNA polymerase, the T7 promoter is used. The SP6 promoter may be used when the enzyme having RNA polymerase activity is SP6 RNA polymerase, and the T3 promoter may be used when the enzyme having RNA polymerase activity is T3 RNA polymerase. In addition to the promoter sequence, an additional sequence related to transcription efficiency such as an enhancer sequence may be included.

  When the amplification is performed using the first primer to which the promoter sequence described above is added and the second primer, it is complementary to a specific base sequence by an enzyme having RNA-dependent DNA polymerase activity using RNA as a template. Prior to or simultaneously with the step (1), a specific nucleic acid around the 5 ′ end of the region homologous to the base sequence of the first primer (step (1)). RNA) is preferably cleaved. By this cleavage, after cDNA synthesis, a DNA strand complementary to the promoter sequence of the first primer hybridized to cDNA can be efficiently synthesized by extending the 3 ′ end of the cDNA, resulting in functional 2 This is because a double-stranded DNA promoter structure can be formed efficiently. The method for cleaving the specific nucleic acid (RNA) is not particularly limited as long as it can specifically cleave the periphery of the 5 'end of the region homologous to the base sequence of the first primer. As an example of a method for cleaving a specific nucleic acid (RNA), it overlaps the 5 ′ end of the region homologous to the base sequence of the first primer of the specific nucleic acid (RNA) and in the 5 ′ end direction of the specific nucleic acid (RNA) By adding an oligonucleotide capable of forming a complementary double strand with a specific nucleic acid (RNA) in an adjacent region (hereinafter referred to as “cleaving oligonucleotide”), an RNA-DNA double strand is formed, and the 2 Examples include a method of cleaving an RNA portion in this strand with an enzyme having ribonuclease H (RNase H) activity, and the like. This cleaving method can be cleaved easily and has a specific cleavage position. It can be said. Incidentally, the 3′-terminal hydroxyl group of the cleaving oligonucleotide may be appropriately modified, for example, by amination, in order to prevent an extension reaction.

  In the case of a method for identifying bacteria or viruses by performing detection with an INAF probe in parallel with an amplification reaction of a specific nucleic acid, which is an example of the identification method of the present invention, an INAF probe, a primer set, an enzyme, an enzyme substrate, etc. A sample containing a specific nucleic acid is dispensed into a container containing the reagents to be contained and reacted at an appropriate temperature, whereby the amplification reaction of the specific nucleic acid and the detection (identification) using the INAF probe can be performed simultaneously. Further, when at least a part of the container is made of a transparent material and fluorescence emitted from the intercalating fluorescent dye introduced into the INAF probe can be detected from the outside, after dispensing the sample containing the specific nucleic acid, Since the amplification reaction of the specific nucleic acid and the detection (identification) by the INAF probe can be performed in a sealed state, contamination between samples can be prevented.

  The present invention uses n types of oligonucleotide probes that can form complementary double strands with a partial base sequence of a specific nucleic acid derived from n types (n is 2 or more) of bacteria or viruses belonging to the same genus. Thus, in the method for identifying n types of bacteria or viruses belonging to the same genus, the nucleotide sequence of the specific nucleic acid is a sequence having high homology among the bacteria or viruses to be identified, and the oligonucleotide probe is one of the specific nucleic acids. When a complementary double strand is formed with the base sequence of the portion, the fluorescence characteristics change compared to before the formation, and the amount of change in the fluorescence characteristics due to the oligonucleotide probe differs depending on the bacteria or virus identified by the oligonucleotide probe It is characterized by that.

  The identification method of the present invention enables identification between bacteria belonging to the same genus even when a specific nucleic acid having high homology among bacteria belonging to the same genus such as ribosomal RNA (rRNA) is used. Therefore, although there is a high necessity for identification, identification between bacteria belonging to the genus Mycobacterium (Mycobacterium), particularly mycobacterial abium having extremely high homology of rRNA base sequence, has not been available. Identification between Mycobacterium avium and Mycobacterium intracellulare can also be performed quickly and easily by the identification method of the present invention.

