JP2003284565A - Oligonucleotide for detecting mycobacterium intracellulare of atypical mycobacterium and method for detecting the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide oligonucleotides useful for specifically cleaving or proliferating an RNA derived from Pst S1b gene of Mycobacterium intracellulare belonging to MAC (Mycobacterium avium complex) causing a disease which is similar to pulmonary tuberculosis to patients having underlying diseases in the lungs at a relatively low temperature and a definite temperature (35°C to 50°C, preferably 41°C) or carrying out detection and identification thereof at a high sensitivity and preferable combinations of these oligonucleotides. <P>SOLUTION: The oligonucleotides are bond to specific parts of Mycobacterium intracellulare and have sequences including at least 10 bases of specific sequences. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an atypical mycobacteria Mycobacterium intracellulare in clinical examination.
The present invention relates to an oligonucleotide for detecting ellulare and a detection method. The oligonucleotide provided by the present invention is used as a reagent for gene diagnosis for performing operations such as cleavage, amplification and detection of RNA and DNA, and R
It is effective as a reagent for inhibiting the reverse transcription and translation of NA.

[0002]

2. Description of the Prior Art Mycobacterium atypical mycobacteria
M intracellularis is a MAC (Mycobacterium avium) together with Mycobacterium avium, which is also an atypical mycobacteria.
belongs to a bacterial species called "complex". MA
C causes pulmonary tuberculosis resemblance in patients with underlying lung diseases, accounting for about 70% of non-tuberculous mycobacterial infections in Japan. MAC is generally resistant to various antibacterial agents.

Conventionally, M. intracellular
The identification of atypical mycobacteria such as e was mainly based on the culture test, but the test was complicated and required 3 to 4 weeks for identification, and also showed typical properties among the same bacterial species. Some strains did not exist, and some skill was required to identify atypical mycobacteria.

[0004]

As described above, the test methods currently used do not necessarily answer the applications for rapid detection and confirmation of therapeutic effect required in actual medical fields. It is desired to develop a highly sensitive and rapid detection method as well as detecting only viable bacteria. Further, in order to make the inspection easier, it is required to develop an automated inspection device.

As a highly sensitive detection method, a method of amplifying a target nucleic acid can be used. Known methods for amplifying a specific RNA sequence include the NASBA method and the 3SR method, which amplify a specific RNA sequence by the concerted action of reverse transcriptase and RNA polymerase. This method uses a target RNA as a template, synthesizes a double-stranded DNA containing a promoter sequence with a primer containing a promoter sequence, reverse transcriptase, and ribonuclease H, and targets the double-stranded DNA with RNA polymerase as a template. It is a method of synthesizing RNA containing a specific base sequence of RNA, and then performing a chain reaction in which the RNA serves as a template for the synthesis of double-stranded DNA containing a promoter sequence.

[0006] As described above, the NASBA method and the 3SR method are considered to be suitable for automation because they allow nucleic acid amplification at a constant temperature. However, since these amplification methods carry out the reaction at a relatively low temperature (for example, 41 ° C.), the target RNA
May form an intramolecular structure, inhibit the binding of the primer, and reduce the reaction efficiency. Therefore,
By performing heat denaturation of the target RNA before the amplification reaction, it was necessary to destroy the intramolecular structure of the target RNA and improve the binding efficiency of the primer. Furthermore,
Even when detecting RNA at low temperature, an oligonucleotide capable of binding to RNA having the above-mentioned molecular structure was required.

Therefore, the present invention is a Mycobacterium
ium intracellulare Pst S1
Useful for specifically cleaving or amplifying RNA derived from b gene at relatively low temperature and constant temperature (35 ° C to 50 ° C, preferably 41 ° C), and performing detection and identification thereof with high sensitivity It is intended to provide various oligonucleotides and suitable combinations.

[0008]

Means for Solving the Problems The invention of claim 1 made in order to achieve the above object is a Mycobacte.
ium intracellulare Pst S
An oligonucleotide required for cleaving, detecting, or amplifying RNA derived from the 1b gene. int
racellulare Pst S1b gene DNA
Alternatively, it is an oligonucleotide consisting of at least 10 consecutive bases in any of the sequences shown in SEQ ID NOS: 1 to 12, which can specifically bind to RNA derived from the gene.

The invention of claim 2 of the present application relates to the invention of claim 1, wherein a part of the oligonucleotide is the RN.
It is characterized by being an oligonucleotide probe for cleaving the RNA at the specific site by binding to the specific site of A. The invention of claim 3 of the present application relates to the invention of claim 1, characterized in that the oligonucleotide is an oligonucleotide primer for a DNA extension reaction. The invention of claim 4 of the present application relates to the invention of claim 1, characterized in that it is an oligonucleotide probe in which a part of the oligonucleotide is modified or labeled with a detectable labeling substance. The invention of claim 5 of the present application is characterized in that it is a synthetic oligonucleotide in which a part of the bases of the oligonucleotide is converted within a range that does not impair the function as an oligonucleotide probe.

The invention according to claim 6 of the present application is based on M. intrac
R derived from the Pst S1b gene of ellulare
It is an amplification process of NA, which is present in the sample. intr
RNA-dependent DNA using a specific sequence of RNA derived from the P. acerulare Pst S1b gene as a template
CDNA is synthesized with a polymerase, RNA-DNA double-stranded RNA is decomposed with ribonuclease H to generate single-stranded DNA, and the single-stranded DNA is used as a template for the specific sequence or A double-stranded DNA having a promoter sequence capable of transcribing RNA consisting of a sequence complementary to a specific sequence is produced, and the double-stranded DNA produces an RNA transcript in the presence of RNA polymerase. Subsequently, cDNA produced by the RNA-dependent DNA polymerase
In an RNA amplification process that serves as a template for synthesis, M.
SEQ ID NO: 13 having a sequence homologous to RNA derived from intracellulare Pst S1b gene
A first primer consisting of at least 10 consecutive bases in the sequence shown in SEQ ID NO: 6 and an at least 10 consecutive bases in the sequence shown in SEQ ID NO: 6 to be amplified. A second primer having a sequence complementary to a part of the RNA sequence derived from the intracellulare Pst S1b gene (wherein either the first primer or the second primer is the promoter sequence of RNA polymerase on the 5 ′ side thereof). (Including) is used.

