JP3677056B2 - Method for detecting or identifying lactic acid bacteria - Google Patents

Description

【００１２】
ＰＣＲ法に用いるＤＮＡポリメラーゼは95℃の耐熱性を有するものであればその起源は問わない。ＰＣＲ法の反応条件として、変性温度は90〜95℃、アニーリング温度は40〜60℃、伸長温度は70〜75℃、サイクル数10回以上が好ましいが、これに限定されず、通常ＰＣＲ法に使用できる条件であればよい。
得られた反応生成物は、アガロースゲルを用いた電気泳動法等の検出または同定手段により分離され、増幅産物の有無が確認される。このような方法により、乳酸桿菌 Lactobacillus sp. DA1、Lactobacillus sp. BG2 、Lactobacillus sp. SE3 と同等な性質、つまり、ビール中で増殖するにもかかわらず、ブレビス等の既知のビール有害乳酸菌プライマーでは検出されない性質の乳酸桿菌を検出または同定することができる。これらの乳酸桿菌は、ビール混濁能を有するものであることがわかっている。従って、本発明の方法により、ビール中に存在する有害な菌を検出または同定することが可能となる。
【００１３】
さらに、本発明の検出または同定方法を従来の検出または同定方法と組み合わせることにより、乳酸菌、特に、ビール混濁能を有する乳酸菌をより完全に検出または同定することができる。
次に本発明を実施例を用いて具体的に説明するが、本発明の範囲はこれに限定されるものではない。
【００１４】
【実施例】
〔実施例１〕
乳酸桿菌 Lactobacillus sp. DA1、sp. BG2 およびsp. SE3 の調製
市販のビールあるいはビール工場から分離されたヘテロ型発酵乳酸菌のうち、ビール中で増殖可能で、かつ、抗 Lactobacillus brevis JCM1170 血清による抗原抗体反応（特開平4-72570号公報）において反応しない菌株３種を選び、Lactobacillus sp. DA1（以下、「ＤＡ１菌」と記す）、sp. BG2（以下、「ＢＧ２菌」と記す）およびsp. SE3（以下、「ＳＥ３菌」と記す）とした。
【００１５】
〔実施例２〕
ＤＡ１菌の１６ＳｒＲＮＡ遺伝子の塩基配列の決定
例１で調製したＤＡ１菌を乳酸菌検出培地Ｍ−ＮＢＢ培地（MBAA Technical Quarterly, 22, 61 (1985))で４日間、25℃で培養した。
菌体培養液１ｍｌを1.5ｍｌ容サンプルチューブに入れ、遠心分離をおこなった。沈殿を回収することにより菌体を集菌した後、滅菌蒸留水100μｌで２回洗浄した。菌体を滅菌蒸留水100μｌに懸濁させ、150μｌのフェノール：クロロホルム（１：１）（以下、「Ｐ／Ｃ」と記す。）と0.4ｇのガラスビーズ（粒径106μｍ以下）の入った1.5ｍｌ容サンプルチューブに加えて、ミキサーで１分間攪拌した。遠心分離して、水層を別チューブに移し、100μｌのＰ／Ｃで再抽出し、水層を得て、これをＤＮＡ抽出液とした。
【００１６】
図１に示した３つのフラグメントＡ、ＢおよびＣを増幅するために、細菌に共通なユニバーサルプライマー（表１参照）を３対（Ａ．２７ｆ（配列番号３）−９０７ｒ（配列番号４）、Ｂ．５３０ｆ（配列番号５）−１３９２ｒ（配列番号６）、およびＣ．１１１４ｆ（配列番号７）−１５２５ｒ（配列番号８））用意した。ＰＣＲ法はGene Amp PCR reagent kit（パーキン エルマー シータス社製）を用いて表２の反応液組成でおこなった。反応は、サーマルサイクラーモデル480（パーキン エルマー シータス社製）中で、変性を94℃、１分間、アニーリングを50℃、１分間、伸長を72℃、１分間順次おこなう工程を25サイクル繰り返すことにより行った。
【００１７】
【表２】

【００１８】
増幅された断片を0.7％アガロースゲルで電気泳動した後切り出し、ＤＮＡＣＥＬＬ（第１科学薬品社製）を用いてゲルから溶出させた。ゲルから溶出させた断片をＰ／Ｃで抽出し、エタノール沈殿により得られたＤＮＡを精製ＤＮＡテンプレートとした。
次に、ＡＬＦ ＤＮＡシーケンサー（ファルマシア社製）を用い、ＦＩＴＣで蛍光標識したユニバーサルプライマー（表１参照）を適宜使用して、常法に従い、Ｄｙｅプライマー法によるジデオキシ法により、得られたＤＮＡの配列決定をおこなった。ジデオキシ法はAuto Cycle Sequencing Kit(ファルマシア社製）を用いておこなった。反応液組成、反応条件は表３に 示す。なお、反応はサーマルサイクラー480中でおこなった。
【００１９】
【表３】

【００２０】
得られた塩基配列を、配列番号１、並びに、図２〜４に示す。比較のために、キリンビール（株）ビール研究所で決定されたラクトバチルス ブレビス Ｌ６３の１６ＳｒＲＮＡ遺伝子の塩基配列を配列番号９、並びに、図２〜４に示す。図２〜４は、ＤＡ１菌の１６ＳｒＲＮＡ遺伝子の塩基配列（上段に示す）およびラクトバチルス ブレビスＬ６３の１６ＳｒＲＮＡ遺伝子の塩基配列（下段に示す）を比較したものである。図２〜４中、線で結んだ部分は、ＤＡ１菌とラクトバチルス ブレビスＬ６３との間で相同性のある配列部分である。
【００２１】
図２〜４からわかるように、ＤＡ１菌の１６ＳｒＲＮＡ遺伝子の塩基配列は、全体にわたってラクトバチルス ブレビスの１６ＳｒＲＮＡ遺伝子の塩基配列と異なっている。特に上流100ｂｐまではかなり異なっている。ラクトバチルス属においては５’末端近辺が可変領域であることが知られている（System Appl. Microbiol.15,123(1992) ）。そこで、ＤＡ１菌の１〜100ｂｐまでの領域について、ジーンバンクに納めれている塩基配列とのホモロジーサーチをおこなったところ、90％以上のマッチングを示すＤＮＡは存在しなかった。従来の、例えばラクトバチルス ブレビスをターゲットにしたプライマーはこの領域の配列を利用したものであり（例えば、前記ASBC Journalに記載のプライマー（配列番号10））、そのプライマーを用いても本菌を検出できないことが予想される。そのほかの既知の乳酸菌検出用プライマーについても同様である。
【００２２】
〔実施例３〕 配列番号２の塩基配列を有するプライマーの製造
配列番号１に示される塩基配列のうち40番目から57番目までの塩基配列を有するプライマーをABI DNA シンセサイザーモデル392 を用いて合成した。
得られたプライマーの塩基配列を配列番号２に示す。
【００２３】
〔実施例４〕 配列番号２の塩基配列を有するプライマーを用いた乳酸菌の検出および同定
ＤＡ１菌、ＢＧ２菌およびＳＥ３菌、ラクトバチルス ブレビス(JCM1170) 、カゼイ(ATCC25303) 、ファーメンタム(JCM1560) およびプランタラム(JCM1149) （これらの菌と同じ種に分類される菌の中に、ビール混濁能を有するものが存在することが報告されている）の各菌をＭ−ＮＢＢ培地で、２〜14日間、25℃で培養した。培養液を例２と同様の方法で処理して、ＤＮＡ抽出液を得た。これについて、例３で製造したプライマー（配列番号２）およびユニバーサルプライマー９０７ｒ（配列番号４）を用いてＰＣＲ法をおこなった。反応条件は例２と同様であった。得られた反応生成物をアガロースゲルによる電気泳動（150Ｖ，１時間）に供した。