  Hereinafter, the present invention will be described in more detail using an example in which a specific nucleic acid is ribosomal RNA (rRNA) and a bacterium belonging to the same genus is a bacterium belonging to the genus Mycobacterium. It is not limited by these examples.

Example 1 Preparation of Oligonucleotide Labeled with Intercalating Fluorescent Dye Oligonucleotide Probes Labeled with Intercalating Fluorescent Dye (hereinafter referred to as INAF Probe) shown in (A) to (G) below are non-patented. It was produced with reference to the method described in Document 1.
(A) It was obtained by labeling the phosphodiester part between the 5th guanine and the 6th adenine from the 5 ′ end in the base sequence described in SEQ ID NO: 1 with oxazole yellow through a linker. , Oligonucleotide probes.
(B) It was obtained by labeling the phosphodiester part between the 14th thymine and the 15th adenine from the 5 ′ end in the base sequence described in SEQ ID NO: 2 with oxazole yellow via a linker. , Oligonucleotide probes. The 12th n from the 5 ′ end of SEQ ID NO: 2 is a 5-nitroindole nucleotide.
(C) Obtained by labeling the phosphodiester moiety between the 7th guanine and the 8th cytosine from the 5 ′ end of the sequence described in SEQ ID NO: 3 with oxazole yellow via a linker, Oligonucleotide probe.
(D) After introducing an amino group using a commercially available reagent (Label-ON Reagents, manufactured by Clontech) at the position of the 12th adenine from the 5 ′ end in the sequence described in SEQ ID NO: 4, An oligonucleotide probe obtained by labeling oxazole yellow and further modifying the 3 ′ end with biotin.
(E) After introducing an amino group using a commercially available reagent (Label-ON Reagents, manufactured by Clontech) at the position of the 13th adenine from the 5 ′ end in the sequence described in SEQ ID NO: 5, An oligonucleotide probe obtained by labeling oxazole yellow and further modifying the 3 ′ end with biotin.
(F) After introducing an amino group using a commercially available reagent (Label-ON Reagents, manufactured by Clontech) at the 7th guanine position from the 5 ′ end in the sequence shown in SEQ ID NO: 6, An oligonucleotide probe obtained by labeling oxazole yellow and further modifying the 3 ′ end with biotin.
(G) After introducing an amino group using a commercially available reagent (Label-ON Reagents, manufactured by Clontech) at the position of the seventh adenine from the 5 ′ end in the sequence described in SEQ ID NO: 7, An oligonucleotide probe obtained by labeling oxazole yellow and further modifying the 3 ′ end with biotin.

Example 2 Simultaneous identification of Mycobacterium avium and Mycobacterium intracellulare (Part 1)
Among the INAF probes prepared in Example 1, (A) and (B) probes were used to detect rRNA derived from bacteria belonging to the genus Mycobacterium, and the detection performance and specificity were evaluated.
(1) The RNA sample used for evaluation was prepared by the following method.
(1-1) M.I. tuberculosis 16S rRNA, M. et al. avium 16S rRNA, M.I. intracellulare 16S rRNA and M. pneumoniae. kansasii 16S rRNA
Each 16S rRNA gene (16S rDNA) is cloned, and after in vitro transcription, purified, a standard RNA is prepared. The standard RNA is diluted with an RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.25 U). RNA samples were prepared by diluting to a constant concentration using / μL ribonuclease inhibitor, 5.0 mM DTT).
(1-2) M.M. florentinum RNA
OD _600nm is 0.1. The RNA sample was prepared by extracting the florentinum culture solution with a commercially available extraction reagent (EXTRAGEN MB, manufactured by Tosoh Corporation).
(2) Dispense 20 μL of the reaction solution having the composition shown below into a commercially available 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and add 5 μL of the RNA sample (when there is one RNA sample) ) Or 2.5 μL of each (when two types of RNA samples are mixed).

