WO2007122940A1 - Method of constructing artificially positive control dnas for multiplex pcr - Google Patents

Method of constructing artificially positive control dnas for multiplex pcr Download PDF

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Publication number
WO2007122940A1
WO2007122940A1 PCT/JP2007/055598 JP2007055598W WO2007122940A1 WO 2007122940 A1 WO2007122940 A1 WO 2007122940A1 JP 2007055598 W JP2007055598 W JP 2007055598W WO 2007122940 A1 WO2007122940 A1 WO 2007122940A1
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Prior art keywords
positive control
primer
dna
artificial positive
multiplex pcr
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PCT/JP2007/055598
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French (fr)
Japanese (ja)
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Koji Suzuki
Shizuka Asano
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Asahi Breweries, Ltd.
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Publication of WO2007122940A1 publication Critical patent/WO2007122940A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention relates to a method for producing an artificial positive control DNA group for multiplex PCR.
  • the multiplex PCR method (see, for example, Patent Document 1), in which a large number of PCR primer pairs are mixed in a single reaction system, has the advantage that multiple bacterial species can be tested simultaneously.
  • the number of reaction tubes for the positive control DNA is required by the number of the corresponding individual bacterial species, and there remains a problem that the number of test points increases.
  • Non-Patent Documents 1 and 2 there is an example (for example, see Non-Patent Documents 1 and 2) in which a plurality of positive controls (DNA of bacteria to be tested) are mixed and subjected to PCR reaction.
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2005-34121
  • Non-Patent Document 1 J. Appl. Microbiol. 2004; 97 (2): 384-394
  • Non-Patent Document 2 Mol. Cell. Probes 2005; 19 (5): 314-322
  • each positive control band can be clearly distinguished, and the reactivity of all primer pairs can be determined.
  • the purpose was to provide a method for preparing an artificial positive control DNA for multiplex PCR that can be confirmed in one reaction system.
  • the present inventors have completed the present invention based on these findings.
  • the present invention according to claim 1 is characterized in that the following steps (a) to (c) are performed for each primer for multiplex PCR, and an artificial positive control for multiplex PCR is provided. This is a method for producing DNA groups.
  • step (c) Artificial positive control DNA, which is an amplification product obtained by performing PCR reaction using the artificial positive control preparation primer pair synthesized in step (b), with DNA having the known available sequence as a saddle type Process
  • the present invention according to claim 2 is a multiplex manufactured by the method according to claim 1. This is a method for confirming the reactivity of primers for multiplex PCR, characterized in that the artificial positive control DNA group for PCR is subjected to PCR reaction in the same reaction solution.
  • the bands of each positive control can be clearly distinguished, and amplification reactions of all the formulated primers can be performed. It can be confirmed in one reaction system. This leads to simplification of test methods, prevention of erroneous operations, and reduction of test costs.
  • the present invention even if a positive control is mixed into sample DNA, the length of the amplified product of the sample and that of the positive control are different, so that erroneous determination can be prevented.
  • FIG. 1 is a diagram showing an electrophoresis result showing a result of PCR reaction using an artificial positive control DNA mixed solution in Examples 1 and 2.
  • FIG. 2 is a diagram showing a region (portion surrounded by the mouth) used for the artificial positive control DNA of Example 2 on the ⁇ phage DNA sequence and the position of the primer for preparing the artificial positive control (underlined portion).
  • FIG. 3 is a diagram showing an electrophoresis result showing a result of PCR reaction using an artificial positive control DNA mixed solution in Example 3.
  • the present invention according to claim 1 is a method for producing an artificial positive control DNA group for multiplex PCR.
  • the artificial positive control DNA for multiplex PCR refers to a multiplex PCR method in which two or more primer pairs are used in one reaction system to carry out a PCR reaction.
  • the multiplex PCR primer used in the present invention may be any type.
  • the step (a) of selecting a region having any number of bases between 50 and 2000 bases is performed.
  • examples of DNA having a known sequence include ⁇ phage DNA having a known base sequence registered in Gen Bank as Accession No .: V 00636, J02459, M17233, X00906, and ⁇ X174 phage Force that can use DNA (Accession No. NC-001422) etc. It is not limited to this. However, in order to prevent false positives that occur when the sample DNA is mixed with the positive control DNA, it is preferable to use the DNA of microorganisms that are closely related to or the same species as the microbial species targeted by the multiplex PCR method.
  • the region used for the artificial positive control DNA of the present invention is arbitrarily selected.
  • the number of bases in the above region for the other primer pairs is completely different so that the bands of PCR amplification products by each primer pair appear to be separated in the electrophoresis result after the PCR reaction.
  • the number of bases in the region is preferably in the range of 50 to 2000 bases. Considering the number of bases in the multiplex PCR primer (usually around 10 to 30 bases), at least 50 bases are required for the amplification product. On the other hand, if it exceeds 2000 bases, the PCR reaction takes time and is not practical.
  • the regions used for the artificial positive control DNA do not overlap with each other in the DNA for which the available sequence is known. This is because a nonspecific amplification reaction may occur between artificial positive controls.
  • Step (a) 10 to 30 bases at the 5 'end of the region selected in step (a) are bound to the 3' end of the forward primer for multiplex PCR.
  • Artificial positive formed by binding a complementary sequence of 10-30 bases at the 3 'end of the region selected in step (a) to the 3rd side of the forward primer for artificial positive control and the reverse primer for multiplex PCR.
  • Step (b) of synthesizing a reverse primer for preparing a control and a primer pair for generating a powerful artificial positive control is performed.
  • artificial positive control DNA is prepared by PCR reaction. Therefore, an artificial positive control primer pair is synthesized by binding a sequence designed based on the DNA sequences at both ends of the region to a multiplex PCR primer pair.
  • each of these has a primer for multiplex PCR on the 5 ′ side and a sequence designed on the 3, side based on the DNA sequence at the 5, 5 ′ or 3 ′ end of the region.
  • the artificial positive control preparation forward primer of the present invention comprises the DNA sequence at the 5th and terminal ends of the region bound to the 3rd side of the multiplex PCR forward primer.
  • the number of bases of the DNA sequence at the 5 ′ end of the region is preferably 10 to 30 bases, preferably 15 to 25 bases.
  • the reverse primer for producing an artificial positive control of the present invention is obtained by binding a complementary DNA sequence to the 3 ′ end of the region on the 3 ′ side of the reverse primer for multiplex PCR.
  • the number of bases of the DNA sequence at the 3 ′ end of the region is preferably 10 to 30 bases, preferably 15 to 25 bases.
  • the number of bases of the sequence based on the DNA sequence at the 5 ′ end or 3 ′ end of the region is preferred, and if it is less than the range, the artificial positive control preparation primer is specific to the region. Cannot be combined. On the other hand, when the number of bases exceeds the preferred range, the cost of primer synthesis increases and is not practical.
  • the primer pair for producing an artificial positive control obtained as described above is specific to the above-mentioned region in the known DNA due to the portion designed based on the DNA sequences at both ends of the above-mentioned region. Can be combined.
  • a PCR reaction is carried out using a primer pair for preparing an artificial positive control, wherein the DNA having an already known sequence is known as a cage and synthesized in step (b).
  • Step (c) of producing an artificial positive control DNA, which is an amplification product obtained by performing the above steps, is performed.
  • the artificial positive control primer pair obtained in the step (b) is obtained by the portion designed based on the DNA sequences at both ends of the region selected in the step (a).
  • a possible sequence can bind specifically to the region in the known DNA. Therefore, by the PCR reaction in this step, in the above region, the DNA sequence to which the complementary sequence strength of the forward primer for multiplex PCR is added at the 5 ′ end and the reverse primer for multiplex PCR is added at the 3 ′ end is amplified product. (Artificial positive control DN A).
  • the artificial positive control DNA of the present invention is When used for CR primer pair reactivity test, artificial positive control DNA with the desired number of bases is amplified, and the primer pair reactivity is confirmed, for example, by examining the presence or absence of amplified product bands by electrophoresis. be able to.
  • the artificial positive control DNA of the present invention is preferably used after being purified by an ordinary nucleic acid purification method.
  • the respective steps (a) to (c) are individually performed to prepare an artificial positive control DNA.
  • the artificial positive control DNA corresponding to each pair can be diluted to an appropriate concentration and used as a mixed solution in the multiplex PCR method, so that the reactivity can be confirmed simultaneously for all primer pairs V. .
