JP4658816B2 - Primer for detection of peanut and detection method thereof - Google Patents
Primer for detection of peanut and detection method thereof Download PDFInfo
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- JP4658816B2 JP4658816B2 JP2006010028A JP2006010028A JP4658816B2 JP 4658816 B2 JP4658816 B2 JP 4658816B2 JP 2006010028 A JP2006010028 A JP 2006010028A JP 2006010028 A JP2006010028 A JP 2006010028A JP 4658816 B2 JP4658816 B2 JP 4658816B2
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Description
本発明は、アレルギーを引き起こす恐れのある落花生属を対象として、落花生属の植物が１種でも食品原料や製品等に含まれていた場合に、その量が微量であっても高感度で検出することを可能とする新しいプライマーおよびその検出方法に関する。 The present invention targets a peanut genus that may cause allergies, and even when a single plant of the peanut genus is contained in a food raw material or product, the amount of the peanut genus is detected with high sensitivity. The present invention relates to a new primer and a detection method thereof.
落花生属の検出用プライマー及びその検出方法については、本願出願人が既に開発し、特許出願を行ったものがある（特許文献１）。
当該検出方法では、３種類のプライマーペアのうちいずれか１種類を用いる。すなわち、１つ目は落花生のITS-１配列中に作製したプライマーと5.8S rRNA遺伝子配列中に作製したプライマーのペア、２つ目は5.8S rRNA遺伝子配列中に作製したプライマーとITS-２配列中に作製したプライマーのペア、３つ目はITS-１配列中に作製したプライマーとITS-２配列中に作製したプライマーのペアである。
１つ目のペアは、検出方法においてはタッチダウンPCR法が採用されており、そのためPCR温度条件が複雑になり、特に検出限界付近の低濃度で落花生属植物が含まれている試料を検査する場合には、PCR装置毎の微妙な温度特性の違い等の影響で異なる検査結果を与える可能性がある。
２つ目のペア及び３つ目のペアは、タッチダウンPCR法を採用していないが、落花生属と同じマメ科に属する一部の近縁植物を誤って検出する可能性がある。また、PCR標的増幅産物も比較的長く、２つ目のペアの場合は２５３〜２５９ｂｐ、３つ目のペアの場合は３８４〜３９０ｂｐと長いため、加工されて鋳型DNAが寸断された試料の場合、PCR標的増幅産物が短い方法に比べて検出感度が低くなることが危惧される。
About the primer for the detection of the peanut genus and its detection method, the applicant of this application has already developed and applied for a patent (patent document 1).
In the detection method, any one of the three types of primer pairs is used. That is, the first is a pair of primers made in the peanut ITS-1 sequence and the primer made in the 5.8S rRNA gene sequence, the second is the primer and ITS-2 sequence made in the 5.8S rRNA gene sequence The pair of primers prepared in the third is the pair of the primer prepared in the ITS-1 sequence and the primer prepared in the ITS-2 sequence.
For the first pair, the touchdown PCR method is used in the detection method, which complicates the PCR temperature conditions, and in particular examines samples containing peanut plants at low concentrations near the detection limit. In some cases, different test results may be given due to the influence of subtle differences in temperature characteristics among PCR devices.
Although the second pair and the third pair do not employ the touchdown PCR method, some related plants belonging to the same leguminous family as the peanut genus may be erroneously detected. In addition, the PCR target amplification product is also relatively long, and in the case of the second pair, it is 253 to 259 bp, and in the case of the third pair, it is 384 to 390 bp. There is a concern that the detection sensitivity will be lower than the method with a short PCR target amplification product.
落花生属の植物を検出するPCR法を行うに当たって、タッチダウンPCR法を採用しないこと、これによって、タッチダウンPCR法に起因する上記問題を解決することを目的とする。
また、加工品においても、落花生属の植物を感度よく検出することを目的とする。
また、落花生属と同じマメ科に属する一部の近縁植物が誤って検出されない様にすることを目的とする。
In carrying out the PCR method for detecting plants belonging to the genus Peanut, the object is to not use the touchdown PCR method and thereby solve the above-mentioned problems caused by the touchdown PCR method.
Another object of the present invention is to detect plants of the genus Peanut with high sensitivity even in processed products.
It is another object of the present invention to prevent some related plants belonging to the same leguminous family as the peanut genus from being erroneously detected.
