JP5605739B2 - A method for detecting batyl score fragrance in a test sample specifically, in a short time, and with high sensitivity - Google Patents
A method for detecting batyl score fragrance in a test sample specifically, in a short time, and with high sensitivity Download PDFInfo
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Description
本発明は、被検試料中のバチルスコアグランス（Bacillus coagulans）を検出する方法に関するものであり、より詳細には被検試料中のバチルスコアグランスを特異的、短時間、かつ、高感度で検出し、その存在の有無を判定する方法に関するものである。 TECHNICAL FIELD The present invention relates to a method for detecting Bacillus coagulans in a test sample, and more specifically, detects Bacillus coagulans in a test sample specifically, in a short time, and with high sensitivity. The present invention relates to a method for determining the presence or absence.
バチルスコアグランスは、缶詰やレトルト製品においてフラットサワーを引き起こす細菌であり、非アルコール飲料（特に低酸性飲料など）において指標菌となっている。バチルスコアグランスによる汚染を確認するためには、一般に培養法などの各種確認試験が行われる。培養法に関して例えば、バチルスコアグランスの生菌数検出用の培地に関する出願がある（特許文献１）。しかしながら、培養法でのバチルスコアグランスの検出には、以下の問題点が挙げられる。
バチルスコアグランスの培養には、温度や培地等の適切な調整が必要であり、さらに本菌の増殖は遅発であるため本菌由来の汚染を確認する場合には、１週間程度は必要である。
したがって、バチルスコアグランスによる汚染の確認試験の操作は煩雑で熟練を求められる。
Bacillus scourance is a bacterium that causes flat sour in canned and retort products, and is an indicator for non-alcoholic beverages (especially low acid beverages). In order to confirm the contamination due to the bacillus scent, generally various confirmation tests such as a culture method are performed. Regarding the culture method, for example, there is an application relating to a medium for detecting the viable count of Bacillus glances (Patent Document 1). However, the following problems can be mentioned in the detection of the bacillus gland by the culture method.
When cultivating Bacillus sorghum, appropriate adjustments such as temperature and culture medium are necessary. Furthermore, since the growth of this bacterium is delayed, it takes about 1 week to confirm contamination from this bacterium. is there.
Therefore, the operation of the contamination confirmation test using the batyl score glance is complicated and requires skill.
微生物の検出方法としては、培養法の他に遺伝子による検出方法がある。遺伝子検出法とは、PCR法で特異的なプライマーによりターゲットの増幅産物を増幅させて検出する方法である。微生物を特定する遺伝子の対象領域としては16SやITSなどさまざまな領域が存在し、種々のプライマーを用いることで対象遺伝子の増幅が試みられている。
しかしながら、製造現場での検出では、バチルスコアグランスの有無を判断することが求められているが、現在までに報告されているPCR法を用いたバチルスコアグランスの検出では、3.9×103cfu/mlでなければ検出できない。したがって、感度を高めることは実用化に向けて課題である（特許文献２）。
As a method for detecting a microorganism, there is a detection method using a gene in addition to a culture method. The gene detection method is a method in which a target amplification product is amplified and detected by a specific primer in the PCR method. Various target regions such as 16S and ITS exist as target regions of genes that specify microorganisms, and amplification of target genes has been attempted using various primers.
However, in the detection at the manufacturing site, it is required to determine the presence or absence of the batyl score glance, but in the detection of the bacil score glance using the PCR method reported so far, 3.9 × 10 3 cfu / Detection is only possible with ml. Therefore, increasing the sensitivity is a problem for practical use (Patent Document 2).
本発明は、被検試料中のバチルスコアグランスを特異的、短時間、かつ、高感度で検出する方法を提供することを目的とする。 An object of the present invention is to provide a method for detecting a batyl score glance in a test sample specifically, in a short time, and with high sensitivity.