【００２４】
その結果、ＤＡ１菌、ＢＧ２菌およびＳＥ３菌のみに約900ｐｂｓのバンドが検出された。
【００２５】
〔比較例１〕
ＤＡ１菌、ＢＧ２菌およびＳＥ３菌のＤＮＡ抽出液について、配列表２の塩基配列を有するプライマーの代わりに、ブレビス特異プライマー（配列番号10）、カゼイ特異プライマー（配列番号11）、ファーメンタム特異プライマー（配列番号12）、プランタラム特異プライマー（配列番号13）を用いてＰＣＲ法を行ったほかは、例４の手順を繰り返した。ここで、いずれのプライマーもＡＢＩ社製ＤＮＡシンセサイザーで合成した。
【００２６】
その結果、どのプライマーを用いた場合にも、バンドは確認できなかった。
従って、配列番号２の塩基配列を有するプライマーを用いることにより、既知のプライマーを用いても検出や同定ができないある種の乳酸菌を検出および同定することができることが明らかとなった。
【００２７】
【発明の効果】
本発明により、既知の乳酸菌検出用プライマーで検出や同定ができない乳酸菌を検出および同定できるＤＮＡ分子が提供された。
また、本発明により、従来の方法では検出できない乳酸菌を迅速に検出または同定することができるようになった。
さらに、本発明により、ある種の乳酸菌の１６ＳｒＲＮＡ遺伝子が提供された。
【００２８】
【配列表】
配列番号：１
配列の長さ：１４７５
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
起源
生物名：乳酸菌
株名：Lactobacillus sp. DA1
配列
CGTTGGCGGC GTGCCTAAAT AACATGCAAG TCGAACGAGG TCTCCTAACT GATAGCTGGT 60
GCTTGCATCA GCTTGACGAT AGATCTGACC GAGTGGCGAA CTGGTGAGTA ACACGTGGGT 120
AACCTGCCCA GAAGAAGGGG ATAACACCTG GAAACAGATG CTAATACCGT ATAACAACGA 180
GAACCACATG GTTCTCGTTT GAAAGATGGC TTTTATGCTA TCGCTTCTGG ATGGACCCGC 240
GGCGTATTAG CTAGTTGGTG AGATAATAGC TCACCAAGGC AATGATACGT AGCAGACCTG 300
AGAGGGTAAT CTGCCACAAT GGGACTGAGA CACGGCCCAT ACTCCTACGG GAGGCAGCAG 360
TAGGGAATCT TCCACAATGG ACGAAAGTCT GATGGAGCAA CGCCGCGTGA GTGAAGAAGG 420
GTTTCGGCTC GTAAAACTCT GTTGTTAGAG AAGAACGACC GTGAGAGCAA CTGCTCACGG 480
TGTGACGGTA TCTAACCAGA AAGTCACGGC TAACTACGTG CCAGCAGCCG CGGTAATACG 540
TAGGTGGCAA ACGTTGTCCG GATTTATTGG GCGTAAAGCG AGCGCAGGCG GTTTTCTAAG 600
TCTGATGTTG AAACTTCGGC TTAACCGGAG AAGTGCATCG GAAACTGGAT AACTTGAGTG 660
CAGAAAAGGA CAGTGGAACT TCATGTGTAG CGGTGAAATG CGTAGATATA TGAAGGAACA 720
CCAGTGGCGA AGGCGGCTGT CTGGTCTGCA TCTGACGCTG AGGCTCGAAA GCATGGGTAG 780
CAAACAGGAT TAGATACCCT GGTAGTCCAT GCCGTAAACG ATGAATGCTA GGTGTTGGGA 840
GGTTTCCGCC TCTCAGTGCC GCAGCTAACG CATTAAGCAT TCCGCCTGGG GAGTACGACC 900
GCAAGGTTGA AACTCAAAGG AATTGACGGG GACCCGCACA AGCGGTGGAG CATGTGGTTT 960
AATTCGATGC TACGCGAAGA ACCTTACCAG GACTTGACAT CTTCTGTTAA CCTAAGAGAT 1020
TAGGTGTCCC CTTCGGGGGC AGAATGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG 1080
TGAGATGTTG GGTTAAGTCC CGCAACGAGC GCAACCCTTG TCTTTAGTTG CCAGCATTAA 1140
GTTGGGCACT CTAGAGAGAC TGCCGGTGAT AAACCGGAGG AAGGTGGGGA TGACGTCAAA 1200
TCATCATGCC CCTTATGTCC TGGGCTACAC ACGTGCTACA ATGGACGGTA CAACGAGTTG 1260
CGAAACCGCA AGGTCAAGCT AATCTCTTAA AGCCGTTCTC AGTTCGGATT GCAGGCTGCA 1320
ACTCGCCTGC ATGAAGTTGG AATCGCTAGT AATCGTGGAT CAGCATGCCA CGGTGAATAC 1380
GTTCCCGGGT CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCAAAGTCG 1440
GTTGGATAAC CTTTACGGAG TCCGCCGCCT AAGGT 1475
【００２９】
配列番号：２
配列の長さ：１８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＧＴＣＴＣＣＴＡＡＣ ＴＧＡＴＡＧＣＴ １８
【００３０】
配列番号：３
配列の長さ：２０
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＡＧＡＧＴＴＴＧＡＴ ＣＣＴＧＧＣＴＣＡＧ ２０
【００３１】
配列番号：４
配列の長さ：１０
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＣＣＧＴＣＡＡＴＴＣ ＣＴＴＴＧＡＧＴＴＴ １０
【００３２】
配列番号：５
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＧＴＧＣＣＡＧＣＡＧ ＣＣＧＣＧＧ １６
【００３３】
配列番号：６
配列の長さ：１５
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＡＣＧＧＧＣＧＧＴＧ ＴＧＴＡＣ １５
【００３４】
配列番号：７
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＧＣＡＡＣＧＡＧＣＧ ＣＡＡＣＣＣ １６
【００３５】
配列番号：８
配列の長さ：１７
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＡＡＧＧＡＧＧＴＧＡ ＴＣＣＡＧＣＣ １７
【００３６】
配列番号：９
配列の長さ：１５０３
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