  • the artificial positive control DNA of the present invention has a different number of bases from the amplification region used for bacterial species identification, even when the positive control is mixed with the sample DNA in the preparation of a PCR reaction solution, etc. Misjudgment does not occur.
  • the present invention according to claim 2 is characterized in that the artificial positive control DNA group for multiplex PCR prepared by the method according to claim 1 is subjected to a PCR reaction in the same reaction solution. This is a method for confirming the reactivity of multiplex PCR primers.
  • the PCR reaction can be carried out by a conventional method, and the composition of the reaction solution, the enzyme used, the reaction conditions, etc. can be appropriately set.
  • a multiplex PCR method was constructed to detect and identify the following 6 Lactobacillus species of lactic acid bacteria that are harmful in beer production. Lactobacillus brevis, L. lindneri, L. paracolli
  • the primers shown in Table 1 were designed based on the 16S rRNA gene of noides, L. casei, L. coryniformis and L. plantarum and the known nucleotide sequence of the ITS region. In each primer pair, the forward primer is listed in the upper row and the reverse primer is listed in the lower row.
  • any region of the known base sequence of ⁇ phage DNA (Takara Bio) (SEQ ID NO: 9, Accession No .: V00636, J02459, M17233, X00906) in the artificial positive control DNA
  • the area to be used was selected for each bacterial species listed in Table 1.
  • a forward primer for preparing an artificial positive control in which about 20 bases at the 5 ′ end of the region selected above were bound to the 3 ′ side of the forward primer, and the 3 ′ side of the reverse primer.
  • a reverse primer for preparing an artificial positive control was designed, to which a complementary sequence of about 20 bases at the 3 ′ end of the region selected above was bound.
  • Table 2 shows the names of multiplex PCR primers corresponding to each bacterial species and the corresponding primer names for artificial positive control preparation.
  • sequences of artificial positive control preparation primers are shown in Table 3 (underlined indicates the primer sequence portion for multiplex PCR in Table 1).
  • the forward primer is described in the upper part and the reverse primer is described in the lower part.
  • a PCR reaction using a diluted solution of ⁇ phage DNA (Takara Bio Inc.) as a cage was performed for each bacterial species.
  • the PCR reaction was performed using the Takara Bio PerfectShot TM Ex Taq with the composition shown in Table 4.
  • the reaction was performed using Applied Biosystems GeneAmp PCR System 9700 under the reaction conditions shown in Table 5.
  • Table 6 shows the estimated base pairs of PCR reaction amplification products contained in the artificial positive control solution when each primer pair was used.
  • the artificial positive control solutions corresponding to each of the 6 lactic acid bacteria Prepare and mix 100-5000-fold diluted solution as an artificial positive control DNA mixture VVV
  • An electrophoretogram is shown in FIG. From the electrophoretic pattern, amplified fragments of 6 different molecular weights were clearly detected. Since the molecular weights of these amplified fragments coincided with the estimated base pairs in Table 5, the reactivity of all primers could be confirmed.
  • a multiplex PCR method was established to detect two Pectinatus species, which are harmful bacteria in beer production.
  • the primers shown in Table 8 were designed based on the 16S rRNA gene of Pectinatus frisingensis and P. cerevisiiphilus and the known nucleotide sequence of the ITS region. In each primer pair, the forward primer is listed in the upper row and the reverse primer is listed in the lower row.
  • P ec t inat us cer ev is i iphi lus 16C-F (SEQ ID NO: 22) CGTATGCAGAGATGCATATT
  • an artificial positive is detected in an arbitrary region (specifically, a portion surrounded by a mouth in FIG. 2) of a known base sequence (SEQ ID NO: 9 in the sequence listing) of ⁇ phage DNA (Takara Bio Inc.).
  • the region used for control DNA was selected for each bacterial species listed in Table 8.
  • the artificial primer control forward primer to which about 20 bases at the 5 and end of the region selected above were bound on the 3 ′ side of the forward primer, on the 3 ′ side of the reverse primer.
  • a reverse primer for preparing an artificial positive control was designed, to which a complementary sequence of about 20 bases at the 3 ′ end of the region selected above was bound.
  • Table 9 shows the multiplex PCR primer names corresponding to each bacterial species and the corresponding artificial positive control primer names.
  • sequences of primers for preparation of artificial positive controls are shown in Table 10 (underlined indicates the primer sequence portion for multiplex PCR in Table 8).
  • the forward primer is listed in the upper row and the reverse primer is listed in the lower row.
  • Fig. 2 shows the region used for each artificial positive control DNA on the ⁇ phage DNA sequence (Accession No .: V00636, J02459, M17233, X0090 6) (the part surrounded by the mouth) and each artificial positive control preparation. Shows the position (underlined part) of the primer.
  • Pectinatus cerev is i i hi lus 16C-F AP0SIFPC2
  • Table 11 shows the estimated base pairs of PCR reaction amplification products contained in each artificial positive control solution.
  • the obtained artificial positive control solution (30 ng / ⁇ 1) was diluted in a range of 100 to 5000 times and mixed to obtain an artificial positive control DNA mixture.
  • the artificial positive control DNA mixture 51 was added to 45 ⁇ 1 of the Pectinatus multiplex PCR reaction solution described above, and a PCR reaction was performed under the PCR reaction conditions shown in Table 7.
  • GeneAmp PCR System 9700 from Applied Biosystems was used for the reaction.
  • the multiplex PCR primer sequences designed for each bacterial species are listed in Table 12, the artificial positive control primer names corresponding to the multiplex PCR primers are listed in Table 13, and the artificial positive control primer The sequences are shown in Table 14 (underlined indicates the primer sequence portion for multiplex PCR in Table 12).
  • Table 15 shows the estimated base pairs of PCR reaction amplification products contained in each artificial positive control solution.
  • the obtained artificial positive control solution (30 ng / ⁇ 1) was diluted in a range of 100 to 5000 times and mixed to obtain an artificial positive control DNA mixture.
  • the electrophoresis photograph is shown in FIG. From the electrophoresis pattern, amplified fragments of three different molecular weights were clearly detected. Since the molecular weights of these amplified fragments were in agreement with the estimated base pairs in Table 15, the reactivity of all primers could be confirmed.

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Abstract

It is intended to provide a method of constructing artificial control DNAs for multiplex PCR characterized by comprising conducting the following steps (a) to (c) for each multiplex PCR primer: (a) the step of selecting a region having an arbitrary number (from 50 to 2000) of bases in an available DNA having a known sequence; (b) the step of synthesizing a primer pair for constructing an artificial positive control consisting of a forward primer for constructing an artificial positive control wherein 10 to 30 bases at the 5’-terminus of the region selected in the above step (a) is attached to the 3’-side of a forward primer for multiplex PCR and a reverse primer for constructing an artificial positive control wherein 10 to 30 bases at the 3’-terminus of the region selected in the above step (a) is attached to the 3’-side of a reverse primer for multiplex PCR; and (c) the step of using the available DNA having a known sequence as described above as a template and conducting PCR by using the primer pair for constructing an artificial positive control having been synthesized in the above step (b) to thereby construct an artificial positive control DNA as the amplification product.

Description

マルチプレックス PCR用人工陽性対照 DNA群の作製法  Preparation of artificial positive control DNA group for multiplex PCR
技術分野  Technical field
[0001] 本発明は、マルチプレックス PCR用人工陽性対照 DNA群の作製法に関する。  [0001] The present invention relates to a method for producing an artificial positive control DNA group for multiplex PCR.
背景技術  Background art
[0002] 微生物の菌種を同定するために用いられる PCR法では、 PCR反応の信頼性を保証 するために、菌種特異的プライマー対により正常に増幅反応が起こることを確認しな ければならない。そのため、従来は、検査対象とする菌の DNAを陽性対照とする PCR 反応によって、プライマー対の反応性検査が行われて 、た。  [0002] In the PCR method used to identify the species of microorganisms, in order to guarantee the reliability of the PCR reaction, it is necessary to confirm that the amplification reaction normally occurs by the species-specific primer pair. . Therefore, conventionally, the reactivity test of primer pairs has been performed by PCR reaction using the DNA of the bacteria to be tested as a positive control.
