JP2008000002A - NUCLEIC ACID AMPLIFICATION PRIMER FOR DETECTING RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN 1A (RBCS-1A) GENE AND/OR mRNA OF THE GENE AND INSPECTION METHOD USING THE GENE AND/OR mRNA OF THE GENE AS INTERNAL STANDARD - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inspection method using a proper gene and/or the mRNA of the gene as an internal standard and to obtain a primer effectively amplifying the cDNA of the gene and/or the mRNA of the gene. <P>SOLUTION: There is provided a nucleic acid-amplifying primer for detecting a ribulose bisphosphate carboxylase small chain 1A (RBCS-1A) gene and/or the mRNA of the gene by a LAMP method. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a nucleic acid amplification primer for detecting the RBCS-1A gene and / or mRNA of the gene (hereinafter also simply referred to as primer), and an inspection using the gene and / or mRNA of the gene as an internal standard. With respect to methods.

  In recent years, genetic testing has rapidly spread in the field of clinical diagnosis. Genetic testing is the analysis of nucleic acids, chromosomes, and the like to test for the presence or absence of mutations or karyotypes associated with genetic diseases. One example of genetic testing is the diagnosis of cancerous lymph node metastasis. Cancer cells leave the primary lesion and metastasize throughout the body via blood vessels and lymphatic vessels. In cancer surgery, it is necessary to remove the lesion as reliably as possible. Therefore, it is required to accurately detect metastasis and perform appropriate treatment according to the degree of metastasis. For this reason, the diagnosis of lymph node metastasis of cancer cells during surgery is extremely important. As a technique for diagnosing lymph node metastasis of cancer, there is a method of detecting a nucleic acid of a cancer marker. With recent developments in gene analysis technology, cancer diagnosis has been carried out effectively by amplifying and detecting nucleic acids related to cancer markers contained in lymph node tissue excised from the living body. Furthermore, when the nucleic acid of the cancer marker is amplified by the nucleic acid amplification method, the amplification reaction proceeds rapidly, and the time until the nucleic acid copy number rapidly increases (amplification rise time) is measured in real time. It is also possible to quantify the nucleic acid of the cancer marker.

  A LAMP method is known as a method for amplifying the above-described nucleic acid (Patent Document 1). The LAMP method is a gene amplification method using a plurality of primers including a primer that forms a hairpin structure at the end of an amplification product when a strand displacement reaction proceeds. First, in the initial reaction, two kinds of inner primers (FIP, RIP), two kinds of outer primers (F3 primer, R3 primer) and a strand displacement type DNA polymerase are used to form a dumbbell having single-stranded loop portions at both ends from the template DNA. Is synthesized. This structure becomes the starting structure of the amplification cycle, and the DNA height / synthesis reaction proceeds from the 3 'end of this structure using itself as a template. The amplification product has a structure in which complementary sequences derived from the base sequence of the target nucleic acid repeat each other. The LAMP method is characterized in that it does not require a heat denaturation operation from a double strand to a single strand of the template DNA, and all amplification reactions proceed continuously at an isothermal temperature. When the template is RNA, the origin structure can be synthesized in the same way by adding reverse transcriptase to the reaction solution composition when the template is DNA, and the amplification can proceed (RT-LAMP method). . In the LAMP method or the RT-LAMP method, an amplification product sufficient for detection can be obtained in about 30 minutes. Therefore, since the time required for nucleic acid detection is shortened, for example, it can be applied to diagnosis of cancer metastasis to a lymph node for the purpose of quickly determining a treatment policy. Moreover, since a result is obtained quickly, it can be applied to intraoperative diagnosis.

  When detecting mRNA corresponding to a cancer marker, a method using mRNA of β-actin gene as an internal standard substance is known (Patent Document 2). By using mRNA of a housekeeping gene such as β-actin gene as an internal standard, it is possible to detect relative cancer marker gene mRNA without considering cancer marker gene mRNA extraction efficiency or cDNA amplification efficiency. Is possible.

  However, since β-actin does not know the original expression level in lymph nodes excised from the living body, even if housekeeping gene mRNA is used as an internal standard, the amplification inhibitor in the lymph node is the cancer marker gene mRNA. It may not be possible to confirm whether the amplification of cDNA is affected. Furthermore, when the amplification inhibitor affects nucleic acid amplification, the degree of inhibition of the cancer marker on nucleic acid amplification differs from the degree of inhibition of the housekeeping gene on nucleic acid amplification. It is difficult to correct the amplification rise time of the cancer marker gene mRNA.

International Publication WO 00/28082 Pamphlet International Publication WO 03/70935 Pamphlet

  An object of the present invention is to provide a test method using an appropriate gene and / or mRNA of the gene as an internal standard, and a primer for effectively amplifying the cDNA of the gene and / or mRNA of the gene. That is.

  As a result of diligent studies to solve the above problems, the present inventors do not exist in the human genome as an internal standard substance, and the degree of inhibition caused by the inhibitory substance is the same as the degree of inhibition caused by nucleic acid amplification of the cancer marker. It has been found that a nucleic acid is used. This internal standard confirms whether or not the inhibitor has an effect on the amplification of the nucleic acid of the cancer marker gene during nucleic acid amplification. Time can be corrected. Furthermore, the RBCS-1A gene and / or the mRNA of the gene are suitable as nucleic acids that are not present in the human genome and have the same degree of inhibition as the inhibition by the cancer marker nucleic acid amplification. It turned out that.

  Therefore, the present inventor has developed an effective primer for detecting the RBCS-1A gene and / or mRNA of the gene that can be used as an internal standard, and a test method using the gene and / or mRNA of the gene, I will provide a.