起源
生物名：ラクトバチルス ブレビス
株名：Ｌ６３
配列
GGCATGCCTA ATACATGCAA GTCGAACGAG CTTCCGTTGA ATGACGTGCT TGCACTGATT 60
TCACCAATGA AGCGAGTGGC GAACTGGTGA GTAACACGTG GGAAATCTGC CCAGAAGCAG 120
GGGATAACAC TTGGAAACAG GTGCTAATAC CGTATAACAA CAAAATCCGC ATGGATTTTG 180
TTTGAAAGGT GGCTTCGGCT ATCACTTCTG GATGATCCCG CGGCGTATTA GTTAGTTGGT 240
GAGGTAAAGG CCCACCAAGA CGATGATACG TAGCCGACCT GAGAGGGTAA TCGGCCACAT 300
TGGGACTGAG ACACGGCCCA AACTCCTACG GGAGGCAGCA GTAGGGAATC TTCCACAATG 360
GACGAAAGTC TGATGGAGCA ATGCCGCGTG AGTGAAGAAG GGTTTCGGCT CGTAAAACTC 420
TGTTGTTAAA GAAGAACACC TTTGAGAGTA ACTGTTCAAG GGTTGACGGT ATTTAACCAG 480
AAAGCCACGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGTGGCA AGCGTTGTCC 540
GGATTTATTG GGCGTAAAGC GAGCGCAGGC GGTTTTTTAA GTCTGATGTG AAAGCCTTCG 600
GCTTAACCGG AGAAGTGCAT CGGAAACTGG GAGACTTGAG TGCAGAAGAG GACAGTGGAA 660
CTCCATGTGT AGCGGTGGAA TGCGTAGATA TATGGAAGAA CACCAGTGGC GAAGGCGGCT 720
GTCTAGTCTG TAACTGACGC TGAGGCTCGA AAGCATGGGT AGCGAACAGG ATTAGATACC 780
CTGGTAGTCC ATGCCGTAAA CGATGAGTGC TAAGTGTTGG AGGGTTTCCG CCCTTCAGTG 840
CTGCAGCTAA CGCATTAAGC ACTCCGCCTG GGGAGTACGA CCGCAAGGTT GAAACTCAAA 900
GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA GCTACGCGAA 960
GAACCTTACC AGGTCTTGAC ATCTTCTGCC AATCTTAGAG ATAAGACGTT CCCTTCGGGG 1020
ACAGAATGAC AGGTGGTGCA TGGTTGTCGT CAGCTCGTGT CGTGAGATGT TGGGTTAAGT 1080
CCCGCAACGA GCGCAACCCT TATTATCAGT TGCCAGCATT CAGTTGGGCA CTCTGGTGAG 1140
ACTGCCGGTG ACAAACCGGA GGAAGGTGGG GATGACGTCA AATCATCATG CCCCTTATGA 1200
CCTGGGCTAC ACACGTGCTA CAATGGACGG TACAACGAGT CGCGAAGTCG TGAGGCTAAG 1260
CTAATCTCTT AAAGCCGTTC TCAGTTCGGA TTGTAGGCTG CAACTCGCCT ACATGAAGTT 1320
GGAATCGCTA GTAATCGCGG ATCAGCATGC CGCGGTGAAT ACGTTCCCGG GCCTTGTACA 1380
CACCGCCCGT CACACCATGA GAGTTTGTAA CACCCAAAGC CGGTGAGATA ACCTTCGGGA 1440
GTCAGCCGTC TAAGGTGGGA CAGATGATTA GGGTGAAGTC GTAACAAGGT AGCCGTAGGA 1500
GAA 1503
【００３７】
配列番号：１０
配列の長さ：１７
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＡＡＧＴＣＧＡＡＣＧ ＡＧＣＴＴＣＣ １７
【００３８】
配列番号：１１
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＧＡＧＡＡＧＡＡＴＧ ＧＴＣＧＧＣ １６
【００３９】
配列番号：１２
配列の長さ：１１
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＣＡＡＴＣＡＡＴＴＧ ＧＧＣＣＡＡＣＧＣＧ Ｔ １１
【００４０】
配列番号：１３
配列の長さ：１９
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成DNA
配列
ＴＡＣＣＣＧＣＡＴＡ ＡＣＡＡＣＴＴＧＧ １９
【図面の簡単な説明】
【図１】図１は、１６ＳｒＤＮＡ；ユニバーサルプライマー２７ｆおよび９０７ｒを用いて増幅される断片Ａ；ユニバーサルプライマー５３０ｆおよび１３９２ｒを用いて増幅される断片Ｂ；およびユニバーサルプライマー１１４ｆおよび１５２５ｒを用いて増幅される断片Ｃの模式図である。
【図２】図２は、乳酸桿菌 Lactobacillus sp. DA1およびラクトバチルス ブレビスＬ６３の１６ＳｒＲＮＡ遺伝子の塩基配列を比較したものである。
【図３】図３は、乳酸桿菌 Lactobacillus sp. DA1およびラクトバチルス ブレビスＬ６３の１６ＳｒＲＮＡ遺伝子の塩基配列を比較したもの（図２の続き）である。
【図４】図４は、乳酸桿菌 Lactobacillus sp. DA1およびラクトバチルス ブレビスＬ６３の１６ＳｒＲＮＡ遺伝子の塩基配列を比較したもの（図３の続き）である。[0001]
[Industrial application fields]
The present invention relates to a DNA molecule that can be used for detection of lactic acid bacteria and a method for detecting lactic acid bacteria using the DNA molecules.
[0002]
[Prior art]
When storing beer, microorganisms mixed in beer, especially certain lactic acid bacteria, grows and causes harmful effects such as turbidity of beer. Yes.
As such beer harmful fungi, several lactic acid bacteria such as Lactobacillus brevis and L. casei have been pointed out, and there are many methods for rapidly detecting or identifying these. It has been studied in the past.
