JP2012135234A - Oligonucleotide probe for detecting bacillus tuberculosis, and detective method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To specifically and highly sensitively detecting Bacillus tuberculosis-derived 16S rRNA or its gene.SOLUTION: A nucleic acid amplification is conducted using a combination of oligonucleotides consisting of a first primer having a sequence homologous to part of Bacillus tuberculosis-derived 16S rRNA or its gene and a second primer having the complementary sequence to detect the thus amplified nucleic acid using an oligonucleotide probe.

Description

  The present invention relates to an oligonucleotide probe for rapidly, sensitively and specifically detecting 16S rRNA of Mycobacterium tuberculosis or a gene thereof, and a detection method using the same.

  Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Since the diagnosis of infectious diseases requires rapid and reliable identification of infection-causing bacteria, the tuberculosis identification test is extremely important clinically.

  Conventionally, the identification test for M. tuberculosis has been performed by a culture method. However, a rapid identification test using a nucleic acid amplification method has been developed, and it has become possible to identify M. tuberculosis in a short time. An example of a nucleic acid amplification method that has been used is the PCR method (see Patent Documents 1, 2, and 3). However, in the PCR method, it is necessary to raise and lower the reaction temperature abruptly, and this has been a barrier for labor saving and cost reduction of the reaction apparatus at the time of automation.

  A method for amplifying a nucleic acid at a constant temperature is also known. As such a method, the LAMP method (see Non-Patent Document 1), the NASBA method (see Patent Documents 4 and 5), the TMA method (see Patent Document 6), and the like have been reported. In these nucleic acid amplification methods, after nucleic acid amplification, the amplified nucleic acid is detected by electrophoresis or a hybridization method using an oligonucleotide probe bound with a detectable label. There is a problem that it is complicated and cannot be quantified with good reproducibility.

  As a method for easily amplifying and detecting RNA, a double-stranded DNA containing a promoter sequence is prepared by using a primer containing a promoter sequence for the target RNA, reverse transcriptase and, if necessary, ribonuclease H (RNase H). There is a method of synthesizing and synthesizing RNA containing a specific base sequence of RNA to be targeted by RNA polymerase, and subsequently performing a chain reaction using the RNA as a template for double-stranded DNA synthesis containing a promoter sequence (Patent Documents 7 and 7) Non-patent document 2). In this method, when labeled with an intercalating fluorescent dye and forms a complementary double strand with the target nucleic acid, the intercalating fluorescent dye is intercalated into a complementary double stranded portion so that the fluorescence characteristics change. Changes in fluorescence characteristics are detected in the presence of the designed oligonucleotide probe, and RNA amplification and detection can be carried out simultaneously in a single step, simply and at a constant temperature.

  Each of the above methods amplifies a nucleic acid using an oligonucleotide probe. However, in Mycobacterium tuberculosis 16S rRNA or its gene, a higher-order structure such as a dimer or loop is present in a region specific to Mycobacterium tuberculosis. A probe that detects M. tuberculosis 16S rRNA or its gene with high sensitivity and specificity, for example, because a region that is easy to form is included (for example, a region represented by base numbers 89008 to 89017 of GenBank number BX842576) Is extremely difficult. In particular, when using an amplification method capable of performing amplification detection of a target RNA under a relatively low temperature (preferably 40 to 50 ° C.), the probe easily forms a higher-order structure. It is particularly difficult to design a probe.

US Pat. No. 4,683,195 U.S. Pat.No. 4,683,202 US Pat. No. 4,965,188 Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent No. 3241717 JP 2000-14400 A

Thai H.H. T.A. C. et al. J. et al. Clin. Microbiol. 42, 1956-61 (2004) Ishiguro T. et al. Anal. Biochem. 314, 77-86 (2003)

  Accordingly, an object of the present invention is to provide an oligonucleotide probe for specifically detecting clinically important M. tuberculosis and a method for detecting M. tuberculosis using the oligonucleotide probe.