[0012] The invention of claim 7 relates to the invention of claim 6, wherein in the RNA amplification step, it can specifically bind to an RNA transcript produced by amplification and is labeled with an intercalating fluorescent dye. It is carried out in the presence of the labeled oligonucleotide, and the change in the fluorescence property of the reaction solution is measured (however, the labeled oligonucleotide has a sequence different from that of the first and second primers). The invention of claim 8 of the present application relates to the invention of claim 7, wherein the oligonucleotide is RNA.
It is characterized in that it is designed to complementarily bind to at least a part of the sequence of the transcript, and the fluorescence property is changed as compared with the case where no complex is formed. The invention of claim 9 of the present application relates to the invention of claim 8, wherein the oligonucleotide comprises at least 10 consecutive bases or more in any of the sequences shown in SEQ ID NO: 14, or a complementary sequence thereof. It is characterized by Hereinafter, the present invention will be described in detail.

[0012] The oligonucleotide of the present invention is an oligonucleotide that specifically forms a complementary bond to the intramolecular structure-free region of the target RNA when the above RNA is amplified, and undergoes the above-described thermal denaturation. It specifically binds to the target RNA without any consequence. As described above, the present invention has a relatively low temperature and a constant temperature (35 ° C to 50 ° C,
Preferably 41 ° C.), M. intracellula
An oligonucleotide that binds to an intramolecular structure free region of RNA derived from the Pst S1b gene of M.re. intracellulare Pst S
It is a useful oligonucleotide for specifically cleaving, amplifying or detecting RNA derived from the 1b gene. More specifically, the present invention is an oligonucleotide probe for cleaving the target RNA at a specific position,
An oligonucleotide primer for amplifying the target DNA by a PCR method, an oligonucleotide primer for amplifying the target RNA by a NASBA method, etc., and for detecting a target nucleic acid without such amplification or after such amplification It is an oligonucleotide for achieving rapid and highly sensitive detection when used as an oligonucleotide probe.

SEQ ID NOs: 1 to 12 are M. intrace
RN derived from the Pst S1b gene of llurale
1 shows an example of the oligonucleotide of the present invention which is useful for cleaving, amplifying or detecting RNA derived from A. Here, M. intracellulare
The RNA derived from the Pst S1b gene also includes RNA produced using these genes as a template. The oligonucleotide of the present invention may contain the entire base sequences described as SEQ ID NOS: 1 to 12, respectively. intracellulare Pst S1
Since about 10 bases are sufficient for the specific binding to RNA derived from the b gene, an oligonucleotide consisting of at least 10 consecutive bases or more in the respective described base sequences may be used.

The oligonucleotide of the present invention can be used, for example, as a probe for cleaving RNA. Target RNA
In order to cleave at a specific position, the oligonucleotide of the present invention may be hybridized with a single-stranded target RNA, and an enzyme that cleaves only the RNA of the RNA-DNA heteronuclear double-stranded portion may act. . As the enzyme, an enzyme generally known to have ribonuclease H activity may be used.

The oligonucleotide of the present invention can be used, for example, as an oligonucleotide primer for nucleic acid amplification. When the nucleic acid amplification method is carried out using the oligonucleotide of the present invention as a primer, the target nucleic acid, that is, M. i
Only the nucleic acid of the Pst S1b gene of Ntracellulare can be amplified. As the amplification method, the PCR method,
LCR method, NASBA method, 3SR method, etc. can be exemplified,
Among them, the nucleic acid amplification method that can be performed at a constant temperature, such as the LCR method, the NASBA method and the 3SR method, is preferable. By detecting this amplification product by various methods, M. intrace
It is possible to detect llurale. In this case, the above-mentioned oligonucleotide other than the oligonucleotide used in the amplification may be used as a probe, and the amplified fragment of the specific sequence may be confirmed by electrophoresis.

The oligonucleotide of the present invention can be used as a probe, for example, by modifying a part thereof or labeling it with a detectable labeling substance. When the target nucleic acid is to be detected, the oligonucleotide of the present invention labeled with a detectable labeling substance may be hybridized with the single-stranded target nucleic acid, and the label may be detected for the hybridized probe.
For detection of the label, a method suitable for the labeling substance may be adopted.For example, when an intercalating fluorescent dye is used for labeling the oligonucleotide, a double-stranded nucleic acid composed of the target nucleic acid and the oligonucleotide probe is used. If a dye or the like having the property of increasing fluorescence intensity by intercalation is used, it is possible to easily detect only the hybridized probe without removing the probe that is not hybridized with the target nucleic acid. When a usual fluorescent dye or the like is used as a label, it may be detected after removing a probe that does not hybridize with the target nucleic acid. In the detection, it is desirable to amplify the target nucleic acid in the sample to a detectable amount by various nucleic acid amplification methods such as PCR method, NASBA method and 3SR method. Above all, constant temperature nucleic acid amplification such as NASBA method and 3SR method. Law is the most desirable. On the other hand, when a probe labeled with the above-mentioned oligonucleotide is allowed to coexist in the reaction solution during amplification, modification such as addition of glycolic acid to the 3 ′ end should be performed so that the probe does not function as a nucleotide primer. Is especially desirable.

