Primer pair for amplifying mecA gene, mecA gene detection kit, and mecA gene detection method
Technical field
[0001]
　TECHNICAL FIELD The present invention relates to a primer pair, a kit and a detection method for detecting the mecA gene, which is a specific genetic element of methicillin resistance, simply, rapidly and with high sensitivity.
Background technology
[0002]
　Methicillin-resistant Staphylococcus aureus (hereinafter abbreviated as "MRSA") is a major causative agent of nosocomial infections worldwide, and is regarded as the most clinically important drug-resistant bacterium in many countries. Has been done. The gene involved in methicillin resistance of MRSA, mecA, encodes PBP (Penicillin Binding Protein)-2′ (also called PBP-2a). Staphylococcus aureus normally produces four major PBPs. PBP is a protein involved in cell wall peptidoglycan synthesis and has transpeptidase activity. β-lactam antibiotics act by binding to the active site of PBP, inhibiting transpeptidase activity, and inhibiting peptidoglycan synthesis. However, PBP-2′ avoids the peptidoglycan synthesis inhibitory effect by reducing the affinity with β-lactam antibiotics (Non-patent Document 1).
　In Staphylococcus aureus and coagulase-negative staphylococci, the presence or absence of methicillin resistance is very important information in diagnosing infected patients and deciding a therapeutic course. Since Staphylococcus aureus is highly pathogenic, it is regarded as important compared with other coagulase-negative staphylococci, but staphylococcus aureus and coagulase-negative staphylococcus that have acquired methicillin resistance are also infected with a compromised host. It's a big problem. Therefore, as a measure against nosocomial infection with methicillin-resistant staphylococcus, rapid detection of methicillin resistance has been desired for prompt diagnosis and treatment.
　As a method for testing methicillin resistance, methods such as drug sensitivity test, immunoassay, and detection by PCR (polymerase chain reaction) have been carried out (Non-patent document 2).
　In drug susceptibility testing, blood collected from patients must be cultured for at least one day to increase staphylococci. Further, since the immunoassay also requires a step of culturing Staphylococcus, it takes more than one day until the result of the methicillin resistance test is obtained.
　Compared to these, nucleic acid amplification tests such as PCR have a very high sensitivity and therefore do not require the process of culture, and it is possible to carry out the test directly from the blood of the patient. Therefore, the test result can be submitted within a few hours after the patient blood sample arrives (Patent Document 1).
Advanced technical literature
Patent literature
[0003]
Patent Document 1: International Publication No. 2007/097323
[0004]
Unlicensed Literature 1: Journal of Bacteriology, May 1980 p. 275 Unlicensed
Literature 2: Journal of Clinical Microbiology, July 1992, p. 1685
Summary of the invention
Problems to be Solved by the Invention
[0005]
　Although several methods for detecting a methicillin resistance gene by nucleic acid amplification such as PCR have been already reported, many reports do not specify specific detection sensitivity. Even if the detection sensitivity is specified, there is only a report that it has not achieved the detection sensitivity sufficient for clinical use (Non-Patent Document 2).
　On the other hand, the detection of the methicillin resistance gene has a great influence on the diagnosis and treatment policy of the patient. Therefore, it is an urgent clinical need to develop a method for detecting the methicillin resistance gene rapidly, accurately and surely. Therefore, an object of the present invention is to detect a methicillin resistance gene rapidly, accurately and surely. It is an object of the present invention to provide a primer pair for realizing particularly sensitive detection of methicillin resistance gene.
Means for solving the problem
[0006]
　The present invention is a method for rapidly identifying a methicillin-resistant gene directly from a patient sample by using the nucleic acid amplification method and surely detecting it with higher detection sensitivity than the conventional method. The present inventors focused on the PCR method with the aim of establishing a highly sensitive and rapid method for detecting a methicillin resistance gene. In particular, the primers used in the PCR method for detecting the methicillin resistance gene with high sensitivity were examined.
　The following conditions were used in designing the primer.
　The size of the DNA fragment to be amplified was increased within the range where the amplification efficiency did not decrease. Specifically, the size of the DNA fragment was set to 150 bp or more and 700 bp or less. By increasing the size of the DNA fragment to be amplified, the fluorescence intensity of the amplification curve during the PCR reaction was increased, and the height of the peak obtained by Melting analysis was also increased.
　The sequence of the mecA homolog is confirmed, and the base sequence of the primer pair is designed only in the part having high conservation. Specifically, a primer containing a low-conservative sequence within 5 bases from the 3'end of the primer was excluded, and the base sequence was modified to obtain a highly-conservative sequence.
　Based on the concept described above, 32 specific Fwd and Rev primers were respectively prepared, and by combining them, 313 primer pairs considered to be promising were prepared, and by examining the optimal combination from them, A primer pair capable of achieving higher detection sensitivity as compared with the prior application of Patent Document 1 was selected.
　That is, the primer pair according to the present invention is a primer pair
　for detecting the mecA gene (methicillin resistance gene),
　It is characterized in that the primer pair is one primer pair selected from the group consisting of the following (a) to (c).