上記目的に対して、本発明者らが鋭意検討を重ねた結果、特定の塩基配列を有するプライマーを用いることによって上記目的を効果的に達成することができることを見出し、本発明を完成させた。すなわち、本発明は、配列番号１又は配列番号２で表される塩基配列からなる落花生属の検出用プライマーを提供する。
また、本発明は、配列番号１で表される塩基配列からなるプライマーと配列番号２で表される塩基配列からなるプライマーとを組み合わせた落花生属の検出用プライマーペアを提供する。
さらに、本発明は、前記プライマーペアを用いて、落花生属のITS-1配列の少なくとも一部を含む増幅産物を得る工程と、該増幅産物の存在を指標として落花生属の存在を判定する工程とを含む落花生属の検出方法を提供する。
As a result of extensive studies by the present inventors for the above object, it was found that the object can be effectively achieved by using a primer having a specific base sequence, and the present invention has been completed. That is, the present invention provides a primer for detecting a peanut genus comprising a base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
The present invention also provides a primer pair for detecting a peanut genus in which a primer comprising the base sequence represented by SEQ ID NO: 1 and a primer comprising the base sequence represented by SEQ ID NO: 2 are combined.
Furthermore, the present invention provides a step of obtaining an amplification product containing at least a part of the ITS-1 sequence of the peanut genus using the primer pair, and a step of determining the presence of the peanut genus using the presence of the amplification product as an index; A method for detecting a peanut genus comprising:
本発明の落花生属の検出用プライマー及びその検出方法によると、PCR法を行うに当たって、タッチダウンPCR法を採用しないことによって、タッチダウンPCR法に起因する、PCR温度条件が複雑になり、特に検出限界付近の低濃度で落花生属植物が含まれている試料を検査する場合には、PCR装置毎の微妙な温度特性の違い等の影響で異なる検査結果を与える可能性を小さくすることができる。また、PCR標的増幅産物を85〜87bpと短くしたことにより、加工品においても、落花生属の植物を感度よく検出することができる。また、本発明のプライマーに変更することによって、落花生属と同じマメ科に属する一部の近縁植物が誤って検出されない様にすることができるという利点がある。 According to the peanut genus detection primer and its detection method of the present invention, the PCR temperature condition resulting from the touchdown PCR method becomes complicated by not using the touchdown PCR method in performing the PCR method. When inspecting a sample containing a peanut genus plant at a low concentration near the limit, it is possible to reduce the possibility of giving different test results due to the subtle differences in temperature characteristics of each PCR device. In addition, by shortening the PCR target amplification product to 85 to 87 bp, it is possible to detect plants of the peanut genus with high sensitivity even in processed products. Further, by changing to the primer of the present invention, there is an advantage that some related plants belonging to the same leguminous family as the peanut genus can be prevented from being erroneously detected.
本発明においては、まず、落花生属の検出用プライマーを設計した。設計されるプライマーは、落花生属のITS-1領域に設計され、落花生属のITS-1配列中の塩基配列を有する核酸分子とストリンジェントな条件下でハイブリダイズし得るプライマーであって、該核酸分子とハイブリダイズした時に3'末端が落花生属のITS-1配列中の塩基と相補的に結合する特性を有するオリゴヌクレオチドであることが必要であり、当該要件を満足する具体的なプライマーとして、下記の配列番号１又は配列番号２で表されるオリゴヌクレオチドを見出した。
ＣＡＡＡＡＣＣＣＣＧＧＣＧＣＧＧＡＡＡ（配列番号１）
ＧＴＣＧＣＣＣＣＧＡＣＣＧＧＡＴＧＣ（配列番号２）
In the present invention, first, a primer for detecting peanuts was designed. The designed primer is a primer designed in the ITS-1 region of peanut genus and capable of hybridizing under stringent conditions with a nucleic acid molecule having a base sequence in the ITS-1 sequence of peanut genus, As a specific primer that satisfies the requirements, it is necessary that the oligonucleotide has a property that the 3 ′ end hybridizes to the base in the ITS-1 sequence of the peanut genus when hybridized with the molecule. The oligonucleotide represented by the following SEQ ID NO: 1 or SEQ ID NO: 2 was found.