The present invention also relates to M. intrac
R derived from the Pst S-3 gene of ellulare
Provided are a nucleic acid amplification step for amplifying NA and a method for detecting an RNA transcript produced by the nucleic acid amplification step. The amplification process of the present invention includes PCR method, NASBA method and 3SR method, but among them, by the concerted action of reverse transcriptase and RNA polymerase (reaction under conditions such that reverse transcriptase and RNA polymerase act cooperatively). Let)
M. NASBA that amplifies a specific RNA sequence derived from the Pst S1b gene of intracellulare
Method, a constant temperature nucleic acid amplification method such as 3SR method is preferable.

For example, the NASBA method is used in M. RNA using a specific sequence of RNA derived from the intracellulare Pst S1b gene as a template
CDNA is synthesized by a dependent DNA polymerase, and RNA-DNA double-stranded RN is synthesized by ribonuclease H.
A is decomposed to generate a single-stranded DNA, and a promoter sequence capable of transcribing an RNA comprising the specific sequence or a sequence complementary to the specific sequence by a DNA-dependent DNA polymerase using the single-stranded DNA as a template Double-stranded D having
An RNA amplification process in which NA is produced, and the double-stranded DNA produces an RNA transcript in the presence of RNA polymerase, and the RNA transcript is subsequently used as a template for cDNA synthesis by the RNA-dependent DNA polymerase. However, the present invention relates to M. Intracellular P
A first primer having at least 10 consecutive bases in the sequence shown in SEQ ID NO: 13 having a sequence homologous to the RNA derived from st S1b gene, and at least 10 consecutive bases in the sequence shown in SEQ ID NO: 6 Amplified M. intracellulare
Second primer having a sequence complementary to a part of the RNA sequence derived from the Pst S1b gene of Escherichia coli (herein, either the first primer or the second primer has a promoter sequence of RNA polymerase on its 5'terminal side). (Including) is used.

An RNA-dependent DNA polymerase,
DNA-dependent DNA polymerase, ribonuclease H
Is not particularly limited, but AMV reverse transcriptase having all of these activities is preferable. The RNA polymerase is also not particularly limited, but T7 phage RNA polymerase and SP6 phage RNA polymerase are preferable.

In the above amplification process, M. intracel
RNA derived from the Llare Pst S1b gene
It overlaps with the 5'end region of the region to be a specific sequence in the sequence (1
(About 10 bases) and an oligonucleotide complementary to the adjacent region is added, and the M. intracellul
Cleavage of RNA derived from are Pst S1b gene at the 5′-terminal region of a specific sequence (by ribonuclease H)
As a template for the initial stage of nucleic acid amplification, the specific sequence is not located at the 5'end. intracellul
RNA derived from the Pst S1b gene of are can also be amplified. For this disconnection, for example,
Oligonucleotides of SEQ ID NOs: 1 to 12 (provided that the oligonucleotides other than the one used as the second primer in the amplification step) can be used. The cleavage oligonucleotide is preferably one in which the 3'terminal hydroxyl group is chemically modified (for example, aminated) in order to suppress the extension reaction from the 3'end.

The amplification product obtained by the above nucleic acid amplification method can be detected by a known nucleic acid detection method. In a preferred embodiment, the nucleic acid amplification is carried out in the presence of an oligonucleotide labeled with an intercalating fluorescent dye. It is desirable to carry out the measurement and measure the change in the fluorescence characteristic of the reaction solution. The oligonucleotide is one in which an intercalating fluorescent dye is bound to phosphorus in the oligonucleotide via a linker. When a double strand is formed with a target nucleic acid (complementary nucleic acid), the intercalator portion becomes a double-stranded portion. It is characterized in that it does not require a separation analysis because the fluorescence characteristics are changed by intercalation (Ishiguro, T. et al.
996) Nucleic Acids Res. 24 (2
4) 4992-4997).

The sequence of the oligonucleotide is not particularly limited as long as it has a sequence complementary to at least a part of the amplification product, but a sequence consisting of at least 10 consecutive bases in the sequence shown in SEQ ID NO: 14, Alternatively, it is preferably its complementary sequence. Further, in order to suppress the extension reaction using the oligonucleotide as a primer, it is desirable that the 3'-terminal hydroxyl group of the oligonucleotide is chemically modified (for example, glycolic acid is added).

Accordingly, M. intracellul
RNA having the same sequence as the specific sequence in the RNA derived from the Pst S1b gene of are in one tube,
It becomes possible to amplify and detect in a single step at a constant temperature, and the application to automation becomes easy.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 Atypical Mycobacteria Mycobacterium intra
An oligonucleotide was selected that specifically binds to RNA derived from the Pst S1b gene of cellulare at 41 ° C.

(1) M. intracellulare
The standard RNA (1204 bases, SEQ ID NO: 15) containing the region derived from the Pst S1b gene in the base sequence of 1. was quantified by ultraviolet absorption at 260 nm, and then diluted with RNA (10 mM Tris-hydrochloric acid buffer (pH 8.0)). 1
mM EDTA, 1 mM DTT, 0.5 U / μL R
It was diluted to 2.236 pmol / μL using Nase inhibitor (Takara Shuzo Co., Ltd.).

(2) Add 14 μL of the reaction solution having the following composition to PC
R tube (capacity 0.5 mL; GeneAmp Th
in-Walled Reaction Tube, manufactured by Perkin Elmer).