(A) a combination of SEQ ID NO: 3 and SEQ ID NO: 7, a combination of SEQ ID NO: 2 and SEQ ID NO: 9, a combination of SEQ ID NO: 1 and SEQ ID NO: 8, a combination of SEQ ID NO: 1 and SEQ ID NO: 9, a SEQ ID NO: 4 and an SEQ ID NO: 11 A primer pair consisting of the combination of SEQ ID NO:5 and SEQ ID NO:12, the combination of SEQ ID NO:6 and SEQ ID NO:10 or the combination of SEQ ID NO:6 and SEQ ID NO:12,
(b) In (a) above, one or both of A primer pair in which 1 to 2 bases are added, deleted or substituted so long as a part of the base sequence of the primer is not impaired,
(c) corresponding to the base sequence of (a) or (b) above A primer pair consisting of complementary sequences.
[0007]
　The mecA gene detection kit according to the present invention is characterized by containing at least one primer pair selected from the group consisting of (a) to (c) above.
　The method for detecting mecA in a sample according to the present invention 　comprises a PCR step of
　performing PCR using DNA prepared from the sample and a primer pair for amplifying the DNA, and an
amplification product obtained by the PCR step. of the detection of the mecA gene, or by analysis of the amplification product, and performing detection of mecA gene in the said sample
has,
　the primer pair used in the PCR process, the (a) ~ (
It is characterized in that it is at least one primer pair selected from the group consisting of c) .
The invention's effect
[0008]
　ADVANTAGE OF THE INVENTION According to this invention, the primer pair for PCR and the mecA gene detection kit for highly sensitive mecA gene detection, and the detection method of a mecA gene can be provided. Therefore, according to the present invention, it is possible to detect a trace amount of mecA gene in a sample in the medical field, the food field, etc. with higher sensitivity and accuracy than in the conventional test kit.
Brief description of the drawings
[0009]
FIG. 1 is a diagram showing a comparison of measurement results of a methicillin-resistant strain and a methicillin-sensitive strain. FIG. 1(a) shows the measurement results of amplification curve and Tm value when a sample from a methicillin-resistant strain was used, and FIG. 1(b) shows the amplification curve and Tm value of a sample from a methicillin-sensitive strain. The measurement results are shown.
MODE FOR CARRYING OUT THE INVENTION
[0010]
　 In the
　present invention, the term “primer” refers to a DNA serving as a starting point during DNA replication in the PCR method. In the present specification, unless otherwise specified, a primer refers to a DNA primer.
　The point of the present invention lies in a specific primer pair that can enjoy the effect of improving detection sensitivity.
　The pair of the primer consisting of the nucleotide sequence of SEQ ID NO: 45 and the primer consisting of the nucleotide sequence of SEQ ID NO: 46 described in Patent Document 1 has a DNA sequence that hybridizes to the sequence in the structural gene of the methicillin resistance gene (mecA). The base sequence of SEQ ID NO: 45 and the base sequence of SEQ ID NO: 46 described in Patent Document 1 are referred to as SEQ ID NOS: 13 and 14 in the present specification, respectively.
　However, even with the above-mentioned primer pair, the detection method using PCR did not have sufficient detection sensitivity, and it was not possible to detect low concentrations of mecA (Example 5 and Table 2 described later). This may show false negatives due to insufficient detection sensitivity when analyzing a sample containing low concentrations of MRSA.
[0011]
　The structural gene of the mecA gene is composed of approximately 2100 bp, and PCR should be performed using each of the primer pairs included in the following group a designed by the present inventors to hybridize within the structural gene. It was found that the mecA gene can be detected with even higher sensitivity.
Group (a):
(a1) A primer pair comprising a combination of the primer consisting of SEQ ID NO : 3 and the primer consisting of SEQ ID NO : 7.
(A2) A primer pair comprising a combination of the primer consisting of SEQ ID NO:2 and the primer consisting of SEQ ID NO:9.
(A3) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 1 and the primer consisting of SEQ ID NO: 8.
(A4) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 1 and the primer consisting of SEQ ID NO: 9.
(A5) A primer pair comprising a combination of the primer consisting of SEQ ID NO:4 and the primer consisting of SEQ ID NO:11.
(A6) A primer pair comprising a combination of the primer consisting of SEQ ID NO:5 and the primer consisting of SEQ ID NO:12.
(A7) A primer pair comprising a combination of the primer consisting of SEQ ID NO:6 and the primer consisting of SEQ ID NO:10.
(A8) A primer pair comprising a combination of the primer consisting of SEQ ID NO:6 and the primer consisting of SEQ ID NO:12.
[0012]
　A modified primer composed of a modified base sequence obtained by converting a part of the bases in the base sequence of the DNA fragment (oligonucleotide) constituting each of the above-mentioned primers can also be applied as long as the effect of the present invention can be enjoyed. .. In principle, a modified primer is considered to be equivalent to each primer if it can hybridize with the complementary DNA strand of each primer. Furthermore, this modified primer considered equivalent has a function as a primer that hybridizes with the complementary DNA strand of the original primer, and one or two bases are added or deleted in the base sequence of the original primer. A primer having a modified or deleted nucleotide sequence is preferred.
　Therefore, the primer pairs included in the following group (b) can also be used as the mecA gene detection primer pairs.
Group (b):
(b1-1) A primer pair comprising a modified primer consisting of the modified nucleotide sequence of SEQ ID NO : 3 and a primer consisting of SEQ ID NO: 7.
(B1-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO:3 and the modified primer consisting of the modified base sequence of SEQ ID NO:7.