CAAAACCCCGGCGCGGAAA (SEQ ID NO: 1)
GTCGCCCCGACCGGATGC (SEQ ID NO: 2)
なお、「ストリンジェントな条件下でハイブリダイズする」とは、２つのDNA断片がSambrook Jらによって記載されたような標準的なハイブリダイゼーション条件下で、相互にハイブリダイズすることを意味する（Expression of cloned genes in E. coli（Molecular Cloning：A laboratory manual（1989））Cold Spring harbor Laboratory Press, New York, USA, 9. 47-9. 62及び11.45-11.61）。より具体的には、例えば以下の式で求められるTm値を基準としてハイブリダイゼーション及び洗浄（例えば約2.0×SSC、50℃）を行うことを意味する。
Tm＝81.5＋16.6（log10[Na+]）＋0.41（fraction G＋C）−（600/N）
また、本明細書でいう属とは、属に含まれる落花生全部を含むもの、又は属に含まれる落花生の中から選んだ幾つかの種を含むものを意味する。
“Hybridize under stringent conditions” means that two DNA fragments hybridize with each other under standard hybridization conditions as described by Sambrook J et al. (Expression of cloned genes in E. coli (Molecular Cloning: A laboratory manual (1989)) Cold Spring harbor Laboratory Press, New York, USA, 9. 47-9.62 and 11.45-11.61). More specifically, for example, hybridization and washing (for example, about 2.0 × SSC, 50 ° C.) are performed based on the Tm value obtained by the following formula.
Tm = 81.5 + 16.6 (log10 [Na +]) + 0.41 (fraction G + C)-(600 / N)
Moreover, the genus as used in this specification means the thing containing all the peanuts contained in a genus, or the thing containing some species selected from the peanuts contained in a genus.
次に、前記のプライマーの一方をセンスプライマー、他方をアンチセンスプライマーとして、PCRを行い、落花生属のITS-1配列の少なくとも一部を含むPCR標的増幅産物の存在を指標として落花生属の存在を検出する。具体的には、配列番号１で表される塩基配列からなるプライマーと配列番号２で表される塩基配列からなるプライマーとを組み合わせた落花生属の検出用プライマーペアを用いて、落花生属のITS-1配列の少なくとも一部を含む増幅産物を得、該増幅産物の存在を指標として落花生属の存在を判定する。
上記PCRに当たっては、例えば、Saiki RK, et al., Science, 230: 1350-1354（1985）や植物細胞工学別冊、植物のPCR実験プロトコール、島本功・佐々木卓治監修（1995年）等に記載されている通常の方法に基づき、変性、アニーリング、伸張の各ステップの温度と時間、酵素（DNA ポリメラーゼ）の種類と濃度、dNTP濃度、プライマー濃度、塩化マグネシウム濃度、鋳型DNA量等の条件を適宜、変更し最良のものを選択する。ただし、アニーリング温度が全てのサイクルにわたって一定である条件を用いた一般的なPCR法を採用することができ、いわゆるタッチダウンPCR法を採用する必要はない。
Next, PCR is performed using one of the primers as a sense primer and the other as an antisense primer, and the presence of a PCR target amplification product containing at least a part of the ITS-1 sequence of the peanut genus is used as an indicator to determine the presence of the peanut genus. To detect. Specifically, using a primer pair for detecting a peanut genus in which a primer composed of the base sequence represented by SEQ ID NO: 1 and a primer composed of the base sequence represented by SEQ ID NO: 2 are combined, the ITS- An amplification product including at least a part of one sequence is obtained, and the presence of the peanut genus is determined using the presence of the amplification product as an index.
The PCR is described in, for example, Saiki RK, et al., Science, 230: 1350-1354 (1985), separate volume of plant cell engineering, plant PCR experiment protocol, supervised by Isao Shimamoto and Takuji Sasaki (1995), etc. Based on the usual methods, conditions such as denaturation, annealing, extension step and temperature, enzyme (DNA polymerase) type and concentration, dNTP concentration, primer concentration, magnesium chloride concentration, template DNA amount, etc. Change and select the best one. However, a general PCR method using conditions in which the annealing temperature is constant over all cycles can be adopted, and there is no need to adopt a so-called touchdown PCR method.