本発明は、バチルスコアグランスの16S rRNA遺伝子に特異的な塩基配列又は前記塩基配列の相補的配列を含むオリゴヌクレオチドであって、前記特異的な塩基配列が配列番号１又は２に示される塩基配列である、前記オリゴヌクレオチドを提供する。
また、本発明は、バチルスコアグランスの16S rRNA遺伝子を鋳型としてPCR法を行うために用いられるオリゴヌクレオチドプライマーであって、バチルスコアグランスの16S rRNA遺伝子に特異的な塩基配列又は前記塩基配列の相補的配列を含むオリゴヌクレオチドを１種以上含み、前記特異的な塩基配列が配列番号１又は２に示される塩基配列である、前記オリゴヌクレオチドプライマーを提供する。
また、本発明は、被検試料におけるバチルスコアグランスの存在の判定方法であって、被検試料から核酸を抽出し、前記オリゴヌクレオチドプライマーを用いてPCR法を行うことを含む前記判定方法を提供する。
また、本発明は、被検試料から抽出した核酸を鋳型としてPCR法を行うことによって得られる特定の増幅産物を指標として、前記被検試料におけるバチルスコアグランスの存在を判定するために用いられる判定キットであって、バチルスコアグランスの16S rRNA遺伝子に特異的な塩基配列又は前記塩基配列の相補的配列を含むオリゴヌクレオチドであって、前記特異的な塩基配列が配列番号１又は２に示される塩基配列である前記オリゴヌクレオチドを１種以上含む前記キットを提供する。
The present invention relates to an oligonucleotide containing a base sequence specific to the 16S rRNA gene of Bacillus glance or a complementary sequence of the base sequence, wherein the specific base sequence is represented by SEQ ID NO: 1 or 2. The oligonucleotide is provided.
The present invention also relates to an oligonucleotide primer used for performing a PCR method using a 16S rRNA gene of Bacillus saccharence as a template, and a base sequence specific to the 16S rRNA gene of Bacillus sagrance or a complement of the base sequence Provided is the oligonucleotide primer comprising at least one oligonucleotide containing a target sequence, wherein the specific base sequence is the base sequence shown in SEQ ID NO: 1 or 2.
In addition, the present invention provides a method for determining the presence of a batyl score glance in a test sample, the method comprising extracting a nucleic acid from the test sample and performing a PCR method using the oligonucleotide primer To do.
Further, the present invention provides a determination used for determining the presence of a batyl score glance in the test sample, using as a marker a specific amplification product obtained by performing PCR using a nucleic acid extracted from the test sample as a template. A kit, which is an oligonucleotide containing a base sequence specific to the 16S rRNA gene of Bacillus glance or a complementary sequence of the base sequence, wherein the specific base sequence is shown in SEQ ID NO: 1 or 2 The kit is provided comprising one or more of the oligonucleotides as sequences.
本発明により、被検試料中のバチルスコアグランスを特異的、短時間、かつ、高感度で検出することができる。 According to the present invention, it is possible to detect a batyl score fragrance in a test sample specifically, in a short time, and with high sensitivity.
本発明の被検試料におけるバチルスコアグランスの存在の判定方法は、被検試料から核酸を抽出し、特定のオリゴヌクレオチドプライマーを用いてPCR法を行うことを含む。
被検試料としては、緑茶、烏龍茶、麦茶などの低酸性飲料をはじめとする飲料のほか、レトルト食品及びこれらの原料などが挙げられる。
被検試料から核酸（RNA）を抽出する方法としては、従来技術において公知の方法を使用することができる。例えば、RNeasy Mini Kit（QIAGEN社）などを用いて行うことができる。また、抽出の際には、RNA分解酵素阻害剤としてジチオトレイトールを用いることが好ましい。従来技術においてよく使用されているβ-メルカプトエタノールを用いる場合と比較して、検出感度が高くなる。また、ジチオトレイトールはβ-メルカプトエタノールのように猛烈な悪臭や異臭を発することも無く、毒物及び劇物に指定されたβ-メルカプトエタノールよりも扱いが容易である。好ましくは、ジチオトレイトールは、20〜80mMの範囲で用いられ、より好ましくは35〜60mMの範囲で用いられる。
また、被検試料から核酸を抽出する前に、例えばメンブレンフィルターを用いて菌体を補足し、これを培養してもよい。これにより検出感度を高めることができる。
The method for determining the presence of a batyl score glance in a test sample of the present invention includes extracting a nucleic acid from the test sample and performing a PCR method using a specific oligonucleotide primer.
