JP4323740B2 - Molecular genetic analysis and evaluation methods for contaminated environments and environmental samples - Google Patents

Description

【００５２】
この算出にあたって、バクテリア、アーキア、ユーカリアの各ドメインの検出・定量には、Amannら（R. I. Amann et al.: 1995、前出）に記載されているEuba338、Arch 915、Euka 502などのDNAプローブを用いることができる。また、全生物共通領域を標的としたDNAプローブには、Univ 1390（D. A. E. Zheng et al.: 1996、前出）などを用いることができる。特定機能微生物の検出・定量には、その微生物に固有の遺伝子塩基配列領域、たとえば16S rDNA,、18S rDNA、23SrDNA、gyrBなどの遺伝子塩基配列情報の中からその種に特異的な塩基配列領域を選定し、その配列を含むプローブを合成すればよい。Non-RI標識核酸プローブは、上述した方法により作製できる。
【００５３】
上記の方法を用いて、自然環境や人工的な環境中に優占している特定機能微生物の優占度を解析することにより、その環境の微生物群集機能を評価することができる。ここで、「微生物群集機能」とは、対象とした環境中に生息する複数種の微生物により営まれる総体としての機能のことをいう。ここで言う微生物の種類とは、通常の分類単位である種であることが好ましいが、実験上の機能単位、たとえば石油分解菌群やPCB分解菌群という一つの機能単位であってもよい。したがって、特定機能微生物一種のみの機能を微生物群集機能とは呼ばない。
【００５４】
例えば、石油の分解をめぐっては、脂肪族炭化水素を分解する微生物や芳香族炭化水素を分解する微生物以外にも、それらの分解中間代謝産物を炭酸ガスまで分解する過程には多くの微生物が関与しており、これらの微生物にビタミン等の微量必須栄養素を供給する微生物等も共存しているものと考えられ、複数種の微生物が協同して石油の無機化、無毒化に関与しているものと考えることができる。この場合、ある分解産物が生育阻害物質になりうる場合には、その分解産物を利用可能な微生物と共存することにより分解効率は上昇するものと考えられ、微生物群集全体として高い分解機能が発現されることになる。
【００５５】
具体的には、前述した方法により微生物群集内の一種または複数種の特定機能微生物、たとえば好気性の炭化水素分解菌、PCB分解菌、タンパク質分解菌、グルコース資化性菌等、および嫌気性の硫酸還元菌やメタン生成菌等、ならびに病原菌等の有害微生物、各種酵素または抗生物質を生産する有用物質生産菌等の優占度を調べておき、その時間変化を調べることにより、各々の特定機能微生物の遷移を解析することができる。その結果、たとえば、沿岸表層水において、ある時期に硫酸還元菌の優占度が上昇していれば、その時の微生物群集は潜在的に硫酸還元機能が高いと評価できるし、その原因として、試料採取以前に台風や地殻活動等による底泥の巻き上がりや嫌気的な排水の流入等により、それらが通常の生息環境から移入してきたものと推定することができる。
【００５６】
また、上記の方法を用いて、汚染環境を解析・評価することができる。たとえば、対象とする特定機能微生物が、あるパルプ工場の排水中に特異的に優占する従属栄養細菌である場合、ある時期にその優占度が上昇していれば、その環境はこの排水に汚染されている可能性が高いと判断でき、その優占度の変化を長期間に渡ってモニターしてその遷移の周期性や季節性を把握していれば、その変化が突発的なものであるかどうか、またその負荷が人為的なものかどうかを評価することができる。また、有用物質生産微生物を含む環境試料の採取場所、時期・季節、および有害微生物の発生場所、時期・季節を推定することができる。
【００５７】
さらに、上記の方法を用いて、有害化学物質汚染環境および特定微生物を含むと考えられる環境試料を解析・評価することができる。たとえば、対象とする特定機能微生物がPCB分解菌である場合、ある時期にその優占度が上昇していれば、その環境はPCB汚染されている可能性が高いと判断でき、その微生物群集は総体としてPCB分解機能が高まっていると判定できる。さらに、その優占度の変化を長期間モニターしその遷移の周期性や季節性を把握していれば、その変化が突発的なものであるかどうか、その負荷が工場排水の流入等人為的なものかどうかを推定することができる。
【００５８】
また、上記の方法を用いて、油濁環境を解析・評価することができる。たとえば、対象とする特定機能微生物が石油成分由来のもの、たとえばテトラデカンやアントラセンの分解菌である場合、ある時期にその優占度が上昇していれば、その環境は、前者の場合であれば石油成分の中でもｎ-アルカン等の飽和炭化水素で、後者の場合は芳香族炭化水素で、それぞれ汚染されている可能性が高いと判断でき、その微生物群集は総体としてその石油成分の分解機能が高まっていると判定できる。また、特定機能微生物の優占度の変遷を調べることにより、石油汚染またはその他の汚染の程度やバイオレメディエーション過程の解析・評価を行うことができる。さらに、その優占度の変化を長期間に渡ってモニターしその遷移の周期性や季節性を把握していれば、その変化が突発的なものであるかどうか、その負荷が船舶事故等に起因した人為的なものかどうかを推定することができる。