(B1-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 3 and a modified primer consisting of the modified base sequence of SEQ ID NO: 7.
(B2-1) A primer pair comprising a modified primer consisting of the modified nucleotide sequence of SEQ ID NO:2 and a primer consisting of SEQ ID NO:9.
(B2-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO:2 and the modified primer consisting of the modified base sequence of SEQ ID NO:9.
(B2-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 2 and a modified primer consisting of the modified base sequence of SEQ ID NO: 9.
(B3-1) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 1 and a primer consisting of the base sequence of SEQ ID NO: 8.
(B3-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 1 and the modified primer consisting of the modified base sequence of SEQ ID NO: 8.
(B3-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 1 and a modified primer consisting of the modified base sequence of SEQ ID NO: 8.
(B4-1) A primer pair comprising a combination of a modified primer having the modified nucleotide sequence of SEQ ID NO: 1 and a primer having SEQ ID NO: 9.
(B4-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 1 and the modified primer consisting of the modified base sequence of SEQ ID NO: 9.
(B4-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 1 and a modified primer consisting of the modified base sequence of SEQ ID NO: 9.
(B5-1) A primer pair consisting of a combination of a modified primer consisting of the modified nucleotide sequence of SEQ ID NO: 4 and a primer consisting of SEQ ID NO: 11.
(B5-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 4 and the modified primer consisting of the modified base sequence of SEQ ID NO: 11.
(B5-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 4 and a modified primer consisting of the modified base sequence of SEQ ID NO: 11.
(B6-1) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 5 and a primer consisting of SEQ ID NO: 12.
(B6-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 5 and the modified primer consisting of the modified base sequence of SEQ ID NO: 12.
(B6-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 5 and a modified primer consisting of the modified base sequence of SEQ ID NO: 12.
(B7-1) A primer pair comprising a combination of a modified primer consisting of the modified nucleotide sequence of SEQ ID NO: 6 and a primer consisting of SEQ ID NO: 10.
(B7-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 6 and the modified primer consisting of the modified base sequence of SEQ ID NO: 10.
(B7-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 6 and a modified primer consisting of the modified base sequence of SEQ ID NO: 10.
(B8-1) A primer pair consisting of a combination of a modified primer consisting of the modified nucleotide sequence of SEQ ID NO:6 and a primer consisting of SEQ ID NO:12.
(B8-2) A primer pair comprising a combination of the primer consisting of SEQ ID NO: 6 and the modified primer consisting of the modified base sequence of SEQ ID NO: 12.
(B8-3) A primer pair comprising a modified primer consisting of the modified base sequence of SEQ ID NO: 6 and a modified primer consisting of the modified base sequence of SEQ ID NO: 12.
[0013]
　Furthermore, a complementary primer pair consisting of a complementary sequence of the primer that constitutes each of the above-mentioned primer pairs can also be used as a mecA gene detection primer pair. That is, the primer pairs included in the following group (c) can also be used as the mecA gene detection primer pairs.
Group (c):
(c1-1) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO : 3 and a primer consisting of the complementary sequence of SEQ ID NO : 7.
(C1-2) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified nucleotide sequence of SEQ ID NO: 3 and a primer consisting of the complementary sequence of SEQ ID NO: 7.
(C1-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:3 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO:7.
(C1-4) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 3 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 7.
(C2-1) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:2 and a primer consisting of the complementary sequence of SEQ ID NO:9.
(C2-2) A primer pair consisting of a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 2 and a primer consisting of the complementary sequence of SEQ ID NO: 9.
(C2-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:2 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO:9.
(C2-4) A primer pair comprising a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 2 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 9.
(C3-1) A primer pair consisting of a combination of a primer consisting of the complementary sequence of SEQ ID NO: 1 and a primer consisting of the complementary sequence of SEQ ID NO: 8.
(C3-2) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 1 and a primer consisting of the complementary sequence of the base sequence of SEQ ID NO: 8.
(C3-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO: 1 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 8.
(C3-4) A primer pair comprising a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 1 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 8.
(C4-1) A primer pair consisting of a combination of a primer consisting of the complementary sequence of SEQ ID NO: 1 and a primer consisting of the complementary sequence of SEQ ID NO: 9.
(C4-2) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 1 and a primer consisting of the complementary sequence of SEQ ID NO: 9.
(C4-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO: 1 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 9.
(C4-4) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 1 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 9.
(C5-1) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO: 4 and a primer consisting of the complementary sequence of SEQ ID NO: 11.
(C5-2) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified nucleotide sequence of SEQ ID NO: 4 and a primer consisting of the complementary sequence of SEQ ID NO: 11.
(C5-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO: 4 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 11.
(C5-4) A primer pair composed of a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 4 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 11.
(C6-1) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:5 and a primer consisting of the complementary sequence of SEQ ID NO:12.
(C6-2) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 5 and a primer consisting of the complementary sequence of SEQ ID NO: 12.
(C6-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:5 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO:12.
(C6-4) A primer pair comprising a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 5 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 12.
(C7-1) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:6 and a primer consisting of the complementary sequence of SEQ ID NO:10.
(C7-2) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 6 and a primer consisting of the complementary sequence of SEQ ID NO: 10.
(C7-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:6 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO:10.
(C7-4) A primer pair comprising a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 6 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 10.
(C8-1) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:6 and a primer consisting of the complementary sequence of SEQ ID NO:12.