Examples of the test sample include beverages including low acid beverages such as green tea, oolong tea and barley tea, retort foods, and raw materials thereof.
As a method for extracting nucleic acid (RNA) from a test sample, a method known in the prior art can be used. For example, RNeasy Mini Kit (QIAGEN) can be used. In the extraction, dithiothreitol is preferably used as an RNase inhibitor. Compared with the case of using β-mercaptoethanol which is often used in the prior art, the detection sensitivity is increased. In addition, dithiothreitol does not emit a foul odor or off-flavor like β-mercaptoethanol, and is easier to handle than β-mercaptoethanol designated as a poisonous or deleterious substance. Preferably, dithiothreitol is used in the range of 20-80 mM, more preferably in the range of 35-60 mM.
Moreover, before extracting a nucleic acid from a test sample, a microbial cell may be supplemented, for example using a membrane filter, and you may culture | cultivate this. Thereby, detection sensitivity can be raised.
本発明の判定方法においては、次いで抽出した核酸を鋳型としてPCR法を行う。PCR法においては、バチルスコアグランスの16S rRNA遺伝子に特異的な塩基配列又は前記塩基配列の相補的配列を含むオリゴヌクレオチドを１種以上含むオリゴヌクレオチドプライマーを使用する。ここで、前記特異的な塩基配列は、下記配列番号１又は２に示される塩基配列である。
(5')GCATGGAGAAAAAGGAAAG(3')（配列番号１）
(5')CCCGAAGGCCTTCTTCAC(3')（配列番号２）
配列番号１又は２で表される塩基配列又はこれに相補的な塩基配列に50以下、好適には20以下の塩基が付加されたオリゴヌクレオチドを含むものであっても、このようなオリゴヌクレオチドがバチルスコアグランスの16S rRNA遺伝子に特異的である限り、本発明においてオリゴヌクレオチドプライマーとして使用しても良い。さらに、配列番号１又は２で表される塩基配列又はこれに相補的な塩基配列において、10以下の塩基が、置換、欠失、挿入及び/又は付加されたオリゴヌクレオチドを含むものであっても、このようなオリゴヌクレオチドがバチルスコアグランスの16S rRNA遺伝子に特異的である限り、本発明においてオリゴヌクレオチドプライマーとして使用しても良い。本発明の判定方法において、好ましくはオリゴヌクレオチドプライマーとして、配列番号１で表される塩基配列からなるオリゴヌクレオチド及び配列番号２で表される塩基配列からなるオリゴヌクレオチドを含むオリゴヌクレオチドプライマーを使用する。
In the determination method of the present invention, PCR is then performed using the extracted nucleic acid as a template. In the PCR method, an oligonucleotide primer containing at least one kind of oligonucleotide containing a base sequence specific to the 16S rRNA gene of Bacillus glance or a complementary sequence of the base sequence is used. Here, the specific base sequence is a base sequence represented by SEQ ID NO: 1 or 2 below.
(5 ′) GCATGGAGAAAAAGGAAAG (3 ′) (SEQ ID NO: 1)
(5 ') CCCGAAGGCCTTCTTCAC (3') (SEQ ID NO: 2)
Even if the oligonucleotide includes 50 or less, preferably 20 or less bases added to the base sequence represented by SEQ ID NO: 1 or 2, or a base sequence complementary thereto, As long as it is specific to the 16S rRNA gene of the bacillus gland, it may be used as an oligonucleotide primer in the present invention. Furthermore, in the base sequence represented by SEQ ID NO: 1 or 2, or a base sequence complementary thereto, it may contain an oligonucleotide wherein 10 or less bases are substituted, deleted, inserted and / or added. As long as such an oligonucleotide is specific to the 16S rRNA gene of Bacillus glucans, it may be used as an oligonucleotide primer in the present invention. In the determination method of the present invention, preferably, an oligonucleotide primer comprising an oligonucleotide having the base sequence represented by SEQ ID NO: 1 and an oligonucleotide having the base sequence represented by SEQ ID NO: 2 is used as the oligonucleotide primer.