【００５９】
特定機能微生物として、多くの海域に分布することが知られている脂肪族炭化水素分解菌に着目した場合、四国沿岸域より集積培養を経て純粋分離された脂肪族炭化水素分解菌GR211-P1菌株（特許寄託番号FERM BP-7788：平成１３年１１月５日付け国際寄託へ移管）の16S rDNAの塩基配列（配列表配列番号５）の一部を有し、Alcanivorax 属の石油分解菌、特にAlcanivorax borkumensisおよびその近縁種に由来する核酸と特異的にハイブリダイズし、それを検出または定量しうる配列番号１の塩基配列領域（配列番号５における塩基番号207〜226の領域。Escherichia coli の16S rDNAの塩基配列における５'末端からの位置を示したナンバーリングシステム[H. F. Noller and C. R. Woese, Science, 212: 403-411, 1981]では、塩基番号222〜241に相当する領域）およびこの領域の塩基配列の全部または一部を含む塩基長10〜50 bp、好ましくは塩基長15〜25を有するDNAプローブ、またはそれらに相当するリボヌクレオチド配列を有するRNAプローブを設計するとよい。一例として、以下のプローブを挙げることができる。
（１）5'-CGA CGC GAG CTC ATC CAT CA-3' (Albo 222, 20 mer)（配列番号１）
3'-GCT GCG CTC GAG TAG GTA GT-5' （配列番号７）
これらのプローブを合成し、Non-RI標識、すなわち、蛍光色素や抗原物質などで標識する場合は、前述した方法を用いて行えばよい。
【００６０】
特定機能微生物として、複数の海域でその分布が確認されている芳香族炭化水素分解菌に着目する場合、以下の塩基配列を有するDNAプローブおよびRNAプローブをを用いることができる。
（１）5'-GGAAACCCGCCCAACAGT-3'（Cypug829-846^*, 18mer：配列番号６における塩基番号823〜840の領域）（配列番号２）
3'-CCTTTGGGCGGGTTGTCA-5' （配列番号８）
（２）5'-TGCACCACTAAGCGGAAACC-3'（Cypug840-859^*, 20mer：配列番号６における塩基番号834〜853の領域）（配列番号３）
3'-ACGTGGTGATTCGCCTTTGG-5' （配列番号９）
（３）5'-TGCACCACTAAGCGGAAACCCGCCCAACAGT-3'
（Cypug829-859^*, 31mer：配列番号６における塩基番号823〜853の領域）（配列番号４）
（なお、^*（数字）はEscherichia coliの16S rDNA塩基配列における5'末端からの位置（ナンバーリングシステム）を示す（Noller H. F. and C. R. Woese, 1981. Science, 212:403-411）。） （３）のプローブの塩基配列は、（１）と（２）のプローブの塩基配列が隣接したものであるが、この場合はプローブが自己結合する可能性があるので注意が必要になる。
【００６１】
これらのプローブは、1997年1月2日のロシアタンカーナホトカ号による日本海での石油流出事故後、船首部が着底して大量の流出油が漂着した福井県三国町沿岸海域において1997年1月15日に採取した試料から見出された石油分解菌に由来する16S rDNA塩基配列（配列表配列番号６）の一部を有し、Cycloclasticus属の芳香族炭化水素分解菌、特にCycloclasticus pugetiiおよびその近縁種を検出するために設計されたものである。
【００６２】
これらの特定機能微生物検出用核酸プローブに加え、その特定機能微生物が属するドメインを対象とした検出用核酸プローブや、それを含む全生物を対象としたユニバーサルプローブを用い、対象とする環境微生物含有試料に上述した分子遺伝学的な検出および定量化手法（相対分子定量法）を適用することにより、Alcanivorax 属の脂肪族炭化水素分解菌、特にAlcanivorax borkumensisおよびその近縁種、およびCycloclasticus属の芳香族炭化水素分解菌、特にCycloclasticus pugetiiおよびその近縁種が、全微生物群集および/またはその両者が属するバクテリアドメインの中で、それぞれどの程度の優占度で存在しているのかを、分子レベルで正確に測定することができる。さらに、この優占度の時間的および/または空間的な変化を解析することにより、自然環境や人工的な環境、特に石油で汚染された油濁環境の状態を、微生物学的な観点から解析・評価することが可能となる。
【００６３】