  As a result of intensive studies in view of the above object, the present inventors have completed the present invention. That is, the present invention is a method for detecting a specific base sequence of Mycobacterium tuberculosis-derived 16S rRNA present in a sample or a gene thereof, comprising the sequence shown in SEQ ID NO: 2, 7, or 9 or a complementary sequence thereof. An oligonucleotide probe is used. The present invention also provides a detection reagent for Mycobacterium tuberculosis-derived 16S rRNA, a first primer comprising the sequence shown in SEQ ID NO: 13, and a second primer comprising the sequence shown in any one of SEQ ID NO: 21, 22 or 23 It comprises a primer and an oligonucleotide probe comprising the sequence shown in any of SEQ ID NOs: 2, 7 or 9 or a complementary sequence thereof. Hereinafter, the present invention will be described in detail.

  The present invention uses nucleic acids extracted from biological samples such as sputum, gastric fluid, blood, urine, feces, body cavity fluid, tissue, bronchial lavage fluid, bronchoalveolar lavage fluid as a sample, or such sample as it is as a sample. Is to detect whether or not a specific base sequence of Mycobacterium tuberculosis-derived 16S rRNA or a gene thereof is present, and the abundance thereof. In both cases where the sample is used as it is and when the nucleic acid is extracted from the sample, the 16S rRNA derived from M. tuberculosis is amplified and detected in the process or later. It is preferable to remove substances that interfere with the activity of the enzyme for amplification.

  The present invention detects the presence of a specific base sequence of Mycobacterium tuberculosis-derived 16S rRNA or a gene thereof, and the specific base sequence is a base sequence specifically found in Mycobacterium tuberculosis-derived 16S rRNA or a gene thereof, And especially in rRNA, it is a base sequence which exists in the area | region where it is hard to take a higher order structure. As described above, the region that is specific to M. tuberculosis on 16S rRNA and is difficult to take higher-order structures is extremely limited. In addition, when rRNA is amplified and detected, it may also be referred to as a base sequence homologous to the base sequence from the 5 ′ end of the homologous region with the first primer to the 3 ′ end of the complementary region with the second primer. it can. In other words, when performing the RNA amplification step, an RNA transcript of a specific base sequence or its complementary sequence is amplified (produced), and a template for transcription of the RNA transcript is used. The resulting double-stranded DNA has a specific base sequence in the sense strand or antisense strand downstream of the RNA polymerase promoter sequence.

  The present invention is an oligonucleotide probe comprising the sequence shown in SEQ ID NO: 2, 7 or 9 or a complementary sequence thereof, and further an oligonucleotide probe having a sequence that can hybridize to these under stringent conditions. Stringent conditions can be selected from known conditions and are not particularly limited. For example, 50% (v / v) formamide at 42 ° C., 0.1% bovine serum albumin, 0.1% It means that it can hybridize under conditions where Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, 75 mM sodium citrate and the like coexist.

  Detection using the oligonucleotide probe of the present invention can be performed by a method using electrophoresis or liquid chromatography, for example. In addition, it is also possible to detect by a hybridization method, for example, by binding an oligonucleotide to a detectable labeling substance. As the labeling substance, known substances such as enzymes, fluorescent dyes, radioisotopes and luminescent dyes can be used. In the present invention, a molecular beacon (US Pat. No. 5,925,517, US Pat. No. 6,103,476), a TaqMan probe (US Pat. No. 5,210,015, US Pat. No. 5,487,972), Q-Probe (US Pat. Patent No. 3437816), a cycling probe (US Pat. No. 5,011,769, US Pat. No. 5,403,711) and an intercalating fluorescent dye-labeled probe (see Patent Document 7 and Non-Patent Document 2) can be exemplified as particularly preferred oligonucleotide probes. Among them, when labeled with an intercalating fluorescent dye and forms a complementary double strand with a target nucleic acid, the fluorescence characteristics are changed by intercalating the intercalating fluorescent dye portion into the complementary double strand portion. Since the designed oligonucleotide probe can be detected in the course of the amplification process described later, the amplification process can be performed simply by putting it in a container together with reagents including oligonucleotide DNA, primers, enzymes, enzyme substrates, and the like described later. It is possible to carry out a detection step. As a result, the present invention can be rapidly implemented only by an operation of previously putting the above-described reagent into a container and dispensing a predetermined amount of sample, and for example, detecting a signal emitted from a fluorescent dye from the outside. If a part of the container is made of a transparent material as much as possible, the container can be hermetically sealed after dispensing the sample to prevent contamination between the samples.