Composition of reaction solution (each concentration is the final reaction solution volume 15
Concentration in μL) 60 mM Tris-hydrochloric acid buffer solution (pH 8.6) 17 mM magnesium chloride 90 mM potassium chloride 6U RNase inhibitor (Takara Shuzo Co., Ltd.)
1 mM DTT 0.066 μM standard RNA 0.2 μM oligonucleotide (any one of the following oligonucleotides was used. The base number is Pst S1b of M. intracellulareure)
The start position of the region gene [52nd base of SEQ ID NO: 15] is set to 1. ) MIP-1Rb (Pst S1b gene base number 60)
To 79, complementary oligonucleotides, SEQ ID NO: 1) MIP-2Rb (base number 21 of Pst S1b gene)
2 to 234, complementary oligonucleotide, SEQ ID NO: 2) MIP-3Rb (Pst S1b gene base number 22)
9 to 249 complementary oligonucleotide, SEQ ID NO: 16) MIP-4Rb (base number 28 of Pst S1b gene)
Oligonucleotides complementary to 9 to 310, SEQ ID NO: 17) MIP-5Rb (base number 40 of Pst S1b gene)
8 to 427, complementary oligonucleotide, SEQ ID NO: 3) MIP-6Rb (base number 43 of Pst S1b gene)
Oligonucleotides complementary to 9 to 461, SEQ ID NO: 4) MIP-7Rb (base number 46 of Pst S1b gene)
Oligonucleotides complementary to 1 to 483, SEQ ID NO: 18) MIP-8Rb (base number 48 of Pst S1b gene)
7 to 507 complementary oligonucleotide, SEQ ID NO: 19) MIP-9Rb (base number 52 of Pst S1b gene)
Oligonucleotides complementary to 4 to 545, SEQ ID NO: 5) MIP-10Rb (base number 5 of Pst S1b gene)
Oligonucleotides complementary to 71 to 590, SEQ ID NO: 6) MIP-11Rb (base number 6 of Pst S1b gene)
Oligonucleotides complementary to 34 to 655, SEQ ID NO: 7) MIP-12Rb (base number 7 of Pst S1b gene)
Oligonucleotides complementary to 04 to 723, SEQ ID NO: 8) MIP-13Rb (base number 7 of Pst S1b gene)
21 to 743 complementary oligonucleotide, SEQ ID NO: 9) MIP-14Rb (base number 7 of Pst S1b gene)
Oligonucleotides complementary to 57 to 776, SEQ ID NO: 20) MIP-15Rb (base number 8 of Pst S1b gene)
Oligonucleotides complementary to 16 to 835, SEQ ID NO: 10) MIP-16Rb (base number 8 of Pst S1b gene)
An oligonucleotide complementary to 53 to 875, SEQ ID NO: 11) MIP-17Rb (base number 9 of Pst S1b gene)
Oligonucleotides complementary to 17 to 938, SEQ ID NO: 21) MIP-18Rb (base number 1 of Pst S1b gene)
Oligonucleotides complementary to 027 to 1049, SEQ ID NO: 12) MIP-19Rb (base number 1 of Pst S1b gene)
Oligonucleotide complementary to 076 to 1098, SEQ ID NO: 22) MIP-20Rb (base number 1 of Pst S1b gene)
Oligonucleotide complementary to 093 to 1115, SEQ ID NO: 23) Distilled water for volume adjustment (3) After incubating the above reaction solution at 41 ° C. for 5 minutes, 8 U / μ
L AMV reverse transcriptase (Takara Shuzo Co., Ltd., this enzyme is DN
1 μL of a solution containing A / RNA, which is an enzyme that cleaves double-stranded RNA).

(4) Subsequently, the PCR tube was kept at 41 ° C. for 10 minutes.

(5) To confirm the cleavage fragment after the reaction,
Urea denaturing polyacrylamide gel (acrylamide concentration 6%, urea 7M) electrophoresis was performed. Staining after electrophoresis was performed with SYBR Green II (Takara Shuzo Co., Ltd.). When the oligonucleotide binds to a specific site of the target RNA, RNA / DNA double-stranded RNA is cleaved by the ribonuclease H activity of AMV reverse transcriptase, and a specific band is detected.

The results of electrophoresis (black and white reversal) are shown in FIG. When the oligonucleotide specifically binds to the standard RNA, the standard RNA is degraded in that region, resulting in a degradation product of a particular chain length. Table 1 shows the position and expected chain length of the band when the oligonucleotide specifically binds to the standard RNA. MIP-1Rb, MIP-2R
b, MIP-5Rb, MIP-6Rb, MIP-9R
b, MIP-10Rb, MIP-11Rb, MIP-1
2Rb, MIP-13Rb, MIP-15Rb, MIP
Specific bands were confirmed in the case of -16Rb and MIP-18Rb. From these results, these oligonucleotides were isolated from M. intracellulare
Was strongly bound to RNA derived from the Pst S1b gene of. The base number in Table 1 is the number when the start position of the Pst S1b gene (the 52nd base of SEQ ID NO: 15) is 1 in the base sequence shown in SEQ ID NO: 15. The circles in the table indicate that only the specific band was observed, the triangle indicates that the specific band was observed but the non-specific band was also observed, and the cross indicates that no specific band was observed.

[0031]

[Table 1] Example 2 M. An RNA amplification reaction was performed using an oligonucleotide that specifically binds to RNA derived from the intracellulare Pst S1b gene.

(1) M.S. intrac
ellulare Pst S1b gene standard RNA
(SEQ ID NO: 15) was quantified by UV absorption at 260 nm, and then diluted with RNA (10 mM Tris-hydrochloric acid buffer (pH 8.0), 1 mM EDTA, 1 mM DTT,
0.5 U / μL RNase inhibitor (manufactured by Takara Shuzo Co., Ltd.) was used to dilute to 10 ⁴ copies / 5 μL. Only the diluted solution was used for the control test section (negative).

(2) Add 20 μL of the reaction solution having the following composition to 0.2
5 mL PCR tube (GeneAmp Thin-
Walled Reaction Tubes (manufactured by PerkinElmer), and 5 μL of the above RNA sample
Was added.