That is, the present invention comprises the following I to XX.
I. A primer for nucleic acid amplification for detecting ribulose bisphosphate carboxylase small chain 1A (RBCS-1A) gene and / or mRNA of said gene by LAMP method.
II. The primer for nucleic acid amplification according to I, wherein the gene is derived from an Arabidopsis plant.
III. RBCS-1A gene comprising an oligonucleotide consisting of a sequence selected from 1) to 5) shown below and / or a nucleic acid amplification primer for detecting mRNA of the gene;
1) An oligonucleotide selected from the 50th to 530th region of the base sequence represented by SEQ ID NO: 1 and the region of its complementary strand, and comprising at least 5 or more consecutive bases of SEQ ID NO: 1 and / or its complementary strand.
2) An oligonucleotide having a base sequence represented by any one of SEQ ID NOs: 2-45.
3) A complementary strand of the oligonucleotide according to 1) or 2).
4) An oligonucleotide that can hybridize with the oligonucleotide according to any one of 1) to 3) under stringent conditions.
5) An oligonucleotide having a primer function, wherein one to seven bases among the oligonucleotides described in 1) to 4) above include a mutated base sequence such as substitution, deletion, insertion, or addition.
IV. A nucleic acid amplification primer for detecting an RBCS-1A gene and / or mRNA of the gene comprising an oligonucleotide selected from any one of the nucleotide sequences represented by SEQ ID NOs: 10 to 45.
V. The primer for nucleic acid amplification according to III or IV, wherein the nucleic acid amplification means is the LAMP method.
VI. Nucleic acid amplification for detecting RBCS-1A gene and / or mRNA of said gene, wherein at least two kinds of primers comprising an oligonucleotide consisting of a sequence selected from 1) to 5) shown below are selected Primer set;
1) An oligonucleotide selected from 50 to 530 of the base sequence represented by SEQ ID NO: 1 and the region of its complementary strand, and comprising at least 5 or more consecutive bases of SEQ ID NO: 1 and / or its complementary strand.
2) An oligonucleotide having a base sequence represented by any one of SEQ ID NOs: 2-45.
3) A complementary strand of the oligonucleotide according to 1) or 2).
4) An oligonucleotide that can hybridize with the oligonucleotide according to any one of 1) to 3) under stringent conditions.
5) An oligonucleotide having a primer function, wherein one to seven bases among the oligonucleotides described in 1) to 4) above contain a mutated base sequence such as substitution, deletion, insertion or addition.
VII. The primer set for nucleic acid amplification according to VI, wherein the nucleic acid amplification means is the LAMP method.
VIII. The primer set for nucleic acid amplification according to VI or VII for detecting an RBCS-1A gene and / or mRNA of the gene, wherein at least four kinds are selected from primers for nucleic acid amplification containing the oligonucleotide.
IX. The nucleic acid amplification primer included in the nucleic acid amplification primer set recognizes at least two regions each of the nucleotide sequence represented by SEQ ID NO: 1 and / or its complementary strand. Item 8. A primer set for nucleic acid amplification according to any one of Items VI to VIII.
X. The nucleic acid amplification primer contained in the primer set for nucleic acid amplification recognizes at least six regions of the base sequence represented by SEQ ID NO: 1 and / or its complementary strand, A primer set for nucleic acid amplification according to any one of the above.
XI. A nucleic acid amplification primer for detecting the RBCS-1A gene and / or mRNA of the gene comprising an oligonucleotide having the nucleotide sequence represented by SEQ ID NOs: 10 to 45, comprising: (a) SEQ ID NOs: 37 to 40 and (b) A primer set for nucleic acid amplification comprising a combination in which one nucleic acid amplification primer is selected from each classification when classified into SEQ ID NOs: 41 to 44.
XII. A primer set for nucleic acid amplification described in XI, and a combination of (c) SEQ ID NOs: 10 to 13 and (d) SEQ ID NOs: 23 to 27 and 45, wherein one nucleic acid amplification primer is selected from each classification; A primer set for nucleic acid amplification, comprising:
XIII. A primer set for nucleic acid amplification described in XII, and a combination of (e) SEQ ID NOs: 28 to 31 and (f) selected one primer from each classification when classified into SEQ ID NOs: 32-36. A primer set for nucleic acid amplification.
XIV. RBCS-1A gene comprising any one of 1) to 5) below and / or a nucleic acid amplification primer set for detecting mRNA of the gene;
1) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 38, 42, 11, and 25 as nucleic acid amplification primers.
2) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, and 24 as nucleic acid amplification primers.
3) A primer set for nucleic acid amplification comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, and 23 as primers for nucleic acid amplification.
4) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 40, 44, 13, and 27 as nucleic acid amplification primers.
5) A primer set for nucleic acid amplification containing oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 39, 43, 12, and 26 as primers for nucleic acid amplification.
XV. RBCS-1A gene comprising any one of 1) to 6) below and / or a primer set for nucleic acid amplification for detecting mRNA of the gene;
1) A primer set for nucleic acid amplification comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 38, 42, 11, 25, 29, and 34 as nucleic acid amplification primers.
2) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 38, 42, 11, 25, 29, and 32 as nucleic acid amplification primers.
3) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, 24, 28, and 33 as nucleic acid amplification primers.
4) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, 23, 28, and 33 as nucleic acid amplification primers.
5) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 40, 44, 13, 27, 31, and 36 as nucleic acid amplification primers.
6) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 39, 43, 12, 26, 30, and 35 as nucleic acid amplification primers.
XVI. Nucleic acid detection by selecting a necessary nucleic acid amplification primer from the nucleic acid amplification primers according to III or IV, or selecting and using the nucleic acid amplification primer set according to any one of claims V to XV Method.
XVII. The nucleic acid detection method according to XVI, which uses the LAMP method for nucleic acid detection.
XVIII. The reagent used for the nucleic acid detection method as described in XVI or XVII.
XIX. A reagent kit used for the nucleic acid detection method according to XVI or XVII.
XX. RBCS-1A gene and / or mRNA of said gene is used as an internal standard.

  According to the present invention, there are provided a more effective primer for detecting the RBCS-1A gene and / or mRNA of the gene, and a test method using the gene and / or mRNA of the gene as an internal standard. The

<About RBCS-1A>
RBCS-1A is one of the small subunits of ribulose diphosphate carboxylase (RubisCO), and the base sequence of the gene has already been determined (Theologis et. Al, Nature, 2000 Dec. 14; 408 (6814) : 816-820. RubisCO is an enzyme that produces organic carbon from inorganic carbon dioxide in the air. This enzyme is not present in humans but is found in large quantities in the cells of many photosynthetic organisms. RubisCO such as plants, algae, and cyanobacteria is composed of one or a plurality of large subunits and one or a plurality of small subunits, and a photosynthetic bacterium RubisCO is composed of only a large subunit. The large subunit is encoded in the chloroplast genome and the small subunit is encoded in the nuclear genome.
The RBCS-1A gene and / or mRNA of the gene used in the present invention is preferably derived from a plant belonging to the genus Arabidopsis spp., And more preferably derived from Arabidopsis thaliana.