  It does not specifically limit as an intercalator fluorescent dye, It can illustrate using oxazole yellow, thiazole orange, ethidium bromide, or these derivatives. For example, oxazole yellow is a dye that significantly increases fluorescence at 510 nm (excitation wavelength: 490 nm) when intercalated into double-stranded DNA. Such a dye may be bound to the oligonucleotide via an appropriate linker at the end of the oligonucleotide probe, the phosphodiester part or the base part. When detection is performed during the amplification step, the oligonucleotide is preferably modified with the hydroxyl group for the purpose of preventing extension from the hydroxyl group at the 3 'end.

  Usually, even if 16S rRNA derived from Mycobacterium tuberculosis or a specific base sequence of the gene thereof is present in the sample, the abundance thereof is very small. Therefore, the present invention amplifies a specific base sequence of 16S rRNA or its gene in addition to the step of detecting 16S rRNA or its gene (hereinafter also referred to as “detection step”) using the oligonucleotide probe as described above. Carrying out a step (hereinafter also referred to as “amplification step”). Examples of the nucleic acid amplification method that can be employed in the amplification step include the PCR method, LAMP method, TRC method, NASBA method, or TMA method, but the specific base present in the region where the oligonucleotide probe of the present invention is difficult to take a higher order structure. The TRC method, NASBA method, and TMA method, which can be carried out simply and quickly at a constant temperature (relatively low temperature), are preferred because they are directed to the arrangement.

A particularly preferred amplification step uses a first primer having a sequence homologous to a part of the specific base sequence, and a second primer having a sequence complementary to a part of the specific base sequence (herein, the first primer). Either one of the first primer and the second primer is obtained by adding an RNA polymerase promoter sequence to the 5 ′ end thereof, and the following steps (1) to (5) are performed.
(1) a step of synthesizing cDNA complementary to a specific base sequence 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) Double-stranded DNA having a promoter sequence capable of transcribing a specific base sequence or RNA complementary to the specific base sequence by an enzyme having DNA-dependent DNA polymerase activity using single-stranded DNA as a template Generating a process,
(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.

  The amplification process as described above includes an enzyme having RNA-dependent DNA polymerase activity (reverse transcriptase) using single-stranded RNA as a template, an enzyme having RNase H activity, and a DNA-dependent DNA polymerase activity using single-stranded DNA as a template. It progresses with an enzyme having an activity and an enzyme having RNA polymerase activity. As these enzymes, enzymes having several activities may be used, or a plurality of enzymes having respective activities may be used. For example, reverse transcriptase having three activities of RNA-dependent DNA polymerase activity using single-stranded RNA as a template, RNase H activity and DNA-dependent DNA polymerase activity using single-stranded DNA as a template, and double-stranded DNA as a template. It is possible to exemplify the use of a combination of RNA synthetases. However, an enzyme having RNase H activity may be further added to the reverse transcriptase and RNA synthase having these three activities as required. As a reverse transcriptase having three activities, for example, AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase or a derivative thereof, among them, AMV reverse transcriptase or a derivative thereof is particularly preferable. Examples of the enzyme having RNA polymerase activity include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase derived from bacteriophage, which are widely used in molecular biological experiments, and derivatives thereof.

  In order to proceed with the amplification step, a buffer, magnesium salt, potassium salt, nucleoside-triphosphate and ribonucleoside-triphosphate are added in addition to the sample and each of the enzymes. In order to adjust the reaction efficiency, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), bovine serum albumin (BSA), sugar and the like are added, and the enzyme reaction is allowed to proceed under appropriate conditions. For example, when using AMV reverse transcriptase and T7 RNA polymerase, the reaction temperature may be set in the range of 35 to 65 ° C, preferably in the range of 40 ° C to 50 ° C.