[0003]
For example, a method using an antigen-antibody reaction (JP-A-4-197167), a method using a PCR method using a specific primer (ASBC Journal. 51, 63 (1993)), and the like have been developed. However, these methods are techniques for detecting specific lactic acid bacteria, and there is a possibility that detection of other harmful bacteria may be missed.
[0004]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a DNA molecule for detecting or identifying a lactic acid bacterium that cannot be detected or identified with a known primer for detecting a lactic acid bacterium. Another object of the present invention is to provide a method for rapidly detecting or identifying lactic acid bacteria that cannot be detected with known primers for detecting lactic acid bacteria. A further object of the present invention is to provide 16S rRNA genes of certain lactic acid bacteria.
[0005]
[Means for Solving the Problems]
The present inventor has clarified the base sequence of a 16S ribosomal RNA (16SrRNA) gene of a certain lactic acid bacterium that cannot be detected with a known primer, and uses a part of the gene as a primer for nucleic acid of harmful microorganisms that may be present in a sample, particularly beer As a result, it was found that specific lactic acid bacteria can be detected, and the present invention has been completed. That is, the present invention provides a DNA molecule for detecting lactic acid bacteria comprising part or all of the base sequence represented by SEQ ID NO: 1. The present invention also provides a method for detecting or identifying lactic acid bacteria in a sample using a DNA molecule comprising the DNA molecule or a base sequence complementary to the base sequence. Furthermore, the present invention provides a 16S rRNA gene of lactic acid bacteria comprising the base sequence represented by SEQ ID NO: 1.
[0006]
Hereinafter, the present invention will be described in detail.
The DNA molecule for detecting lactic acid bacteria of the present invention contains a part or all of the base sequence shown in SEQ ID NO: 1. Here, “DNA molecule” refers to a molecule containing two or more nucleotides. SEQ ID NO: 1 shows the base sequence of the 16S rRNA gene of Lactobacillus sp. DA1. Lactobacillus sp. DA1 is a lactobacilli isolated from a beer factory, and was deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology on April 22, 1994, and the microorganism accession number is FERM BP-4652 It is. Any location can be selected from the above base sequence, but the number of bases in the sequence needs to be at least about 10 bp. In order to obtain high detection sensitivity, it is preferably about 15 to about 25 bp. Further, in order to increase the selectivity to bacteria, among the base sequence shown in SEQ ID NO: 1, the first to 100th base sequences corresponding to the variable region, particularly one of the 30th to 100th base sequences. A DNA molecule containing part or all is desirable. As a specific example, a DNA molecule containing the 40th to 57th base sequences (shown in SEQ ID NO: 2) of the base sequence shown in SEQ ID NO: 1 can be mentioned.