多数の PCRプライマー対を単一の反応系で混合するマルチプレックス PCR法（例え ば特許文献 1参照)では、複数の菌種を一斉に検査できる利点がある。しかし、各プ ライマー対の反応を確認するためには、対応する個々の菌種の数だけ陽性対照 DN A用の反応チューブ数が必要とされ、試験点数が多くなるという問題点が残る。  The multiplex PCR method (see, for example, Patent Document 1), in which a large number of PCR primer pairs are mixed in a single reaction system, has the advantage that multiple bacterial species can be tested simultaneously. However, in order to confirm the reaction of each primer pair, the number of reaction tubes for the positive control DNA is required by the number of the corresponding individual bacterial species, and there remains a problem that the number of test points increases.
また、陽性対照 (検査対象とする菌の DNA)を複数混合して PCR反応に供して ヽる 例 (例えば非特許文献 1、 2参照)もある。  In addition, there is an example (for example, see Non-Patent Documents 1 and 2) in which a plurality of positive controls (DNA of bacteria to be tested) are mixed and subjected to PCR reaction.
し力しながら、マルチプレックス PCR法において、人工陽性対照を使用した例は未 だ報告がない。  However, there have been no reports on the use of artificial positive controls in the multiplex PCR method.
[0003] 特許文献 1 :特開 2005— 34121号公報 [0003] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2005-34121
非特許文献 1 :J. Appl. Microbiol. 2004; 97(2): 384-394  Non-Patent Document 1: J. Appl. Microbiol. 2004; 97 (2): 384-394
非特許文献 2 :Mol. Cell. Probes 2005; 19(5): 314-322  Non-Patent Document 2: Mol. Cell. Probes 2005; 19 (5): 314-322
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0004] このように、従来、微生物種同定用プライマー対の反応性検査における陽性対照と しては、検査対象とする微生物自体の DNAが用いられて 、た。 [0004] Thus, conventionally, as a positive control in the reactivity test of a primer pair for identifying a microorganism species, the DNA of the microorganism to be tested has been used.
しカゝしながら、 PCR反応を行う際、操作上のミスによりサンプル DNAに陽性対照 DNA が混入した場合は、サンプルが本来は陰性であるにもかかわらず、陽性と誤判定され るおそれがあった。 また、各プライマー対の増幅産物の長さが類似しているため、複数のプライマー対 を同時に検査する場合には、電気泳動によりそれぞれのバンドが明確に分離しない という問題もあった。 However, when a PCR reaction is performed, if a positive control DNA is mixed into the sample DNA due to an operational error, the sample may be falsely judged positive even though it is originally negative. It was. In addition, since the lengths of the amplification products of each primer pair are similar, there is also a problem that when examining a plurality of primer pairs at the same time, the respective bands are not clearly separated by electrophoresis.
[0005] したがって、本発明では、誤判定を防止でき、かつ、任意の塩基数を有して 、るた め、それぞれの陽性対照のバンドが明確に区別でき、全てのプライマー対の反応性 を 1反応系で確認できるマルチプレックス PCR用人工陽性対照 DNAの作製方法を提 供することを目的とした。  [0005] Therefore, in the present invention, misjudgment can be prevented and an arbitrary number of bases can be used. Therefore, each positive control band can be clearly distinguished, and the reactivity of all primer pairs can be determined. The purpose was to provide a method for preparing an artificial positive control DNA for multiplex PCR that can be confirmed in one reaction system.
課題を解決するための手段  Means for solving the problem
[0006] そこで、本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、任意の塩 基数を有する人工陽性対照 DNAを用いることにより、複数のプライマー対の反応性を 1反応系で確認できることを見出した。さらに、 λファージ DNAなど容易に入手可能 な汎用の DNAを利用して、そのような人工陽性対照 DNAを簡便に作製できる方法を 開発した。 [0006] Therefore, as a result of intensive studies to solve the above problems, the present inventors have made the reactivity of a plurality of primer pairs one reaction system by using an artificial positive control DNA having an arbitrary number of base groups. I found out that In addition, we developed a method that allows simple production of such artificial positive control DNA by using readily available general-purpose DNA such as λ phage DNA.
本発明者らは、これらの知見に基づいて本発明を完成させるに至ったのである。  The present inventors have completed the present invention based on these findings.
[0007] すなわち、請求項 1に係る本発明は、マルチプレックス PCR用の各プライマーにつ いて、以下の工程 (a)〜(c)を行うことを特徴とする、マルチプレックス PCR用人工陽性 対照 DNA群の作製法である。 That is, the present invention according to claim 1 is characterized in that the following steps (a) to (c) are performed for each primer for multiplex PCR, and an artificial positive control for multiplex PCR is provided. This is a method for producing DNA groups.
(a)入手可能な配列が既知の DNAにお!/、て、塩基数 50〜2000の間で任意の塩基数 を有する領域を選択する工程、  (a) a step of selecting a region having an arbitrary number of bases between 50 and 2000 bases in DNA having an available sequence known!
(b)マルチプレックス PCR用フォワードプライマーの 3，側に、工程 (a)で選択した領域の 5'末端の 10〜30塩基を結合させてなる人工陽性対照作製用フォワードプライマーと 、マルチプレックス PCR用リバースプライマーの 3，側に、工程 (a)で選択した領域の 3， 末端の 10〜30塩基の相補的配列を結合させてなる人工陽性対照作製用リバースプ ライマーと、力もなる人工陽性対照作製用プライマー対を合成する工程、および、 (b) An artificial positive control preparation forward primer in which 10 to 30 bases at the 5 ′ end of the region selected in step (a) are bound to the 3rd side of the forward primer for multiplex PCR, and for multiplex PCR. A reverse primer for preparing an artificial positive control in which a complementary sequence of 10 to 30 bases at the end and 3 of the region selected in step (a) is bound to the third side of the reverse primer, and an artificial positive control that has strength. Synthesizing a primer pair; and
(c)前記入手可能な配列が既知の DNAを铸型とし、工程 (b)で合成した人工陽性対照 作製用プライマー対を用いて PCR反応を行って得られる増幅産物である人工陽性対 照 DNAを作製する工程 (c) Artificial positive control DNA, which is an amplification product obtained by performing PCR reaction using the artificial positive control preparation primer pair synthesized in step (b), with DNA having the known available sequence as a saddle type Process
請求項 2に係る本発明は、請求項 1に記載の方法により作製されたマルチプレック ス PCR用人工陽性対照 DNA群を同一反応液中で PCR反応に供することを特徴とする 、マルチプレックス PCR用プライマーの反応性を確認する方法である。 The present invention according to claim 2 is a multiplex manufactured by the method according to claim 1. This is a method for confirming the reactivity of primers for multiplex PCR, characterized in that the artificial positive control DNA group for PCR is subjected to PCR reaction in the same reaction solution.
発明の効果  The invention's effect
[0008] 本発明によれば、任意の塩基数を有する人工陽性対照 DNAを用いるため、それぞ れの陽性対照のバンドが明確に区別でき、配合されたすベてのプライマーの増幅反 応を 1反応系で確認することが可能となる。このことにより、試験法の簡易化、誤操作 の防止、試験費用の低減につながる。  [0008] According to the present invention, since an artificial positive control DNA having an arbitrary number of bases is used, the bands of each positive control can be clearly distinguished, and amplification reactions of all the formulated primers can be performed. It can be confirmed in one reaction system. This leads to simplification of test methods, prevention of erroneous operations, and reduction of test costs.
また、本発明によれば、万一サンプル DNAに陽性対照が混入した場合でも、サンプ ルと陽性対照の増幅産物の長さが異なるため、誤判定を防ぐことができる。  Furthermore, according to the present invention, even if a positive control is mixed into sample DNA, the length of the amplified product of the sample and that of the positive control are different, so that erroneous determination can be prevented.
図面の簡単な説明  Brief Description of Drawings
[0009] [図 1]実施例 1、 2における人工陽性対照 DNA混合液を用いた PCR反応の結果を示 す電気泳動結果を示す図である。  FIG. 1 is a diagram showing an electrophoresis result showing a result of PCR reaction using an artificial positive control DNA mixed solution in Examples 1 and 2.
[図 2] λファージ DNA配列上における実施例 2の人工陽性対照 DNAに用いる領域（ 口で囲んだ部分)および人工陽性対照作製用プライマーの位置 (下線部分)を示す 図である。  FIG. 2 is a diagram showing a region (portion surrounded by the mouth) used for the artificial positive control DNA of Example 2 on the λ phage DNA sequence and the position of the primer for preparing the artificial positive control (underlined portion).