Composition of reaction solution (each concentration is 30 times the final reaction solution volume)
Concentration in μL) 60 mM Tris-hydrochloric acid buffer (pH 8.6) 17 mM Magnesium chloride 150 mM Potassium chloride 6U RNase Inhibitor 1 mM DTT 0.25 mM each of dATP, dCTP, dGTP, dT
TP 3.6 mM ITP 3.0 mM each ATP, CTP, GTP, UTP 0.16 μM first oligonucleotide 1.0 μM second oligonucleotide 1.0 μM third oligonucleotide 13% DMSO Distilled water for volume adjustment ( 3) As the first, second and third oligonucleotides, the oligonucleotides having the sequences shown in the explanation of Table 1 were used.
A amplification reaction was performed. The solutions were prepared so that the combinations of the first, second, and third oligonucleotides would be the combinations shown in Table 2.

(4) After incubating the above reaction solution at 41 ° C. for 5 minutes, 5 μL of an enzyme solution having the following composition was added.

Composition of enzyme solution (each value is the final reaction solution volume of 30
Value in μL) 2.0% Sorbitol 3.6 μg Bovine serum albumin 142U T7 RNA polymerase (manufactured by Gibco) 8U AMV reverse transcriptase (manufactured by Takara Shuzo Co., Ltd.) Distilled water for volume adjustment (5) Continue to PCR tube at 41 ° C. at 30 ° C. Keep warm for minutes.

(6) In order to confirm the RNA amplified portion after the reaction, agarose gel (agarose concentration 4%) electrophoresis was performed. Staining after electrophoresis is SYBR Green
II (manufactured by Takara Shuzo). When the oligonucleotide probe binds to the specific site of the target RNA, the RNA sandwiched between the second oligonucleotide and the third oligonucleotide is amplified and a specific band is observed.

A photograph of the result of electrophoresis (black and white inversion) is shown in FIG. The chain length of the specific band amplified in this reaction is shown in Table 2.
As shown in. From this, in the RNA amplification reaction using the combination of oligonucleotides shown in Table 2, a specific band was confirmed in the combination (b). Therefore, the oligonucleotides used in this combination were M. It was shown to be effective in detecting intracellulare.

[0039]

[Table 2] Table 2 shows the combination of the first, second, and third oligonucleotides used in this experimental system, and R
The chain length of a specific band amplified when the NA amplification reaction is performed is shown. Of the base sequence of the first oligonucleotide,
The hydroxyl group at the 3'end is aminated. In the base sequence of the second oligonucleotide, the region from the first "A" to the 22nd "A" at the 5'end is the T7 promoter region, and the following 23th "G" to 28
The region up to the second "A" is an enhancer sequence. In addition, the base number is the number when the start position of the Pst S1b gene is set to 1 in SEQ ID NO: 15.

First oligonucleotide MIP-5Sb (SEQ ID NO: 24, base numbers 404 to 4)
27) MIP-6Sb (SEQ ID NO: 25, base numbers 438 to 4)
61) MIP-21Sb (SEQ ID NO: 26, base numbers 779 to 802) MIP-16Sb (SEQ ID NO: 27, base numbers 852 to 875) Second oligonucleotide MIP-5Fb (SEQ ID NO: 28, base numbers 422 to 4)
44) MIP-6Fb (SEQ ID NO: 29, base numbers 456 to 4)
78) MIP-21Fb (SEQ ID NO: 30, base numbers 794 to 816) MIP-16Fb (SEQ ID NO: 31, base numbers 870 to 892) Third oligonucleotide MIP-9Rb (SEQ ID NO: 5, base numbers 524 to 54)
5) MIP-10Rb (SEQ ID NO: 6, base numbers 571 to 5)
90) MIP-12Rb (SEQ ID NO: 8, base numbers 704 to 7)
23) MAP-22Rb (SEQ ID NO: 32, base numbers 958 to 977) MAP-18Rb (SEQ ID NO: 12, base numbers 1027 to 1049) Example 3 Using the combination of oligonucleotides according to the present invention, target M. intracellulare Pst
Detection at various initial copy numbers of RNA from the S-3 gene was performed.

(1) The same M.S. intrac
ellulare Pst S1b gene standard RNA
(SEQ ID NO: 15) was used as an RNA diluent (10 mM Tris
-HCl (pH 8.0), 1 mM EDTA, 0.5 U
/ ΜL RNase Inhibitor (Takara Shuzo Co., Ltd., 5 mM DTT) was used and 10 ⁵ copies /
Diluted from 5 μL to 30 copies / 5 μL. Only the diluted solution was used for the control test section (negative).

(2) 20 μL of the reaction solution having the following composition was applied to PC
R tube (capacity 0.5 mL; GeneAmp T
hin-Walled Reaction Tube
s, manufactured by Perkin Elmer), and the RNA
5 μL of sample was added.

Composition of reaction solution (each concentration is 30 times the final reaction solution volume)
Concentration in μL) 60 mM Tris-hydrochloric acid buffer solution (pH 8.6) 17 mM magnesium chloride 120 mM potassium chloride 6U RNase Inhibitor 1 mM DTT 0.25 mM each of dATP, dCTP, dGTP, dT
TP 3.6 mM ITP 3.0 mM each ATP, CTP, GTP, UTP 0.16 μM first oligonucleotide (MIP-5S
b, SEQ ID NO: 24, the hydroxyl groups at the 3'end are aminated. ) 1.0 μM second oligonucleotide (MIP-5F
b, SEQ ID NO: 28) 1.0 μM third oligonucleotide (MIP-10
R, SEQ ID NO: 6) Oligonucleotide labeled with 25 nM intercalating fluorescent dye (YO-intra-SG, SEQ ID NO: 14, with 7'A 'and 8'G' from the 5'end) The intercalating fluorescent dye is labeled on the phosphorus between them, and the hydroxyl group at the 3'end is modified with a glycol group.) 13% DMSO Distilled water for volume adjustment (3) After 5 minutes of incubation, 5 μL of enzyme solution having the following composition and preliminarily kept at 41 ° C. for 2 minutes was added.

