JP2001353000A - Detection of mec a gene of methicilinresistant staphyrococcus aureus(mrsa) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a combination of oligonucleotides that can specifically achieve the amplification, detection and identification of the RNA originating from the mec A gene of MRSA with high sensitivity. SOLUTION: In the detection process utilizing the RNA amplification technique, characteristically the oligonucleotide of sequence number 1 is used as the first primer, any of oligonucleotides of the sequence number 2 to 4 is used as the second primer, or No.5 oligonucleotide is used as the first primer and either of No.6 or 1 oligonucleotide, as the second primer or No.8 oligonucleotide is used as the first primer and either of No.6 or 7 oligonucleotide, as the second primer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting methicillin-resistant Staphylococcus aureus in a clinical test.

[0002]

2. Description of the Related Art MRSA is a β-protein produced by staphylococci.
It means a resistant strain of Staphylococcus aureus that is resistant to β-lactamase resistant penicillins such as methicillin, which is stable to lactamase. It is a major pathogen of nosocomial infections,
Strains that show mild resistance to vancomycin, which is regarded as a trump card of therapeutics, have also been detected. in this way,
MRSA is a major medical problem due to the lack of definitive antimicrobial agents. Therefore, accurate and rapid detection in clinical tests is a major issue in diagnosis and treatment.

[0003] Usually, a cell wall synthetic protein PBP (penicillin-Bind) produced by Staphylococcus aureus is used.
ing Protein) is from PBP-1 to PBP-4
However, MRSA also produces a new PBP named PBP-2 '. This PBP is β-
It is a specific protein with a weak binding affinity for lactam antibiotics, and is known to play a central role in resistance. Gene mecA encoding PBP-2 '
Is already known (FEBS Lett. 22
1, 167-171, 1987, etc.). So MR
For detection and identification of SA, a hybridization method using a gene probe specific to the mecA gene has been attempted.

[0004]

As described above, the MRS
Attempts have been made to detect A at the gene level, but the preparation of the sample requires cultivation of a bacterium collected from a patient sample, and thus has a problem with rapidity. Since the detection and identification of MRSA requires a long time for culture and it is difficult to detect a very small amount of the mecA gene present in a sample in a short time, it is a rapid and highly sensitive method in the field of clinical diagnosis. The emergence of detection methods is desired. Furthermore, development of an automatic inspection apparatus is desired in order to make inspection easier.

In order to perform detection with high sensitivity, a gene to be detected and identified and RNA derived from the gene are required.
It is preferable to amplify and detect specific sequences in the following (hereinafter, these are referred to as target nucleic acids).

[0006] As an amplification method when the target nucleic acid is DNA, a polymerase chain reaction (PCR) method is known. In this method, a cycle consisting of thermal denaturation, primer annealing, and extension is repeated in the presence of a set of primers complementary and homologous to both ends of a specific sequence in a target DNA and a thermotolerant DNA polymerase. Amplifying the specific sequence.
At this time, in order to amplify the specific sequence by the PCR method, an oligonucleotide having high specificity to the specific sequence is required. Oligonucleotides with high specificity are required. Furthermore, it is necessary to consider the optimal combination of those oligonucleotides. Therefore, the mecA gene is converted to P using an oligonucleotide having a specific sequence.
Attempts have been made to detect S. aureus on chromosomal DNA by the CR method. However, since the preparation of the sample requires cultivation of bacteria collected from a patient sample, there was a problem in the speed as in the above-described hybridization method. Further, even if the mecA gene on the chromosomal DNA is detected, the expression of PBP-2 'is not actually identified, and therefore, there is a problem in clinical significance. In addition, the PCR method requires a complicated operation of repeatedly increasing and decreasing the temperature rapidly, which is an obstacle to automation.

On the other hand, when the target nucleic acid is RNA, amplification methods include RT-PCR, reverse transcriptase and R-PCR.
The NASBA method, the 3SR method, and the like that amplify the specific sequence by the concerted action of NA polymerase are known. This method uses a double-stranded DNA containing a promoter sequence with a primer containing a promoter sequence, a reverse transcriptase, and ribonuclease H for a specific sequence of a target RNA.
And synthesizing RNA containing the specific sequence by RNA polymerase using the double-stranded DNA as a template, and performing a chain reaction in which the RNA is subsequently used as a template for double-stranded DNA synthesis including a promoter sequence. is there.
The NASBA method and the 3SR method can perform nucleic acid amplification at a constant temperature, and are considered to be suitable methods for automation. However, since these amplification methods carry out the reaction at a relatively low temperature (for example, 41 ° C.), there is a possibility that the target RNA forms an intramolecular structure, inhibits the binding of the primer, and lowers the reaction efficiency. Therefore, before the amplification reaction, the target RNA
Heat denaturation destroys the intramolecular structure of the target RNA and requires an operation to improve the binding efficiency of the primer.