[図 3]実施例 3における人工陽性対照 DNA混合液を用いた PCR反応の結果を示す電 気泳動結果を示す図である。  FIG. 3 is a diagram showing an electrophoresis result showing a result of PCR reaction using an artificial positive control DNA mixed solution in Example 3.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0010] 以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
請求項 1に記載の本発明は、マルチプレックス PCR用人工陽性対照 DNA群の作製 法である。  The present invention according to claim 1 is a method for producing an artificial positive control DNA group for multiplex PCR.
本発明にお 、てマルチプレックス PCR用人工陽性対照 DNAとは、 2種以上のプライ マー対を 1反応系で使用して PCR反応を行うマルチプレックス PCR法にお!、て、各プ ライマー対の反応性を確認するために用いられる人工の陽性対照 DNAである。 なお、本発明において用いられるマルチプレックス PCR用プライマーは、どのような ものであっても良い。  In the present invention, the artificial positive control DNA for multiplex PCR refers to a multiplex PCR method in which two or more primer pairs are used in one reaction system to carry out a PCR reaction. Is an artificial positive control DNA used to confirm the reactivity of The multiplex PCR primer used in the present invention may be any type.
[0011] 請求項 1に記載の本発明では、はじめに、入手可能な配列が既知の DNAにおいて 、塩基数 50〜2000の間で任意の塩基数を有する領域を選択する工程 (a)を行う。 本発明において、入手可能な配列が既知の DNAとしては、例えば Accession No. :V 00636, J02459, M17233, X00906として Gen Bankに登録されている公知の塩基配列 を有する λファージ DNAや、 φ X174ファージ DNA (Accession No.NC- 001422)などを 用いることができる力 これに限定されない。ただし、サンプル DNAに陽性対照 DNA が混入した場合に起こる擬陽性を防ぐため、マルチプレックス PCR法の対象とされる 微生物種と近縁もしくは同一種の微生物の DNAを用いることは好ましくな 、。 [0011] In the present invention according to claim 1, first, in the DNA having a known sequence, the step (a) of selecting a region having any number of bases between 50 and 2000 bases is performed. In the present invention, examples of DNA having a known sequence include λ phage DNA having a known base sequence registered in Gen Bank as Accession No .: V 00636, J02459, M17233, X00906, and φX174 phage Force that can use DNA (Accession No. NC-001422) etc. It is not limited to this. However, in order to prevent false positives that occur when the sample DNA is mixed with the positive control DNA, it is preferable to use the DNA of microorganisms that are closely related to or the same species as the microbial species targeted by the multiplex PCR method.
[0012] 上記の入手可能な配列が既知の DNAにおいて、本発明の人工陽性対照 DNAに用 Vヽる領域を任意に選択する。 [0012] In the DNA having the known sequence, the region used for the artificial positive control DNA of the present invention is arbitrarily selected.
その際、 PCR反応後の電気泳動結果において、各プライマー対による PCR増幅産 物のバンドが分離して見えるように、他のプライマー対に対する前記領域の塩基数と 全く異なるようにすることが好ま 、。  At that time, it is preferable that the number of bases in the above region for the other primer pairs is completely different so that the bands of PCR amplification products by each primer pair appear to be separated in the electrophoresis result after the PCR reaction. .
また、前記領域の塩基数は 50〜2000塩基の範囲であることが好ましい。マルチプレ ックス PCR用プライマーの塩基数 (通常 10〜30塩基程度）を考慮すると、増幅産物の 塩基数として少なくとも 50塩基は必要である。一方、 2000塩基を超えると PCR反応に 時間がかかり、実用的ではない。  The number of bases in the region is preferably in the range of 50 to 2000 bases. Considering the number of bases in the multiplex PCR primer (usually around 10 to 30 bases), at least 50 bases are required for the amplification product. On the other hand, if it exceeds 2000 bases, the PCR reaction takes time and is not practical.
なお、入手可能な配列が既知の DNAにおいて、人工陽性対照 DNAに用いる領域 は互いに重複していないことが望ましい。なぜなら、人工陽性対照同士で非特異的 な増幅反応が起こる可能性があるからである。  In addition, it is desirable that the regions used for the artificial positive control DNA do not overlap with each other in the DNA for which the available sequence is known. This is because a nonspecific amplification reaction may occur between artificial positive controls.
[0013] 請求項 1に記載の本発明では、次に、マルチプレックス PCR用フォワードプライマー の 3'側に、工程 (a)で選択した領域の 5 '末端の 10〜30塩基を結合させてなる人工陽 性対照作製用フォワードプライマーと、マルチプレックス PCR用リバースプライマーの 3，側に、工程 (a)で選択した領域の 3 '末端の 10〜30塩基の相補的配列を結合させて なる人工陽性対照作製用リバースプライマーと、力 なる人工陽性対照作製用プライ マー対を合成する工程 (b)を行う。 [0013] In the present invention according to claim 1, next, 10 to 30 bases at the 5 'end of the region selected in step (a) are bound to the 3' end of the forward primer for multiplex PCR. Artificial positive formed by binding a complementary sequence of 10-30 bases at the 3 'end of the region selected in step (a) to the 3rd side of the forward primer for artificial positive control and the reverse primer for multiplex PCR. Step (b) of synthesizing a reverse primer for preparing a control and a primer pair for generating a powerful artificial positive control is performed.
[0014] 本発明では、 PCR反応によって人工陽性対照 DNAを作製する。そのため、前記領 域の両端の DNA配列に基!、て設計した配列を、マルチプレックス PCR用プライマー 対に結合させてなる人工陽性対照作製用プライマー対を合成する。 [0014] In the present invention, artificial positive control DNA is prepared by PCR reaction. Therefore, an artificial positive control primer pair is synthesized by binding a sequence designed based on the DNA sequences at both ends of the region to a multiplex PCR primer pair.
人工陽性対照作製用プライマー対は、フォワードプライマーとリバースプライマーか らなる。これらはいずれも、マルチプレックス PCR用プライマーを 5'側に、前記領域の 5，末端又は 3 '末端の DNA配列に基 、て設計した配列を 3，側に有する。 Is the primer pair for artificial positive control production a forward primer and a reverse primer? It becomes. Each of these has a primer for multiplex PCR on the 5 ′ side and a sequence designed on the 3, side based on the DNA sequence at the 5, 5 ′ or 3 ′ end of the region.
[0015] すなわち、本発明の人工陽性対照作製用フォワードプライマーは、マルチプレック ス PCR用フォワードプライマーの 3，側に、前記領域の 5，末端の DNA配列を結合させ てなる。この際、前記領域の 5'末端の DNA配列の塩基数は 10〜30塩基、好ましくは 1 5〜25塩基とすることが好まし 、。 [0015] That is, the artificial positive control preparation forward primer of the present invention comprises the DNA sequence at the 5th and terminal ends of the region bound to the 3rd side of the multiplex PCR forward primer. At this time, the number of bases of the DNA sequence at the 5 ′ end of the region is preferably 10 to 30 bases, preferably 15 to 25 bases.
また、本発明の人工陽性対照作製用リバースプライマーは、マルチプレックス PCR 用リバースプライマーの 3'側に、前記領域の 3'末端に相補的な DNA配列を結合さ せてなる。この際、前記領域の 3'末端の DNA配列の塩基数は 10〜30塩基、好ましく は 15〜25塩基とすることが好まし 、。  The reverse primer for producing an artificial positive control of the present invention is obtained by binding a complementary DNA sequence to the 3 ′ end of the region on the 3 ′ side of the reverse primer for multiplex PCR. At this time, the number of bases of the DNA sequence at the 3 ′ end of the region is preferably 10 to 30 bases, preferably 15 to 25 bases.
上記にお!、て、前記領域の 5 '末端又は 3 '末端の DNA配列に基く配列の塩基数が 好ま 、範囲未満である場合は、人工陽性対照作製用プライマーが前記領域に特 異的に結合することができない。また、当該塩基数が好ましい範囲を超えた場合は、 プライマー合成のコストが高くなり、実用的でない。  As described above, the number of bases of the sequence based on the DNA sequence at the 5 ′ end or 3 ′ end of the region is preferred, and if it is less than the range, the artificial positive control preparation primer is specific to the region. Cannot be combined. On the other hand, when the number of bases exceeds the preferred range, the cost of primer synthesis increases and is not practical.