(C8-2) A primer pair comprising a combination of a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 6 and a primer consisting of the complementary sequence of SEQ ID NO: 12.
(C8-3) A primer pair comprising a combination of a primer consisting of the complementary sequence of SEQ ID NO:6 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO:12.
(C8-4) A primer pair comprising a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 6 and a modified primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 12.
[0014]
　Among these primer pairs, the primer pair (a7) and the primer pairs (b7-1) to (b7-3) and (c7-1) to (c7-4) are preferable.
　The primer pairs included in the above groups (a) to (c) are common in that they hybridize specifically within the structural gene of the mecA gene to amplify a DNA fragment specific to the mecA gene. It has the function of.
[0015]
　It should be noted that the primer pair of the present invention was first discovered after confirming the presence or absence of the effect based on the combination of various primers in a considerable amount, and cannot be achieved at a mere confirmation level of the effect. In addition, it is added that the result of the sequence search confirms that the primer pair for detecting the mecA gene is a novel combination.
　The present invention does not prevent the combined use of a plurality of known primers for bacteria, fungi, and viruses in addition to the primer pair of the present invention.
　The Primer concentration in PCR is not particularly limited, but is generally in the range of 0.05 μM to 1.0 μM, and is appropriately set according to the examination.
[0016]

　The enzyme for PCR used in the present invention is preferably a thermostable DNA polymerase derived from an organism, more preferably methane bacterium, thermophilic acidophilic bacterium, thermophilic bacterium, It is derived from prokaryotes such as hyperthermophiles.
　Specific examples of the thermostable DNA polymerase used in the present invention include Thermus aquaticus, Thermus thermophilus, Bacillus stearothermophilus, and Thermococcus gossypii. Thermococcus gorgonarius), Thermococcus-kodakaraensis KOD1 (Thermococcus kodakaraensis KOD1), Pyrococcus Woesei (Pyrococcus woesei), Pyrococcus Furioshisu (Pyrococcus furiosus), Earopairamu-pernix (Aeropyrum pernix), Aku Ifekkusu-Aeorikasu (Aquifex aerobicus, Sulfolobus tokodaii, Pyrolobus fumarii, or Methananopyrus kandleri.
　Further, the thermostable DNA polymerase may be a thermostable DNA polymerase artificially synthesized by genetic engineering.
[0017]
As
　the reagents other than the primer pair used for PCR analysis in the present invention, known reagents can be used in known combinations. Reagents necessary for the measurement include PCR enzyme, pH buffer, dNTP, Mg 2+ source, sterile water and the like. Further, in real-time PCR analysis, a labeling substance such as a fluorescent dye is required in addition to the above.
　The pH buffer is used as a sample to be subjected to PCR analysis in order to adjust the pH to 7.0 to 10.0 (more preferably pH 8.0 to 9.0). Specific examples include Tris hydrochloric acid buffer. For example, in the case of Tris hydrochloric acid buffer, it is used at 5 mM to 100 mM.
　dNTP is a nucleoside source for DNA amplification by PCR, and four kinds of dATP, dCTP, dGTP and dTTP are required. In addition, dNTP is chemically modified for use in the hot start method, for example, TriLink BioTechnologies, Inc. CleanAmp ™　dNTPs manufactured by Sigma may be used. The amount of each of dATP, dCTP, dGTP and dTTP used is about 0.1 to 0.2 mM.
　Mg 2+ is required for DNA amplification by PCR . Examples of the Mg 2+ source include MgCl 2 and MgSO 4 . MgCl 2 is preferred The amount used is in the range of 0.5 mM to 5.0 mM and is appropriately used depending on the study.
　The fluorescent dye is used for the purpose of detecting a DNA amplification product by real-time PCR and further for measuring the Tm value of the amplified DNA fragment, and examples thereof include a method using an intercalator having a labeling function. Examples of intercalators include ethidium bromide, Cyber ​​Green I (SYBR Green I), Resolight (manufactured by Roche), EvaGreen (manufactured by Biotium), and BOXTO (manufactured by taata biocenter). Preferred intercalators are EvaGreen and Resolight. The usage amount is in accordance with the manufacturer's recommendation of the fluorescent dye used.
　In addition, the kit may contain bacterial genomic DNA used as a positive control for PCR and sterilized water used as a negative control.
[0018]
　As the PCR analysis/measurement container (eg, analysis/measurement tube) used in the present invention, a container having a shape and a structure suitable for the measuring instrument can be selected and used. If the one recommended by the supplier of the measuring instrument is used, it can be used for the measurement without any particular problem.
　Other necessary instruments for carrying out the present invention include instruments widely used in experiments of molecular biology such as pipettes, pipette tips, and 1.5 ml microtubes. Examples of instruments include PCR and clean bench. Examples include equipment that is widely used in molecular biology experiments, such as tube centrifuges.
[0019]

　The mecA gene detection kit of the present invention is a kit for detecting the mecA gene by analyzing a DNA fragment amplified by the primer pair of the present invention. The analysis of the DNA fragment as the amplification product can be performed by a known method. For example, the DNA fragment as an amplification product can be analyzed by molecular weight analysis of the DNA fragment, Tm value measurement (melting curve analysis) and the like. You may combine two or more types of analysis methods. The measurement of Tm value is preferable, and real-time PCR is preferably used for this purpose. The molecular weight can be analyzed by electrophoresis or a mass spectrometer.