  In the amplification step described above, when a promoter sequence is added to the first primer, the RNA transcript contains a sequence homologous to the template RNA, and when a promoter sequence is added to the second primer, RNA The transcript will contain the complementary sequence of the template RNA. The promoter sequence may be any sequence that can bind to RNA polymerase and initiate transcription, and known promoter sequences specific for various RNA polymerases can be used. For example, promoter sequences usually used in molecular biological experiments such as T7 promoter, SP6 promoter, T3 promoter and the like can be used without particular limitation. In addition to the promoter sequence, an additional sequence related to transcription efficiency such as an enhancer may be further included.

  First and second primers for amplifying a specific base sequence to be detected by an oligonucleotide probe comprising the sequence shown in SEQ ID NO: 2, 7 or 9 or a complementary sequence thereof by the amplification step described above Examples of combinations include a combination of a first primer consisting of 23 or more consecutive bases in the sequence shown in SEQ ID NO: 17 and a second primer consisting of 18 or more consecutive bases in the sequence shown in SEQ ID NO: 19. Preferably, a first primer comprising a sequence consisting of 23 or more consecutive bases in the sequence shown in SEQ ID NO: 16 and comprising the sequence shown in SEQ ID NO: 13 and a sequence in the sequence shown in SEQ ID NO: 21 It is a combination of a second primer comprising a sequence consisting of 18 bases or more and comprising the sequence shown in SEQ ID NO: 20, more preferably It is a combination of a second primer consisting of any of the sequences shown in the first primer with SEQ ID NO: 21, 22 or 23 comprising a sequence shown in SEQ ID NO: 13.

  When the amplification step described above is performed, a promoter sequence may be added to either the first or second primer. However, when a promoter sequence is added to the first primer, the tuberculosis-derived 16S Prior to or simultaneously with the step of synthesizing cDNA using rRNA as a template (step (1) described above), it is preferable to cleave the RNA at the 5 ′ end site of the specific base sequence. By cleaving at the 5 ′ end of the specific nucleotide sequence in this way, after cDNA synthesis, the DNA strand complementary to the promoter sequence of the first primer hybridized to the cDNA is extended, and the 3 ′ end of the cDNA is extended. It is because it can synthesize | combine efficiently and can form a functional double stranded DNA promoter structure efficiently as a result. The cleavage method is not particularly limited as long as the site can be specifically cleaved, but it is not limited to the 5 ′ end site of the specific base sequence in the 16S rRNA derived from Mycobacterium tuberculosis (the partial sequence including the 5 ′ end site in the specific base sequence). An oligonucleotide DNA having a sequence that overlaps and is complementary to a region adjacent to the 5 ′ direction (hereinafter referred to as “cleaving oligonucleotide DNA”) is added to form an RNA-DNA duplex, A method of cleaving the RNA portion in the double strand with an enzyme having ribonuclease H (RNase H) activity is preferable from the viewpoint of cleavage specificity and simplicity. In addition, the hydroxyl group at the 3 ′ end of the cleavage oligonucleotide DNA is preferably subjected to appropriate modification such as amination in order to prevent the elongation reaction.

  Examples of the cleavage oligonucleotide DNA include those consisting of 24 or more consecutive bases in the sequence shown in SEQ ID NO: 29, more preferably those consisting of the sequence shown in SEQ ID NO: 30.

  When the amplification step is further carried out, the detection step comprises an oligonucleotide probe comprising the sequence shown in any one of SEQ ID NO: 2, 7 or 9 or its complementary sequence as described above during or after amplification. Use and detect RNA transcripts. Further, in order to perform the detection step in the process of amplification, that is, to simultaneously perform RNA amplification and RNA transcript detection, it is particularly preferable to use the above-described intercalator fluorescent dye-labeled probe as the oligonucleotide probe. .