上記のようにして得られた人工陽性対照作製用プライマー対は、前記領域の両端 の DNA配列に基 、て設計された部分によって、入手可能な配列が既知の DNAにお ける前記領域に特異的に結合することができる。  The primer pair for producing an artificial positive control obtained as described above is specific to the above-mentioned region in the known DNA due to the portion designed based on the DNA sequences at both ends of the above-mentioned region. Can be combined.
[0016] 請求項 1に記載の本発明では、続、て、前記入手可能な配列が既知の DNAを铸型 とし、工程 (b)で合成した人工陽性対照作製用プライマー対を用いて PCR反応を行つ て得られる増幅産物である人工陽性対照 DNAを作製する工程 (c)を行う。 [0016] In the present invention according to claim 1, subsequently, a PCR reaction is carried out using a primer pair for preparing an artificial positive control, wherein the DNA having an already known sequence is known as a cage and synthesized in step (b). Step (c) of producing an artificial positive control DNA, which is an amplification product obtained by performing the above steps, is performed.
上記したように、上記工程 (b)で得られた人工陽性対照作製用プライマー対は、上 記工程 (a)で選択した領域の両端の DNA配列に基 ヽて設計された部分によって、入 手可能な配列が既知の DNAにおける前記領域に特異的に結合することができる。 したがって、本工程の PCR反応により、前記領域において、 5'末端にマルチプレツ タス PCR用フォワードプライマーカ、 3，末端にマルチプレックス PCR用リバースプライ マーの相補的配列力 それぞれ付加された DNA配列が増幅産物（人工陽性対照 DN A)として得られる。  As described above, the artificial positive control primer pair obtained in the step (b) is obtained by the portion designed based on the DNA sequences at both ends of the region selected in the step (a). A possible sequence can bind specifically to the region in the known DNA. Therefore, by the PCR reaction in this step, in the above region, the DNA sequence to which the complementary sequence strength of the forward primer for multiplex PCR is added at the 5 ′ end and the reverse primer for multiplex PCR is added at the 3 ′ end is amplified product. (Artificial positive control DN A).
このようにして得られた本発明の人工陽性対照 DNAを、対応するマルチプレックス P CR用プライマー対の反応性検査に用いると、所望の塩基数を有する人工陽性対照 DNAが増幅され、例えば電気泳動法で増幅産物のバンドの有無を調べることにより、 プライマー対の反応性を確認することができる。 The artificial positive control DNA of the present invention thus obtained is When used for CR primer pair reactivity test, artificial positive control DNA with the desired number of bases is amplified, and the primer pair reactivity is confirmed, for example, by examining the presence or absence of amplified product bands by electrophoresis. be able to.
なお、本発明の人工陽性対照 DNAは、通常の核酸精製法により精製して用いるこ とが好ましい。  The artificial positive control DNA of the present invention is preferably used after being purified by an ordinary nucleic acid purification method.
[0017] 請求項 1に記載の本発明では、マルチプレックス PCR用の各プライマー対について 、それぞれ個別に上記工程 (a)〜(c)の操作を行って、人工陽性対照 DNAを作製する 各プライマー対に対応する人工陽性対照 DNAは、それぞれ適当な濃度に希釈して 混合液として、マルチプレックス PCR法に用いることにより、全てのプライマー対につ V、て一斉に反応性を確認することができる。  [0017] In the present invention according to claim 1, for each primer pair for multiplex PCR, the respective steps (a) to (c) are individually performed to prepare an artificial positive control DNA. The artificial positive control DNA corresponding to each pair can be diluted to an appropriate concentration and used as a mixed solution in the multiplex PCR method, so that the reactivity can be confirmed simultaneously for all primer pairs V. .
このとき、各プライマー対に対応する PCR増幅産物の分子量は互いに全く異なるよ うに設定しているため、電気泳動結果において全てのバンドを明瞭に区別することが できる。  At this time, since the molecular weights of the PCR amplification products corresponding to each primer pair are set to be completely different from each other, all bands can be clearly distinguished in the electrophoresis result.
また、本発明の人工陽性対照 DNAは、菌種の同定に利用する増幅領域とは異なる 塩基数を有するため、 PCR反応液の調製の際などに万一サンプル DNAに陽性対照 が混入した場合でも、誤判定が起こることがない。  In addition, since the artificial positive control DNA of the present invention has a different number of bases from the amplification region used for bacterial species identification, even when the positive control is mixed with the sample DNA in the preparation of a PCR reaction solution, etc. Misjudgment does not occur.
[0018] 請求項 2に記載の本発明は、請求項 1に記載の方法により作製されたマルチプレツ タス PCR用人工陽性対照 DNA群を同一反応液中で PCR反応に供することを特徴とす る、マルチプレックス PCR用プライマーの反応性を確認する方法である。 [0018] The present invention according to claim 2 is characterized in that the artificial positive control DNA group for multiplex PCR prepared by the method according to claim 1 is subjected to a PCR reaction in the same reaction solution. This is a method for confirming the reactivity of multiplex PCR primers.
本発明において、 PCR反応は常法により行うことができ、反応液組成、使用酵素、 反応条件等は適宜設定することができる。  In the present invention, the PCR reaction can be carried out by a conventional method, and the composition of the reaction solution, the enzyme used, the reaction conditions, etc. can be appropriately set.
実施例  Example
[0019] 以下、本発明を実施例によってさらに具体的に説明するが、本発明はこれに限定さ れるものではない。  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.
[0020] <実施例 1 > <Example 1>
ビール製造において有害である下記の Lactobacillus属乳酸菌 6菌種を検出同定す るマルチプレックス PCR法を構築した。 Lactobacillus brevis、 L. lindneri、 L. paracolli noides、 L. casei、 L. coryniformis及び L. plantarumの 16SrRNA遺伝子及び ITS領域 の公知塩基配列をもとに、表 1に示すプライマーを設計した。なお、各プライマー対 において、上段にフォワードプライマーを、下段にリバースプライマーを記載した。 A multiplex PCR method was constructed to detect and identify the following 6 Lactobacillus species of lactic acid bacteria that are harmful in beer production. Lactobacillus brevis, L. lindneri, L. paracolli The primers shown in Table 1 were designed based on the 16S rRNA gene of noides, L. casei, L. coryniformis and L. plantarum and the known nucleotide sequence of the ITS region. In each primer pair, the forward primer is listed in the upper row and the reverse primer is listed in the lower row.
[表 1]  [table 1]
Figure imgf000008_0001
Figure imgf000008_0001
[0022] これらのプライマーを 0.1 μ Μから 10 μ Μの範囲の最終濃度となるように、 PerfectSho t™ Ex Taq (Loading dye mix) (タカラバイオ社）と混合したものを乳酸菌マルチプレツ タス PCR反応液とした。これを用いた PCR検査の結果、それぞれの供試菌株で特異 的に感度良く反応することを確認した。 [0022] A mixture of these primers and PerfectShot ™ Ex Taq (Loading dye mix) (Takara Bio Inc.) to a final concentration in the range of 0.1 µm to 10 µm. It was. As a result of PCR using this, it was confirmed that each test strain reacts specifically with high sensitivity.
[0023] 次に、 λファージ DNA (タカラバイオ社）の公知の塩基配列（配列表の配列番号 9、 Accession No. :V00636, J02459, M17233, X00906)の任意の領域に、人工陽性対照 DNAに用いる領域を表 1に記載の各菌種ごとに選定した。そして、各菌種について、 フォワードプライマーの 3 '側に、上記で選択した領域の 5 '末端の約 20塩基を結合さ せた人工陽性対照作製用フォワードプライマーを、リバースプライマーの 3 '側に、上 記で選択した領域の 3 '末端の約 20塩基の相補的配列を結合させた人工陽性対照 作製用リバースプライマーを、それぞれ設計した。  [0023] Next, in any region of the known base sequence of λ phage DNA (Takara Bio) (SEQ ID NO: 9, Accession No .: V00636, J02459, M17233, X00906) in the artificial positive control DNA The area to be used was selected for each bacterial species listed in Table 1. Then, for each bacterial species, a forward primer for preparing an artificial positive control in which about 20 bases at the 5 ′ end of the region selected above were bound to the 3 ′ side of the forward primer, and the 3 ′ side of the reverse primer. A reverse primer for preparing an artificial positive control was designed, to which a complementary sequence of about 20 bases at the 3 ′ end of the region selected above was bound.