Accordingly, the present invention relates to a cell wall synthesis protein PBP-2 'produced by mesitillin-resistant Staphylococcus aureus.
The purpose of the present invention is to provide a suitable combination of oligonucleotides useful for specifically amplifying RNA derived from the mecA gene encoding at a relatively low temperature (for example, 41 ° C.) and performing detection and identification with high sensitivity. Things.

[0009]

Means for Solving the Problems In order to achieve the above object, the invention of claim 1 of the present application is directed to a method wherein a specific sequence of an RNA derived from the mecA gene of mesitillin-resistant Staphylococcus aureus is used as a template and homologous to the specific sequence. A first primer having a specific sequence and a second primer having a sequence complementary to the specific sequence (wherein one of the first and second primers has a 5 ′ RNA polymerase promoter (With sequence added)
And cDNA by RNA-dependent DNA polymerase
A to form a double-stranded RNA-DNA, and the RNA of the double-stranded RNA-DNA is decomposed by ribonuclease H to produce a single-stranded DNA.
A having a promoter sequence capable of transcribing RNA comprising the RNA sequence or a sequence complementary to the RNA sequence by DNA-dependent DNA polymerase using A as a template
Producing a single-stranded DNA, and the double-stranded DNA producing an RNA transcript in the presence of the RNA polymerase;
In a detection method using an RNA amplification step in which a transcript is subsequently used as a template for the production of single-stranded DNA by the RNA-dependent DNA polymerase, the oligonucleotide of SEQ ID NO: 1 is used as the first primer, and the sequence is used as the second primer. Using any one of the oligonucleotides of Nos. 2 to 4, using the oligonucleotide of SEQ ID NO: 5 as the first primer, using the oligonucleotide of SEQ ID NO: 6 or 7 as the second primer, or using the oligonucleotide of the first primer The oligonucleotide of No. 8 and the oligonucleotide of SEQ ID No. 6 or 7 are used as the second primer.

[0010] The invention of claim 2 of the present application is directed to the invention of claim 1, wherein the first primer is SEQ ID NO: 1, 5 or 8.
Characterized in that it is an oligonucleotide consisting of at least 10 consecutive nucleotides or more in the sequence of The invention of claim 3 of the present application is the invention according to claim 1, wherein the second primer is an oligonucleotide consisting of at least 10 consecutive nucleotides or more of the sequence of SEQ ID NO: 2, 3, 4, 6, or 7. It is characterized by. The invention according to claim 4 of the present application is directed to the invention according to claim 1, wherein the RNA amplification step is performed in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye,
Here, the sequence of the probe is complementary to at least a part of the RNA transcript, and the probe has a fluorescent property that is changed by complementary binding to the RNA transcript as compared with a case where no complex is formed. This is a method for detecting the mecA gene of mesitillin-resistant Staphylococcus aureus, comprising measuring the fluorescence intensity of a reaction solution. Hereinafter, the present invention will be described in detail.

The present invention provides a relatively low temperature and constant temperature (3
5 ° C. to 50 ° C., preferably 41 ° C.) to provide a combination of oligonucleotides for amplifying and detecting RNA derived from the mecA gene of mesicillin-resistant Staphylococcus aureus, ie, RN derived from the mecA gene.
By providing a combination of an oligonucleotide primer for amplification of A and an oligonucleotide probe for detection, a simple, rapid and highly sensitive mec
A method for detecting the A gene and a detection kit are provided for clinical tests and the like.

In one example of the embodiment of the present invention, a second primer (a 3 ′ terminal region of a specific sequence of a target RNA) is used as a template with a specific sequence of an RNA derived from the mecA gene of methicillin-resistant Staphylococcus aureus present in a sample. Is complementary to the RNA, and cDNA is generated from an elongation reaction by RNA-dependent DNA polymerase to produce RNA.
Forming a double-stranded DNA, and then decomposing the RNA-DNA double-stranded RNA with ribonuclease H to produce single-stranded DNA. Thereafter, a first primer (having a sequence homologous to the 5′-terminal region of the target RNA and having a promoter sequence of RNA polymerase added to the 5′-terminal) complementarily binds to the single-stranded DNA, and the DNA-dependent Double-stranded DNA having a promoter capable of transcribing RNA consisting of a sequence homologous to the target RNA sequence is produced by sex DNA polymerase. And the double-stranded DN
In the presence of RNA polymerase, an RNA transcript consisting of a sequence homologous to the target RNA is amplified. The invention of the present application is characterized in that the oligonucleotide of SEQ ID NO: 1, 5, or 8 is used as the first primer, and the oligonucleotide of SEQ ID NO: 2, 3, 4, 6, or 7 is used as the second primer. The first and second primers may be the full length of each SEQ ID NO, but a combination of oligonucleotides consisting of at least 10 consecutive nucleotides or more in each sequence may also be used.