本発明の方法によると、石油などの有害化学物質で汚染された環境を、その環境中に生息する微生物を、従来の分離・培養手法、およびRI（放射性同位体）ならびにPCR法を用いずに、簡便、迅速に分子遺伝学的に評価することができる。また、本発明の方法は、特に有用酵素や抗生物質等の有用物質生産微生物などの特定機能微生物の検出・定量、およびスクリーニングに応用することができる。さらに、本発明の方法は、病原体等の有害微生物などの特定機能微生物の検出・定量、およびスクリーニングに応用することもできる。したがって、本発明の方法に必要な試薬および器具をキットとして提供すること、ならびに本発明の方法を自動化することが可能かつ有益である。さらに、本発明の方法は、該有害化学物質汚染環境の評価及びバイオ環境修復（バイオレメディエーション）過程の解析や修復効果を評価するための診断ツールとしてきわめて有益である。また、本発明は、自然環境や人工的な環境中に含まれる、有用物質生産微生物、および病原微生物等の有害微生物などの特定機能微生物を検出・定量、およびスクリーニングするために有益である。したがって、本発明は、かかる特定機能微生物を含む環境試料の解析・評価において極めて有益である。
【００６４】
また、本発明で開示したDNAプローブは、近年多くの海域で出現することが報告されている特定の脂肪族炭化水素分解菌を特異的に識別し得るものであり、その検出および定量にきわめて有益である。
【００６５】
【実施例】
本発明を以下の実施例により具体的に説明する。これらの実施例は本発明をより具体的に説明するためのものであって、本発明の範囲を限定するものではない。
〔実施例１〕
（１）Alcanivorax borkumensis 検出用DNAプローブの開発
四国沿岸域より採取した試料から、集積培養を経て純粋分離に成功した脂肪族炭化水素分解菌GR211-P1菌株（特許寄託番号FERM BP-7788：平成１３年１１月５日付け国際寄託へ移管）の16S rDNA塩基配列情報（配列番号５）の中から、Stahl and Amann （Development and Application of Nucleic Acid Probes. In: Nucleic Acid Techniques in Bacterial Systematics. Ed.: E. Stackebrandt and M. Goodfellow, John Wiley and Sons, Chichester, pp. 205-248, 1991）により示された高次構造による障害が見られないと考えられる領域から、この菌種に特異的な配列を選抜し、該配列を有するオリゴヌクレオチドを合成し、その５'末端をFITC、TRITCまたはCy 5の蛍光色素により標識化し、最終的に配列番号１に示した配列を有するNon-RI標識DNAプローブ（以下、Albo 222ということがある）を作製した。
【００６６】
このプローブの配列は、国際塩基配列データベース上でAlcanivorax borkumensis SK2（基準菌株；M. M. Yakimov et al.: 1998、前出）の配列とのみ100％の相同性を示すことが確認され、Alcanivorax borkumensis検出用のDNAプローブとして有効であることが示された。
【００６７】
本発明に示したDNAプローブの使用にあたっては、Alcanivorax borkumensis DSM11573（標準菌株）を標的微生物、Pseudomonas aeruginosa IFO12689（標準菌株）などを対象微生物とし、Maruyama and Sunamura（Simultaneous direct counting of total and specific microbial cells in seawater, using a deep-sea microbe as biomarker. Applied and Environmental Microbiology, 66: 2211-2215, 2000）に記載した装置や手法を用い、FISH法にて実際にその有効性を確認した。この際のハイブリダイゼーションは、20 ％ホルムアミド存在下で４３℃で行い、洗浄処理は４４℃で行った。
【００６８】
（２）Cycloclasticus pugeti検出用DNAプローブ
もう一つの標的微生物としたCycloclasticus pugetiiを検出するためのDNAプローブとしては、Cyclopug 829-846（配列番号２：以下Cypug829ということがある）を用いた。このプローブの配列は、1997年1月2日に発生したロシアタンカーナホトカ号による日本海での石油流出事故後、船首部が着底して大量の流出油が漂着した福井県三国町沿岸海域において1月15日に採取した試料から、Ｃ重油を唯一の炭素源として用いた石油分解菌の段階希釈培養計数（MPN）試料中の増殖陽性フロント試料を得、そこから直接DNAを抽出し、その配列決定により明らかにされたCycloclasticus pugetii 16S rDNA塩基配列情報から、Cycloclasticus pugetiiに特異的な配列として見出したものである。
【００６９】
〔実施例２〕
これらのDNAプローブを用い、実際の石油汚染海域を対象とし、Cycloclasticus pugetiiおよびAlcanivorax borkumensisの優占度解析を実施した結果を以下に示す。
【００７０】