本発明の判定方法において、PCR法は公知の方法を用いて行われる。例えば、抽出された核酸（RNAまたはDNA）を鋳型としてcDNAを合成し、熱変性によりcDNAを一本鎖にした後（例えば94℃）、これにプライマーをアニーリングさせ（例えば、60℃）、耐熱性DNAポリメラーゼを用いて相補鎖を合成する（例えば、72℃）、というサイクルを、例えば20〜40回繰返すことにより、目的とする遺伝子領域だけを増幅させる。このようにして得られた増幅産物を、例えば電気泳動やクロマトグラフィー、DNAチップ（マイクロアレイ）、抗原抗体反応などによって検出することができる。 In the determination method of the present invention, the PCR method is performed using a known method. For example, cDNA is synthesized using the extracted nucleic acid (RNA or DNA) as a template, the cDNA is made into a single strand by heat denaturation (for example, 94 ° C.), and then annealed with a primer (for example, 60 ° C.). By repeating a cycle of synthesizing a complementary strand using a sex DNA polymerase (for example, 72 ° C.), for example, 20 to 40 times, only the target gene region is amplified. The amplification product thus obtained can be detected by, for example, electrophoresis, chromatography, DNA chip (microarray), antigen-antibody reaction or the like.
１．前培養
あらかじめ適宜希釈した菌体１mLを500mLの緑茶に摂取して菌懸濁液を得た。この菌懸濁液500mLをメンブレンフィルターにより吸引ろ過して菌体を捕捉し、このメンブレンフィルターを平板寒天培地上に載せた。次いで、バチルスコアグランスの最適な増殖温度である45℃で６時間の培養を行った。使用した寒天培地は以下のとおりである。
製品名：パールコア標準寒天培地（栄研化学株式会社製）
組成（培地：1,000mLあたり）：
酵母エキス：2.5ｇ
リプトン：5.0ｇ
ブドウ糖：1.0ｇ
カンテン：15.0ｇ
1. Pre-culture 1 mL of bacterial cells diluted appropriately in advance was taken into 500 mL of green tea to obtain a bacterial suspension. 500 mL of this bacterial suspension was suction filtered through a membrane filter to capture the bacterial cells, and this membrane filter was placed on a flat plate agar medium. Subsequently, the cells were cultured for 6 hours at 45 ° C., which is the optimum growth temperature for the bacillus glance. The agar medium used was as follows.
Product name: Pearl core standard agar medium (Eiken Chemical Co., Ltd.)
Composition (medium: per 1,000 mL):
Yeast extract: 2.5g
Lipton: 5.0g
Glucose: 1.0g
Kanten: 15.0g
２．核酸（RNA）抽出
上記で示す培養したメンブレンフィルターを回収し、RNeasy Mini Kit（QIAGEN社）を使用して、次のようにして核酸の抽出を行った。
・溶菌工程
（１）ハイブリダイゼーション用バックにメンブレンフィルターを挿入した。
（２）リゾチーム（15mg/mL）を添加したTEバッファーを400μL添加後、ヒートシールした。
（３）室温で10分間静置した（ただし、２分間隔で10秒間揉む）。
（４）バッファーRLTにRNA分解酵素阻害剤としてDTTの終濃度が40mMとなるように加えたものを1.4mL添加した。
（５）混合液を２mLチューブに回収した。
（６）18900×ｇで２分間遠心し、上清1.6mLを除き、エタノールを１mL添加し、ピペッテイングにて激しく撹拌した。
・精製工程
（１）得られた溶菌液をカラムにアプライし、8000×ｇで15秒間遠心した後、流下液を廃棄した。
（２）700μLのバッファーRW1を加え、≧8000×ｇで15秒間遠心し、流下液とチューブを廃棄した。
（３）新しいチューブにカラムを移し、500μLのバッファーRPEを加え、≧8000×ｇで15秒間遠心し、流下液を廃棄した。
（４）500μlのバッファーRPEを加え、≧8000×ｇで２分間遠心した。
（５）新しい1.5mLチューブにカラムを移し、30〜50μLのRNase Free Waterを添加し、8000×ｇで１分間遠心してRNAを抽出した。
2. Nucleic acid (RNA) extraction The cultured membrane filter shown above was collected, and nucleic acid was extracted using RNeasy Mini Kit (QIAGEN) as follows.