Composition of reaction solution: concentration is final concentration after addition of enzyme solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
17 mM magnesium chloride 100 mM potassium chloride 1 mM DTT
0.25 mM dATP, dCTP, dGTP, dTTP each
Each 3 mM ATP, CTP, UTP, GTP
3.6 mM ITP
0.16 μM Cleavage oligonucleotide DNA (3 ′ end hydroxyl group modified with amino group)
1 μM first primer (added T7 promoter sequence at 5 ′ end)
1 μM Second primer 5 nM (A) INAF probe 21 nM (B) INAF probe 10% to 13% DMSO
(4) After the reaction solution was kept at 43 ° C. for 5 minutes, 5 μL of an enzyme solution (see below for the composition) that was kept warm at 43 ° C. for 2 minutes was added.

Composition of enzyme solution: final concentration at the time of reaction (in 30 μL) 2.0% sorbitol 0.21 U / μL AMV reverse transcriptase (Life Science)
2.37 or 4.73 U / μL T7 RNA polymerase 0.12 mg / mL bovine serum albumin (5) Next, subject the PCR tube to a fluorescence spectrophotometer with a temperature control function that can directly detect the PCR tube, and a constant temperature of 43 ° C. to 46 ° C. Simultaneously with the RNA amplification reaction, the fluorescence intensity (excitation wavelength: 470 nm, fluorescence wavelength: 520 nm) of the reaction solution was detected over time for 30 minutes.

In Table 1, M.M. avium and M.M. detection performance evaluation for intracellulare and M. The result of the specificity evaluation with respect to florentinum is shown. The RNA samples used in this example were (i) M.M. avium 16S rRNA 50 copies / test (ii) avium 16S rRNA 5 × 10 ⁷ copies / test (iii) intracellulare 16S rRNA 100 copies / test (iv) intracellulare 16S rRNA 5 × 10 ⁷ copies / test (v) avium 16S rRNA 50 copies / test mixed sample with intracellulare 16S rRNA 100 copies / test (vi) avium 16S rRNA 5 × 10 ⁷ copies / test and Intracellulare 16S rRNA 5 × 10 ⁷ copies / test mixed sample (vii) The fluorescence intensity ratio is a value obtained by dividing the fluorescence intensity value of the reaction solution 30 minutes after the addition of the enzyme by the fluorescence intensity value (background) immediately after the addition of the enzyme (0 minute). is there.

  As a result of detecting amplified RNA using the INAF probe of (A) and (B), M.P. The Avium 16S rRNA sample ((i) and (ii)) has a fluorescence intensity ratio of about 1.9 after 30 minutes regardless of the amount thereof. Intracellulare 16S rRNA samples ((iii) and (iv)) had a fluorescence intensity ratio of about 2.8 after 30 minutes regardless of the amount of M. avium 16S rRNA and M. The mixed sample of intracellulare 16S rRNA ((v) and (vi)) had a fluorescence intensity ratio of about 3.3 or more after 30 minutes regardless of the amount. On the other hand, M.M. avium 16S rRNA and M. Intracellulare 16S rRNA has a very high nucleotide sequence homology. Fluorentinum sample (vii) has almost no increase in fluorescence intensity, and the INAF probes in (A) and (B) are M.P. avium 16S rRNA and M. It can be seen that intracellular 16S rRNA is specifically detected.