<Primer design>
The present invention is applicable to the LAMP method and provides a primer for detecting the RBCS-1A gene and / or mRNA of the gene.
The basic concept of primer design used in the LAMP method is as described in Patent Document 1. Specifically, the nucleic acid regions F3c, F2c, F1c from the 3 ′ end side and the nucleic acid regions R3, R2, R1 from the 5 ′ end side of the target nucleic acid to be amplified are set, and at least these six nucleic acid regions are set. On the other hand, an oligonucleotide chain containing a substantially identical base sequence or a substantially complementary base sequence is selected, and at least four kinds of primers are designed.
The complementary strand of the target nucleic acid is also the target nucleic acid. This complementary strand has nucleic acid regions R3c, R2c and R1c from the 3 ′ end side and nucleic acid regions F3, F2 and F1 from the 5 ′ end side. Here, each nucleic acid region of R3c, R2c, R1c, F3, F2, and F1 has a base sequence complementary to the nucleic acid amount region of R3, R2, R1, F3c, F2c, and F1c of the target nucleic acid, respectively.

  The substantially identical base sequence is used in the following meaning. When a complementary strand synthesized using a certain base sequence as a template hybridizes to the target base sequence and gives a starting point for complementary strand synthesis, this base sequence is substantially identical to the target base sequence. is there. For example, a base sequence that is substantially identical to R2 is a base sequence that functions as a template that provides a base sequence that can hybridize to R2 and serve as a starting point for complementary strand synthesis in addition to the completely same base sequence as R2. including.

  On the other hand, a substantially complementary nucleotide sequence means a nucleotide sequence that can hybridize under stringent conditions and can provide a 3 'end that serves as a starting point for complementary strand synthesis.

  The terms identical or complementary used for the characterization of the base sequences that make up the oligonucleotides according to the invention need not be completely identical or completely complementary. That is, the same as a certain sequence can also include a sequence complementary to a base sequence capable of hybridizing to a certain sequence.

  The primer of the present invention has a chain length that allows base pairing with a complementary strand while maintaining the necessary specificity under the given environment in various nucleic acid synthesis reactions described below. Specifically, it is 5 to 200 bases, more preferably 10 to 50 bases. Since the chain length of a primer recognized by a known polymerase that catalyzes a sequence-dependent nucleic acid synthesis reaction is at least about 5 bases, the chain length of the hybridizing portion needs to be longer. In addition, in order to maintain specificity as a base sequence, it is preferable to maintain a length of 10 bases or more. On the other hand, since it is difficult to prepare an excessively long base sequence by chemical synthesis, the chain length as described above is exemplified as a desirable range.

  The term template used in the present invention means a nucleic acid on the side serving as a template for complementary strand synthesis. A complementary strand having a base sequence complementary to the template has a meaning as a strand that can hybridize to the template, but the relationship between them is only relative. That is, the strand synthesized as a complementary strand can function as a template again. That is, the complementary strand can also serve as a template.

  Each of the primers selected in the present invention constitutes one of FIP (Forward Inner Primer), F3 primer (Forward Outer Primer), RIP (Reverse Inner Primer) and R3 primer (Reverse Outer Primer).

  FIP has a base sequence of the F2 region substantially complementary to the F2c region of the target nucleic acid to be amplified on the 3 ′ end side and a base sequence substantially the same as the F1c region of the target nucleic acid on the 5 ′ end side. Design to have. In this case, a sequence independent of the target nucleic acid may be interposed between the sequences of F2 and F1c. The length of the sequence independent of the target nucleic acid is 0 to 50 bases, preferably 0 to 40 bases.

  The F3 primer is designed to have substantially the same base sequence as the F3 region that is substantially complementary to the F3c region of the target nucleic acid.

  RIP is designed so that the base sequence of the R2 region substantially complementary to the R2c region of the target nucleic acid is at the 3 'end, and the base sequence is substantially the same as the R1c region of the target nucleic acid at the 5' end. Similarly to FIP, RIP may have a sequence that does not depend on the target nucleic acid between R2 and R1c sequences.

  The R3 primer is designed to have substantially the same base sequence as the R3 region that is substantially complementary to the R3c region of the target nucleic acid.

  Further, in the LAMP method, the amplification time can be shortened by using at least one kind of loop primer (see International Publication WO 02/24902 pamphlet). The loop primer is a primer having a complementary sequence in a single-stranded portion of the loop on the 5 ′ end side of the dumbbell structure, specifically, for example, between the R1 region and the R2 region or between the F1 region and the F2 region. Say. By using a loop primer, it is possible to increase the starting point of nucleic acid synthesis. This loop primer is designed so as to hybridize to a loop region where FIP or RIP formed in the nucleic acid synthesis process does not hybridize.

<Design of RBCS-1A gene or / and mRNA detection primer>
The primer for detecting RBCS-1A gene and / or mRNA (hereinafter referred to as RBCS-1A primer) is continuous from the base sequence consisting of 878 bases represented by the known SEQ ID NO: 1 and / or its complementary sequence. It is designed by selecting an appropriate oligonucleotide containing at least 5 bases. In this embodiment, RBCS-1A (Genbank Accession No. NM_105379) derived from Arabidopsis thaliana is used, but the organism derived therefrom is not particularly limited.

  The RBCS-1A primer pays attention to the base composition, GC content, secondary structure, Tm value, etc., and the length of the DNA region of the target nucleic acid recognized by the primer is at least 5 bases, preferably 10-30. A base, more preferably 17-25 bases can be selected. The Tm value can be generally obtained by the Nearest Neighbor method. The DNA region recognized by the primer may be selected from those having a Tm value of 55 to 65 ° C, preferably 58 to 64 ° C, and a GC content of 40 to 70%, preferably 50 to 65%. it can.

  Under such conditions, the primer region selected in the present invention is included in the 50th to 540th region of the base sequence represented by SEQ ID NO: 1 and / or the region of its complementary strand.