上記PCR後に、85〜87bpサイズのPCR標的増幅産物が存在することを指標として、落花生属の混入を検出する。この検出に当たっては、PCR後の反応液中に標的とするサイズ（85〜87bp）のPCR増幅産物が存在するか否か、すなわち、落花生属のITS-1配列の少なくとも一部を含む標的とするサイズのPCR増幅産物が存在すれば、被検査対象試料中に落花生属の植物が混入していることになり、反対に、PCR増幅産物が存在しないか、又はPCR増幅産物が存在しても落花生属のITS-1配列の少なくとも一部を含む標的とするサイズのPCR増幅産物が存在しなければ、被検査対象試料中に落花生属の植物が混入していないことになる。そして、85〜87bpサイズのPCR標的増幅産物が存在するかどうかの確認は、食品原料や製品等の被検査対象試料から抽出したDNAのPCR後の反応液を、例えば電気泳動によって解析して、増幅産物サイズ85〜87bpに単一バンドが現れているかどうかによって、落花生属が被検査対象試料中に存在するか否かを検出する。本発明では、この検出を高感度、例えば２ppmレベルでの検出を可能とした。 After the PCR, contamination of the peanut genus is detected using as an index the presence of a PCR target amplification product having a size of 85 to 87 bp. In this detection, whether or not a PCR amplification product of a target size (85 to 87 bp) exists in the reaction solution after PCR, that is, a target containing at least a part of the ITS-1 sequence of the peanut genus If there is a PCR amplification product of the size, it means that a plant belonging to the genus Peanut is mixed in the sample to be examined, and conversely, the peanut is absent even if the PCR amplification product is not present or present. If there is no target-sized PCR amplification product containing at least a portion of the genus ITS-1 sequence, then the peanut plant is not contaminated in the sample to be examined. And confirmation of whether the PCR target amplification product of 85-87 bp size exists, the reaction solution after PCR of DNA extracted from the sample to be inspected such as food materials and products, for example, by electrophoresis, Whether or not a peanut genus is present in the sample to be examined is detected depending on whether or not a single band appears in the amplification product size of 85 to 87 bp. In the present invention, this detection can be performed with high sensitivity, for example, at a level of 2 ppm.
（実施例１）
（試料の調製）
種子を1％ SDS溶液中で超音波洗浄後、蒸留水中で超音波洗浄し、50℃で風乾した。2ml容チューブ（eppendorf社製）に種子約0.3gとφ7mm径のジルコニアビーズ（株式会社ニッカトー製）1粒を入れ、Retsch MM 300（QIAGEN社製）により細かく粉砕した。粉砕後のチューブに1mlのバッファー G2（QIAGEN社製）、10μlのProteinase K（20mg／ml）（QIAGEN社製）、1μlのRNase A（100mg／ml）（QIAGEN社製）を加え、混合した後、50℃で1時間保温した。その後、約3,000×gで10分間遠心分離し、その上清液を得た。得られた上清液を、予め1mlのバッファー QBT（QIAGEN社製）で平衡化したGenomic-tip 20／G（QIAGEN社製）に供してDNAをtipに吸着させた。その後、4mlのバッファーQC（QIAGEN社製）でtipを洗浄し、予め50℃に加温してある1mlのバッファーQF（QIAGEN社製）でDNAを溶出させた。溶出液に4容量のバッファーNT2（MACHEREY-NAGEL社製）を加えて混合した後、二本のNucleoSpin Extract Column（MACHEREY-NAGEL社製）に一回に650μlずつ供し、約6,000×gで1分間遠心分離してDNAをColumnに吸着させた。これを全液量処理するまで繰り返した。その後、Columnに600μlのバッファー NT3（MACHEREY-NAGEL社製）を加え、約6,000×gで1分間遠心分離してColumnを洗浄、再度600μlのバッファーNT3を加え、最高速度で1分間遠心分離して、Columnに残っているバッファーNT3を完全に除去した。最終的に、予め70℃に加温してある100μlのバッファーNE（MACHEREY-NAGEL社製）をColumnに加え、室温で１分静置後最高速度で1分間遠心分離してDNAをColumnから溶出し、イソプロパノール沈澱により回収した沈澱物を50μlの滅菌超純水に溶解した。溶液中のDNA濃度を測定し、適宜滅菌超純水で希釈したものをPCRの鋳型DNA試料とした。なお、鋳型DNAは、DNA溶液を分光光度計で測定し、220〜350nmのスペクトルを測定し、260nmに極大値がみられるものを使用した。
Example 1
(Sample preparation)
The seeds were ultrasonically washed in 1% SDS solution, then ultrasonically washed in distilled water, and air-dried at 50 ° C. About 0.3 g of seeds and one zirconia bead of φ7 mm diameter (manufactured by Nikkato Co., Ltd.) were placed in a 2 ml tube (manufactured by eppendorf), and finely pulverized with Retsch MM 300 (manufactured by QIAGEN). After adding 1 ml of buffer G2 (QIAGEN), 10 μl Proteinase K (20 mg / ml) (QIAGEN) and 1 μl RNase A (100 mg / ml) (QIAGEN) to the tube after grinding And kept at 50 ° C. for 1 hour. Thereafter, the mixture was centrifuged at about 3,000 × g for 10 minutes to obtain the supernatant. The obtained supernatant was subjected to Genomic-tip 20 / G (QIAGEN) equilibrated with 1 ml of buffer QBT (QIAGEN) in advance to adsorb the DNA to the tip. Thereafter, the tip was washed with 4 ml of buffer QC (QIAGEN), and the DNA was eluted with 1 ml of buffer QF (QIAGEN) preheated to 50 ° C. After adding 4 volumes of buffer NT2 (MACHEREY-NAGEL) to the eluate and mixing, apply 650 μl at a time to two NucleoSpin Extract Columns (MACHEREY-NAGEL) at approximately 6,000 × g for 1 minute. The DNA was adsorbed on the column by centrifugation. This was repeated until the entire liquid volume was processed. Then, add 600 μl of buffer NT3 (manufactured by MACHEREY-NAGEL) to the column, centrifuge at about 6,000 × g for 1 minute to wash the column, add 600 μl of buffer NT3 again, and centrifuge at maximum speed for 1 minute. The buffer NT3 remaining in the Column was completely removed. Finally, 100 µl of Buffer NE (manufactured by MACHEREY-NAGEL) preheated to 70 ° C is added to the Column, left at room temperature for 1 minute, and then centrifuged at maximum speed for 1 minute to elute the DNA from the Column. The precipitate recovered by isopropanol precipitation was dissolved in 50 μl of sterilized ultrapure water. The DNA concentration in the solution was measured, and appropriately diluted with sterile ultrapure water was used as a PCR template DNA sample. The template DNA used was a DNA solution measured with a spectrophotometer, a spectrum of 220 to 350 nm was measured, and a maximum value was observed at 260 nm.
（PCR反応条件）
PCR反応条件は、表１のとおりである。なお、Taq polymeraseには、QIAGEN社製のHotStarTaq PCR Master Mixを使用した。
The PCR reaction conditions are as shown in Table 1. For Taq polymerase, HotStarTaq PCR Master Mix manufactured by QIAGEN was used.
（検出）
得られたPCR反応液をエチジウムブロマイド含有の3％アガロースゲル電気泳動に供して確認した。
PCRサーマルサイクラーは、通常はGeneAmp PCR System 9600（Applied Biosystems社製）を用いるが、他にGeneAmp PCR System 2400（Applied Biosystems社製）、DNA Engine（MJ Research社製）も使用可能であることを確認している。
上記電気泳動による検出結果を図１に示す。
図１の結果から明らかなように、落花生については、約86bpのサイズにはっきりと標的増幅産物が存在しているのに対し、小麦やとうもろこし等の落花生以外の植物の場合は、約86bpのサイズに標的増幅産物が存在していないことがわかる。このことより、プライマーペアは、落花生を特異的に検出することが示された。また、サケ精子DNA50ngに添加した落花生DNA100fg（2ppm重量/重量）から確実に約86bpのサイズの標的増幅産物が存在していることから、2ppmレベルの感度で落花生を検出できることが示された。
(detection)
The obtained PCR reaction solution was confirmed by subjecting to ethidium bromide-containing 3% agarose gel electrophoresis.
The PCR thermal cycler usually uses GeneAmp PCR System 9600 (Applied Biosystems), but it is confirmed that GeneAmp PCR System 2400 (Applied Biosystems) and DNA Engine (MJ Research) can also be used. is doing.
The detection result by the electrophoresis is shown in FIG.
As is clear from the results of FIG. 1, for peanuts, the target amplification product is clearly present at a size of about 86 bp, while for plants other than peanuts such as wheat and corn, the size is about 86 bp. It can be seen that there is no target amplification product. This showed that the primer pair specifically detects peanuts. In addition, the presence of a target amplification product of about 86 bp in size was confirmed from 100 fg (2 ppm weight / weight) of peanut DNA added to 50 ng salmon sperm DNA, indicating that peanut can be detected with a sensitivity of 2 ppm level.
（実施例２）
落花生近縁種、落花生以外の特定原材料、マメ科植物、主要食品原料植物、ナッツ類のITS-1領域を対象としてAmplify 1.0（Bill Engels）によるPCRシミュレーションを行った。その結果を表２に示す。
PCR simulations using Amplify 1.0 (Bill Engels) were conducted for ITS-1 areas of peanut-related species, specific raw materials other than peanuts, legumes, major food ingredients, and nuts. The results are shown in Table 2.
本発明のプライマー及び当該プライマーを使用したPCR法を活用することによって、食品や食品原材料等の中に微量の落花生が混入していないかどうかを検出するのに大いに役立たせることができる。 By utilizing the primer of the present invention and the PCR method using the primer, it can be greatly useful for detecting whether or not a trace amount of peanut is mixed in food or food raw materials.
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