In one embodiment of the present invention, the target RN
A needs to be truncated at the 5 'end of the specific sequence. As a method for cleaving the target RNA in this way, the target RNA is added by adding an oligonucleotide having a sequence complementary to a region overlapping and adjacent to the 5 ′ end of the specific sequence (cleavage oligonucleotide probe). A method of cleaving RNA with ribonuclease H or the like is preferred.
It is desirable to use a 3′-end of the cleavage oligonucleotide that has been treated so as not to function as an oligonucleotide primer, for example, an aminated one.

In one embodiment of the present invention, an oligonucleotide probe (detection oligonucleotide probe) labeled with an intercalating fluorescent dye having a sequence complementary to at least a part of the sequence of the RNA transcript to be amplified. ) It is preferred to carry out this amplification step in the presence. At this time, the fluorescence characteristic changes as compared with the case where the probe does not form a complex due to the complementary binding with the RNA transcript, and the fluorescence intensity of the reaction solution may be measured. Furthermore, when a labeled oligonucleotide probe is allowed to coexist during the amplification step,
It is particularly desirable to make modifications such as adding glycolic acid to the 3 'end of the probe so that the probe does not function as a primer for the extension reaction. As the oligonucleotide probe for detection, use of an oligonucleotide having the sequence described in SEQ ID NO: 3 or 12 can be exemplified.

According to another aspect of the present invention, a second primer (a sequence complementary to a target RNA, which is a sequence complementary to a target RNA, is used as a template) with a specific sequence of an RNA derived from the mecA gene of mesicillin-resistant Staphylococcus aureus present in a sample as a template. RNA polymerase is added to the '-' side, and cDNA is generated from an elongation reaction by RNA-dependent DNA polymerase to produce RNA-DNA.
And then decompose the RNA of the RNA-DNA double-strand with ribonuclease H to form a single-strand D
Generate NA. Thereafter, a first primer (having a sequence homologous to the 5′-terminal region of the target RNA) complementarily binds to the single-stranded DNA, and a DNA-dependent DNA polymerase is used to separate from the sequence homologous to the target RNA sequence. Become R
A double-stranded DNA having a promoter capable of transcribing NA is generated. And the RN consisting of a sequence homologous to the target RNA in the presence of RNA polymerase.
A transcript is amplified. The invention of the present application provides an oligonucleotide of SEQ ID NO: 1, 5 or 8 as a first primer, and SEQ ID NOs: 2, 3, 4,
It is characterized in that an oligonucleotide having a sequence of 6 or 7 is used. The first and second primers may be the full length of each SEQ ID NO, but a combination of oligonucleotides consisting of at least 10 consecutive nucleotides or more in each sequence may also be used.

In one embodiment of the present invention, an oligonucleotide probe labeled with an intercalating fluorescent dye having a sequence complementary to at least a part of the sequence of an RNA transcript to be amplified (oligonucleotide probe for detection) ) It is preferred to carry out this amplification step in the presence. At this time, the fluorescence characteristic changes as compared with the case where the probe does not form a complex due to the complementary binding with the RNA transcript, and the fluorescence intensity of the reaction solution may be measured. Furthermore, when a labeled oligonucleotide probe is allowed to coexist during the amplification step,
It is particularly desirable to make modifications such as adding glycolic acid to the 3 'end of the probe so that the probe does not function as a primer for the extension reaction. As the oligonucleotide probe for detection, use of an oligonucleotide having a sequence complementary to the sequence described in SEQ ID NO: 3 or 12 can be exemplified.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Specific amplification of a target RNA was performed using a combination of oligonucleotide primers according to the present invention. Note that m
ecA-RNA is RNA synthesized and purified by in vitro transcription using a double-stranded DNA containing the base sequence of mecA as a template.