-Lysis step (1) A membrane filter was inserted into the hybridization bag.
(2) After adding 400 μL of TE buffer added with lysozyme (15 mg / mL), heat sealing was performed.
(3) It was allowed to stand at room temperature for 10 minutes (however, it was held for 10 seconds at 2 minute intervals).
(4) 1.4 mL of a buffer RLT added as an RNase inhibitor so that the final concentration of DTT was 40 mM was added.
(5) The mixed solution was collected in a 2 mL tube.
(6) Centrifugation was performed at 18900 × g for 2 minutes, 1.6 mL of the supernatant was removed, 1 mL of ethanol was added, and the mixture was vigorously stirred by pipetting.
Purification step (1) The obtained lysate was applied to a column and centrifuged at 8000 × g for 15 seconds, and then the falling solution was discarded.
(2) 700 μL of buffer RW1 was added, centrifuged at ≧ 8000 × g for 15 seconds, and the falling liquid and the tube were discarded.
(3) The column was transferred to a new tube, 500 μL of buffer RPE was added, and the mixture was centrifuged at ≧ 8000 × g for 15 seconds, and the falling solution was discarded.
(4) 500 μl of buffer RPE was added and centrifuged at ≧ 8000 × g for 2 minutes.
(5) The column was transferred to a new 1.5 mL tube, 30-50 μL of RNase Free Water was added, and RNA was extracted by centrifugation at 8000 × g for 1 minute.
３．バチルスコアグランスの特異的なヌクレオチド配列の特定
バチルスコアグランスを特異的に検出するために、他の微生物と異なる特異的なバチルスコアグランスの16S rRNA遺伝子をコードするヌクレオチド配列を特定し、そのヌクレオチド配列と相補的になるように化学合成された以下のオリゴヌクレオチドをRT-PCR法のプライマーセットとして用いた。
(5')GCATGGAGAAAAAGGAAAG(3')（配列番号１）
(5')CCCGAAGGCCTTCTTCAC(3')（配列番号２）
3. Identification of a specific nucleotide sequence of the bacillus gland In order to specifically detect the bacillus gland, a nucleotide sequence encoding a specific 16S rRNA gene of a specific bacillus gland that is different from other microorganisms is identified, and the nucleotide sequence The following oligonucleotides chemically synthesized so as to be complementary to each other were used as primer sets for the RT-PCR method.
(5 ′) GCATGGAGAAAAAGGAAAG (3 ′) (SEQ ID NO: 1)
(5 ') CCCGAAGGCCTTCTTCAC (3') (SEQ ID NO: 2)
４．RT-PCR増幅
上記プライマーセットを用いてRT−PCRによる増幅を行った。反応は、サンプル５μLに対して、逆転写酵素：Prime Script Rtase 、DNA 合成酵素：Takara Ex TaqTM及びRNase Inhibitorを含むPrime Script One Step RT-PCR Kit Ver.2（タカラバイオ株式会社）を２μL、反応バッファーにdNTP Mixture及びOne Step Enhancer Solutionを含む「2×1 step buffer」（タカラバイオ株式会社）を25μL、プライマーとして配列番号１及び配列番号２に示すプライマーセットを20pmol加え、最終的に滅菌水で50μLに調整して行った。 RT-PCR条件を以下に示す。
（ｉ）逆転写：50℃/30分〔１サイクル〕
（ii）熱変性：94℃/２分〔１サイクル〕
（iii）熱変性：94℃/１分、アニーリング：60℃/１分、伸長反応：72℃/１分〔30サイクル〕
（iv）伸長反応：72℃/２分〔１サイクル〕
4). RT-PCR amplification RT-PCR amplification was performed using the above primer set. For the reaction, 5 μL of the sample, 2 μL of Prime Script One Step RT-PCR Kit Ver.2 (Takara Bio Inc.) containing reverse transcriptase: Prime Script Rtase, DNA synthase: Takara Ex Taq ™ and RNase Inhibitor, Add 25 μL of “2 × 1 step buffer” (Takara Bio Inc.) containing dNTP Mixture and One Step Enhancer Solution to the reaction buffer, add 20 pmol of the primer set shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers, and finally sterilized water Adjusted to 50 μL. RT-PCR conditions are shown below.