　The mecA gene detection kit according to the present invention is configured to include one or more of the primer pairs of groups (a) to (c) described above. In addition to this primer, if necessary, a PCR enzyme, pH buffer, MgCl 2 , dNTP (or CleanAmp ™　dNTP) and a reagent such as sterilized water, a PCR analysis measurement container (for example, an analysis measurement tube), In addition, at least one reagent and/or component selected from components such as necessary instruments can be included. Furthermore, a fluorescent dye can be added as a component in the analysis by real-time PCR. Further, depending on various aspects of the PCR method, components necessary for the PCR method used in addition to these components may be added to the kit. Each of these reagents or parts may be packaged in any form, such as individually, partly, or integrally. Specifically, for example, the following modes are available.
1) Separate for each reagent used.
2) pH buffer, MgCl 2, DNTP (or CleanAmp ™　dNTP), a mixture in which fluorescent dyes are premixed in the analysis by real-time PCR, a primer pair and an enzyme for PCR are divided into three parts, that is, separately packaged.
3) pH buffer, MgCl 2 , dNTP (or CleanAmp ™　dNTP), a fluorescent dye for analysis by real-time PCR, a mixture in which one primer pair is premixed, and an enzyme for PCR, which are divided into two parts, namely, Package separately.
4) Package a mixture of pH buffer, MgCl 2 , dNTP (or CleanAmp ™　dNTP), a fluorescent dye for analysis by real-time PCR, a primer pair, and an enzyme for PCR all pre-mixed.
　When the kit has a plurality of primer pairs, the plurality of primer pairs are separately packaged. Even when a plurality of mixtures containing different primer pairs are used in the construction of the kit, each mixture is packaged separately.

　The mecA gene detection kit of the present invention can be used for detecting mecA gene in various fields such as medical field, food field, and environmental analysis. In addition, it can be used in combination with the rapid identification method of infectious disease-causing bacteria described in Patent Document 1, and can be used for simultaneous and rapid identification of causative bacteria and mecA gene detection.
　The sample used for the inspection is not particularly limited and can be applied to a wide range of samples. Specifically, in the medical field, blood, cerebrospinal fluid, lacrimal fluid, aqueous humor, amniotic fluid, synovial fluid, saliva, nasal swab, urine, other body fluids, and catheter attachments such as catheters can be mentioned. In the food field, food itself, liquid in the process of production, or solid feed, equipment deposits in the manufacturing process and the like can be mentioned.
[0020]
When
　the mecA gene detection kit of the present invention is used to detect a mecA gene in a sample, it is necessary to extract DNA in advance from bacteria that may be present in the sample. As a DNA extraction method, an alkali dissolution method, a boiling method, a phenol extraction method, a bead crushing method, etc. are currently known, and various DNA extraction kits are also sold by manufacturers.
　The DNA extraction method in the present invention is not particularly limited, but the optimum method differs depending on the sample, and therefore it is desirable to select a method suitable for the target sample.
　
　The primer pair of the present invention is used in the PCR method. As the PCR method, various PCR methods can be used as long as they are PCR methods for amplifying a target gene for detecting the mecA gene. A preferred analysis method is real-time PCR, more preferably a combination of real-time PCR and melting curve analysis for Tm value measurement. In this case, the real-time PCR device used is not particularly limited. The Rotor Gene Q manufactured by Qiagen Co., Ltd. used in the examples of the present application is an example of a real-time PCR device that can be suitably used.
　In PCR and real-time PCR, known devices such as devices and methods may be used.
　The conditions of temperature cycle in PCR (temperature, time, rate of temperature increase/decrease, number of cycles) are not particularly limited, and may be appropriately selected depending on the properties of the primer to be used, the enzyme for PCR, the template, etc. and the sensitivity of the DNA detection method after PCR. Just set it. Many documents are already known about setting such conditions.
　In PCR, generally, a heat denaturation step of a template double-stranded DNA, an annealing step of a primer, and a DNA extension step with an enzyme for PCR are repeated. The heat denaturation step may be carried out at a temperature and time at which the template double-stranded DNA is dissociated into single strands, and is set, for example, at 90° C. to 98° C. for several seconds to several minutes. In addition, a heat denaturation process of several minutes to 10 minutes is often added only to the first cycle at the start of PCR. The annealing step of the primer is set according to the base sequence and the number of bases of the primer, but it is often set at 40°C to 72°C for several seconds to several tens of seconds. In the DNA extension step, the temperature is generally, for example, 58° C. to 76° C. according to the properties such as the optimum temperature of the enzyme for PCR, and the time of the DNA extension step depends on the chain length of the DNA to be amplified. The required time is roughly estimated and set from the DNA synthesis rate of the enzyme for PCR. The target DNA is amplified by repeating the steps of heat denaturation, annealing, and extension. The number of repetitions depends on the amount of template DNA, the amount of enzyme for PCR, and the sensitivity of the DNA detection method after PCR. The number may be appropriately changed, but a typical example is 10 to 50 times. Further, when the annealing temperature and the DNA elongation temperature are about the same, both steps can be performed simultaneously.