  The RBCS-1A primer is 1) an oligonucleotide which is contained in the 50th to 540th region of the base sequence represented by SEQ ID NO: 1 and / or its complementary strand region and has at least 5 bases, and 2) a sequence Oligonucleotide having the nucleotide sequence represented by Nos. 2-45, 3) the complementary strand of the oligonucleotide according to 1) or 2), 4) the oligonucleotide according to any one of 1) to 3) Or oligonucleotides that can hybridize under stringent conditions, or 5) the oligonucleotides described in 1) to 4) above, wherein one to seven bases are mutated such as substitutions, deletions, insertions or additions. Oligonucleotides comprising a base sequence selected and designed.

  Oligonucleotides can be produced by a method known per se, for example, chemically synthesized. Alternatively, natural nucleic acids can be cleaved with a restriction enzyme or the like, modified so as to have the above base sequence, or ligated. Specifically, it can be synthesized using an oligonucleotide synthesizer (Expedite Model 8909 DNA synthesizer manufactured by ABI) or the like. In addition, as a method for synthesizing an oligonucleotide in which one to seven bases are mutated such as substitution, deletion, insertion or addition, a method known per se can be used. For example, site-directed mutagenesis, gene homologous recombination, primer extension, or PCR can be used alone or in appropriate combination.

  Stringent hybridization conditions can be selected from generally known conditions. Examples include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate, pH 7.6, 5 × Denhart's solution, 10% dextran sulfate, and 20 μg / ml DNA. Examples of the conditions include overnight hybridization in solution at 42 ° C., followed by secondary washing with 2 × SSC / 0.1% SDS at room temperature.

<Primer set>
When nucleic acid amplification is performed using the primer of the present invention, a primer set is used in combination of at least two kinds. In the LAMP method or the RT-LAMP method, at least four kinds of primers (FIP, F3 primer, RIP, and R3 primer) are used in combination as a primer set. Further, one or more loop primers can be combined to be used as a primer set.

<RT-LAMP method>
The LAMP method is a nucleic acid amplification method using DNA as a template, whereas the RT-LAMP method is a kind of LAMP method using RNA as a template. The basic idea of the LAMP method is as described in Patent Document 1. In the RT-LAMP method, the starting point structure of the LAMP method is synthesized while synthesizing cDNA from the template RNA in one solution. Specifically, after the following step (1), the starting structure of the amplification cycle of the LAMP method is formed by repeating the steps (2) to (5).
(1) The 3 ′ end of FIP anneals to the F2c region of the template RNA strand, and reverse transcriptase extends a DNA strand complementary to the template RNA strand. As the reverse transcriptase, for example, one derived from avian myeloblastosis virus (AMV) can be used.
(2) The F3 primer anneals to the F3c region of the template RNA strand, and the reverse transcriptase extends the DNA strand complementary to the template RNA strand while peeling the DNA strand from the FIP synthesized in (1) above from the template RNA. Let
(3) The 3 ′ end of RIP anneals to the R2c region (region complementary to R2 of the template RNA strand) of the DNA strand peeled off in (2) above, so that the DNA strand extends.
(4) The R3 primer anneals to the R3c region of the DNA strand (region complementary to the R3 region of the template RNA strand), and the R3 primer peels off the DNA strand from the RIP extended in (3) above. By the action, a DNA strand complementary to the DNA strand from FIP is elongated, and the starting structure of the LAMP method is synthesized.
(5) Since the sequences at both ends of the DNA strands peeled off in (4) above have complementary sequences in the same DNA strand sequence, each sequence is self-annealed to form an origin structure having loop portions at both ends. .
(5) Thereafter, this origin structure becomes the origin of the amplification cycle, and the height / synthesis reaction of DNA proceeds from the 3 ′ end side of this structure using itself as a template. As the amplification cycle progresses, amplification products are synthesized in various sizes in a structure in which these sequences repeat each other with a complementary sequence derived from the base sequence of the target nucleic acid as a unit.
There are enzymes having both reverse transcriptase activity and DNA polymerase activity (for example, Bca DNA polymerase). When such an enzyme is used, the above reaction can be carried out with one enzyme.

<Measurement method>
In the LAMP method, the amplified DNA strand has a sequence complementary to its own sequence, and most of it forms a base pair bond. This feature can be used to detect amplification products. When nucleic acid amplification is performed using the primer of the present invention in the presence of a fluorescent dye that is a double-stranded intercalator such as ethidium bromide, SYBER GREEN I, or Pico Green, the fluorescence intensity increases as the product increases. An increase is observed. If this is monitored in real time, DNA amplification and increase in fluorescence can be tracked simultaneously in a closed system.
In the LAMP method, insoluble magnesium pyrophosphate is generated as a by-product during the amplification reaction and becomes cloudy. Therefore, check the turbidity of the reaction solution visually, or measure the turbidity by measuring the absorbance or scattered light intensity of the reaction solution, or filter the reaction solution with a colored filter and check the residue on the filter. Can detect a target nucleic acid (see International Publication WO 01/83817 pamphlet). When turbidity measurement is performed by measuring the absorbance or scattered light intensity of the reaction solution, DNA amplification and turbidity are monitored in a closed system by monitoring the turbidity change in real time, as in the case of using the fluorescent dye. Increases can be tracked simultaneously.

<Reagents, reagent kits, etc.>
Various reagents necessary for detecting nucleic acid using the primer of the present invention can be packaged in advance to form a kit. Specifically, various oligonucleotides necessary as a primer for complementary strand synthesis of the present invention or as a primer for substitution, an enzyme having reverse transcription activity, dNTP serving as a substrate for complementary strand synthesis, and a strand displacement type complementary strand A DNA polymerase for synthesis, a buffer solution that provides conditions suitable for the enzyme reaction, and reagents necessary for detection of reaction products as required are provided as a kit.
Of the reagents described above, an enzyme having reverse transcription activity and a DNA polymerase may be accommodated in the same container.
Moreover, you may accommodate dNTP, a buffer solution, and various oligonucleotides in the same container among the above-mentioned reagents.

  Hereinafter, selection of the RBCS-1A gene region and primer design in the present embodiment will be described more specifically.

