JP2005117980A - 食品変敗性乳酸菌の検出用pcrプライマー - Google Patents
食品変敗性乳酸菌の検出用pcrプライマー Download PDFInfo
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Abstract
【解決手段】特定の塩基配列からなるフォワードプライマーと、別の特定の塩基配列からなるリバースプライマーとを組み合わせたプライマーセットからなり、16SrDNA遺伝子に由来するV3領域配列をPCRによって増殖可能な食品変敗性乳酸菌の検出用PCRプライマーとする。食品から採取された乳酸菌を培養してDNAを分離し、このDNAに対して上記のプライマーセットを用いてPCRを行い、該V3領域配列を増殖させ、この増殖配列をDGGEにより分離してバンドを形成させ、同条件で形成したDNA既知乳酸菌のバンドと移動距離を比較することにより乳酸菌種を判定する。
【選択図】なし
Description
そこで、生化学的性状による判別法に代わるものとして、遺伝子レベルでの判別法が注目されている。
先ず、PCR−DGGE法に使用する乳酸菌検出用プライマーを16SrDNAのV3領域にて作成する。
また、リバースプライマーV3−53Rプライマーは、Lane, D. J.: 16S/23S rRNA sequencing: Nucleic Acid Techniques in Bacterial Systematics. Stackebrandt, E. and Goodfellow, M.(eds.), p. 115-136 (1991)の530Rプライマーの3’側を一塩基欠損したものである。
このプライマーペアを用いてPCR条件を設定し、食肉製品から分離された乳酸菌を用いてDGGE条件を決定する。
まず、食肉などを原料とする種々の食品から採取した乳酸菌を培養してDNAを分離する。
この発明でいう食品変敗性乳酸菌の検出対象の食品とは、乳酸菌の検出の有無によって食品の変敗を判別しようとする検査対象物であり、ラクトバチルス属、ストレプトコッカス属、エンテロコッカス属、ロイコノストック属などの乳酸菌が増殖する可能性のある食品である他に、特に条件を限定するものではない。例えば、食肉、食肉加工品、乳製品などが代表例として挙げられる。
菌株は、過去に食肉製品から検出された表1に示す保存菌株を用い、乳酸菌の培地は2%D−グルコースを添加したGAM broth(日水)にて30℃で培養した。
なお、表1および図1中に示したサンプル番号またはレーン番号に対応する菌種(学名の日本語表記)は以下の通りである。
(1)ロイコノストック・カルノーサム
(2)エンテロコッカス・フェカリス
(3)ラクトバチルス・サケ
(4)ロイコノストック・アルゼンチナム
(5)エンテロコッカス・マロドレイタス
(6)ロイコノストック・メセンテロイデス
(7)ラクトバチルス・フェルメンタム
(8)ロイコノストック・サイトリウム
試料の調製;増菌液を1.5mlエッペンドルフチューブに取り、冷却遠心分離(15000×g, 5min)によって集菌し、菌体のペレットにLysozyme-TE buffer(5mg/ml)567μlを加え、37℃で60分間反応させた。反応後、10%SDSsolution30μlと20mg/mlのプロテイナーゼK3μlを加え、さらに37℃で60分間反応させた。
[PCR反応液組成] 10×PCRバッファ(1.5mM MgCl2 ) (Takara)5μl、dNTP mixture (Takara)4μl、20pmol/μlGC40−339fプライマー1.25μl、20pmol/μlV3−53Rプライマー1.25μl、TaqDNAポリメラーゼ(Takara)0.25μl、templateDNA(5ng/μl)5μl、D.W.33.25μl。
DGGE解析は、Bio-Rad D-code Systemを用いて行った。泳動には変性剤濃度(100%変性剤濃度を7M尿素,40%ホルムアミドとした。)30%から60%のグラジェントをつけた8%ポリアクリルアミドゲルを使用した。泳動条件は、200V,3時間、60℃で行った。泳動終了後ゲルをエチジウムブロマイドで染色し、バンドパターンの解析を行った。
まずPCR−DGGE法に使用するための配列番号1および2に示す乳酸菌検出用プライマーを、16SrDNAのV3領域にて作成した。
Claims (2)
- 配列番号1で示される塩基配列からなるフォワードプライマーと、配列番号2で示される塩基配列からなるリバースプライマーとを組み合わせたプライマーセットからなり、16SrDNA遺伝子に由来するV3領域配列をポリメラーゼ連鎖反応(PCR)によって増殖可能な食品変敗性乳酸菌の検出用PCRプライマー。