　Also in real-time PCR, the conditions for heat denaturation, annealing, DNA extension necessary for DNA amplification, and the number of times these steps are repeated are the same as those described above for PCR. In real-time PCR, the amount of amplified DNA can be quantified or estimated by measuring the fluorescence intensity derived from the intercalator before and after the DNA extension step. The temperature at which the fluorescence intensity is measured may be the temperature at the time of DNA extension as it is, for example, when carrying out using an intercalator, and the chain length of the target DNA to be amplified is relatively long and its Tm value is relatively large. If it is high, non-specifically amplified non-specifically amplified DNA (also referred to as non-specifically amplified DNA) having a relatively short chain length such as primer dimer has a relatively low Tm value. The temperature between these values ​​may be set, including the intermediate value between the Tm value of 1 and the Tm value of non-specifically amplified DNA. By doing so, especially when carried out using an intercalator, only non-specifically amplified DNA such as primer dimer is dissociated from double-stranded to single-stranded, and the amount of DNA quantified or estimated from the fluorescence intensity depends on the target DNA. It can be.
　Furthermore, in real-time PCR, the Tm value of the amplified DNA can be measured by melting curve analysis after the completion of the DNA amplification step. In melting curve analysis, the dissociation of DNA from double-stranded to single-stranded depending on the temperature change is observed, but the temperature and detection conditions are not particularly limited. Generally, heat denaturation (90° C. to 98° C.), duplex formation (Annealing) (40° C. to 80° C.), melting (double strand formation temperature to about 98° C. gradually By raising the temperature rise step and monitoring the change in fluorescence intensity in the melting step, a melting curve can be obtained, and the Tm value can be obtained therefrom. Such measurement can be performed by many types of real-time PCR devices, and can be performed according to the usage method of the device.
[0021]
　 As a
　method for detecting the mecA gene in a sample according to the present invention, a method having the following steps can be used.
(1) A step of performing PCR using bacterial genomic DNA prepared from the sample, a primer pair for obtaining an amplification product containing the mecA gene, and a PCR reagent such as a thermostable DNA polymerase.
(2) A step of detecting the mecA gene in the amplification product in the PCR, or detecting the mecA gene in the sample by analyzing the amplification product.
　When using a plurality of primer pairs, the PCR step (1) is performed separately for each of the plurality of primer pairs. That is, one primer pair is added to one reaction solution for PCR, and the PCR step (1) is performed using each of a plurality of reaction solutions to which different primer pairs have been added.
　By using at least one primer pair according to the present invention as a primer pair for obtaining an amplification product containing the mecA gene, highly sensitive detection of the methicillin resistance gene (mecA) can be performed.
　In this detection method, it is preferable that the amplification step is carried out under the suppression of amplification of a gene (non-target gene) other than the mecA gene as the target gene. The hot start PCR can be used to suppress the amplification of this non-target gene. An example is the hot start method using an anti-DNA polymerase antibody. In that case, it is preferable to use an excessive amount of anti-DNA polymerase antibody with respect to 1 U of thermostable DNA polymerase. Further, as disclosed in Patent Document 1 and International Publication No. 2010/082640, the hot start method by subjecting a DNA polymerase to reversible chemical modification can also be suitably used. Furthermore, the hot start method using a chemically modified dNTP or a chemically modified primer as described in US Patent Application Publication No. 20070281308 (US2007/0281308A1) can also be preferably used. In addition, a hot start method by physically separating the DNA polymerase from the components essential for DNA amplification by the DNA polymerase (eg, primer, dNTP and Mg 2+ salt) by using a wax that melts by heating Can also be suitably used.
[0022]
　Detection of the target gene in the amplification product in the amplification product detection step can be carried out by measuring the Tm value by real-time PCR using an intercalator having a fluorescent label for detection. Preferred intercalators are Cyber ​​Green I, EvaGreen, Resolight and BOXTO, more preferably EvaGreen and Resolight.
　In the detection step by measuring the Tm value using real-time PCR, the amplification product of the target gene can be detected, and the amplification products of other non-target genes can be detected undetected. As a method therefor, the conditions under which the
amplification product of the target gene can be detected and the amplification products of other non-target genes are not detected are as follows: (1) If the melting temperature (TmA) of the target gene amplification product is A primer that is higher than the melting temperature (TmB) of the gene amplification product is selected, and
(2) detection of the amplification product is
set by performing the detection at a temperature between TmA and TmB, and only the amplification product of the target gene is set. The method of detection is preferred.
　Furthermore, a method can be used in which the amplification step and the detection step are performed by real-time PCR using a display device that displays the amount of the amplification product, and the amplification product of the non-target gene is not displayed on the display device.
　The analysis of the amplification product in the detection step can be performed by analyzing the amplification product, which is developed and visualized by electrophoresis on a gel or the like. The analysis of the amplification product can also be performed by analyzing the amplification product by decoding the base sequence of the amplification product. Furthermore, the analysis of the amplification product can also be performed by a method in which the molecular weight of the amplification product is measured by a mass spectrometer and analyzed.
Example
[0023]
(Example 1) Primary screening
　Using genomic DNA extracted from a culture solution of MRSA (RIKEN consignment strain, JCM31453) using a high pure PCR template preparation kit (Roche), the primers of SEQ ID NO: 15 and SEQ ID NO: 16 were used. PCR was performed with the composition of the reaction solution shown in Table 1 to amplify the full length mecA ORF. Next, a dilution series of amplification products of mecA was prepared, and PCR was carried out with the reaction solution composition shown in Table 1 using a comparison primer pair consisting of a forward primer consisting of SEQ ID NO: 13 and a reverse primer consisting of SEQ ID NO: 14. Then, the concentration of the amplification product of mecA at which the number of rising cycles was approximately 20 was determined and used as a template. In addition, methicillin-resistant Staphylococcus aureus, JCM31453 is a microbial material development room (RIKEN BRC-JCM), RIKEN BioResource Center, RIKEN BioResource Center a more publicly available.