  The detection of 16S rRNA derived from Mycobacterium tuberculosis of the present invention in which the amplification step is performed is preferably performed by using an oligonucleotide consisting of 24 or more consecutive nucleotides in the sequence shown in SEQ ID NO: 29 as a cleavage oligonucleotide DNA, as a first primer. An oligonucleotide consisting of 23 or more consecutive bases in the sequence shown in SEQ ID NO: 17, an oligonucleotide consisting of 19 or more consecutive bases in the sequence shown in SEQ ID NO: 19 as a second primer, and an intercalating fluorescent dye This is carried out using an oligonucleotide consisting of the sequence shown in any one of SEQ ID NOs: 2, 7 and 9 labeled with. More preferably, the oligonucleotide consisting of 24 or more consecutive nucleotides in the sequence shown in SEQ ID NO: 29 as the cleavage oligonucleotide DNA, and the 23 or more consecutive nucleotides in the sequence shown in SEQ ID NO: 16 as the first primer An oligonucleotide comprising the sequence shown in SEQ ID NO: 13 and a sequence consisting of 18 or more consecutive bases in the sequence shown in SEQ ID NO: 21 as the second primer, including the sequence shown in SEQ ID NO: 20 This is carried out using an oligonucleotide consisting of the sequence shown in SEQ ID NO: 2, 7 or 9 labeled with an oligonucleotide or an intercalating fluorescent dye. Particularly preferably, it is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 30 as the cleavage oligonucleotide DNA, and an oligonucleotide consisting of the sequence shown in SEQ ID NO: 13 as the first primer, and a promoter sequence at its 5 ′ end Labeled with an intercalating fluorescent dye as an oligonucleotide probe, an oligonucleotide consisting of the sequence shown in any of SEQ ID NO: 21, 22 or 23 as a second primer It is carried out using an oligonucleotide consisting of the sequence shown in any of SEQ ID NO: 2, 7 or 9.

  By using the oligonucleotide probe of the present invention, it is possible to detect a sequence (specific base sequence) specific to M. tuberculosis found in 16S rRNA derived from M. tuberculosis. Moreover, since this oligonucleotide probe is difficult to form higher-order structures such as dimers and loops even at relatively low temperatures of 40 ° C. to 50 ° C., an amplification method that specifically amplifies RNA that does not require temperature change is used. This is particularly effective when an amplification step is performed and a specific base sequence is detected in the process.

  As the oligonucleotide probe of the present invention, particularly preferably, a significant increase in fluorescence is observed if the amplification step is performed in the presence of an oligonucleotide labeled with an intercalating fluorescent dye and the fluorescence intensity is detected over time in the process. In addition, the detection can be completed at an arbitrary time, and the tuberculosis-derived 16S rRNA can be completed in about 30 minutes even if the time required for amplification is added.

  Thus, according to the present invention, it is possible to rapidly, sensitively and specifically detect M. tuberculosis-derived 16S rRNA or its gene contained in a sample.

  Hereinafter, embodiments of the present invention will be described by way of examples. However, the present invention is not limited to these examples.

Example 1 Preparation of oligonucleotide labeled with an intercalating fluorescent dye An oligonucleotide probe labeled with an intercalating fluorescent dye was prepared with reference to the method described in Non-Patent Document 2.

  10th C from the 5 ′ end of the sequence shown in SEQ ID NO: 1, 7th A from the 5 ′ end of the sequence shown in SEQ ID NO: 2, and 6th C from the 5 ′ end of the sequence shown in SEQ ID NO: 3 12th C from the 5 ′ end of the sequence shown in SEQ ID NO: 4, 14th T from the 5 ′ end of the sequence shown in SEQ ID NO: 6, 7th from the 5 ′ end of the sequence shown in SEQ ID NO: 8 G, 6th A from 5 'end of sequence shown in SEQ ID NO: 9, 11th A from 5' end of sequence shown in SEQ ID NO: 10, 14th from 5 'end of sequence shown in SEQ ID NO: 11 An amino group was introduced using a commercially available reagent (Label-ON Reagents, manufactured by Clontech) at the 9th C position from the 5 ′ end of the sequence shown in T of SEQ ID NO: 12, and the 3 ′ end was further biotinylated. Qualified with Oxazole yellow, which is an intercalating fluorescent dye, was bound to the introduced amino group to prepare oligonucleotides (SEQ ID NOs: 1 to 4, 6, 8 to 12) labeled with oxazole yellow.