[0007]
Lactic acid bacteria in a sample can be detected using a DNA molecule containing the base sequence as described above or a DNA molecule containing a base sequence complementary to the base sequence of the DNA molecule. The DNA molecule or a DNA molecule containing a base sequence complementary to the base sequence of the DNA molecule should be prepared by chemical synthesis according to a known method described in the ABI Model 392 and 394 DNA / RNA Synthesizer operation manuals. Can do.
[0008]
In order to detect or identify lactic acid bacteria in a sample, a DNA molecule containing a base sequence complementary to the above DNA molecule or its base sequence is labeled with a radioactive element, a fluorescent dye, etc., and then used as a probe in the sample. Hybridization method that hybridizes with the nucleic acid of the microorganism present in the sample, and a method of amplifying the nucleic acid of the microorganism present in the sample using these DNA molecules as a primer can be used. It is preferable to use the latter method.
[0009]
Hereinafter, an example of the detection or identification method of the present invention will be described. This method is present in a sample using a DNA molecule containing a base sequence complementary to the above DNA molecule or its base sequence as a primer. A step of amplifying the nucleic acid of the microorganism.
First, microorganisms are separated from a sample by a method such as membrane filtration. Examples of the sample include all brewing process samples such as product beer, young beer before aging, and recovered yeast. Nucleic acids are extracted from the separated microorganisms as they are or after they are further cultured and grown. The nucleic acid may be any of DNA, RNA, a hybrid of DNA and RNA, and a mixture thereof, but is preferably DNA. The nucleic acid may be either double-stranded or single-stranded, but preferably is double-stranded. Since even a very small amount of sample can be detected by using the PCR method, for example, at least 20 cells can be detected sufficiently.
[0010]
As a method for extracting a nucleic acid from a microorganism, any conventionally known method may be used. For example, a method in which a microorganism is pulverized with glass beads and then extracted with phenol / chloroform, an extraction method by a lytic enzyme treatment, or an autoclave. For example, a method of extracting after lysis with an aqueous solution or an extraction method using a surfactant treatment can be used.
The extracted nucleic acid is amplified by the PCR method using a primer composed of the DNA molecule of the present invention or a DNA molecule containing a base sequence complementary to the base sequence, and at least one universal primer. The primer may be double-stranded or single-stranded, but is preferably single-stranded to increase the efficiency of nucleic acid amplification by the PCR method. Moreover, when a primer is a double strand, it is preferable to perform the process which isolate | separates the strand before using for PCR method. The universal primer is a primer having a sequence common to all bacteria. As an example, the 16S rRNA sequencing primer described in TABLE 6.3 of Nucleic Acid Techniques in Bacterial Systematics John Wiley & Sons Ltd. (1991) P.133 is used. Can be mentioned, but is not limited thereto. Table 1 below shows some of the 16S rRNA sequencing primers described in TABLE 6.3 of the above document. A combination of a primer composed of a DNA molecule of the present invention or a DNA molecule comprising a base sequence complementary to the base sequence thereof, and a universal primer may be such that one is a sense primer and the other is an antisense primer. For example, a sense primer can be set from the 1st to 100th nucleotide sequences shown in SEQ ID NO: 1 and combined with the antisense primer of the universal primer shown in Table 1. Of these, a combination of a DNA molecule containing the base sequence shown in SEQ ID NO: 2 and 907r is preferred.
[0011]
[Table 1]

[0012]
The DNA polymerase used in the PCR method may be of any origin as long as it has a heat resistance of 95 ° C. As reaction conditions for the PCR method, a denaturation temperature of 90 to 95 ° C., an annealing temperature of 40 to 60 ° C., an extension temperature of 70 to 75 ° C., and a cycle number of 10 times or more are preferable, but not limited thereto. Any condition that can be used is acceptable.
The obtained reaction products are separated by detection or identification means such as electrophoresis using an agarose gel, and the presence or absence of amplification products is confirmed. By this method, Lactobacillus sp. DA1, Lactobacillus sp. BG2 and Lactobacillus sp. SE3 have the same properties as those of Lactobacillus sp. It is possible to detect or identify lactobacilli that are not. These lactobacilli are known to have beer turbidity. Therefore, the method of the present invention makes it possible to detect or identify harmful bacteria present in beer.
[0013]
Furthermore, by combining the detection or identification method of the present invention with a conventional detection or identification method, lactic acid bacteria, particularly lactic acid bacteria having beer turbidity, can be detected or identified more completely.
Next, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited thereto.
[0014]
【Example】
[Example 1]
Preparation of Lactobacillus sp. DA1, sp. BG2 and sp. SE3 Hetero-type fermenting lactic acid bacteria isolated from commercial beer or beer factories that can grow in beer and have antigen antibodies from anti-Lactobacillus brevis JCM1170 serum Three strains which do not react in the reaction (Japanese Patent Laid-Open No. 4-72570) are selected, and Lactobacillus sp. DA1 (hereinafter referred to as “DA1 bacteria”), sp. BG2 (hereinafter referred to as “BG2 bacteria”) and sp. SE3 (hereinafter referred to as “SE3 bacteria”).
[0015]
[Example 2]
Determination of the base sequence of 16S rRNA gene of DA1 strain The DA1 strain prepared in Example 1 was cultured at 25 ° C. for 4 days in lactic acid bacteria detection medium M-NBB medium (MBAA Technical Quarterly, 22, 61 (1985)).