Also, the results of Table 1 can be summarized as follows:
(I) A sample having a fluorescence intensity ratio of 1.2 or less after 30 minutes avium and M.M. intracellulare negative,
(II) A sample having a fluorescence intensity ratio after 1.8 minutes of 1.8 to 2.3 avium positive,
(III) A sample having a fluorescence intensity ratio of 2.7 to 3.0 after 30 minutes was measured by M.C. intracellular positive,
(IV) A sample having a fluorescence intensity ratio of 3.3 or more after 30 minutes avium and M.M. intracellular positive,
By providing the determination criteria, the M.A. and B. avium and M.M. It can be seen that intracellare can be identified simultaneously. In addition, the RNA amplification detection method using the INAF probe of (A) and (B) is rapid because the result can be understood 30 minutes after the start of the reaction, and the RNA amount is as low as 50 to 100 copies / test. Since it can be amplified and detected, it is highly sensitive, and the fluorescence intensity hardly increases even for highly homologous bacteria in the same genus.

Example 3 Simultaneous identification of Mycobacterium avium and Mycobacterium intracellulare (Part 2)
Among the RNA amplification detection methods (condition [1]) described in Example 2,
The INAF probe and its concentration,
(C) INAF probe 12.5 nM and (D) INAF probe 12.5 nM (condition [2]),
(D) INAF probe 12.5 nM and (E) INAF probe 12.5 nM (condition [3]),
(C) INAF probe 12.5 nM only (condition [4]) or (D) INAF probe 12.5 nM only (condition [5])
The RNA amplification detection reaction was carried out in the same manner as in Example 2 except that. Note that the INAF probe in (C) is M.M. avium and M.M. It is a probe that specifically detects intracellularis, and the INAF probe in (D) is M.I. It is a probe that specifically detects intracellularis.

In Table 2, M.M. avium and M.M. detection performance evaluation for intracellulare and M. The result of specificity evaluation with respect to tuberculosis is shown. The RNA sample used in this example was (viii) M.I. avium 16S rRNA 10 ⁴ copies / test (ix) intracellulare 16S rRNA 10 ⁴ copies / test (x) tuberculosis 10 ⁸ copies / test, and the fluorescence intensity ratio is obtained by dividing the fluorescence intensity value of the reaction solution 30 minutes after the enzyme addition by the fluorescence intensity value (background) immediately after the enzyme addition (0 minutes). is there.

M.M. avium 16S rRNA and M. Intracellulare 16S rRNA has a very high nucleotide sequence homology. The tuberculosis 16S rRNA sample (x) has little increase in fluorescence intensity, and conditions [2] and [3] avium 16S rRNA and M. It can be seen that intracellular 16S rRNA is specifically detected. On the other hand, M.M. avium 16S rRNA and M. Regarding the identification with intracellularella 16S rRNA, the condition [2] can be simultaneously identified by the criteria (I) to (III) above, but the condition [3] can be identified as M.I. Fluorescence intensity ratio and M. aurium 16S rRNA sample (viii) It can be seen that simultaneous identification is difficult because the fluorescence intensity ratio when measuring intracellulare 16S rRNA sample (ix) is 1.66.

  In addition, when the INAF probe used in the condition [2] is used alone (conditions [4] and [5]), the M.P. Fluorescence intensity ratio and M. aurium 16S rRNA sample (viii) Since there is almost no difference in the fluorescence intensity ratio when measuring intracellulare 16S rRNA sample (ix), condition [2] is the first time that the combination of the INAF probe in (C) and the INAF probe in (D) . avium 16S rRNA and M. It can be seen that simultaneous identification with intracellulare 16S rRNA is possible.

Example 4 Simultaneous identification of Mycobacterium tuberculosis and Mycobacterium intracellulare Among the RNA amplification detection methods described in Example 2 (condition [1]), the INAF probe and its concentration were determined using the INAF probe 25nM in (D) and the INAF in (F) An RNA amplification detection reaction was performed in the same manner as in Example 2 except that the probe was set to 50 nM (condition [6]).