  In addition, referring to the same document, the 13th C and 14th A from the 5 ′ end of the sequence shown in SEQ ID NO: 5 and the 4th C and 5th from the 5 ′ end of the sequence shown in SEQ ID NO: 7 An oligonucleotide labeled with oxazole yellow in which oxazol yellow was bonded to the phosphodiester moiety between A and A via a linker was prepared.

Example 2 Evaluation of Mycobacterium tuberculosis-derived 16S rRNA Detection Oligonucleotides Labeled with Intercalator Fluorescent Dyes of Combinations Shown in Table 1 (hereinafter referred to as “INAF probe”), first primer, RNA was detected by the methods shown in (1) to (5) using the second primer and the cleavage oligonucleotide, and the detection performance and specificity were evaluated.

  (1) Mycobacterium tuberculosis 16S rRNA, M. avium 16S rRNA and M. p. Each of the kansasii 16S rRNA was prepared by cloning the 16S rRNA gene, performing in vitro transcription, and purification (hereinafter referred to as “standard RNA”). M.M. The marine RNA was prepared by extracting a culture solution having an OD600 = 0.1 with a commercially available extraction reagent (EXTRAGEN MB, manufactured by Tosoh Corporation).

(2) Using a diluted RNA (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.25 U / μL ribonuclease inhibitor, 5.0 mM DTT), 10 ³ copies / 5 μL of Mycobacterium tuberculosis 16S rRNA, M.M. Avium 16S rRNA is 10 ⁸ copies / 5 μL. Kansasii 16S rRNA was diluted to 10 ⁸ copies / 5 μL, and these were used as samples. On the other hand, M.M. marinum used the stock solution of the extract as the RNA sample.

  (3) 20 μL of the reaction solution having the following composition was dispensed into a commercially available 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and 5 μL of the RNA sample was added thereto. A case where 5 μL of water for injection was added instead of the RNA sample was used as a negative control (hereinafter referred to as “Nega”).

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 Oligonucleotide DNA for cleavage
(Modified 3 'terminal hydroxyl group with amino group)
1 μM first primer (T7 promoter sequence (SEQ ID NO: 15) added to the 5 ′ end of the base sequence described in each SEQ ID NO)
1 μM second primer 10 nM INAF probe (prepared in Example 1)
10.4% DMSO
(4) After the above reaction solution was kept at 43 ° C. for 5 minutes, 5 μL of an enzyme solution (the composition is described below) that was kept warm at 43 ° C. for 2 minutes in advance was added.

Composition of enzyme solution: final concentration during reaction (in 30 μL) 2.0% sorbitol 6.4 U AMV reverse transcriptase (Life Science)
142U T7 RNA polymerase (Invitrogen)
3.6 μg Bovine Serum Albumin (5) Next, subject the PCR tube to a fluorescence spectrophotometer with a temperature control function that can directly detect and react at 43 ° C. and simultaneously measure the fluorescence intensity of the reaction solution (excitation wavelength: 470 nm, fluorescence wavelength: 520 nm). Detection was performed for 30 minutes over time.

  Table 1 shows the results of the detection performance evaluation for M. tuberculosis and the specificity evaluation for non-tuberculous mycobacteria (M. avium, M. kansasii, M. marinum). Regarding the detection performance evaluation, an RNA sample and Nega of Mycobacterium tuberculosis are detected, and the fluorescence intensity ratio of the reaction solution (a ratio obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity value of the background) is 1 when the enzyme is added. .3 or more cases were positive, and the time at that time was taken as the detection time. When the fluorescence intensity ratio was less than 1.3, it was regarded as negative and indicated as “(−)”. Moreover, the value of the fluorescence intensity ratio after 30 minutes in the standard RNA of Mycobacterium tuberculosis is also shown.