1 ml of the bacterial cell culture solution was placed in a 1.5 ml sample tube and centrifuged. The cells were collected by collecting the precipitate, and then washed twice with 100 μl of sterilized distilled water. The cells were suspended in 100 μl of sterile distilled water, and 1.5 μl containing 150 μl of phenol: chloroform (1: 1) (hereinafter referred to as “P / C”) and 0.4 g of glass beads (particle size of 106 μm or less). In addition to the ml sample tube, the mixture was stirred with a mixer for 1 minute. After centrifuging, the aqueous layer was transferred to a separate tube and re-extracted with 100 μl of P / C to obtain an aqueous layer, which was used as a DNA extract.
[0016]
In order to amplify the three fragments A, B and C shown in FIG. 1, three pairs of universal primers (see Table 1) common to bacteria (A.27f (SEQ ID NO: 3) -907r (SEQ ID NO: 4), B.530f (SEQ ID NO: 5) -1392r (SEQ ID NO: 6) and C. 1114f (SEQ ID NO: 7) -1525r (SEQ ID NO: 8)) were prepared. The PCR method was performed using the Gene Amp PCR reagent kit (manufactured by Perkin Elmer Cetus) with the reaction solution composition shown in Table 2. The reaction was performed in a thermal cycler model 480 (Perkin Elmer Citas) by repeating 25 cycles of denaturation at 94 ° C for 1 minute, annealing at 50 ° C for 1 minute, and extension at 72 ° C for 1 minute. It was.
[0017]
[Table 2]

[0018]
The amplified fragment was electrophoresed on a 0.7% agarose gel, cut out, and eluted from the gel using DNACELL (Daiichi Kagaku Yakuhin). Fragments eluted from the gel were extracted with P / C, and DNA obtained by ethanol precipitation was used as a purified DNA template.
Next, using an ALF DNA sequencer (manufactured by Pharmacia), appropriately using a universal primer (see Table 1) fluorescently labeled with FITC, and according to the conventional method, the DNA sequence obtained by the dideoxy method using the Dye primer method Made a decision. The dideoxy method was performed using an Auto Cycle Sequencing Kit (Pharmacia). The reaction solution composition and reaction conditions are shown in Table 3. The reaction was carried out in a thermal cycler 480.
[0019]
[Table 3]

[0020]
The obtained base sequence is shown in SEQ ID NO: 1 and FIGS. For comparison, the base sequence of the 16S rRNA gene of Lactobacillus brevis L63 determined at Kirin Brewery Co., Ltd. Beer Laboratory is shown in SEQ ID NO: 9 and FIGS. 2-4 compares the base sequence of the 16S rRNA gene of DA1 bacteria (shown in the upper part) and the base sequence of the 16S rRNA gene of Lactobacillus brevis L63 (shown in the lower part). 2-4, the part connected with the line | wire is an arrangement | sequence part with homology between DA1 microbe and Lactobacillus brevis L63.
[0021]
As can be seen from FIGS. 2 to 4, the base sequence of the 16S rRNA gene of DA1 bacterium is totally different from the base sequence of the 16S rRNA gene of Lactobacillus brevis. Especially up to 100 bp upstream is quite different. In the genus Lactobacillus, it is known that the vicinity of the 5 ′ end is a variable region (System Appl. Microbiol. 15, 123 (1992)). Thus, when a homology search was performed for the region of 1 to 100 bp of DA1 bacteria with the base sequence stored in Genebank, no DNA showing 90% or more matching was present. Conventional primers targeting Lactobacillus brevis, for example, use the sequence of this region (for example, the primer described in the ASBC Journal (SEQ ID NO: 10)). It is expected not to be possible. The same applies to other known lactic acid bacteria detection primers.
[0022]
[Example 3] Production of primer having the nucleotide sequence of SEQ ID NO: 2 Primers having the 40th to 57th nucleotide sequences of the nucleotide sequence shown in SEQ ID NO: 1 were synthesized using ABI DNA synthesizer model 392.
The base sequence of the obtained primer is shown in SEQ ID NO: 2.
[0023]
[Example 4] Detection and identification of lactic acid bacteria using a primer having the nucleotide sequence of SEQ ID NO: 2 DA1, BG2 and SE3, Lactobacillus brevis (JCM1170), casei (ATCC25303), fermentum (JCM1560) and plan Each bacterium of Talam (JCM1149) (which has been reported to have beer turbidity among the bacteria classified as the same species as these bacteria) in M-NBB medium for 2 to 14 days And cultured at 25 ° C. The culture solution was treated in the same manner as in Example 2 to obtain a DNA extract. About this, PCR method was performed using the primer (sequence number 2) manufactured in Example 3, and the universal primer 907r (sequence number 4). The reaction conditions were the same as in Example 2. The obtained reaction product was subjected to electrophoresis (150 V, 1 hour) using an agarose gel.
[0024]
As a result, a band of about 900 pbs was detected only in DA1, BG2 and SE3.
[0025]
[Comparative Example 1]
For DNA extracts of DA1, BG2 and SE3 bacteria, instead of the primer having the base sequence of Sequence Listing 2, a brevis-specific primer (SEQ ID NO: 10), a casei-specific primer (SEQ ID NO: 11), a fermentum-specific primer ( The procedure of Example 4 was repeated except that PCR was performed using SEQ ID NO: 12) and plantarum specific primer (SEQ ID NO: 13). Here, all the primers were synthesized with a DNA synthesizer manufactured by ABI.
[0026]
As a result, no band was confirmed when any primer was used.
Therefore, it was revealed that by using a primer having the base sequence of SEQ ID NO: 2, it is possible to detect and identify certain lactic acid bacteria that cannot be detected or identified using a known primer.