(1) mecA- derived from PBP-2 '
Nucleotide numbers 1 to 2013 of RNA (nucleotide numbers of RNA are as follows: Matsuhashi et al., "FEBS Lett. 221, 167 to 171"
(1987) ").
mer) as a sample, quantified by ultraviolet absorption at 260 nm, and then diluted with an RNA diluent (10 mM Tris-HCl).
(PH 8.0), 0.1 mM EDTA, 0.5 U / μ
l RNase Inhibitor, 5.0 mM D
(TT) and diluted to 1.0 × 10 ⁴ copies / 2.5 μl. In the control test group (Nega), only the diluent was used.

(2) 23.3 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (Gene Amp T
Hin-Walled Reaction Tube
s, manufactured by PerkinElmer), and
2.5 μl of sample was added.

Composition of reaction solution (concentration is final concentration of reaction system after addition of enzyme solution) 60.0 mM Tris-HCl buffer (pH 8.6) 13.0 mM magnesium chloride 90.0 mM potassium chloride 1.0 mM DTT 0 each .25 mM dATP, dCTP, dGTP, dT
TP 3.0 mM ATP, CTP, UTP 2.25 mM GTP 3.6 mM ITP 1.0 μM each of the first primer and the second primer 0.16 μM of the oligonucleotide probe for cleavage (target RNA is used for the first primer) Oligonucleotide for cleavage at the position where it can bind, 3 'end is aminated) 39 U Ribonuclease inhibitor (Takara Shuzo Co., Ltd.) 15.0% distilled water for DMSO volume adjustment Note that the first primer, As the combination of the second primer and the probe for cleavage, among the following numbers,
Any one was used.

1. Oligonucleotides from the 4th to the 15th end of the sequence described as SEQ ID NO: 1 as the first primer (provided that the promoter sequence of T7 polymerase shown in SEQ ID NO: 13 has been added to the 5 'end thereof) 1. the oligonucleotide described in SEQ ID NO: 2 as a second primer, and the oligonucleotide described in SEQ ID NO: 9 as a cleavage probe The first to fourth oligonucleotides from the 5 ′ end to the 15th end of the sequence described as SEQ ID NO: 1 (provided that the promoter sequence of T7 polymerase of SEQ ID NO: 13 was added to the 5 ′ end), An oligonucleotide described in SEQ ID NO: 3 as a primer,
2. The oligonucleotide described in SEQ ID NO: 9 as a cleavage probe The first to fourth oligonucleotides from the 5 ′ end to the 15th end of the sequence described as SEQ ID NO: 1 (provided that the promoter sequence of T7 polymerase of SEQ ID NO: 13 was added to the 5 ′ end), An oligonucleotide described in SEQ ID NO: 4 as a primer,
3. oligonucleotide described in SEQ ID NO: 9 as a cleavage probe The first to fourth oligonucleotides from the 5 'end to the 28th position of the sequence described as SEQ ID NO: 1 (provided with a promoter sequence of T7 polymerase of SEQ ID NO: 13 at the 5' end), An oligonucleotide described in SEQ ID NO: 3 as a primer,
4. The oligonucleotide described in SEQ ID NO: 9 as a cleavage probe The first to fourth oligonucleotides from the 5 'end to the 28th position of the sequence described as SEQ ID NO: 1 (provided with a promoter sequence of T7 polymerase of SEQ ID NO: 13 at the 5' end), An oligonucleotide described in SEQ ID NO: 4 as a primer,
5. Oligonucleotide described in SEQ ID NO: 9 as cleavage probe The first to 25th oligonucleotides at the 5 ′ end of the sequence described as SEQ ID NO: 1 (provided with a T7 polymerase promoter sequence of SEQ ID NO: 13 at the 5 ′ end) as the first primer, An oligonucleotide described in SEQ ID NO: 3 as a primer,
6. Oligonucleotide described in SEQ ID NO: 9 as a cleavage probe The first to 25th oligonucleotides at the 5 ′ end of the sequence described as SEQ ID NO: 1 (provided with a T7 polymerase promoter sequence of SEQ ID NO: 13 at the 5 ′ end) as the first primer, An oligonucleotide described in SEQ ID NO: 4 as a primer,
7. Oligonucleotide described in SEQ ID NO: 9 as cleavage probe The first to fourth oligonucleotides from the 5 'end to the 28th position of the sequence described as SEQ ID NO: 5 (provided with a promoter sequence of T7 polymerase of SEQ ID NO: 13 at the 5'end); An oligonucleotide described in SEQ ID NO: 6 as a primer,
8. Oligonucleotide set forth in SEQ ID NO: 10 as a cleavage probe The first to fourth oligonucleotides from the 5 'end to the 28th position of the sequence described as SEQ ID NO: 5 (provided with a promoter sequence of T7 polymerase of SEQ ID NO: 13 at the 5'end); An oligonucleotide described in SEQ ID NO: 7 as a primer,
9. Oligonucleotide described in SEQ ID NO: 10 as a cleavage probe The first to 25th oligonucleotides at the 5 ′ end of the sequence described as SEQ ID NO: 5 (the T7 polymerase promoter sequence of SEQ ID NO: 13 was added to the 5 ′ end) as the first primer, 10. The oligonucleotide described in SEQ ID NO: 6 as a primer and the oligonucleotide described in SEQ ID NO: 10 as a cleavage probe The first to 25th oligonucleotides at the 5 ′ end of the sequence described as SEQ ID NO: 5 (the T7 polymerase promoter sequence of SEQ ID NO: 13 was added to the 5 ′ end) as the first primer, 11. The oligonucleotide described in SEQ ID NO: 7 as a primer and the oligonucleotide described in SEQ ID NO: 10 as a cleavage probe As the first primer, the 4th to 28th oligonucleotides at the 5 'end of the sequence described as SEQ ID NO: 8 (provided that the promoter sequence of T7 polymerase of SEQ ID NO: 13 was added to the 5' end), 12. The oligonucleotide described in SEQ ID NO: 6 as a primer and the oligonucleotide described in SEQ ID NO: 11 as a cleavage probe As the first primer, the 4th to 28th oligonucleotides at the 5 'end of the sequence described as SEQ ID NO: 8 (provided that the promoter sequence of T7 polymerase of SEQ ID NO: 13 was added to the 5' end), 13. The oligonucleotide described in SEQ ID NO: 7 as a primer and the oligonucleotide described in SEQ ID NO: 11 as a cleavage probe The first to 25th oligonucleotides at the 5 ′ end of the sequence described as SEQ ID NO: 8 (provided with a T7 polymerase promoter sequence of SEQ ID NO: 13 at the 5 ′ end) as the first primer, 14. The oligonucleotide described in SEQ ID NO: 6 as a primer and the oligonucleotide described in SEQ ID NO: 11 as a cleavage probe The first to 25th oligonucleotides at the 5 ′ end of the sequence described as SEQ ID NO: 8 (provided with a T7 polymerase promoter sequence of SEQ ID NO: 13 at the 5 ′ end) as the first primer, Oligonucleotide described in SEQ ID NO: 7 as a primer, oligonucleotide described in SEQ ID NO: 11 as a cleavage probe (3) After keeping the above reaction solution at 41 ° C. for 5 minutes, preliminarily the following composition at 41 ° C. for 2 minutes 4.2 μl of the incubated enzyme solution was added.