Composition of enzyme solution (each concentration is the final reaction solution volume of 30
Concentration in μL) 2.0% Sorbitol 3.6 μg Bovine serum albumin 142U T7 RNA polymerase (manufactured by Gibco) 8U AMV reverse transcriptase (manufactured by Takara Shuzo) Distilled water for volume adjustment (4) Temperature control function that can directly measure PCR tubes Using a fluorescence spectrophotometer with
The reaction solution was measured over time at an excitation wavelength of 470 nm and a fluorescence wavelength of 510 nm.

The time course of the fluorescence intensity ratio of the sample (fluorescence intensity value at a predetermined time / background fluorescence intensity value) is shown in FIG. Also,
The result of the relationship between the logarithmic value of the initial RNA amount and the rising time (the time until the fluorescence increase ratio becomes a value obtained by adding three times the standard deviation to the negative mean value: 1.2) is shown in FIG. 3 (B). Indicated.
The initial RNA amount is 30 copies / test to 10 ⁵ copies / test.

From FIG. 3, 10 ³ copies were detected in about 17 minutes. A fluorescence profile and a calibration curve depending on the initial concentration of the target RNA were obtained, and the M. in
tracellulare Pst S1b region RNA
It was shown that the quantification of From the above, according to this method, M. It was shown that rapid and highly sensitive detection of intracellulare is possible.

Example 4 The combination of oligonucleotides according to the present invention
M. It was confirmed whether it is specific to intracellulare.

(1) M. intracellular
e, M .; avium, M.A. kansas ii and B
As a CG RNA sample, a liquid obtained by extracting nucleic acid from a colony of each bacterial species was used.

(2) Add 20 μL of the reaction solution having the following composition to PC
R tube (capacity 0.5 mL; GeneAmp Th
in-Walled Reaction Tubes,
(Manufactured by Perkin Elmer), and 5 μL of the above RNA sample was added thereto.

Composition of reaction solution (each concentration is 30 times the final reaction solution volume)
Concentration in μL) 60 mM Tris-hydrochloric acid buffer solution (pH 8.6) 17 mM magnesium chloride 120 mM potassium chloride 6U RNase Inhibitor 1 mM DTT 0.25 mM each of dATP, dCTP, dGTP, dT
TP 3.6 mM ITP 3.0 mM each ATP, CTP, GTP, UTP 0.16 μM first oligonucleotide (MIP-5S
b, SEQ ID NO: 24, the hydroxyl groups at the 3'end are aminated. ) 1.0 μM second oligonucleotide (MIP-5F
b, SEQ ID NO: 28) 1.0 μM third oligonucleotide (MIP-10
R, SEQ ID NO: 6) Oligonucleotide labeled with 25 nM intercalating fluorescent dye (YO-intra-SG, SEQ ID NO: 14, with 7'A 'and 8'G' from the 5'end) The intercalating fluorescent dye is labeled on the phosphorus between them, and the hydroxyl group at the 3'end is modified with a glycol group.) 13% DMSO Distilled water for volume adjustment (3) After incubating for 5 minutes at 5 ° C., 5 μL of an enzyme solution having the following composition and previously incubating at 41 ° C. for 2 minutes was added.

Composition of enzyme solution (each concentration is 30 times the final reaction volume)
Concentration in μL) 2.0% Sorbitol 3.6 μg Bovine serum albumin 142U T7 RNA polymerase (manufactured by Gibco) 8U AMV reverse transcriptase (manufactured by Takara Shuzo) Distilled water for volume adjustment (4) Temperature control function that can directly measure PCR tubes Using a fluorescence spectrophotometer with
The reaction solution was measured over time at an excitation wavelength of 470 nm and a fluorescence wavelength of 510 nm.

The time course of the fluorescence intensity ratio of the sample (fluorescence intensity value at a predetermined time / background fluorescence intensity value) is shown in FIG. 4, with the time of enzyme addition as 0 minutes. As a result, the combination of oligonucleotides of the present invention was determined to be M. int
It was shown that it was specifically detecting racellulare.

[0053]

EFFECT OF THE INVENTION As described above, the oligonucleotide of the present invention has a relatively low temperature (from 35 ° C. to 50 ° C.) at which RNA forms an intramolecular structure and may inhibit the binding of a primer or a probe. (.Degree. C., preferably 41.degree. C.) conditions. intracellulare Pst
It is an oligonucleotide that complementarily binds to RNA derived from the S1b gene. Therefore, it becomes possible to specifically bind the oligonucleotide without thermally denaturing the target RNA. Thus, the oligonucleotides of the present invention are intracellulare Ps
It is useful for cleaving, amplifying or detecting RNA derived from the t S1b gene, that is, as an oligonucleotide primer or an oligonucleotide probe used for RNA amplification.

In addition to the above, the oligonucleotides of the present invention include M. intracellulare Pst
It is also useful for amplifying or detecting the S1b gene.

The oligonucleotide of the present invention is not limited to the one having the base sequence (20 to 23 bases) shown in the sequence listing, and may be any oligonucleotide having at least 10 consecutive bases in these sequences. It is clear from these that a base sequence of about 10 bases is sufficient to ensure the specificity of the primer or probe for the target nucleic acid under relatively low temperature (preferably 41 ° C.) conditions.