For specificity evaluation, positive when the fluorescence intensity ratio after 30 minutes when detecting an RNA sample of nontuberculous mycobacteria is greater than or equal to 0.2+ the fluorescence intensity ratio after 30 minutes when detecting Nega , If less than +0.2 is negative, if positive for any non-tuberculous mycobacteria, "+", if negative for any bacteria, "(-) ".

  From the results in Table 1, the combination of 2, 6 or 8 has good detection performance against Mycobacterium tuberculosis, is negative with Nega, and further does not show cross-reactivity with high concentration RNA of non-tuberculous mycobacteria. I understand that there is. The oligonucleotide probe of combination 2 (oligonucleotide consisting of the sequence shown in SEQ ID NO: 2) was obtained by cutting 3 bases of the 3 ′ end side of the oligonucleotide probe of combination 1 (oligonucleotide consisting of the sequence shown in SEQ ID NO: 1) The oligonucleotide probe of combination 6 (the oligonucleotide consisting of the sequence shown in SEQ ID NO: 7) and the oligonucleotide probe of combination 5 (the oligonucleotide consisting of the sequence shown in SEQ ID NO: 6) have the same sequence of 17 bases. Yes, the oligonucleotide probe of combination 8 (oligonucleotide consisting of the sequence shown in SEQ ID NO: 9) has 2 bases deleted from the 5 ′ end side of the oligonucleotide probe of combination 7 (oligonucleotide consisting of the sequence shown in SEQ ID NO: 8). It is a thing. Thus, in order to detect the 16S rRNA derived from Mycobacterium tuberculosis in a specific and highly sensitive manner even with oligonucleotide probes having similar sequences, the detection performance and specificity vary greatly. It can be seen that the oligonucleotide probe is essential.

Example 3 Examination of Mycobacterium tuberculosis-derived 16S rRNA detection primer set and cleaving oligonucleotide DNA Using the combination of the INAF probe, the first primer, the second primer and the cleaving oligonucleotide DNA shown in Table 2 Detection of standard RNA as in Example 2 (5 copies / 5 μL, 10 copies / 5 μL, 30 copies / 5 μL, 100 copies / μL, 5 and 10 copies n = 4, 30 and 100 copies n = 3) And the detection rate was investigated. The results are shown in Table 2.

From Table 2, combination 12 (cleaving oligonucleotide DNA consisting of the sequence shown in SEQ ID NO: 30, first primer consisting of the sequence shown in SEQ ID NO: 13), combination 13 (cleaving consisting of the sequence shown in SEQ ID NO: 31) Oligonucleotide DNA, first primer comprising the sequence shown in SEQ ID NO: 16), combination 14 (cleaving oligonucleotide DNA comprising the sequence shown in SEQ ID NO: 32, first sequence comprising the sequence shown in SEQ ID NO: 17 With the primer), the detection rate is 100% at 30 copies / test or more, and it can be seen that the combination of these cleavage oligonucleotide DNAs and the first primer can favorably detect M. tuberculosis-derived 16S rRNA. Further, it can be seen that 10 copies / test can be detected in the combination 12 and the combination 13, and in particular, the combination 12 can be detected with high sensitivity.

Example 4
In order to search for the second primer that is an optimal combination with the oligonucleotide DNA for cleavage of the combination 12 and the first primer in Example 3, the combination of oligonucleotides shown in Table 3 was used. detection against Mycobacterium tuberculosis in the same manner as in example 2 performance (10 ² copies / 5 [mu] L) and M. The specificity for avium (10 ³ copies / 5 μL) was investigated. The results are shown in Table 3.