Composition of enzyme solution (re-final concentration during reaction) 1.7% sorbitol 8 units AMV reverse transcriptase (Takara Shuzo Co., Ltd.) 142 units T7 RNA polymerase (GIB)
(Manufactured by CO) 3 μg bovine serum albumin distilled water for adjusting the volume (4) Subsequently, the PCR tube was kept at 41 ° C. for 90 minutes, and then the specific amplification product was analyzed by electrophoresis using a 4% agarose gel.

(5) For staining after electrophoresis, a commercially available staining solution (trade name: SYBR Green II, Takara Shuzo Co., Ltd.)
Was used.

FIG. 1 shows the result of electrophoresis (a black and white inverted photograph).
It was shown to. In any combination, mecA-
In the system to which RNA was added, specific RNA amplification products (arrows) were obtained. This indicates that the combination of these oligonucleotide primers is useful for amplification and detection of RNA derived from the mecA gene of mesitillin-resistant Staphylococcus aureus.

Example 2 It was confirmed that specific detection of target RNA, mecA-RNA, was possible using the combination of oligonucleotide primers according to the present invention.

(1) mecA- derived from PBP-2 '
Nucleotide numbers 1 to 2013 of RNA (nucleotide numbers of RNA are as follows: Matsuhashi et al., "FEBS Lett. 221, 167 to 171"
(1987) ").
mer) as a sample, quantified by ultraviolet absorption at 260 nm, and then diluted with an RNA diluent (10 mM Tris-HCl).
(PH 8.0), 0.1 mM EDTA, 0.5 U / μ
l RNase Inhibitor, 5.0 mM D
Using TT), the dilution was made to be 1.0 × 10 ⁶ copies / 2.5 μl or 1.0 × 10 ⁴ copies / 2.5 μl. In the control test group (Nega), only the diluent was used.