各菌種に対応するマルチプレックス PCR用プライマー名と、それに対応する人工陽 性対照作製用プライマー名を表 2に示した。また、人工陽性対照作製用プライマーの 配列を表 3に示した（下線は表 1のマルチプレックス PCR用プライマー配列部分を示 す)。表 2中、上段にフォワードプライマーを、下段にリバースプライマーを記載した。 [0024] [表 2] Table 2 shows the names of multiplex PCR primers corresponding to each bacterial species and the corresponding primer names for artificial positive control preparation. In addition, the sequences of artificial positive control preparation primers are shown in Table 3 (underlined indicates the primer sequence portion for multiplex PCR in Table 1). In Table 2, the forward primer is described in the upper part and the reverse primer is described in the lower part. [0024] [Table 2]
Figure imgf000009_0001
Figure imgf000009_0001
[0025] [表 3] [0025] [Table 3]
Figure imgf000009_0002
上記の人工陽性対照作製用プライマーを用いて、 λファージ DNA (タカラバイオ社 )の希釈溶液を铸型とした PCR反応を、菌種ごとに行った。 PCR反応は、タカラバイオ 社の PerfectShot™ Ex Taqを用いて表 4の反応液組成で行った。反応には Applied Bi osystems社の GeneAmp PCR System 9700を用いて、表 5の反応条件により実施した
Figure imgf000009_0002
Using the above artificial positive control preparation primer, a PCR reaction using a diluted solution of λ phage DNA (Takara Bio Inc.) as a cage was performed for each bacterial species. The PCR reaction was performed using the Takara Bio PerfectShot ™ Ex Taq with the composition shown in Table 4. The reaction was performed using Applied Biosystems GeneAmp PCR System 9700 under the reaction conditions shown in Table 5.
[0027] [表 4] 滅菌 7 ? δ 85蒸留水 19 a 1 [0027] [Table 4] Sterilized 7? Δ 85 Distilled water 19 a 1
フォ ρ ρワ ρ C Cードプライマ一 （ΙΟΟ μ Μ) 0. 5 μ 1  Pho ρ ρ War ρ CC C Primer (ΙΟΟ μ Μ) 0.5 μ 1
リバースプライマー 〔100 μ Μ) 0. 5 μ 1  Reverse primer (100 μΜ) 0.5 μ 1
λファージ^ ηυ πυ πυ DNA ( 1000倍希釈) 5 μ 1  λ phage ^ ηυ πυ πυ DNA (1000-fold dilution) 5 μ 1
PerfectShot™ ο Ex Taq (. Load ing dye mi x) 25 μ 1  PerfectShot ™ ο Ex Taq (. Load ing dye mix) 25 μ 1
計 50 μ 1  50 μ 1 total
[0028] [表 5] [0028] [Table 5]
30サイクル 30 cycles
[0029] それぞれの菌種に対応する人工陽性対照作製用プライマー対カゝら得られた PCR反 応増幅産物を、 QlAquick PCR Purification Kit (キアゲン社）を用いて、キットに添付 のプロトコールに従 ヽ精製した。精製された DNA溶液を分光光度計 NanoDrop (登録 商標） ND- 1000 (NanoDrop社)の核酸測定モードで測定し、核酸濃度が 30ng/ μ 1に なるように滅菌蒸留水で調整し、人工陽性対照溶液とした。 [0029] Use the QlAquick PCR Purification Kit (Qiagen) according to the protocol attached to the kit, using the PCR reaction amplification product obtained from the pair of artificial positive control primers corresponding to each bacterial species. Purified. The purified DNA solution was measured in the nucleic acid measurement mode of the spectrophotometer NanoDrop (registered trademark) ND-1000 (NanoDrop), adjusted with sterile distilled water to a nucleic acid concentration of 30 ng / μ 1, and an artificial positive control. It was set as the solution.
各プライマー対を用いた際の人工陽性対照溶液に含まれる PCR反応増幅産物の 推定塩基対を、表 6に示した。  Table 6 shows the estimated base pairs of PCR reaction amplification products contained in the artificial positive control solution when each primer pair was used.
[0030] [表 6] [0030] [Table 6]
Figure imgf000010_0001
Figure imgf000010_0001
[0031] 対象とする乳酸菌 6菌種のそれぞれに対応する人工陽性対照溶液を上 作製し、 100-5000倍の範囲で希釈したものを混合し、人工陽性対照 DNA混合液とし V V V [0031] The artificial positive control solutions corresponding to each of the 6 lactic acid bacteria Prepare and mix 100-5000-fold diluted solution as an artificial positive control DNA mixture VVV
た。  It was.
上記した乳酸菌マルチプレックス PCR反応液 45 1に、人工陽性対照 DNA混合液 5 μ 1を加え、表 7に示す PCR反応条件で PCR反応を行った。反応には Applied Biosyst ems社の GeneAmp PCR System 9700を用いた。  An artificial positive control DNA mixture 5 μ1 was added to the lactic acid bacterium multiplex PCR reaction solution 45 1 described above, and a PCR reaction was performed under the PCR reaction conditions shown in Table 7. GeneAmp PCR System 9700 (Applied Biosyst ems) was used for the reaction.
[0032] [表 7]  [0032] [Table 7]
2分 30秒 2 minutes 30 seconds
1 δ秒  1 δ seconds
16# 30サィクル  16 # 30 cycle
？ € 30秒 —J  ? € 30 seconds —J
？ ΓΟ 3分  ? ΓΟ 3 minutes
[0033] 上記の PCR反応により得られた反応液 5 μ 1を、ァガロース ·ゲル電気泳動（2% W/V in TAEバッファー（組成： 1Lあたり Tris4.8g,酢酸 1.14ml, 0.5mM EDTA(pH8.0))に供 した (100V、 30分間)。電気泳動後、サイバーグリーンにより 30分間染色した。ァガロー スはシグマ社の A-9539を用い、 TAEバッファ一はナカライテスタ社のものを用いた。ま た、サイバーグリーン染色液は、 BMA社の SYBR Green I Nucleic Acid Gel Stain 10 μ 1を、 100mlの TAEバッファーに添カ卩して作製した。  [0033] 5 μl of the reaction solution obtained by the PCR reaction described above was added to agarose gel electrophoresis (2% W / V in TAE buffer (composition: Tris 4.8 g per 1 L, acetic acid 1.14 ml, 0.5 mM EDTA (pH 8 0)) (100V, 30 minutes) After electrophoresis, stained with Cyber Green for 30 minutes, Sigma A-9539 was used for the agarose, and Nacalai Tester was used for the TAE buffer. The Cyber Green staining solution was prepared by adding SYBR Green I Nucleic Acid Gel Stain 10 μ1 from BMA to 100 ml of TAE buffer.
電気泳動写真を図 1に示した。泳動パターンから、 6つの異なる分子量の増幅断片 が明瞭に検出された。これらの増幅断片の分子量は、表 5の推定塩基対と一致する ことから、すべてのプライマーの反応性を確認することができた。  An electrophoretogram is shown in FIG. From the electrophoretic pattern, amplified fragments of 6 different molecular weights were clearly detected. Since the molecular weights of these amplified fragments coincided with the estimated base pairs in Table 5, the reactivity of all primers could be confirmed.
[0034] <実施例 2>  <Example 2>
ビール製造における有害菌である Pectinatus属 2菌種を検出するマルチプレックス P CR法を構築した。 Pectinatus frisingensis及び P. cerevisiiphilusの 16SrRNA遺伝子及 び ITS領域の公知塩基配列をもとに、表 8に示すプライマーを設計した。なお、各ブラ イマ一対において、上段にフォワードプライマーを、下段にリバースプライマーを記載 した。  A multiplex PCR method was established to detect two Pectinatus species, which are harmful bacteria in beer production. The primers shown in Table 8 were designed based on the 16S rRNA gene of Pectinatus frisingensis and P. cerevisiiphilus and the known nucleotide sequence of the ITS region. In each primer pair, the forward primer is listed in the upper row and the reverse primer is listed in the lower row.