<Selection of gene region from RBCS-1A base sequence>
From the base sequence described in SEQ ID NO: 1, the position of the gene region of RBCS-1A suitable for the LAMP method was examined using probe design software. In the gene region, F1c and R1c have Tm values of 58.5 to 63.5 ° C, F2 and R2 have Tm values of 61.5 to 62.5 ° C, and F3 and R3 have Tm values of 58.5 to 62.5 ° C. The region was selected so that it was within the range of ° C. Each selected area is shown below.

F1c: gene region in the complementary strand of the sequence shown in SEQ ID NO: 1
148-129 5'-gttagccttgcgggtggctg-3 '(SEQ ID NO: 2)
179-158 5'-cgccgttgcttgtgatggaagt-3 '(SEQ ID NO: 3)
403-382 5'-ccacattgtccagtaccgtcca-3 '(SEQ ID NO: 4)
421-400 5'-accgaacaagggaagcttccac-3 '(SEQ ID NO: 5)

F2: gene region on the sequence described in SEQ ID NO: 1
87-104 5'-tggtcgctcctttcaacg-3 '(SEQ ID NO: 6)
107-125 5'-cttaagtcctccgctgcct-3 '(SEQ ID NO: 7)
324-343 5'-tcgagttggagcacggattt-3 '(SEQ ID NO: 8)
352-370 5'-tgagcacggtaactcaccc-3 '(SEQ ID NO: 9)

F3: gene region on the sequence described in SEQ ID NO: 1
53-72 5'-gctactatggttgcctctcc-3 '(SEQ ID NO: 10)
80-97 5'-gccactatggtcgctcct-3 '(SEQ ID NO: 11)
294-313 5'-tccgcaacaagtggattcct-3 '(SEQ ID NO: 12)
330-349 5'-tggagcacggatttgtgtac-3 '(SEQ ID NO: 13)

R1c: gene region on the sequence described in SEQ ID NO: 1
186-207 5'-ttaactgcatgcaggtgtggcc-2 '(SEQ ID NO: 14)
186-209 5'-ttaactgcatgcaggtgtggcctc-3 '(SEQ ID NO: 15)
196-215 5'-gcaggtgtggcctccgattg-3 '(SEQ ID NO: 16)
407-428 5'-cttcccttgttcggttgcaccg-3 '(SEQ ID NO: 17)
425-445 5'-accgactccgctcaagtgttg-3 '(SEQ ID NO: 18)

R2: gene region in the complementary strand of the sequence shown in SEQ ID NO: 1
265-248 5'-ggaatcggtaaggtcagg-3 '(SEQ ID NO: 19)
267-248 5'-tcggaatcggtaaggtcagg-3 '(SEQ ID NO: 20)
484-465 5'-ggcattggggtactccttct-3 '(SEQ ID NO: 21)
494-475 5'-tcctaatgaaggcattgggg-3 '(SEQ ID NO: 22)

R3: gene region in the complementary strand of the sequence shown in SEQ ID NO: 1
296-276 5'-ggataaggtagtcaacttcct-3 '(SEQ ID NO: 23)
307-288 5'-ccacttgttgcggataaggt-3 '(SEQ ID NO: 24)
307-286 5'-ccacttgttgcggataaggtag-3 '(SEQ ID NO: 25)
514-495 5'-ggtgttgtcgaatccgatga-3 '(SEQ ID NO: 26)
528-511 5'-cactggacttgacgggtg-3 '(SEQ ID NO: 27)

loop F: gene region in the complementary strand of the sequence shown in SEQ ID NO: 1
125-107 5'-aggcagcggaggacttaag-3 '(SEQ ID NO: 28)
154-136 5'-gtcgttgttagccttgcgg-3 '(SEQ ID NO: 29)
366-349 5'-gagttaccgtgctcacgg-3 '(SEQ ID NO: 30)
394-376 5'-ccagtaccgtccatcatag-3 '(SEQ ID NO: 31)

loop R: gene region on the sequence described in SEQ ID NO: 1
220-240 5'-gaagaagtttgagactctctc-3 '(SEQ ID NO: 32)
223-244 5'-gaagtttgagactctctcttac-3 '(SEQ ID NO: 33)
226-245 5'-gtttgagactctctcttacc-3 '(SEQ ID NO: 34)
434-453 5'-gctcaagtgttgaaggaagt-3 '(SEQ ID NO: 35)
449-468 5'-gaagtggaagagtgcaagaa-3 '(SEQ ID NO: 36)

<Design of RBCS-1A primer>
An RBCS-1A primer to be applied to the LAMP method was obtained from the sequence of the selected gene region described above.

FIP: (Concatenation of base sequence of region F1c and base sequence of region F2)
ARFI-25 (SEQ ID NO: 37) Linking the sequences of SEQ ID NOs: 2 and 6
ARFI-936 (SEQ ID NO: 38) Linking the sequences of SEQ ID NOs: 3 and 7
ARB-D2-FAd1 (SEQ ID NO: 39) Linking the sequences of SEQ ID NOs: 4 and 8
ARB-D1-FAd1 (SEQ ID NO: 40) Linking the sequences of SEQ ID NOs: 5 and 9

RIP: (Concatenated region R1c base sequence and region R2 base sequence
ARRI-25 (SEQ ID NO: 41) Link SEQ ID NO: 14 and 20 or 15 and 19
ARRI-936 (SEQ ID NO: 42) Linking the sequences of SEQ ID NOs: 16 and 19
ARB-D2-RAd1 (SEQ ID NO: 43) Linking the sequences of SEQ ID NOs: 17 and 21
ARB-D1-RAd1 (SEQ ID NO: 44) Linking the sequences of SEQ ID NOs: 18 and 22

F3 primer: (Same sequence as the base sequence of F3 region)
ARF0-25 (SEQ ID NO: 10)
ARF0-936 (SEQ ID NO: 11)
ARB-D2-F3d1 (SEQ ID NO: 12)
ARB-D1-F3d1 (SEQ ID NO: 13)