[0027]
【The invention's effect】
According to the present invention, DNA molecules capable of detecting and identifying lactic acid bacteria that cannot be detected or identified by known lactic acid bacteria detection primers have been provided.
In addition, the present invention makes it possible to rapidly detect or identify lactic acid bacteria that cannot be detected by conventional methods.
Furthermore, according to the present invention, 16S rRNA genes of certain lactic acid bacteria are provided.
[0028]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 1475
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: Genomic DNA
Origin organism name: Lactobacillus strain name: Lactobacillus sp. DA1
Array
CGTTGGCGGC GTGCCTAAAT AACATGCAAG TCGAACGAGG TCTCCTAACT GATAGCTGGT 60
GCTTGCATCA GCTTGACGAT AGATCTGACC GAGTGGCGAAAA CTGGTGAGTA ACACGTGGGT 120
AACCTGCCCA GAAGAAGGGG ATAACACCTG GAAACAGATG CTAATACCGT ATAACAACGA 180
GAACCACATG GTTCTCGTTT GAAAGATGGC TTTTATGCTA TCGCTTCTGG ATGGACCCGC 240
GGCGTATTAG CTAGTTGGTG AGATAATAGC TCACCAAGGC AATGATACGT AGCAGACCTG 300
AGAGGGTAAT CTGCCACAAT GGGACTGAGA CACGGCCCAT ACTCCTACGG GAGGCAGCAG 360
TAGGGAATCT TCCACAATGG ACGAAAGTCT GATGGAGCAA CGCCGCGTGA GTGAAGAAGG 420
GTTTCGGCTC GTAAAACTCT GTTGTTAGAG AAGAACGACC GTGAGAGCAA CTGCTCACGG 480
TGTGACGGTA TCTAACCAGA AAGTCACGGC TAACTACGTG CCAGCAGCCG CGGTAATACG 540
TAGGTGGCAA ACGTTGTCCG GATTTATTGG GCGTAAAGCG AGCGCAGGCG GTTTTCTAAG 600
TCTGATGTTG AAACTTCGGC TTAACCGGAG AAGTGCATCG GAAACTGGAT AACTTGAGTG 660
CAGAAAAGGA CAGTGGAACT TCATGTGTAG CGGTGAAATG CGTAGATATA TGAAGGAACA 720
CCAGTGGCGA AGGCGGCTGT CTGGTCTGCA TCTGACGCTG AGGCTCGAAA GCATGGGTAG 780
CAAACAGGAT TAGATACCCT GGTAGTCCAT GCCGTAAACG ATGAATGCTA GGTGTTGGGA 840
GGTTTCCGCC TCTCAGTGCC GCAGCTAACG CATTAAGCAT TCCGCCTGGG GAGTACGACC 900
GCAAGGTTGA AACTCAAAGG AATTGACGGG GACCCGCACA AGCGGTGGAG CATGTGGTTT 960
AATTCGATGC TACGCGAAGA ACCTTACCAG GACTTGACAT CTTCTGTTAA CCTAAGAGAT 1020
TAGGTGTCCC CTTCGGGGGC AGAATGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG 1080
TGAGATGTTG GGTTAAGTCC CGCAACGAGC GCAACCCTTG TCTTTAGTTG CCAGCATTAA 1140
GTTGGGCACT CTAGAGAGAC TGCCGGTGAT AAACCGGAGG AAGGTGGGGA TGACGTCAAA 1200
TCATCATGCC CCTTATGTCC TGGGCTACAC ACGTGCTACA ATGGACGGTA CAACGAGTTG 1260
CGAAACCGCA AGGTCAAGCT AATCTCTTAA AGCCGTTCTC AGTTCGGATT GCAGGCTGCA 1320
ACTCGCCTGC ATGAAGTTGG AATCGCTAGT AATCGTGGAT CAGCATGCCA CGGTGAATAC 1380
GTTCCCGGGT CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA CCCAAAGTCG 1440
GTTGGATAAC CTTTACGGAG TCCGCCGCCT AAGGT 1475
[0029]
SEQ ID NO: 2
Sequence length: 18
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence GTCTCCTAAC TGATAGCT 18
[0030]
SEQ ID NO: 3
Sequence length: 20
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence AGAGTT TG AT CCTGGCTCAG 20
[0031]
SEQ ID NO: 4
Sequence length: 10
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence CCGTCAATTC CTTTGAGTT 10
[0032]
SEQ ID NO: 5
Sequence length: 16
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence GT G CCAGCAG CC G C GG 16
[0033]
SEQ ID NO: 6
Sequence length: 15
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence ACGGGGCGTG TGTAC 15
[0034]
SEQ ID NO: 7
Sequence length: 16
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence GCAACGAGCG CAACCC 16
[0035]
SEQ ID NO: 8
Sequence length: 17
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence AAGGAGGGTGA TCCAGCC 17
[0036]
SEQ ID NO: 9
Sequence length: 1503
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: Genomic DNA
Origin organism name: Lactobacillus brevis strain name: L63
Array
GGCATGCCTA ATACATGCAA GTCGAACGAG CTTCCGTTGA ATGACGTGCT TGCACTGATT 60
TCACCAATGA AGCGAGTGGC GAACTGGTGA GTAACACGTG GGAAATCTGC CCAGAAGCAG 120
GGGATAACAC TTGGAAACAG GTGCTAATAC CGTATAACAA CAAAATCCGC ATGGATTTTG 180
TTTGAAAGGT GGCTTCGGCT ATCACTTCTG GATGATCCCG CGGCGTATTA GTTAGTTGGT 240
GAGGTAAAGG CCCACCAAGA CGATGATACG TAGCCGACCT GAGAGGGTAA TCGGCCACAT 300
TGGGACTGAG ACACGGCCCA AACTCCTACG GGAGGCAGCA GTAGGGAATC TTCCACAATG 360
GACGAAAGTC TGATGGAGCA ATGCCGCGTG AGTGAAGAAG GGTTTCGGCT CGTAAAACTC 420
TGTTGTTAAA GAAGAACACC TTTGAGAGTA ACTGTTCAAG GGTTGACGGT ATTTAACCAG 480
AAAGCCACGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGTGGCA AGCGTTGTCC 540
GGATTTATTG GGCGTAAAGC GAGCGCAGGC GGTTTTTTAA GTCTGATGTG AAAGCCTTCG 600
GCTTAACCGG AGAAGTGCAT CGGAAACTGG GAGACTTGAG TGCAGAAGAG GACAGTGGAA 660
CTCCATGTGT AGCGGTGGAA TGCGTAGATA TATGGAAGAA CACCAGTGGC GAAGGCGGCT 720
GTCTAGTCTG TAACTGACGC TGAGGCTCGA AAGCATGGGT AGCGAACAGG ATTAGATACC 780
CTGGTAGTCC ATGCCGTAAA CGATGAGTGC TAAGTGTTGG AGGGTTTCCG CCCTTCAGTG 840
CTGCAGCTAA CGCATTAAGC ACTCCGCCTG GGGAGTACGA CCGCAAGGTT GAAACTCAAA 900
GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA GCTACGCGAA 960
GAACCTTACC AGGTCTTGAC ATCTTCTGCC AATCTTAGAG ATAAGACGTT CCCTTCGGGG 1020
ACAGAATGAC AGGTGGTGCA TGGTTGTCGT CAGCTCGTGT CGTGAGATGT TGGGTTAAGT 1080
CCCGCAACGA GCGCAACCCT TATTATCAGT TGCCAGCATT CAGTTGGGCA CTCTGGTGAG 1140
ACTGCCGGTG ACAAACCGGA GGAAGGTGGG GATGACGTCA AATCATCATG CCCCTTATGA 1200
CCTGGGCTAC ACACGTGCTA CAATGGACGG TACAACGAGT CGCGAAGTCG TGAGGCTAAG 1260
CTAATCTCTT AAAGCCGTTC TCAGTTCGGA TTGTAGGCTG CAACTCGCCT ACATGAAGTT 1320
GGAATCGCTA GTAATCGCGG ATCAGCATGC CGCGGTGAAT ACGTTCCCGG GCCTTGTACA 1380
CACCGCCCGT CACACCATGA GAGTTTGTAA CACCCAAAGC CGGTGAGATA ACCTTCGGGA 1440
GTCAGCCGTC TAAGGTGGGA CAGATGATTA GGGTGAAGTC GTAACAAGGT AGCCGTAGGA 1500
GAA 1503
[0037]
SEQ ID NO: 10
Sequence length: 17
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence AAGTCGAACG AGCTTCC 17
[0038]
SEQ ID NO: 11
Sequence length: 16
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence GAGAAGAATG GTCGGC 16
[0039]
SEQ ID NO: 12
Sequence length: 11
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence CAATCAATTG GGCCAACGCG T 11
[0040]
SEQ ID NO: 13
Sequence length: 19
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Sequence TACCCGCATA ACACTTGG 19
[Brief description of the drawings]
FIG. 1 shows 16S rDNA; fragment A amplified using universal primers 27f and 907r; fragment B amplified using universal primers 530f and 1392r; and amplified using universal primers 114f and 1525r 2 is a schematic diagram of a fragment C. FIG.
FIG. 2 is a comparison of the base sequences of 16S rRNA genes of Lactobacillus sp. DA1 and Lactobacillus brevis L63.
FIG. 3 is a comparison of the nucleotide sequences of 16S rRNA genes of Lactobacillus sp. DA1 and Lactobacillus brevis L63 (continuation of FIG. 2).
FIG. 4 is a comparison of the nucleotide sequences of 16S rRNA genes of Lactobacillus sp. DA1 and Lactobacillus brevis L63 (continuation of FIG. 3).

Claims (8)

DNA molecule or complementary DNA molecules dairy acid bacteria detection comprising the nucleotide sequence shown in SEQ ID NO: 1. A DNA molecule for detecting lactic acid bacteria, or a complementary DNA molecule thereof , comprising the entire base sequence from the first to the 100th of the base sequence shown in SEQ ID NO: 1 or a part consisting of 15 to 25 consecutive bases thereof . A DNA molecule for detecting lactic acid bacteria or a complementary DNA molecule thereof comprising the entire base sequence from the 30th to the 100th of the base sequence shown in SEQ ID NO: 1 or a part consisting of 15 to 25 consecutive bases thereof . The DNA molecule or the complementary DNA molecule thereof according to claim 2 or 3 , comprising at least the 40th to 57th nucleotide sequences of the nucleotide sequence shown in SEQ ID NO: 1. Using a DNA molecule comprising a nucleotide sequence complementary to DNA molecules or nucleotide sequence of any one of claims 1 to 4, a method of detecting or identifying lactic acid bacteria in a sample. 5. A primer comprising a DNA molecule according to any one of claims 1 to 4 or a DNA molecule comprising a base sequence complementary to the base sequence, and at least one universal primer, and present in a sample 6. The method of claim 5 , comprising amplifying the nucleic acid of the microorganism. The method according to any one of claims 5 to 6 , wherein the lactic acid bacterium to be detected has beer turbidity.   A lactic acid bacterium 16S rRNA gene comprising the base sequence represented by SEQ ID NO: 1.

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