In Table 3, M.M. tuberculosis and M. et al. detection performance evaluation for intracellulare and M. The result of specificity evaluation with respect to avium is shown. The RNA sample used in this example was (xi) M.I. tuberculosis 16S rRNA 10 ⁴ copies / test (ix) intracellulare 16S rRNA 10 ⁴ copies / test (xii) avium 16S rRNA 10 ⁸ copies / test (xiii) kansasii 16SrRNA 10 ⁸ copies / test, and the fluorescence intensity ratio is a value obtained by dividing the fluorescence intensity value of the reaction solution 30 minutes after the enzyme addition by the fluorescence intensity value (background) immediately after the enzyme addition (0 minute) It is.

As a result of detection of amplified RNA using the INAF probe of (D) and (F), M.M. tuberculosis 16S rRNA sample (xi) has a fluorescence intensity ratio of about 2.4 after 30 minutes. Intracellulare 16S rRNA sample (ix) has a fluorescence intensity ratio of about 1.9 after 30 minutes, whereas M. tuberculosis 16S rRNA or M. tuberculosis. Intracellulare 16S rRNA has a very high nucleotide sequence homology. avium 16S rRNA sample (xii) and Since the Kansasii 16S rRNA sample (xiii) has almost no increase in fluorescence intensity, the INAF probes in (D) and (F) are M.P. tuberculosis 16S rRNA and M. coli. It can be seen that intracellular 16S rRNA is specifically detected.

The results of Table 3 can be summarized as follows:
(V) A sample having a fluorescence intensity ratio of 1.2 or less after 30 minutes tuberculosis and M. et al. intracellulare negative,
(VI) A sample having a fluorescence intensity ratio after 30 minutes of about 1.9 intracellular positive,
(VII) A sample having a fluorescence intensity ratio of about 2.4 after 30 minutes was measured by M.C. tuberculosis positive,
By providing the determination criteria, the INA probes (D) and (F) tuberculosis and M. et al. It can be seen that intracellare can be identified simultaneously.

Example 5 Simultaneous identification of Mycobacterium tuberculosis and Mycobacterium avium Among the RNA amplification detection methods described in Example 2 (condition [1]), the INAF probe and its concentration
(E) INAF probe 25 nM and (G) INAF probe 10 nM (condition [7])
(E) INAF probe 25 nM only (condition [8]), or (G) INAF probe 10 nM only (condition [9]),
The RNA amplification detection reaction was carried out in the same manner as in Example 2 except that. In addition, the INAF probe of (E) is M.M. a probe that specifically detects tuberculosis; It is a probe that specifically detects avium.

In Table 4, M.M. tuberculosis and M. et al. Detection performance evaluation for Avium and M. The result of specificity evaluation with respect to intracellare is shown. The RNA sample used in this example was (xi) M.I. tuberculosis 16S rRNA 10 ⁴ copies / test (viii) avium 16S rRNA 10 ⁴ copies / test (xiv) intracellulare 16S rRNA 10 ⁸ copies / test (xiii) Kansasii 16S rRNA 10 ⁸ copies / test (xv) tuberculosis 16S rRNA 10 ³ copies / test, and the fluorescence intensity ratio is a value obtained by dividing the fluorescence intensity value of the reaction solution 30 minutes after the enzyme addition by the fluorescence intensity value (background) immediately after the enzyme addition (0 minutes). It is.

As a result of detection of amplified RNA using the INAF probe (condition [7]) of (E) and (G), M.M. The tuberculosis 16S rRNA sample (xi) has a fluorescence intensity ratio of about 1.6 after 30 minutes. The Avium 16S rRNA sample (viii) has a fluorescence intensity ratio of about 2.0 after 30 minutes, while tuberculosis 16S rRNA or M. tuberculosis. M. avium 16S rRNA has very high nucleotide sequence homology. intracellulare 16S rRNA sample (xiv) and Since the Kansasii 16S rRNA sample (xiii) has almost no increase in fluorescence intensity, the INAF probes in (E) and (G) are M.P. tuberculosis 16S rRNA and M. coli. It can be seen that avium 16SrRNA is specifically detected.