R3 primer: (sequence that is the same as or partially mutated with the base sequence of the R3 region)
ARRO-936-dai2 (SEQ ID NO: 23)
ARRO-25 (SEQ ID NO: 24)
ARRO-936-F6 (SEQ ID NO: 25)
ARB-D2-R3d1 (SEQ ID NO: 26)
ARB-D1-R3d1 (SEQ ID NO: 27)
ARB-D1-R3d2 5'-cactgggcttgacgggtg-3 '(SEQ ID NO: 45)
(ARB-D1-R3d2 is obtained by replacing the seventh base a of ARB-D1-R3d1 with g)

Loop primer F: (same sequence as the base sequence of loop F region)
ARFL-25c-F2 (SEQ ID NO: 28)
ARFL-936c (SEQ ID NO: 29)
ARB-D2-LPFd1 (SEQ ID NO: 30)
ARB-D1-LPFd1 (SEQ ID NO: 31)

Loop primer R: (same sequence as the base sequence of the loop R region)
ARRL-936-d2 (SEQ ID NO: 32)
ARRL-936-F8R4 (SEQ ID NO: 33)
ARRL-936 (SEQ ID NO: 34)
ARB-D2-LPRd1 (SEQ ID NO: 35)
ARB-D1-LPRd1 (SEQ ID NO: 36)

(Experimental example 1)
In Experimental Example 1, RT-LAMP was performed using various RBCS-1A primers, and it was confirmed whether these primers could detect RBCS-1A.

i) Preparation of RNA sample By performing PCR using a primer for PCR designed based on the base sequence of RBCS-1A, a part of RBCS-1A cDNA (specifically, from Arabidopsis thaliana cDNA (manufactured by Nippon Gene)) , 28th to 571th positions of the nucleotide sequence of SEQ ID NO: 1, hereinafter referred to as RBCS-1A amplification region). This RBCS-1A amplification region was incorporated into a plasmid vector (pCRII-TOPO (manufactured by Invitrogen)), and this plasmid vector was transformed into E. coli. Furthermore, after culturing this Escherichia coli and purifying a plasmid vector from Escherichia coli, an RNA containing a transcription product (mRNA corresponding to the RBCS-1A amplification region) using an in vitro transcription system (Riboprobe in vitro transcription system (manufactured by Promega)). A solution was obtained. The RNA concentration of this solution was calculated by absorbance measurement (260 nm), and based on this value, 50 ng / mL yeast RNA (manufactured by Amibon) was used so that the copy numbers of RBCS-1A mRNA were 37000000, 370000, and 3700. This was used as a template used in this experimental example.

ii) Primer set Various primers were used in the combinations shown in Table 1 (primer sets 1 to 6). The numbers shown in the column of each primer in the table indicate the SEQ ID No., indicating that a primer comprising an oligonucleotide having the base sequence represented by each SEQ ID No. was used as a set.

iii) RT-LAMP reaction solution composition Each component having the concentration shown below was mixed in the volume shown below to prepare an RT-LAMP reaction solution.
Purified water for molecular biology (Eppendorf) 3.97μL
750 mM (pH 8.0) Tris-HCl (Nippon Gene) 1.00 μL
10 × ThermoPol Buffer (Daiichi Kagaku) 2.50 μL
10 mM dNTPs (manufactured by Invitrogen) 2.00 μL
100 mM MgSO ₄ (manufactured by Nacalai tex) 0.75 μL
100 mM dithiothreitol (Wako Pure Chemical Industries) 1.25 μL
2% Tergitol (manufactured by Sigma) 2.50 μL
10 U / μL AMV reverse transcriptase (manufactured by Promega) 0.14 μL
8U / μL BstDNA polymerase (manufactured by New England)
2.27 μL
RNase inhibitor (manufactured by Promega) 0.63 μL
Primer 80 pmol / μL FIP 1.00 μL
80 pmol / μL RIP 1.00 μL
5 pmol / μL F3 primer 1.00 μL
5 pmol / μL R3 primer 1.00 μL
60 pmol / μL Loop primer F 1.00 μL
60 pmol / μL Loop primer R 1.00 μL
23.00μL in total

iv) Nucleic acid amplification by RT-LAMP method and measurement of amplification product To 23 μL of the RT-LAMP reaction solution containing the above six primers, 2 μL of the RNA sample described above is added and heated at 65 ° C. for 1 hour for nucleic acid amplification. went. The white turbidity of insoluble magnesium pyrophosphate produced as a byproduct simultaneously with nucleic acid amplification was measured in real time. LA-200 manufactured by Teramex was used for nucleic acid amplification and measurement.
In the amplification of the RBCS-1A amplification region by the RT-LAMP method when using primer sets 1 to 6, the time when the turbidity reached 0.1 was measured. Nucleic acid amplification and turbidity measurement of the amplification product were performed four times, and the average value was also calculated.

v) Results Table 2 shows the measurement results.

  From Table 2, the amplification rise time was 8.7 to 11.4 minutes when an RNA sample containing 37000000 copies of mRNA in the RBCS-1A amplification region was used as a template. The amplification rise time was 10.3 to 13.3 minutes when an RNA sample containing 370000 copies of mRNA in the RBCS-1A amplification region was used as a template. When an RNA sample containing 3700 copies of mRNA in the RBCS-1A amplification region was used as a template, the amplification rise time was 11.5 to 16.3 minutes.

  From the above, it was found that the RBCS-1A amplification region was amplified within 17 minutes in any case. That is, it was confirmed that when each primer set shown in Table 1 was used, the RBCS-1A amplification region could be rapidly amplified and RBCS-1A could be detected.

(Experimental example 2)
In Experimental Example 2, RT-LAMP is performed using an R3 primer (SEQ ID NO: 45) obtained by adding a mutation to a part of the R3 primer of SEQ ID NO: 27, and RBCS-1A is amplified even using a primer having a mutation. Confirmed whether or not.

i) Preparation of RNA sample As in Experimental Example 1, except that the RBCS-1A mRNA was prepared by dilution with 50 ng / mL yeast RNA (manufactured by Amibon) so that the copy number of mRNA was 11000000, 11000000, and 110,000. RNA samples were prepared.

ii) Primer set Primer set 5 in Table 1 and a primer set (hereinafter referred to as primer set 7) using the R3 primer of primer set 5 as the primer of SEQ ID NO: 45 were used.
The R3 primer of SEQ ID NO: 45 is obtained by replacing adenine, which is the seventh base of the R3 primer of SEQ ID NO: 27, with guanine. That is, the seventh base sequence of the R3 primer of SEQ ID NO: 45 is not identical to the seventh base sequence of the R3 region of RBCS-1A.

iii) RT-LAMP reaction solution composition The same RT-LAMP reaction solution as in Experimental Example 1 was used.

iv) Nucleic acid amplification by RT-LAMP method and measurement of amplification product The nucleic acid amplification and amplification product were measured in the same manner as in Experimental Example 1 except that the measurement was performed twice.

v) Results Table 3 shows the measurement results.