(2) 23.3 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (Gene Amp T
Hin-Walled Reaction Tube
s, manufactured by PerkinElmer Co., Ltd.)
2.5 μl of A sample (mecA-RNA) was added.

Composition of reaction solution (concentration is final concentration of reaction system after addition of enzyme solution) 60.0 mM Tris-HCl buffer (pH 8.6) 13.0 mM magnesium chloride 90.0 mM potassium chloride 1.0 mM DTT 0 each .25 mM dATP, dCTP, dGTP, dT
TP 3.0 mM ATP, CTP, UTP 2.25 mM GTP 3.6 mM ITP 1.0 μM first oligonucleotide primer 1.0 μM second oligonucleotide primer 0.16 μM cleavage oligonucleotide probe (target RNA For cleaving at the position where the first primer can bind, 3 'end is aminated) 25.0 nM intercalating fluorescent dye (FIG. 2)
Oligonucleotide probe for detection (MR
SH-YO, the 3 'end is modified with glycolic acid).

39U Ribonuclease inhibitor (Takara Shuzo Co., Ltd.) 15.0% DMSO Distilled water for adjusting the volume Note that any one of the following numbers was used as a combination of primer and probe.

1. An oligonucleotide from the 4th to the 15th end of the sequence described as SEQ ID NO: 1 as the first primer (provided that the promoter sequence of T7 polymerase shown in SEQ ID NO: 13 has been added to its 5 'end); 1. The oligonucleotide described in SEQ ID NO: 2 as the second primer, the oligonucleotide sequence described in SEQ ID NO. 9 as the cleavage probe, and the oligonucleotide sequence described in SEQ ID NO. 3 as the detection probe The first to 25th oligonucleotides at the 5 ′ end of the sequence described as SEQ ID NO: 8 (provided with a T7 polymerase promoter sequence of SEQ ID NO: 13 at the 5 ′ end) as the first primer, An oligonucleotide described in SEQ ID NO: 7 as a primer,
The oligonucleotide sequence described in SEQ ID NO: 11 as a cleavage probe and the oligonucleotide sequence described in SEQ ID NO: 12 as a detection probe (3) After the above reaction solution was incubated at 41 ° C. for 5 minutes, 3. Enzyme solution pre-incubated at 41 ° C. for 2 minutes
2 μl was added.

Composition of enzyme solution (re-final concentration during reaction) 1.7% sorbitol 8 units AMV reverse transcriptase (Takara Shuzo Co., Ltd.) 142 units T7 RNA polymerase (GIB)
3 μg distilled water for bovine serum albumin volume adjustment (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function capable of directly measuring a PCR tube, keeping the temperature at 41 ° C., at an excitation wavelength of 470 nm and a fluorescence wavelength of 510 nm, The fluorescence intensity of the reaction solution was measured over time. FIG. 3 shows the time-dependent change of the fluorescence intensity ratio of the sample (the fluorescence intensity value at a predetermined time / the fluorescence intensity value of the background), with the time at the time of addition of the enzyme being 0 minute. The RNA sample concentration was 10 ⁶ copies / 30 μl for combination 1 and 10 ⁴ copies / 30 μl for combination 2.
l.

In the system in which mecA-RNA was added to the target RNA, specific fluorescence sensitization was obtained. From the above, the combination of oligonucleotides of the present invention is me
It was shown that cA gene-derived RNA can be specifically amplified and detected.

Example 3 Detection of target RNA at various initial copy numbers was performed using a combination of oligonucleotide primers according to the present invention.

(1) mecA- derived from PBP-2 '
Nucleotide numbers 1 to 2013 of RNA (nucleotide numbers of RNA are as follows: Matsuhashi et al., "FEBS Lett. 221, 167 to 171"
(1987) ").
mer) as a sample, quantified by ultraviolet absorption at 260 nm, and then diluted with an RNA diluent (10 mM Tris-HCl).
(PH 8.0), 0.1 mM EDTA, 0.5 U / μ
l RNase Inhibitor, 5.0 mM D
TT) from 1.0 × 10 ⁴ copies / 2.5 μl to 1
It was diluted to be 0 copies / 2.5 μl. In the control test group (Nega), only the diluent was used.

(2) 23.3 μl of the reaction solution having the following composition was placed in a 0.5 ml PCR tube (Gene Amp T
Hin-Walled Reaction Tube
s, manufactured by PerkinElmer Co., Ltd.)
2.5 μl of A sample (mecA-RNA) was added.