- 食品から採取された乳酸菌を培養してDNAを分離し、このDNAに対して配列番号1で示される塩基配列からなるフォワードプライマーと、配列番号2で示される塩基配列からなるリバースプライマーとを組み合わせたプライマーセットを用いてポリメラーゼ連鎖反応(PCR)を行い、これにより16SrDNA遺伝子に由来するV3領域配列を増殖させ、この増殖した配列を変性剤濃度勾配ゲル電気泳動法(DGGE)により分離してバンドを形成させ、同条件で形成したDNA既知乳酸菌のバンドと移動距離を比較することにより乳酸菌種を判定することからなる食品変敗性乳酸菌の検出方法。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006136640A1 (es) * | 2005-06-22 | 2006-12-28 | Consejo Superior De Investigaciones Científicas | Detección e identificación de bacteriófagos de bacterias del ácido láctico mediante reacción en cadena de la polimerasa múltiple (multi-pcr) y sus aplicaciones. |
CN101792760A (zh) * | 2010-02-25 | 2010-08-04 | 中国农业科学院饲料研究所 | 获取细菌16S rRNA基因的V3区核苷酸序列的方法及其专用引物 |
CN113324818A (zh) * | 2021-08-04 | 2021-08-31 | 潍坊市检验检测中心 | 一种基于凝胶电泳原理的发酵食品活菌分离装置 |
CN116228772A (zh) * | 2023-05-09 | 2023-06-06 | 聊城市检验检测中心 | 一种生鲜食品变质区域快速检测方法及系统 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101649352B (zh) * | 2009-09-21 | 2011-11-09 | 内蒙古农业大学 | 益生菌乳制品中发酵乳杆菌的快速定性、定量测定方法 |
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2003
- 2003-10-17 JP JP2003357565A patent/JP4191003B2/ja not_active Expired - Fee Related
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006136640A1 (es) * | 2005-06-22 | 2006-12-28 | Consejo Superior De Investigaciones Científicas | Detección e identificación de bacteriófagos de bacterias del ácido láctico mediante reacción en cadena de la polimerasa múltiple (multi-pcr) y sus aplicaciones. |
ES2276597A1 (es) * | 2005-06-22 | 2007-06-16 | Consejo Superior Investig. Cientificas | Deteccion e identificacion de bacteriofagos de bacterias del acido lactico mediante reaccion en cadena de la polimerasa multiple (multi-pcr) y sus aplicaciones. |
CN101792760A (zh) * | 2010-02-25 | 2010-08-04 | 中国农业科学院饲料研究所 | 获取细菌16S rRNA基因的V3区核苷酸序列的方法及其专用引物 |
CN101792760B (zh) * | 2010-02-25 | 2015-03-11 | 中国农业科学院饲料研究所 | 获取细菌16S rRNA基因的V3区核苷酸序列的方法及其专用引物 |
CN113324818A (zh) * | 2021-08-04 | 2021-08-31 | 潍坊市检验检测中心 | 一种基于凝胶电泳原理的发酵食品活菌分离装置 |
CN113324818B (zh) * | 2021-08-04 | 2021-09-28 | 潍坊市检验检测中心 | 一种基于凝胶电泳原理的发酵食品活菌分离装置 |
CN116228772A (zh) * | 2023-05-09 | 2023-06-06 | 聊城市检验检测中心 | 一种生鲜食品变质区域快速检测方法及系统 |
CN116228772B (zh) * | 2023-05-09 | 2023-07-21 | 聊城市检验检测中心 | 一种生鲜食品变质区域快速检测方法及系统 |
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