(I) Reverse transcription: 50 ° C / 30 minutes [1 cycle]
(Ii) Thermal denaturation: 94 ° C / 2 minutes [1 cycle]
(Iii) Thermal denaturation: 94 ° C./1 min, annealing: 60 ° C./1 min, extension reaction: 72 ° C./1 min [30 cycles]
(Iv) Extension reaction: 72 ° C / 2 minutes [1 cycle]
５．検出
３％アガロースゲルを用いてTAE Buffer中で120Ｖ及び40分間の条件で電気泳動し、臭化エチジウム溶液で15分染色した。流水で15分間洗浄した後、トランスイルミネーターにより発色させ、カメラで撮影した。
5. Detection Using 3% agarose gel, electrophoresis was performed in TAE Buffer under conditions of 120 V and 40 minutes, and stained with ethidium bromide solution for 15 minutes. After washing with running water for 15 minutes, the color was developed with a transilluminator and photographed with a camera.
６．検出可能な菌体濃度
被検試料中の菌体濃度を変えた結果を図１に示す。Sample 1は、1.1×104cfu/mLの菌体濃度のサンプルから核酸（RNA）を抽出したものである。Sample 2〜5は、段階希釈（数字が増えるに従い菌体数は1/10ずつ減少する）したサンプルである。なお、Sample 5は、約1cfuに相当する。
この結果より、本発明の方法は、バチルスコアグランスを14時間で、かつ、ほぼ１菌体からの検出が可能であることを示した。
6). Detectable bacterial cell concentration The results of changing the bacterial cell concentration in the test sample are shown in FIG. Sample 1 is obtained by extracting nucleic acid (RNA) from a sample having a cell concentration of 1.1 × 10 4 cfu / mL. Samples 2 to 5 are samples that are serially diluted (the number of cells decreases by 1/10 as the number increases). Sample 5 corresponds to about 1 cfu.
From this result, it was shown that the method of the present invention can detect the batyl score glance in 14 hours and from almost one microbial cell.
７．β-MEとDTTの比較
55cfuを含む菌体液10μLをチューブに取り、リゾチーム（15mg/mL）を添加したTEバッファーを100μL添加し、上記核酸抽出操作・溶菌工程（３）以降と同様の操作を続けてサンプルを調整した。この際、核酸抽出操作・溶菌工程（４）において、RNA分解酵素阻害剤をβ-メルカプトエタノール（β-ME）109mM、及びジチオトレイトール（DTT）を20〜76mMとして同様の検出を行った。その結果を図２に示す。
図２から明らかなように、39mM〜60mMのジチオトレイトールを用いることにより、β-メルカプトエタノール109mMよりも抽出効率が格段に高くなる。
7). Comparison of β-ME and DTT
10 μL of the bacterial cell solution containing 55 cfu was taken into a tube, 100 μL of TE buffer added with lysozyme (15 mg / mL) was added, and the same operation as in the nucleic acid extraction operation / lysis step (3) and subsequent steps was continued to prepare a sample. At this time, in the nucleic acid extraction operation / bacterial lysis step (4), the same detection was performed with the RNA-degrading enzyme inhibitor β-mercaptoethanol (β-ME) 109 mM and dithiothreitol (DTT) 20-76 mM. The result is shown in FIG.
As is apparent from FIG. 2, the extraction efficiency is significantly higher than that of 109 mM β-mercaptoethanol by using 39 mM to 60 mM dithiothreitol.