  From Table 3, when performing nucleic acid amplification of RBCS-1A with primer set 7 containing an R3 primer having a single base substitution in the base sequence, than when performing nucleic acid amplification with primer set 5 containing an R3 primer without mutation, It was confirmed that the nucleic acid of RBCS-1A shown in SEQ ID NO: 1 could be amplified only when the time until the turbidity reached 0.1 was the same or slightly delayed.

(Experimental example 3)
In Experimental Example 3, human cytokeratin 19 (hereinafter referred to as CK19) mRNA, which is a cancer marker, and RBCS-1A mRNA were used as templates in the presence or absence of an inhibitor by the RT-LAMP method. Each cDNA was amplified and analyzed how the inhibitory substance affects each nucleic acid amplification.

i) Preparation of solubilizing reagent A solubilizing solution containing the following components was prepared.
200 mM (pH 3.0) Glycin-HCl
(Glycin and HCl are both manufactured by Wako Pure Chemical Industries, Ltd.)
5% Brij35 (manufactured by Sigma)
0.05% KS-538 (manufactured by Shin-Etsu Chemical)

ii) Preparation of lysate First, 4 mL of a solubilizing reagent was added to a human lymph node without cancer metastasis, and homogenized at 25000 rpm with an electric homogenizer. The obtained homogenate was centrifuged at 15000 rpm, and the supernatant was used as a lysate in this experimental example. This lysate contains a substance that inhibits nucleic acid amplification by the RT-LAMP method (hereinafter referred to as an inhibitory substance).

iii) Preparation of measurement samples a to h Measurement samples a to h were prepared with the compositions shown below.
Sample for measurement a: Prepared by adding 2 μL of a solubilizing reagent containing 1300000 copies of CK19 mRNA to 23 μL of the RT-LAMP reaction solution prepared in Experimental Example 1.
Sample b for measurement: Prepared by adding 2 μL of a solubilizing reagent containing 13,000 copies of CK19 mRNA to 23 μL of the RT-LAMP reaction solution.
Sample c for measurement: Prepared by adding 2 μL of a solubilizing reagent containing 1100000 copies of RBCS-1A mRNA to 23 μL of RT-LAMP reaction solution.
Measurement sample d: Prepared by adding 2 μL of a solubilizing reagent containing 11,000 copies of RBCS-1A mRNA to 23 μL of RT-LAMP reaction solution.
Sample for measurement e: prepared by adding 2 μL of lysate containing 1300000 copies of CK19 mRNA to 23 μL of RT-LAMP reaction solution.
Sample f for measurement: Prepared by adding 2 μL of lysate containing 13,000 copies of CK19 mRNA to 23 μL of RT-LAMP reaction solution.
Sample for measurement g: Prepared by adding 2 μL of lysate containing 1100000 copies of RBCS-1A mRNA to 23 μL of RT-LAMP reaction solution.
Sample for measurement h: Prepared by adding 2 μL of lysate containing 11000 copies of RBCS-1A mRNA to 23 μL of RT-LAMP reaction solution.
That is, since the measurement samples a to d do not contain lysate, no inhibitor is contained, and the measurement samples e to h contain lysate, and thus contain an inhibitor.
The outline of the composition of the measurement samples a to h is shown in FIG.

iv) Nucleic acid amplification by RT-LAMP method and measurement of amplification product Using LA-200 manufactured by Teramex, white turbidity of insoluble magnesium pyrophosphate produced as a by-product simultaneously with nucleic acid amplification was measured.
Time was measured by the RT-LAMP method until cDNA corresponding to mRNA contained in each measurement sample was amplified and turbidity reached 0.1. The measurement was performed twice for each measurement sample, and the average value was also calculated.

v) Results Table 4 shows the measurement results.

A graph of Table 4 is shown in FIG.
In FIG. 2, ● indicates the time when the turbidity reached 0.1 when the CK19 nucleic acid was amplified in the absence of the inhibitor (hereinafter referred to as turbidity 0.1 arrival time).
(Circle) shows turbidity 0.1 arrival time when the nucleic acid of RBCS-1A is amplified in the absence of an inhibitor.
(2) shows the time to reach turbidity of 0.1 when the nucleic acid of CK19 is amplified in the presence of an inhibitor.
□ indicates the time to reach turbidity of 0.1 when RBCS-1A nucleic acid is amplified in the presence of an inhibitor.
Comparing ● and ■, it can be seen how much time the turbidity 0.1 arrival time was delayed in the nucleic acid amplification of CK19 by the inhibitor.
In addition, when ◯ and □ are compared, it can be seen how much time the turbidity 0.1 arrival time was delayed in the nucleic acid amplification of RBCS-1A by the inhibitor.
From Table 4 and FIG. 2, it was confirmed that the influence of the inhibitor on the nucleic acid amplification of CK19 and the influence of the inhibitor on the nucleic acid amplification of RBCS-1A are comparable.

Conventionally, when CK19 nucleic acid of unknown concentration contained in a sample suspected of having an inhibitory substance was measured, it was difficult to determine whether the CK19 nucleic acid was affected by the inhibitory substance. In addition, it was not possible to know how much the inhibitor was affected when it was affected by the inhibitor.
By using RBCS-1A nucleic acid of known concentration from the results of Experimental Example 3 as an internal standard, it can be confirmed whether or not the inhibitor has an effect on CK19 nucleic acid amplification and RBCS-1A nucleic acid amplification. Has affected the nucleic acid amplification, the amplification rise time of the CK19 nucleic acid can be corrected based on the degree of the influence of the nucleic acid amplification of RBCS-1A. That is, even when the inhibitory substance affects the nucleic acid amplification of CK19, the copy number of the nucleic acid of CK19 contained in the measurement sample before the nucleic acid amplification reaction is accurately calculated based on the corrected amplification rise time. Will be able to.