　Next, in order to evaluate the primers prepared, PCR was carried out in a reaction solution composition shown in Table 1 for the 313 primer pairs. Real-time PCR apparatus rotor Gene Q MDx 5plex HRM (QIAGEN) was used under the reaction conditions of PCR, after heating at 95° C. for 5 minutes, 94° C. for 10 seconds, 65° C. for 10 seconds, and 72° C. for 30 seconds were repeated 40 times. Improvement in the shape of peaks in resolution melting analysis (HRM), band intensity during electrophoresis, and the presence or absence of non-specific products during electrophoresis compared with the results of the comparative primer pair, 88 pairs Was selected.
[0024]
(Example 2) Secondary screening
　The concentration of genomic DNA extracted from a culture solution of MRSA (RIKEN consignment strain, JCM31453) using a High pure PCR template preparation kit (Roche) was adjusted to 50 μg/ml human genomic DNA solution (invitrogen). The solution adjusted so that the number of rising cycles was about 20 was used as a template.
　The 88 primer pairs were evaluated by performing PCR with the reaction solution composition shown in Table 1. Rotagene Q MDx 5plex HRM (QIAGEN) was used as a real-time PCR device. Regarding the PCR reaction conditions, after heating at 95° C. for 5 minutes, 94° C. for 10 seconds, 65° C. for 10 seconds, and 72° C. for 30 seconds were repeated 40 times. In the evaluation, the number of rising cycles, the shape of the peak in HRM, the intensity of the band in electrophoresis, and the presence or absence of nonspecific products in electrophoresis were compared with the results of the comparative primer pair, and improvement was observed 57. Selected a pair.
[0025]
(Example 3) Third screening
　The concentration of genomic DNA extracted from a culture solution of MRSA (RIKEN consignment strain, JCM31453) using a High pure PCR template preparation kit (Roche) was adjusted to a concentration of 50 µg/ml human genomic DNA solution (invitrogen). A solution adjusted to 10.4 molecules/reaction solution was used as a template. The 57 primer pairs were evaluated by performing PCR in triplicate using the reaction solution composition shown in Table 1. Rotagene Q MDx 5plex HRM (QIAGEN) was used as a real-time PCR device. Regarding the PCR reaction conditions, after heating at 95° C. for 5 minutes, 94° C. for 10 seconds, 65° C. for 10 seconds, and 72° C. for 30 seconds were repeated 40 times. In the evaluation, whether amplification was seen in all of the triplicate measurements, dF/dT Height (channel A) in HRM, presence/absence of primer dimer, and presence/absence of peak of non-specific product were compared with the results of the comparison primer pair. By comparison, 9 pairs were selected that showed improvement.
[0026]
(Example 4) Final screening
　Genomic DNA extracted from a culture solution of MRSA (RIKEN consignment strain, JCM31453) by using a high pure PCR template preparation kit (Roche) was added to each concentration (in virogen) at 50 μg/ml human genomic DNA solution. Copy number/reaction solution: In Table 2, a solution adjusted to be Copies/Reaction was used as a template. The 9 primer pairs were evaluated by performing PCR in triplicate with the reaction solution composition shown in Table 1. Rotagene Q MDx 5plex HRM (QIAGEN) was used as a real-time PCR device. Regarding the PCR reaction conditions, after heating at 95° C. for 5 minutes, 94° C. for 10 seconds, 65° C. for 10 seconds, and 72° C. for 30 seconds were repeated 40 times. The lowest concentration at which amplification was observed in all triplicate measurements was taken as the detection limit. It was confirmed by measuring the Tm value whether the desired amplification product was obtained.
　As a result, 8 pairs were selected in which the detection limit was improved as compared with the case where the detection was performed using the comparative primer pair shown in the prior patent document 1. The results are shown in Table 2. Table 3 shows SEQ ID NOS: 13 and 14 of the primer pair for comparison and the respective base sequences of the selected 8 pairs.
[0027]
(Example 5) Comparison between methicillin-resistant strain and methicillin-sensitive strain
　Staphylococcus aureus (methicillin-sensitive RIKEN consignment strain, JCM2151) and MRSA (methicillin-resistant RIKEN consignment strain, JCM16555) culture solution High pure PCR template (R). The genomic DNA solution extracted with was used as a template. The primer pairs of SEQ ID NO: 6 and SEQ ID NO: 10 were evaluated by performing PCR with the reaction solution composition shown in Table 1. Rotagene Q MDx 5plex HRM (QIAGEN) was used as a real-time PCR device. Regarding the PCR reaction conditions, after heating at 95° C. for 5 minutes, 94° C. for 10 seconds, 65° C. for 10 seconds, and 72° C. for 30 seconds were repeated 40 times. Whether or not the desired amplification product was obtained was confirmed by measuring the Tm value based on the presence or absence of a peak near 81°C. The results for the methicillin-resistant strain are shown in FIG. 1(a), and the results for the methicillin-sensitive strain are shown in FIG. 1(b).