[0056]

[Sequence list] SEQUENCE LISTING    <110> TOSOH corporation    <120> Atypical Mycobacterium Mycobacterium intracellulare e Oligonucleotide for detection and detection method    <130> PA211-0737    <160> 32    <210> 1 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> MIP-1Rb    <400> 1 tcgaaccaca cgcagccgcg 20    <210> 2 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-2Rb    <400> 2 gacgttcgag tacttgtcgt gat 23    <210> 3 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> MIP-5Rb    <400> 3 tgatgtgttc ggtgacaccg 20    <210> 4 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-6Rb    <400> 4 ttgtacatgc cggctagcac ctt 23    <210> 5 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> MIP-9Rb <400> 5 agcggaacca ccggtgtggc ag 22    <210> 6 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> MIP-10Rb    <400> 6 tactgggtga acaggaaggt 20    <210> 7 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> MIP-11Rb    <400> 7 ggaaggcgac ggtggtgccg aa 22    <210> 8 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> MIP-12Rb    <400> 8 cggggtgtcc gcgcagccgg 20    <210> 9 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-13Rb    <400> 9 ccgatgtagg ccacgcagcc cgg 23    <210> 10 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> MIP-15Rb    <400> 10 tcttggcgtc gggcagcagg 20    <210> 11 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-16Rb    <400> 11 ggggttttcg aggcgaatcc ggc 23    <210> 12 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-18Rb    <400> 12 ttgtccagga acgaggagtt gtt 23    <210> 13 <211> 23 <212> DNA <213> Artificial Sequence    <220> 23 <223> MIP-5b <400> 13 acacatcaag ctcaacggca agg 23    <210> 14 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> YO-intra-SG    <400> 14 cgtcggagcg gtgcagcgga 20    <210> 15 <211> 1204 <212> RNA <213> Mycobacterium intracellulare    <220> <223> Atypical mycobacteria Mycobacterium intracellulare RNA derived from the Pst S1b gene of e    <400> 15 gggagacaaa gggacgcaag ggaccuaaag gaaagagaaa ggaagaaggc cgugaaaguu 60 cguaugagua gguugcuggc gauggccgcg accgcgcccc ugguuuucgc cgcggcugcg 120 ugugguucga acucgcagac cggugcgccg agucagggcg gcggcggccc ggucgcgacg 180 acgccggcgu caucgccggu gacguugucg gagacgggca gcacgcugcu guacccgcug 240 uucaaccugu ggggccccgc cuaucacgac aaguacucga acgucacgau caccacccag 300 ggcaccggcu ccgggaccgg gaucucccag gccgcggccg gggcggucga gaucggcgcc 360 uccgacgccu accuguccga gggcgacaug gccgcgcaca agggacugau gaacaucgcg 420 cuggccauuu ccgcgcagca ggucaacuac aaccugcccg gugucaccga acacaucaag 480 cucaacggca aggugcuggc cggcauguac aacggcuccg ucaagaccug gaacgacccg 540 cagaucgccg ggcugaaccc cggcgugaau cugccugcca caccgguggu uccgcugcac 600 cgcuccgacg gcuccgguga caccuuccug uucacccagu accuguccaa gcaggacccc 660 gacggcuggg gcaaaucgcc cggcuucggc accaccgucg ccuucccgac ggugcccggc 720 gcgcucgggg agaacggcaa cggcggcaug gugaccggcu gcgcggacac cccgggcugc 780 guggccuaca ucggcaucag cuuccucgac caagcgcagg gcaaggggcu cggcgaggcg 840 cagcuggcca acgccuccga caaguaccug cugcccgacg ccaagagcau ucaggccgcg 900 gccgccggau ucgccucgaa aaccccggcg aaccaagcga ucucgcugau caacggcccg 960 gcgccggacg gcuauccgau cgucaacuac gaguacgcca ucgucaacag cggucagaag 1020 gacgccgcca ccgcccagac ccugcaggcc uuccugcacu gggcgaucag cgacggcaac 1080 aacgccucgu uccuggacaa ggugcacuuc cagccgcugc cggccgacgu ggcgaagcug 1140 ucggacgccc agaucgccaa gaucagcggc caguaggggu uuggugaccg acaucgcgga 1200 auuc 1204    <210> 16 <211> 21 <212> DNA <213> Artificial Sequence    <220> <223> MIP-3Rb    <400> 16 ctgggtggtg atcgtgacgt t 21    <210> 17 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> MIP-4Rb    <400> 17 aggcgccgat cgcgaccgcc cc 22    <210> 18 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-7Rb    <400> 18 gttccaggtc ttgacggagc cgt 23    <210> 19 <211> 21 <212> DNA <213> Artificial Sequence    <220> <223> MIP-8Rb <400> 19 gttcagcccg gcgatctgcg g 21    <210> 20 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> MAP-14Rb    <400> 20 cccttgccct gcgcttggtc 20    <210> 21 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> MIP-17Rb    <400> 21 tagttgacga tcggatagcc gt 22    <210> 22 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-19Rb    <400> 22 ggcgtccgac agcttcgcca cgt 23    <210> 23 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> MIP-20Rb    <400> 23 ctgatcttgg cgatctgggc gtc 23    <210> 24 <211> 24 <212> DNA <213> Artificial Sequence    <220> <223> MIP-5Sb    <400> 24 tgatgtgttc ggtgacaccg ggca 24    <210> 25 <211> 24 <212> DNA <213> Artificial Sequence    <220> <223> MIP-6Sb    <400> 25 ttgtacatgc cggccagcac cttg 24    <210> 26 <211> 24 <212> DNA <213> Artificial Sequence    <220> <223> MIP-21Sb    <400> 26  cgttggccag ctgcgcctcg ccga 24    <210> 27 <211> 24 <212> DNA <213> Artificial Sequence    <220> <223> MIP-16Sb    <400> 27 ggggttttcg aggcgaatcc ggcg 24    <210> 28 <211> 51 <212> DNA <213> Artificial Sequence    <220> <223> MIP-5Fb    <400> 28 aattctaata cgactcacta tagggagaac acatcaagct caacggcaag g 51    <210> 29 <211> 51 <212> DNA <213> Artificial Sequence    <220> <223> MIP-6Fb    <400> 29 aattctaata cgactcacta tagggagagt acaacggctc cgtcaagacc t 51    <210> 30 <211> 51 <212> DNA <213> Artificial Sequence    <220> <223> MIP-21Fb    <400> 30 aattctaata cgactcacta tagggagatg gccaacgcct ccgacaagta c 51    <210> 31 <211> 51 <212> DNA <213> Artificial Sequence    <220> <223> MIP-16Fb    <400> 31 aattctaata cgactcacta tagggagaaa ccccggcgaa ccaagcgatc t 51    <210> 32 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> MIP-22Rb    <400> 32 gcggcgtcct tctgaccgct 20