From Table 3, the combination 12, 16, 17, 18 or 19 (second primer comprising the sequence shown in any of SEQ ID NOs: 21 to 25) has no difference in detection time and is good as the second primer. I understand. It can also be seen that the combination 12, 16 or 17 is particularly good in terms of specificity.

Claims (8)

A method for detecting a specific nucleotide sequence of Mycobacterium tuberculosis-derived 16S rRNA present in a sample or a gene thereof, wherein an oligonucleotide probe comprising the sequence shown in SEQ ID NO: 2, 7 or 9 or a complementary sequence thereof is used. A method for detecting Mycobacterium tuberculosis-derived 16S rRNA or a gene thereof. The method for detecting Mycobacterium tuberculosis-derived 16S rRNA according to claim 1, comprising: a first primer having a sequence homologous to a part of the specific base sequence; and a sequence complementary to a part of the specific base sequence. Using a second primer (wherein either one of the first and second primers has an RNA polymerase promoter sequence added to its 5 ′ end), and the following (1) to (5) RNA is amplified by a process, and the RNA transcript is detected using an oligonucleotide probe comprising the sequence shown in SEQ ID NO: 2, 7 or 9 or a complementary sequence thereof during or after amplification. A method characterized by that.
(1) a step of synthesizing cDNA complementary to a specific base sequence 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) Double-stranded DNA having a promoter sequence capable of transcribing a specific base sequence or RNA complementary to the specific base sequence by an enzyme having DNA-dependent DNA polymerase activity using single-stranded DNA as a template Generating a process,
(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. 16S rRNA derived from Mycobacterium tuberculosis using a first primer consisting of 23 or more consecutive bases in the sequence shown in SEQ ID NO: 17 and a second primer consisting of 18 or more consecutive bases in the sequence shown in SEQ ID NO: 19 The method for detecting Mycobacterium tuberculosis-derived 16S rRNA according to claim 1 or 2, comprising a step of amplifying the specific nucleotide sequence of M. tuberculosis. A first primer comprising a sequence of 23 or more bases in the sequence shown in SEQ ID NO: 16 and comprising the sequence shown in SEQ ID NO: 13, and a sequence of 18 or more bases in the sequence shown in SEQ ID NO: 21 The method further comprises a step of amplifying a specific base sequence of Mycobacterium tuberculosis-derived 16S rRNA using a second primer comprising the sequence shown in SEQ ID NO: 20. A method for detecting Mycobacterium tuberculosis-derived 16S rRNA. A specific base sequence of Mycobacterium tuberculosis-derived 16S rRNA is amplified using a first primer consisting of the sequence shown in SEQ ID NO: 13 and a second primer consisting of any one of the sequences shown in SEQ ID NO: 21, 22 or 23 A method for detecting Mycobacterium tuberculosis-derived 16S rRNA according to claim 1 or 2, comprising a step. The specific base sequence is obtained by cleaving oligonucleotide DNA complementary to a region adjacent to the 5 ′ end of the homologous region with the first primer in the specific base sequence in the 5 ′ direction and RNase H. Cleaving the RNA at the 5′-end site, amplifying a specific base sequence of Mycobacterium tuberculosis-derived 16S rRNA using the first primer and the second primer having a promoter sequence added to the 5′-end. The method according to any one of claims 3 to 5, wherein the cleaving oligonucleotide DNA comprises 24 or more consecutive bases in the sequence shown in SEQ ID NO: 29. A method for detecting Mycobacterium tuberculosis-derived 16S rRNA. The method for detecting Mycobacterium tuberculosis-derived 16S rRNA according to claim 6, wherein the cleavage oligonucleotide DNA comprises the sequence shown in SEQ ID NO: 30. A detection reagent for Mycobacterium tuberculosis-derived 16S rRNA, the first primer consisting of the sequence shown in SEQ ID NO: 13, the second primer consisting of the sequence shown in any of SEQ ID NO: 21, 22 or 23, and the sequence The above-mentioned reagent comprising an oligonucleotide probe comprising the sequence shown in any of Nos. 2, 7 or 9 or a complementary sequence thereof.