Composition of reaction solution (concentration is final concentration of reaction system after addition of enzyme solution) 60.0 mM Tris-HCl buffer (pH 8.6) 13.0 mM magnesium chloride 90.0 mM potassium chloride 1.0 mM DTT 0 each .25 mM dATP, dCTP, dGTP, dT
TP 3.0 mM ATP, CTP, UTP 2.25 mM GTP 3.6 mM ITP 1.0 μM first oligonucleotide primer (28 from the 4 ′ 5 ′ end of the sequence described as SEQ ID NO: 8)
Oligonucleotides up to the th. However, a T7 polymerase promoter sequence of SEQ ID NO: 13 was added to the 5 'end. 1.0 μM second oligonucleotide primer (oligomers from the 1st to 18th 5 ′ end of the sequence described in SEQ ID NO: 6) Nucleotide) 0.16 μM cleavage oligonucleotide probe (SEQ ID NO: 11, oligonucleotide for cleaving target RNA at a position where the first primer can bind, 3 ′ end is aminated) 25.0 nM internucleotide Calorigenic fluorescent dye (Figure 2)
Oligonucleotide probe for detection (MR
SA-YO), the oligonucleotide sequence of which is SEQ ID NO: 12, and the 3 'end modified with glycolic acid.) 39 U Ribonuclease inhibitor (Takara Shuzo) 15.0% distilled water for DMSO volume adjustment (3) After keeping the above reaction solution at 41 ° C. for 4 minutes, an enzyme solution having the following composition and previously kept at 41 ° C. for 2 minutes.
2 μl was added.

Composition of enzyme solution (re-final concentration during reaction) 1.7% sorbitol 8 units AMV reverse transcriptase (Takara Shuzo Co., Ltd.) 142 units T7 RNA polymerase (GIB)
3 μg distilled water for bovine serum albumin volume adjustment (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function capable of directly measuring a PCR tube, keeping the temperature at 41 ° C., at an excitation wavelength of 470 nm and a fluorescence wavelength of 510 nm, The fluorescence intensity of the reaction solution was measured over time. FIG. 4 shows the time-dependent change of the fluorescence intensity ratio of the sample (the fluorescence intensity value at a predetermined time / the fluorescence intensity value of the background), with the time at the time of addition of the enzyme being 0 minute. RNA sample concentration from 10 copies / 30 μl to 1
0 of ⁴ copies / 30μl.

FIG. 4 shows that a fluorescence profile depending on the initial concentration of the target RNA was obtained,
It was suggested that it is possible to estimate the amount of RNA derived from the ecA gene.

[0040]

As described above, according to the present invention,
Even under relatively low temperature and a constant temperature (35 to 50 ° C, preferably 41 ° C), RNA in a sample forms an intramolecular structure and may inhibit the binding of primers and probes.
RN derived from mecA gene encoding BP-2 '
It is useful as a combination of an oligonucleotide primer and an oligonucleotide probe for specifically binding to A, rapidly amplifying a target RNA, and detecting it.

In addition to the above, the combination of oligonucleotides of the present invention is not limited to mecA-RNA,
In order to detect cDNA obtained by reverse transcription of A,
Sequences complementary to the oligonucleotides described above are also useful.

The base length of the oligonucleotide in the combination of the present invention is not limited to the specifically described length, but includes an oligonucleotide consisting of at least 10 consecutive bases or more in these sequences. This is 10 me under relatively low temperature (preferably 41 ° C.) conditions to ensure the specificity of the primer or probe to the target nucleic acid.
It is clear from the fact that a base sequence of about r is sufficient.

[0043]

[Sequence list] SEQUENCE LISTING <110> TOSOH Corporation <120> Method for detecting mecA gene of mesitillin-resistant Staphylococcus aureus <130> PA211-0199 <160> 13 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 aaattgggta caagatgata ccttcgtt 28 <210> 2 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gaaggtgtgc ttac 14 <210 > 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ttttcttttt ctctattaat g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gttagttgaa tatctttgcc 20 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 aaagaaaaaa gatggcaaag atattcaa 28 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ttctttttta tcttcggtta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400 > 7 tcattgctgt taatattttt 20 <210> 8 <211> 28 <212> D NA <213> Artificial Sequence <220> <223> Primer <400> 8 caactaacta ttgatgctaa agttcaaa 28 <210> 9 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 9 cccaattttg atccatttgt tgttgatata gtcttcaga 39 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 10 ttttcttttt ctctattaat gtatgtgcga ttgtattgc 39 <210> 11 <211> 39 <212> DNA < 213> Artificial Sequence <220> <223> Probe <400> 11 gttagttgaa tatctttgcc atcttttttc tttttctct 39 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 tgtttgaggg tggatagcag 20 <210> 13 <211> 28 <212> DNA <213> T7 phage <220> <221> promoter <223> Promoter sequence of T7 polymerase <400> 13 aattctaata cgactcacta tagggaga 28