The outline of the composition of measurement samples a to h. Effect of inhibitors on nucleic acid amplification of CK19 and RBCS-1A.

Claims (20)

  A primer for nucleic acid amplification for detecting ribulose bisphosphate carboxylase small chain 1A (RBCS-1A) gene and / or mRNA of said gene by LAMP method.   The primer for nucleic acid amplification according to claim 1, wherein the gene is derived from a plant belonging to the genus Arabidopsis. RBCS-1A gene comprising an oligonucleotide consisting of a sequence selected from 1) to 5) shown below and / or a nucleic acid amplification primer for detecting mRNA of the gene;
1) An oligonucleotide selected from the 50th to 530th region of the base sequence represented by SEQ ID NO: 1 and the region of its complementary strand, and comprising at least 5 or more consecutive bases of SEQ ID NO: 1 and / or its complementary strand.
2) An oligonucleotide having a base sequence represented by any one of SEQ ID NOs: 2-45.
3) A complementary strand of the oligonucleotide according to 1) or 2).
4) An oligonucleotide that can hybridize with the oligonucleotide according to any one of 1) to 3) under stringent conditions.
5) An oligonucleotide having a primer function, wherein one to seven bases among the oligonucleotides described in 1) to 4) above include a mutated base sequence such as substitution, deletion, insertion, or addition.   A nucleic acid amplification primer for detecting an RBCS-1A gene and / or mRNA of the gene comprising an oligonucleotide selected from any one of the nucleotide sequences represented by SEQ ID NOs: 10 to 45.   The nucleic acid amplification primer according to claim 3 or 4, wherein the nucleic acid amplification means is a LAMP method. Nucleic acid amplification for detecting RBCS-1A gene and / or mRNA of said gene, wherein at least two kinds of primers comprising an oligonucleotide consisting of a sequence selected from 1) to 5) shown below are selected Primer set;
1) An oligonucleotide selected from 50 to 530 of the base sequence represented by SEQ ID NO: 1 and the region of its complementary strand, and comprising at least 5 or more consecutive bases of SEQ ID NO: 1 and / or its complementary strand.
2) An oligonucleotide having a base sequence represented by any one of SEQ ID NOs: 2-45.
3) A complementary strand of the oligonucleotide according to 1) or 2).
4) An oligonucleotide that can hybridize with the oligonucleotide according to any one of 1) to 3) under stringent conditions.
5) An oligonucleotide having a primer function, wherein one to seven bases among the oligonucleotides described in 1) to 4) above contain a mutated base sequence such as substitution, deletion, insertion or addition.   The nucleic acid amplification primer set according to claim 6, wherein the nucleic acid amplification means is a LAMP method.   The primer set for nucleic acid amplification according to claim 6 or 7 for detecting RBCS-1A gene and / or mRNA of said gene, wherein at least four kinds are selected from primers for nucleic acid amplification containing said oligonucleotide. .   The nucleic acid amplification primer included in the nucleic acid amplification primer set recognizes at least two regions each of the nucleotide sequence represented by SEQ ID NO: 1 and / or its complementary strand. Item 9. A primer set for nucleic acid amplification according to any one of Items 6 to 8.   The nucleic acid amplification primer contained in the nucleic acid amplification primer set recognizes at least six regions of the base sequence represented by SEQ ID NO: 1 and / or its complementary strand. A primer set for nucleic acid amplification according to any one of the above.   A nucleic acid amplification primer for detecting an RBCS-1A gene and / or mRNA of the gene comprising an oligonucleotide having a base sequence represented by SEQ ID NOs: 10 to 45, wherein (a) SEQ ID NOs: 37 to 40 and (b) A primer set for nucleic acid amplification comprising a combination in which one nucleic acid amplification primer is selected from each classification when classified into SEQ ID NOs: 41 to 44.   A primer set for nucleic acid amplification according to claim 11, and when classifying into (c) SEQ ID NO: 10 to 13 and (d) SEQ ID NO: 23 to 27 and 45, one nucleic acid amplification primer was selected from each classification A primer set for nucleic acid amplification comprising the combination.   A primer set for nucleic acid amplification according to claim 12, and a combination of (e) SEQ ID NOs: 28 to 31 and (f) selected one primer from each classification when classified into SEQ ID NOs: 32-36. A primer set for nucleic acid amplification characterized by the above. RBCS-1A gene comprising any one of 1) to 5) below and / or a nucleic acid amplification primer set for detecting mRNA of the gene;
1) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 38, 42, 11, and 25 as nucleic acid amplification primers.
2) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, and 24 as nucleic acid amplification primers.
3) A primer set for nucleic acid amplification comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, and 23 as primers for nucleic acid amplification.
4) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 40, 44, 13, and 27 as nucleic acid amplification primers.
5) A primer set for nucleic acid amplification containing oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 39, 43, 12, and 26 as primers for nucleic acid amplification. RBCS-1A gene comprising any one of 1) to 6) below and / or a primer set for nucleic acid amplification for detecting mRNA of the gene;
1) A primer set for nucleic acid amplification comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 38, 42, 11, 25, 29, and 34 as nucleic acid amplification primers.
2) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 38, 42, 11, 25, 29, and 32 as nucleic acid amplification primers.
3) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, 24, 28, and 33 as nucleic acid amplification primers.
4) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 37, 41, 10, 23, 28, and 33 as nucleic acid amplification primers.
5) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 40, 44, 13, 27, 31, and 36 as nucleic acid amplification primers.
6) A nucleic acid amplification primer set comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 39, 43, 12, 26, 30, and 35 as nucleic acid amplification primers.   By selecting a necessary nucleic acid amplification primer from the nucleic acid amplification primers according to claim 3 or 4, or by selecting and using the nucleic acid amplification primer set according to any one of claims 5 to 15. Nucleic acid detection method.   The nucleic acid detection method according to claim 16, wherein the LAMP method is used for nucleic acid detection.   The reagent used for the nucleic acid detection method of Claim 16 or 17.   The reagent kit used for the nucleic acid detection method of Claim 16 or 17. RBCS-1A gene and / or mRNA of said gene is used as an internal standard.

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