８．バチルスコアグランスの特異的な検出
下記に示す菌体の核酸（DNA）抽出サンプル（No.１〜18）について増幅産物の検出を行った。核酸濃度は200ng/mL〜1400ng/mLである。
核酸（DNA）のPCR増幅反応において、手順４を以下のように変更した。サンプル５μLに対して、DNA合成酵素：Takara Ex TaqTMを0.25μL、10xbufferを５μL、dNTP Mixture及びMgCl2を4μL（タカラバイオ株式会社）、プライマーとして手順３で特定したものと同じ配列番号１及び配列番号２に示すプライマーセットを20pmol加え、最終的に滅菌水で50μLに調整して行った。PCR条件は、逆転写反応を含まないことを除いて手順４のRT-PCR条件と同様である。
結果を図３に示す。この結果より、バチルスコアグランスを特異的に検出できることがわかる。
8). Specific Detection of Bacilscoreance The amplification product was detected for nucleic acid (DNA) extracted samples (Nos. 1 to 18) of the bacterial cells shown below. The nucleic acid concentration is 200 ng / mL to 1400 ng / mL.
In the PCR amplification reaction of nucleic acid (DNA), the procedure 4 was changed as follows. DNA synthase: Takara Ex Taq TM 0.25 μL, 10x buffer 5 μL, dNTP Mixture and MgCl 2 4 μL (Takara Bio Inc.), and the same SEQ ID NO: 1 as those specified in step 3 as primers The primer set shown in SEQ ID NO: 2 was added at 20 pmol, and finally adjusted to 50 μL with sterilized water. The PCR conditions are the same as the RT-PCR conditions in Procedure 4 except that the reverse transcription reaction is not included.
The results are shown in FIG. From this result, it can be seen that the bacil score glance can be specifically detected.
・サンプル
1: Alicyclobacillus acidocaldarius
2: Bacillus licheniformis
3: バチルスコアグランス（B.coagulans）
4: Bacillus subtilis
5: Bacillus cereus
6: Bacillus megaterium
7: Paenibacillus polymyxa
8: Bacillus smithii
9: Brevibacillus agri
10: Paenibacillus chibensis
11: Staphylococcus aureus
12: Bacillus thruringiensis
13: Sporolactobacillus inulinus
14: Aeromonas hydrophila
15: Escherichia coli
16: Paenibacillus macerans
17: Brevibacillus brevis
18: バチルスコアグランス（B.coagulans）
·sample
1: Alicyclobacillus acidocaldarius
2: Bacillus licheniformis
3: Bacil score glance (B.coagulans)
4: Bacillus subtilis
5: Bacillus cereus
6: Bacillus megaterium
7: Paenibacillus polymyxa
8: Bacillus smithii
9: Brevibacillus agri
10: Paenibacillus chibensis
11: Staphylococcus aureus
12: Bacillus thruringiensis
13: Sporolactobacillus inulinus
14: Aeromonas hydrophila
15: Escherichia coli
16: Paenibacillus macerans
17: Brevibacillus brevis
18: Bacil score glance (B.coagulans)
９．バチルスコアグランスの確実な検出
バチルスコアグランスを確実に検出できることを確認するために、下記の計32株の核酸（DNA）抽出サンプルを入手して上記８と同様の方法で増幅産物の検出を行い、擬陰性を確認した。その結果を図４に示す。この結果より、擬陰性は認められず、すべてのバチルスコアグランスを検出することができた。
9. Reliable detection of Bacilscore glands In order to confirm that Bacilscore glances can be detected reliably, the following 32 strains of nucleic acid (DNA) extracted samples were obtained, and amplification products were detected in the same manner as above 8. A false negative was confirmed. The result is shown in FIG. From this result, false negatives were not recognized, and all bacillus scent glands could be detected.