[Brief description of the drawings]

FIG. 1 shows the results of performing RNA amplification reactions in Example 1 using various combinations of primers. In the figure, P indicates the case where the initial RNA amount of 10 ⁵ copies / 30 μl was used as the RNA sample, and N indicates the case where only the diluent was used instead of the RNA sample. Lane M is a molecular weight marker, and 1 to 15 are primer / probe combination numbers shown in Example 1.

FIG. 2 is a chemical structure of an oligonucleotide intercalating fluorescent dye labeled with an intercalating fluorescent dye used in Example 2. B _{1 to} B ₃ represent nucleobases.

FIG. 3 shows a reaction time and a time of a fluorescence increase rate in Example 2. 1 and 2 are examples 2
Is the combination number of the primer / probe indicated by. In the figure, P indicates the case where the initial RNA amount of 10 ⁶ copies / 30 μl was used as the RNA sample in combination 1.
In combination 2, the initial RNA amount is 10 ⁴ copies / 30 μl
Is used as an RNA sample. N is the case where only the diluent was used instead of the RNA sample.

FIG. 4 shows the initial RNA amount of 10 ⁴ performed in Example 3.
From 10 copies / 30 μl to 10 copies / 30 μl,
It is a graph of the fluorescence increase rate which increases with reaction time and RNA production. Nega used only diluent instead of RNA samples.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/58 C12N 15/00 ZNAA F-term (Reference) 2G045 AA25 AA28 AA35 CB21 DA12 DA13 DA14 DA77 FB01 FB02 FB07 FB12 GC15 4B024 AA13 CA01 CA09 CA11 HA14 4B063 QA01 QA07 QA18 QA19 QQ06 QQ52 QR07 QR08 QR32 QR35 QR39 QR42 QR56 QS24 QS34 QX02

Claims (4)

[Claims] 1. A methicillin-resistant Staphylococcus aureus (Methicillin-Resistant) present in a sample.
A first primer having a sequence homologous to the specific sequence and a first primer having a sequence complementary to the specific sequence, using a specific sequence of RNA derived from the mecA gene which is a genetic element of Staphylococcus aureus (hereinafter, MRSA) as a template. Two primers (where one of the first and second primers is
RNA-D-DNA polymerase is used to generate cDNA by using an RNA-dependent DNA polymerase promoter sequence on the 5 ′ side.
NA double-stranded, and RNA is formed by ribonuclease H
-DNA can be decomposed to generate double-stranded RNA to generate single-stranded DNA, and the single-stranded DNA can be used as a template to transcribe RNA comprising the RNA sequence or a sequence complementary to the RNA sequence by DNA-dependent DNA polymerase. Producing a double-stranded DNA having a unique promoter sequence, and the double-stranded DNA produces an RNA transcript in the presence of the RNA polymerase, and the RNA transcript is subsequently produced by the RNA-dependent DNA polymerase. In the detection method using an RNA amplification step as a template for the method, the oligonucleotide of SEQ ID NO: 1 is used as the first primer, and the oligonucleotide of any of SEQ ID NOs: 2 to 4 is used as the second primer, The oligonucleotide of SEQ ID NO: 5 as the primer of SEQ ID NO: 5 as the second primer Or 7 using oligonucleotide or oligonucleotide of SEQ ID NO: 8 as the first primer, which comprises using the second primer as SEQ ID NO: 6 or 7 oligonucleotides, detection methods. 2. The method according to claim 1, wherein the first primer is an oligonucleotide consisting of at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 1, 5 or 8. 3. The method according to claim 2, wherein the second primer has SEQ ID NO: 2, 3,
2. The detection method according to claim 1, wherein the oligonucleotide is an oligonucleotide consisting of at least 10 consecutive nucleotides of at least 4, 6, or 7 sequences. 4. The method according to claim 1, wherein the RNA amplification step is performed in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye.
Here, the sequence of the probe is complementary to at least a part of the RNA transcript, and the probe has a fluorescent property that is changed by complementary binding to the RNA transcript as compared with a case where no complex is formed. A method for detecting mesitillin-resistant Staphylococcus aureus, comprising measuring the fluorescence intensity of a reaction solution.