[0028]
[Table 1]

[0029]
[Table 2]

[0030]
[table 3]

Industrial availability
[0031]
　From the test results shown in Table 2, it was found that the detection method of the present invention using the primer pair of the present invention can detect an extremely minute amount of the mecA gene, which is 10 copies or less in one reaction. INDUSTRIAL APPLICABILITY The detection method of the present invention is capable of detecting extremely dilute mecA gene in a very small amount of sample with high sensitivity, high accuracy, and swiftness, and thus has sufficient practicality and wide applicability. INDUSTRIAL APPLICABILITY The primer pair, the detection method and the detection kit of the present invention can be used for detection of a trace amount of mecA gene in various specimens generated in the medical field, food field and environmental analysis.
The scope of the claims
[Request 1]
　A primer pair for detecting the mecA gene (methicillin resistance gene),
　wherein the primer pair is one primer pair selected from the group consisting of the following (a) to (c): A primer pair for detecting the mecA gene.
(A) A combination of SEQ ID NO: 3 and SEQ ID NO: 7, a combination of SEQ ID NO: 2 and SEQ ID NO: 9, a combination of SEQ ID NO: 1 and SEQ ID NO: 8, a combination of SEQ ID NO: 1 and SEQ ID NO: 9, a SEQ ID NO: 4 and a SEQ ID NO: 11 A combination of SEQ ID NO: 5 and SEQ ID NO: 12, a combination of SEQ ID NO: 6 and SEQ ID NO: 10 or a combination of SEQ ID NO: 6 and SEQ ID NO: 12,
(b) In (a) above, one or both of A primer pair in which 1 or 2 bases are added, deleted or substituted within a range that does not impair the function of the primer as a part of the base sequence of the primer,
(c) Corresponds to the base sequence of (a) or (b) above A primer pair consisting of complementary sequences.
[Request 2]
　The primer pair is a primer pair consisting of a combination of SEQ ID NO: 6 and SEQ ID NO: 10, or a part of the base sequence of one or both of the combinations is 1 or 2 bases within a range not impairing the function as a primer. The primer pair according to claim 1, which is an added, deleted or substituted primer pair.
[Request 3]
　A mecA gene detection kit
　comprising a primer pair , wherein the primer pair is at least one primer pair selected from the group consisting of the following (a) to (c): ..
(A) a combination of SEQ ID NO: 3 and SEQ ID NO: 7, a combination of SEQ ID NO: 2 and SEQ ID NO: 9, a combination of SEQ ID NO: 1 and SEQ ID NO: 8, a combination of SEQ ID NO: 1 and SEQ ID NO: 9, a SEQ ID NO: 4 and an SEQ ID NO: 11 A primer pair consisting of the combination of SEQ ID NO:5 and SEQ ID NO:12, the combination of SEQ ID NO:6 and SEQ ID NO:10 or the combination of SEQ ID NO:6 and SEQ ID NO:12,
(b) In (a) above, one or both of A primer pair in which 1 or 2 bases are added, deleted or substituted within a range that does not impair the function of the primer as a part of the base sequence of the primer,
(c) corresponds to the base sequence of (a) or (b) above A primer pair consisting of complementary sequences.
[Request 4]
　The primer pair is a primer pair consisting of a combination of SEQ ID NO: 6 and SEQ ID NO: 10, or a part of the base sequence of one or both of the combinations is 1 or 2 bases within a range not impairing the function as a primer. The kit according to claim 3, which is an added, deleted, or substituted primer pair.
[Request 5]
　The kit according to claim 3 or 4, comprising at least one of a PCR enzyme, a PCR reagent, and a PCR instrument.
[Request 6]
　A method for detecting mecA in a
　sample,
　comprising a PCR step of performing PCR using DNA prepared from the sample and a primer pair for amplifying the DNA , and mecA in the amplification product obtained by the PCR step. the detection of gene, or by analysis of the amplification product, and performing detection of mecA gene in the said sample
has,
　the primer pair used in the PCR step, the following (a) ~ (c) A method for detecting a mecA gene, which comprises at least one primer pair selected from the group consisting of:
(A) a combination of SEQ ID NO: 3 and SEQ ID NO: 7, a combination of SEQ ID NO: 2 and SEQ ID NO: 9, a combination of SEQ ID NO: 1 and SEQ ID NO: 8, a combination of SEQ ID NO: 1 and SEQ ID NO: 9, a SEQ ID NO: 4 and an SEQ ID NO: 11 A primer pair consisting of the combination of SEQ ID NO:5 and SEQ ID NO:12, the combination of SEQ ID NO:6 and SEQ ID NO:10 or the combination of SEQ ID NO:6 and SEQ ID NO:12,
(b) In (a) above, one or both of A primer pair in which 1 or 2 bases are added, deleted or substituted within a range not impairing the function of the primer as a part of the base sequence of the primer,
(c) corresponding to the base sequence of (a) or (b) above A primer pair consisting of complementary sequences.
[Request 7]
　The primer pair is a primer pair consisting of a combination of SEQ ID NO: 6 and SEQ ID NO: 10, or a part of the base sequence of one or both of the combinations is 1 or 2 bases within a range not impairing the function as a primer. The detection method according to claim 6, which is a primer pair added, deleted or substituted.