・サンプル
上の段、左から 下の段、左から
Bacillus coagulans TIFT 115001 Bacillus coagulans TIFT 115017
Bacillus coagulans TIFT 115004 Bacillus coagulans TIFT 115018
Bacillus coagulans TIFT 115005 Bacillus coagulans TIFT 115019
Bacillus coagulans TIFT 115006 Bacillus coagulans TIFT 115020
Bacillus coagulans TIFT 115007 Bacillus coagulans TIFT 115021
Bacillus coagulans TIFT 115008 Bacillus coagulans TIFT 115022
Bacillus coagulans TIFT 115012 Bacillus coagulans TIFT 115025
Bacillus coagulans TIFT 115013 Bacillus coagulans TIFT 115026
Bacillus coagulans TIFT 115014 Bacillus coagulans TIFT 115027
Bacillus coagulans TIFT 115015 Bacillus coagulans TIFT 115028
Bacillus coagulans TIFT 115016 Bacillus coagulans TIFT 115029
No Sample Bacillus coagulans TIFT 115030
Maker Bacillus coagulans TIFT 115031
Bacillus coagulans ATCC 8038 Bacillus coagulans NBRC 3557
Bacillus coagulans ATCC 11014 Bacillus coagulans NBRC 3886
Bacillus coagulans ATCC 11369 Bacillus coagulans NBRC 3887
Bacillus coagulans ATCC 12245 Bacillus coagulans NBRC 12583
Bacillus coagulans ATCC 15949 Bacillus coagulans NBRC 12714
Bacillus coagulans ATCC 23498
Bacillus coagulans ATCC 31284
Bacillus coagulans ATCC BAA-748
TIFT：東洋食品研究所
NBRC：独立行政法人 製品評価技術基盤機構 生物遺伝資源部門
ATCC：American Type Culture Collection
・ Upper sample, from left to lower, from left
Bacillus coagulans TIFT 115001 Bacillus coagulans TIFT 115017
Bacillus coagulans TIFT 115004 Bacillus coagulans TIFT 115018
Bacillus coagulans TIFT 115005 Bacillus coagulans TIFT 115019
Bacillus coagulans TIFT 115006 Bacillus coagulans TIFT 115020
Bacillus coagulans TIFT 115007 Bacillus coagulans TIFT 115021
Bacillus coagulans TIFT 115008 Bacillus coagulans TIFT 115022
Bacillus coagulans TIFT 115012 Bacillus coagulans TIFT 115025
Bacillus coagulans TIFT 115013 Bacillus coagulans TIFT 115026
Bacillus coagulans TIFT 115014 Bacillus coagulans TIFT 115027
Bacillus coagulans TIFT 115015 Bacillus coagulans TIFT 115028
Bacillus coagulans TIFT 115016 Bacillus coagulans TIFT 115029
No Sample Bacillus coagulans TIFT 115030
Maker Bacillus coagulans TIFT 115031
Bacillus coagulans ATCC 8038 Bacillus coagulans NBRC 3557
Bacillus coagulans ATCC 11014 Bacillus coagulans NBRC 3886
Bacillus coagulans ATCC 11369 Bacillus coagulans NBRC 3887
Bacillus coagulans ATCC 12245 Bacillus coagulans NBRC 12583
Bacillus coagulans ATCC 15949 Bacillus coagulans NBRC 12714
Bacillus coagulans ATCC 23498
Bacillus coagulans ATCC 31284
Bacillus coagulans ATCC BAA-748
TIFT: Toyo Food Research Laboratory
NBRC: National Institute for Product Evaluation Technology Biological Genetic Resources Division
ATCC: American Type Culture Collection
Claims (4)
被検試料から核酸を抽出し、請求項１に記載のオリゴヌクレオチドプライマーを用いてPCR法を行うことを含む前記判定方法。 A method for determining the presence of a batyl score glance in a test sample,
The said determination method including extracting a nucleic acid from a test sample, and performing PCR method using the oligonucleotide primer of Claim 1 .
配列番号１に示される塩基配列からなるオリゴヌクレオチド及び配列番号２に示される塩基配列からなるオリゴヌクレオチドを含む前記キット。 Using a specific amplification product obtained by performing a PCR method using a nucleic acid extracted from a test sample as a template, a determination kit used to determine the presence of a batyl score glance in the test sample,
The kit comprising an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 .
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