[Brief description of drawings]

FIG. 1 shows Mycobac at 41 ° C. using MIP-1Rb to MIP-20Rb oligonucleotides.
terium intracellulare Pst
It is a urea denaturation 6% polyacrylamide electrophoresis photograph of the sample after performing the binding test to the standard RNA derived from the S1b gene (black and white inversion). The position of the specific band is shown by the arrow. Lanes 1 to 20 are MIPs
It is the result when a binding test was performed using -1Rb to MIP-20Rb, and Lane N is Nega (using a diluent instead of the oligonucleotide). The molecular weight marker is RNA Marker (0.1-1
kb and 0.2-10 kb) were used (lane M1,
M2).

FIG. 2 shows the initial RNA amount 10 ⁴ performed in Example 2.
In the copy / test, the combination (a) of Table 2 ~
It is an electrophoresis photograph (black-and-white inversion) at the time of carrying out RNA amplification reaction using the oligonucleotide of (f). Lane 1 and 2
Is combination (a), lanes 4 and 5 are combination (b), lanes 7 and 8 are combination (c), lane 10
11 is the result of the combination (d), lanes 13 and 14 are the combination (e), and lanes 16 and 17 are the combination (f), and lanes 3, 6, 9, 12, and 18 are negative controls (RNA sample). Instead of using only the diluent). The molecular weight marker is φX174
/ HaeIII digest (Marker 4) was used (lane M). Of these, a specific band could be confirmed when the combination (b) was used.

FIG. 3 is a graph (A) of the fluorescence intensity ratio which increases with the reaction time and the production of RNA in the initial RNA amount of 30 copies / test to 10 ⁵ copies / test carried out in Example 3 and the initial RNA amount. It is a calibration curve (B) obtained between the logarithmic value and the rise time. Initial copy number 10
^Three copies / test RNA were detectable in about 17 minutes of reaction, indicating a correlation between initial RNA amount and rise time.

FIG. 4 shows the results of M.M. intrac
ellulare, M .; avium, M.A. kansas
ii and BCG RNA samples, reaction time and R
It is a graph of the fluorescence intensity ratio which increases with the generation of NA. Sample numbers 1 to 5 are M.I. avium, 6 to 10
Is M. intracellulare, 11 is M. k
ansasii and Posi are M.M. intracellu
lare PstS1b gene standard RNA (SEQ ID NO: 1
5) Shows 10 ⁵ copies / test respectively. The oligonucleotides of the present invention are M. intracellul
It was shown to specifically detect are.

Claims (9)

[Claims] 1. Atypical mycobacteria Mycobacterium
An oligonucleotide for detecting the Pst S1b gene of intracellulare or RNA derived from the gene, comprising: Mycobacterium
An oligonucleotide characterized by a sequence comprising at least 10 bases or more of any of the sequences shown in SEQ ID NOS: 1 to 12, which binds to a specific site of intracellular. 2. The oligonucleotide according to claim 1, wherein the oligonucleotide is an oligonucleotide probe for cleaving the RNA at the specific site by binding to the specific site of the RNA. 3. The oligonucleotide according to claim 1, wherein the oligonucleotide is an oligonucleotide primer for a DNA extension reaction. 4. The oligonucleotide according to claim 1, wherein the oligonucleotide is an oligonucleotide probe partially modified or labeled with a detectable labeling substance. 5. The above-mentioned oligonucleotide is a synthetic oligonucleotide in which a part of its base is converted within a range not impairing the function as an oligonucleotide probe,
The oligonucleotide of claim 1. 6. An atypical mycobacteria Mycob present in a sample
P of the bacterium intracellulare
Using a specific sequence of RNA derived from st S1b gene as a template, RNA-dependent DNA polymerase is used to generate cDNA.
A-synthesized with RNA-DN by ribonuclease H
A Double-stranded RNA can be decomposed to generate single-stranded DNA, and RNA composed of the specific sequence or a sequence complementary to the specific sequence can be transcribed by the DNA-dependent DNA polymerase using the single-stranded DNA as a template. A double-stranded DNA having a different promoter sequence, and the double-stranded DNA produces an RNA transcript in the presence of RNA polymerase,
An RN in which the RNA transcript is subsequently used as a template for cDNA synthesis by the RNA-dependent DNA polymerase.
In the detection method using the A amplification step, Mycobacterium umimintra that is amplified and consists of at least 10 consecutive bases in the sequence shown in SEQ ID NO: 13.
A first primer having a sequence homologous to a part of the RNA sequence derived from the cellulare Pst S1b gene, and Mycobacterium consisting of at least 10 consecutive bases in the sequence shown in SEQ ID NO: 6.
A second primer having a sequence complementary to a part of the sequence of RNA derived from the intracelulare Pst S1b gene (wherein either the first or the second primer is a promoter sequence of RNA polymerase on the 5 ′ side). Is a primer having a sequence to which is added).
um intracellulare Pst S1b
Amplification process of RNA derived from a gene. 7. The RNA amplification step is carried out in the presence of an oligonucleotide capable of specifically binding to an RNA transcript produced by amplification and labeled with an intercalating fluorescent dye, and 7. The step of claim 6 which comprises measuring the change, provided that the labeled oligonucleotide is a different sequence than the first and second primers. 8. The probe is designed to complementarily bind to a sequence of at least a part of an RNA transcript, and has a change in fluorescence property as compared with the case where no complex is formed. The detection method according to claim 7. 9. The detection method according to claim 8, wherein the probe is a sequence consisting of at least 10 consecutive bases in the sequence shown in SEQ ID NO: 14, or its complementary sequence.