Also, the results of Table 4 can be summarized as follows:
(VIII) A sample having a fluorescence intensity ratio of 1.2 or less after 30 minutes was measured by M.C. tuberculosis and M. et al. avium negative,
(IX) A sample having a fluorescence intensity ratio of about 1.6 after 30 minutes was measured by M.C. tuberculosis positive,
(X) A sample having a fluorescence intensity ratio of about 2.0 after 30 minutes was measured by M.C. avium positive,
By providing the determination criterion, the M.I. tuberculosis and M. et al. It can be seen that avium can be identified simultaneously.

  When the INAF probe used in condition [7] is used alone (conditions [8] and [9]), M.I. tuberculosis 16S rRNA sample (xv) and fluorescence intensity ratio when measured. Since there is almost no difference in the fluorescence intensity ratio when measuring the Avium 16S rRNA sample (viii), the condition [7] is the first to combine M with the INAF probe in (E) and the INAF probe in (G). . tuberculosis 16S rRNA and M. tuberculosis 16S rRNA. It can be seen that simultaneous identification with avium 16S rRNA is possible.

  As described above, based on the results of Examples 2 to 5, simultaneous identification between Mycobacterium species is performed by performing an RNA amplification detection reaction by combining two or more types of the INAF probes (A) to (G). It is understood that is possible.

Claims (5)

Using n types of oligonucleotide probes capable of forming a complementary double strand with a part of the base sequence of specific nucleic acids derived from n types (n is 2 or more) of bacteria or viruses belonging to the same genus, the same genus A method for identifying n types of fungi or viruses belonging to
The base sequence of the specific nucleic acid is a sequence having high homology among the bacteria or viruses to be identified,
The oligonucleotide probe is a probe whose fluorescence characteristics change when it forms a complementary double strand with a part of the base sequence of the specific nucleic acid, compared to before formation,
The amount of change in fluorescence characteristics by the oligonucleotide probe varies depending on the bacteria or virus identified by the oligonucleotide probe.
Said method. Using n types of oligonucleotide probes capable of forming a complementary double strand with a partial base sequence of a specific nucleic acid derived from n types (n is 2 or more) belonging to the genus Mycobacterium , A method for identifying n types of fungi belonging to the genus Mycobacterium,
The specific nucleic acid is ribosomal RNA or ribosomal DNA,
The oligonucleotide probe is a probe whose fluorescence characteristics change when it forms a complementary double strand with a part of the base sequence of the ribosomal RNA, compared to before formation,
The amount of change in fluorescence characteristics due to the oligonucleotide probe varies depending on the bacteria identified by the oligonucleotide probe.
Said method. N types of bacteria belonging to the genus Mycobacterium are Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare species selected from Mycobacterium intracellulare. The method of claim 2, wherein: When n kinds of oligonucleotide probes are labeled with a common intercalating fluorescent dye and form a complementary double strand with a part of the base sequence of the specific nucleic acid, the fluorescent dye is formed on the formed complementary double strand portion. The method according to any one of claims 1 to 3, which is a probe whose fluorescence characteristics change by intercalating as compared with before formation. The specific nucleic acid is RNA, and the RNA is
(1) a step of synthesizing cDNA complementary to a base sequence of a specific nucleic acid by an enzyme having RNA-dependent DNA polymerase activity using RNA as a template;
(2) a step of degrading RNA-DNA double-stranded RNA by an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA),
(3) having a promoter sequence capable of transcribing a base sequence of a specific nucleic acid or a sequence complementary to the base sequence of the specific nucleic acid by an enzyme having DNA-dependent DNA polymerase activity using a single-stranded DNA as a template Generating double-stranded DNA;
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity; and
(5) a step of generating an RNA transcript in a chain by using the RNA transcript as a template for cDNA synthesis in the step of generating the RNA-DNA duplex;
The method according to any one of claims 1 to 4, which is RNA amplified by the method.