[0035] [表 8] 対象菌種 プライマー名 プライマー配列（5'→3')[0035] [Table 8] Target bacterial species Primer name Primer sequence (5 '→ 3')
P ec t inat us cer ev is i iphi lus 16C-F (配列番号 22) CGTATGCAGAGATGCATATT P ec t inat us cer ev is i iphi lus 16C-F (SEQ ID NO: 22) CGTATGCAGAGATGCATATT
IC-R (配列番号 23) CACTCTTACAAGTATCTAC IC-R (SEQ ID NO: 23) CACTCTTACAAGTATCTAC
P . f ri si ngens i s 16F-F (配列番号 24) CGTATCCAGAGATGGATATT P. F ri si ngens i s 16F-F (SEQ ID NO: 24) CGTATCCAGAGATGGATATT
IF- R (配列番号 25) CCATCCTCTTGAAAATCTC  IF- R (SEQ ID NO: 25) CCATCCTCTTGAAAATCTC
[0036] これらのプライマーを、 0.1 μ Μから 10 μ Μの範囲の最終濃度となるように PerfectSho t™ Ex Taq (Loading dye mix) (タカラバイオ社）と混合したものを、 Pectinatus属菌マ ルチプレックス PCR反応液とした。これを用いた PCR検査の結果、それぞれの供試菌 株で特異的に感度良く反応することを確認した。 [0036] These primers were mixed with PerfectShot ™ Ex Taq (Loading dye mix) (Takara Bio Inc.) to a final concentration in the range of 0.1 μΜ to 10 μΜ. A plex PCR reaction solution was used. As a result of PCR using this, it was confirmed that each test strain specifically reacted with high sensitivity.
[0037] 次に、 λファージ DNA (タカラバイオ社）の公知の塩基配列（配列表の配列番号 9) の任意の領域 (具体的には図 2において口で囲まれた部分）に、人工陽性対照 DNA に用 、る領域を表 8に記載の各菌種ごとに選定した。  [0037] Next, an artificial positive is detected in an arbitrary region (specifically, a portion surrounded by a mouth in FIG. 2) of a known base sequence (SEQ ID NO: 9 in the sequence listing) of λ phage DNA (Takara Bio Inc.). The region used for control DNA was selected for each bacterial species listed in Table 8.
そして、各菌種について、フォワードプライマーの 3'側に、上記で選択した領域の 5 ，末端の約 20塩基を結合させた人工陽性対照作製用フォワードプライマーを、リバ一 スプライマーの 3'側に、上記で選択した領域の 3'末端の約 20塩基の相補的配列を 結合させた人工陽性対照作製用リバースプライマーを、それぞれ設計した。  Then, for each bacterial species, the artificial primer control forward primer to which about 20 bases at the 5 and end of the region selected above were bound on the 3 ′ side of the forward primer, on the 3 ′ side of the reverse primer. A reverse primer for preparing an artificial positive control was designed, to which a complementary sequence of about 20 bases at the 3 ′ end of the region selected above was bound.
各菌種に対応するマルチプレックス PCR用プライマー名と、それに対応する人工陽 性対照作製用プライマー名を表 9に示した。また、人工陽性対照作製用プライマーの 配列を表 10に示した（下線は表 8のマルチプレックス PCR用プライマー配列部分を示 す)。表 9中、上段にフォワードプライマーを、下段にリバースプライマーを記載した。 尚、図 2は、 λファージ DNA配列（Accession No.:V00636, J02459, M17233, X0090 6)上での各人工陽性対照 DNAに用いる領域（口で囲まれた部分)および各人工陽 性対照作製用プライマーの位置 (下線部分)を示す。  Table 9 shows the multiplex PCR primer names corresponding to each bacterial species and the corresponding artificial positive control primer names. In addition, the sequences of primers for preparation of artificial positive controls are shown in Table 10 (underlined indicates the primer sequence portion for multiplex PCR in Table 8). In Table 9, the forward primer is listed in the upper row and the reverse primer is listed in the lower row. Fig. 2 shows the region used for each artificial positive control DNA on the λ phage DNA sequence (Accession No .: V00636, J02459, M17233, X0090 6) (the part surrounded by the mouth) and each artificial positive control preparation. Shows the position (underlined part) of the primer.
[0038] [表 9] 対象菌種 マルチズレックス用フ。ライマー 人工陽性対照作製用フ 'ライマ- [0038] [Table 9] Target bacterial species Limer Artificial positive control preparation 'Lima-
Pectinatus cerev is i i hi lus 16C-F AP0SIFPC2 Pectinatus cerev is i i hi lus 16C-F AP0SIFPC2
IC-R AP0SIRPC  IC-R AP0SIRPC
P. f ri si ngens ι s 16F-F AP0SIFPF  P. f ri si ngens ι s 16F-F AP0SIFPF
IF-R AP0SIRPF [0039] [表 10] IF-R AP0SIRPF [0039] [Table 10]
Figure imgf000013_0001
Figure imgf000013_0001
[0040] 上記の人工陽性対照作製用プライマーを用いて、 λファージ DNA (タカラバイオ社 )の希釈溶液を铸型とした PCR反応を、菌種ごとに行った。 PCR反応は、タカラバイオ 社の PerfectShot™ Ex Taqを用いて表 4の反応液組成で行った。反応には Applied Bi osystems社の GeneAmp PCR System 9700を用いて、表 5の反応条件により実施した それぞれの菌種に対応する人工陽性対照作製用プライマー対カゝら得られた PCR反 応増幅産物を、実施例 1と同様の方法で精製及び調製を行い、 Pectinatus属 2菌種 に対する人工陽性対照溶液を作製した。 [0040] Using the artificial positive control preparation primer described above, a PCR reaction using a diluted solution of λ phage DNA (TAKARA BIO INC.) As a cage was performed for each bacterial species. The PCR reaction was performed using the Takara Bio PerfectShot ™ Ex Taq with the composition shown in Table 4. The PCR reaction amplification product obtained by using a pair of artificial positive control primers corresponding to each bacterial species was performed according to the reaction conditions shown in Table 5 using Applied Biosystems GeneAmp PCR System 9700. Then, purification and preparation were performed in the same manner as in Example 1 to prepare an artificial positive control solution for two species of the genus Pectinatus.
各人工陽性対照溶液に含まれる PCR反応増幅産物の推定塩基対を表 11に示した  Table 11 shows the estimated base pairs of PCR reaction amplification products contained in each artificial positive control solution.
[0041] [表 11] [0041] [Table 11]
Figure imgf000013_0002
Figure imgf000013_0002
[0042] 得られた人工陽性対照溶液 (30ng/ μ 1)をそれぞれ 100-5000倍の範囲で希釈して 混合し、人工陽性対照 DNA混合液とした。 [0042] The obtained artificial positive control solution (30 ng / μ1) was diluted in a range of 100 to 5000 times and mixed to obtain an artificial positive control DNA mixture.
上記した Pectinatus属菌マルチプレックス PCR反応液 45 μ 1に、人工陽性対照 DNA 混合液 5 1を加え、表 7に示す PCR反応条件で PCR反応を行った。反応には Applied Biosystems社の GeneAmp PCR System 9700を用いた。  The artificial positive control DNA mixture 51 was added to 45 μ1 of the Pectinatus multiplex PCR reaction solution described above, and a PCR reaction was performed under the PCR reaction conditions shown in Table 7. GeneAmp PCR System 9700 from Applied Biosystems was used for the reaction.
電気泳動写真を図 1に示した。泳動パターンから、 2つの異なる分子量の増幅断片 が明瞭に検出された。これらの増幅断片の分子量は、表 11の推定塩基対と一致する ことから、すべてのプライマーの反応性を確認することができた。 An electrophoretogram is shown in FIG. From the electrophoresis pattern, two amplified fragments with different molecular weights were clearly detected. The molecular weights of these amplified fragments are consistent with the estimated base pairs in Table 11. Therefore, the reactivity of all primers could be confirmed.
[0043] <実施例 3 > <Example 3>
実施例 1及び 2と同様に、ビール製造において有害である球菌、 Pediococcus属 3菌 種および MegasphaeraJ禹 1菌種 (Pediococcus clausseni P. damnosus P. inopinatus Similar to Examples 1 and 2, cocci that are harmful in beer production, 3 species of Pediococcus sp. And 1 species of MegasphaeraJ 禹 (Pediococcus clausseni P. damnosus P. inopinatus
、及び Megasphaera cerevisiae)を検出するためのマルチプレックス PCR法の構築を 行い、人工陽性対照 DNA混合溶液を作製した。 And a multiplex PCR method for detecting Megasphaera cerevisiae) to prepare an artificial positive control DNA mixed solution.
各菌種に対して設計したマルチプレックス PCR用プライマーの配列を表 12に、マル チプレックス PCR用プライマーに対応する人工陽性対照作製用プライマー名を表 13 に、また、人工陽性対照作製用プライマーの配列を表 14に示した（下線は表 12のマ ルチプレックス PCR用プライマー配列部分を示す)。  The multiplex PCR primer sequences designed for each bacterial species are listed in Table 12, the artificial positive control primer names corresponding to the multiplex PCR primers are listed in Table 13, and the artificial positive control primer The sequences are shown in Table 14 (underlined indicates the primer sequence portion for multiplex PCR in Table 12).
[0044] [表 12] [0044] [Table 12]
Figure imgf000014_0001
Figure imgf000014_0001
[0045] [表 13] [0045] [Table 13]
Figure imgf000014_0002
Figure imgf000014_0002
[0046] [表 14]
Figure imgf000015_0001
[0046] [Table 14]
Figure imgf000015_0001
[0047] 上記の人工陽性対照作製用プライマーを用いて、 λファージ DNA (タカラバイオ社 )の希釈溶液を铸型とした PCR反応を、菌種ごとに行った。 PCR反応は、タカラバイオ 社の PerfectShot™ Ex Taqを用いて表 4の反応液組成で行った。反応には Applied Bi osystems社の GeneAmp PCR System 9700を用いて、表 5の反応条件により実施した それぞれの菌種に対応する人工陽性対照作製用プライマー対カゝら得られた PCR反 応増幅産物を、実施例 1と同様の方法で精製及び調製を行い、球菌 4菌種に対する 人工陽性対照溶液を作製した。 [0047] Using the artificial positive control preparation primer described above, a PCR reaction using a diluted solution of λ phage DNA (Takara Bio Inc.) as a cage was performed for each bacterial species. The PCR reaction was performed using the Takara Bio PerfectShot ™ Ex Taq with the composition shown in Table 4. The PCR reaction amplification product obtained by using a pair of artificial positive control primers corresponding to each bacterial species was performed according to the reaction conditions shown in Table 5 using Applied Biosystems GeneAmp PCR System 9700. Then, purification and preparation were performed in the same manner as in Example 1 to prepare an artificial positive control solution for 4 species of cocci.
各人工陽性対照溶液に含まれる PCR反応増幅産物の推定塩基対を表 15に示した  Table 15 shows the estimated base pairs of PCR reaction amplification products contained in each artificial positive control solution.
[0048] [表 15] [0048] [Table 15]
Figure imgf000015_0002
Figure imgf000015_0002
[0049] 得られた人工陽性対照溶液 (30ng/ μ 1)をそれぞれ 100-5000倍の範囲で希釈して 混合し、人工陽性対照 DNA混合液とした。 [0049] The obtained artificial positive control solution (30 ng / μ1) was diluted in a range of 100 to 5000 times and mixed to obtain an artificial positive control DNA mixture.
表 12のプライマーを、 0.1 μ Μから 10 μ Μの範囲の最終濃度となるように PerfectShot ™ Ex Taq (Loading dye mix) (タカラバイオ社）と混合した球菌マルチプレックス PCR 反応液 45 μ 1に、人工陽性対照 DNA混合液 5 μ 1を加え、表 7に示す PCR反応条件で PCR反応を行った。反応には Applied Biosystems社の GeneAmp PCR System 9700を 用いた。 In 45 μ 1 of the cocci multiplex PCR reaction mixture of the primers in Table 12 mixed with PerfectShot ™ Ex Taq (Loading dye mix) (Takara Bio) to a final concentration in the range of 0.1 μΜ to 10 μΜ, Add 5 μ1 of the artificial positive control DNA mixture and use the PCR reaction conditions shown in Table 7. PCR reaction was performed. For the reaction, GeneAmp PCR System 9700 from Applied Biosystems was used.
電気泳動写真を図 3に示した。泳動パターンから、 3つの異なる分子量の増幅断片 が明瞭に検出された。これらの増幅断片の分子量は、表 15の推定塩基対と一致する ことから、すべてのプライマーの反応性を確認することができた。  The electrophoresis photograph is shown in FIG. From the electrophoresis pattern, amplified fragments of three different molecular weights were clearly detected. Since the molecular weights of these amplified fragments were in agreement with the estimated base pairs in Table 15, the reactivity of all primers could be confirmed.

Claims

請求の範囲 The scope of the claims
[1] マルチプレックス PCR用の各プライマーについて、以下の工程 (a)〜(c)を行うことを 特徴とする、マルチプレックス PCR用人工陽性対照 DNA群の作製法。  [1] A method for producing an artificial positive control DNA group for multiplex PCR, comprising performing the following steps (a) to (c) for each primer for multiplex PCR.
(a)入手可能な配列が既知の DNAにお!/、て、塩基数 50〜2000の間で任意の塩基数 を有する領域を選択する工程、  (a) a step of selecting a region having an arbitrary number of bases between 50 and 2000 bases in DNA having an available sequence known!
(b)マルチプレックス PCR用フォワードプライマーの 3，側に、工程 (a)で選択した領域の 5'末端の 10〜30塩基を結合させてなる人工陽性対照作製用フォワードプライマーと 、マルチプレックス PCR用リバースプライマーの 3，側に、工程 (a)で選択した領域の 3， 末端の 10〜30塩基の相補的配列を結合させてなる人工陽性対照作製用リバースプ ライマーと、力もなる人工陽性対照作製用プライマー対を合成する工程、および、 (b) An artificial positive control preparation forward primer in which 10 to 30 bases at the 5 ′ end of the region selected in step (a) are bound to the 3rd side of the forward primer for multiplex PCR, and for multiplex PCR. A reverse primer for preparing an artificial positive control in which a complementary sequence of 10 to 30 bases at the end and 3 of the region selected in step (a) is bound to the third side of the reverse primer, and an artificial positive control that has strength. Synthesizing a primer pair; and
(c)前記入手可能な配列が既知の DNAを铸型とし、工程 (b)で合成した人工陽性対照 作製用プライマー対を用いて PCR反応を行って得られる増幅産物である人工陽性対 照 DNAを作製する工程 (c) Artificial positive control DNA, which is an amplification product obtained by performing PCR reaction using the artificial positive control preparation primer pair synthesized in step (b), with DNA having the known available sequence as a saddle type Process
[2] 請求項 1に記載の方法により作製されたマルチプレックス PCR用人工陽性対照 DN A群を同一反応液中で PCR反応に供することを特徴とする、マルチプレックス PCR用 プライマーの反応性を確認する方法。  [2] Confirmation of reactivity of primers for multiplex PCR, characterized in that the multiplex PCR artificial positive control DNA group prepared by the method of claim 1 is subjected to PCR reaction in the same reaction solution how to.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020983A1 (en) * 2001-08-30 2003-03-13 Virginia Commonwealth University Allele specific pcr for genotyping
WO2004092420A1 (en) * 2003-04-17 2004-10-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Method and kit for molecular identification of smallpox
JP2005095025A (en) * 2003-09-22 2005-04-14 Shimadzu Corp Primer panel for multiplex pcr, multiplex pcr method using the same, and gene analyzing method

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Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020983A1 (en) * 2001-08-30 2003-03-13 Virginia Commonwealth University Allele specific pcr for genotyping
WO2004092420A1 (en) * 2003-04-17 2004-10-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Method and kit for molecular identification of smallpox
JP2005095025A (en) * 2003-09-22 2005-04-14 Shimadzu Corp Primer panel for multiplex pcr, multiplex pcr method using the same, and gene analyzing method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MARCOBAL A. ET AL.: "Multiplex PCR Method for the Simultaneous Detection of Histamine-, Tyramine-, and Putrescine-Producing Lactic Acid Bacteria in Foods", JOURNAL OF FOOD PROTECTION, vol. 68, no. 4, 2005, pages 874 - 878, XP003012938 *
MORA D. ET AL.: "Identification of Pediococcus acidilactici and Pediococcus pentosaceus based on 16S rRNA and idhD gene-targeted multiplex PCR analysis", FEMS MICROBIOLOGY LETTERS, vol. 151, no. 2, 1997, pages 231 - 236, XP002241673 *

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