１）試料の採取と保存
分析対象には、1997年1月2日に発生したロシアタンカーナホトカ号による日本海での石油流出事故後、船首部が着底して大量の流出油が漂着した福井県三国町沿岸海域において、1997年1月15日に採取し凍結保存しておいた試料を用いた。すなわち、現地で採取した油濁海水約３リットルをすみやかに孔径0.2マイクロメーターの中空糸膜フィルターHF400（Millipore社製）を用いて該フィルター上にてろ過濃縮し、該フィルターごと凍結保存した。
【００７１】
２）細胞の破砕とRNAの抽出、保存
得られた環境微生物濃縮試料を中空糸膜フィルターごと12 ml容硬質ガラスチューブに移し、Qiagen社製RNeasy Midi Kitに含まれるRLT緩衝液を添加した。180℃で１時間以上乾熱滅菌しておいたガラスビーズ（Sigma社製GLASS BEADS 106 microns and finer）を、用いたRLT緩衝液1.5 mlに対して1.5 gとなる量で添加した後、細胞破砕器（B.Braun社製 MSK Cell Homogenizer）にて５分間の往復振とう処理により細胞破砕を行った。この処理は、電子温度計でモニターして、液体二酸化炭素を噴霧して試料の温度を5℃程度に維持しながら行った。また、この処理時間は、グラム陰性細菌より細胞壁が堅く、陸上に多く生息するグラム陽性細菌に属するBacillus subtilis IFO13719細胞を用い、顕微鏡下でその細胞破砕状況を観察し、かつ、緩衝液中に溶出された核酸をアガロースゲル電気泳動にて解析し、核酸の溶出量が高く、かつ過度の核酸の断片化を起こさない時間を選定し、処理条件とした。得られた細胞破砕試料より、Qiagen社製RNeasy Midi Kitを用いてRNAを抽出・精製し、各々50-100 μlずつ小分けして-80℃で保存した。
【００７２】
３）全RNA量の測定
ハイブリダイゼーション解析の前に、試料中の全RNA量を、RiboGreen RNA Quantitation Kit（Molecular Probes社製）を用い、蛍光分光光度計RF-5300PC（島津社製）にて励起波長480 nm、蛍光波長520 nm、バンド幅5 nmの設定条件で測定した。
【００７３】
４）Non-RI法でのハイブリダイゼーション
蛍光プローブを用いたNon-RI法でのハイブリダイゼーションは、以下のようにして実施した。まず、凍結保存しておいた試料を緩衝液（Dimethyl pyrocarbonate (DMPC)処理した水、20xSSC [0.15 M NaCl and 0.015 M sodium citrate]、フォルムアルデヒドを5:3:2の割合で混合した溶液）中にて溶解させ、試料中の核酸をポジティブチャージナイロン膜フィルター（Roche Diagnostics社製）上に吸着させ、UV Stratalinker（Stratagene社製）を用いて該フィルター上に固定化した。次に、ハイブリダイゼーション溶液（Raskin L, S. J., Rittmann BE, Stahl DA: Group-Specific 16S rRNA Hybridization Probes to Describe Natural Communities of Methanoges. Applied and Environmental Microbiology, 60: 1232-1240, 1994）を用い、35℃で30分間プレハイブリダイゼーションを行った。その後、終濃度10 pmol/mlとなるように上記Cy 5標識DNAプローブを添加した上記ハイブリダイゼーション溶液を用い、35℃で一晩のハイブリダイゼーションをおこなった後、該フィルターを洗浄溶液（1 % Sodium Dodecyl Sulfateを添加した1xSSC [0.15 M NaCl and 0.015 M sodium citrate]）中で、30分間、合計２回、以下の方法で決定された各々のプローブの至適洗浄温度で洗浄処理に供した。
【００７４】
５）Non-RI法でのプローブ遊離曲線測定と至適洗浄温度の決定
上記の至適洗浄温度は、Zhengら（D. A. E. Zheng et al.: 1996、前出）を参考に、Non-RI法により決定した。すなわち、各々の標準菌株（Cycloclasticus pugetii ATCC51542およびAlcanivorax borkumensis DSM11573）の核酸抽出試料を、上記膜フィルター上にドッドブロット（1ドットあたり約2 μgのRNAを添加）して固定化し、上述した方法により各々のDNAプローブとハイブリダイズさせ、35℃で一晩ハイブリダイゼーションを行った。その後、膜フィルターを8ドット分ずつに切り、その断片を2 mlの洗浄溶液を入れた5ml 用のプラスチックチューブに移した。次に、洗浄温度を35℃から80℃まで3℃毎に上げ、温度を上げるごとに新たな洗浄溶液に交換して、洗浄処理を行った。得られた各々の洗浄溶液中に遊離してくるプローブの蛍光シグナルの量を、蛍光分光光度計RF-5300PC（島津社製）にて測定した（測定条件は、励起波長640 nm、蛍光波長662 nm、バンド幅5nmとした）。最後に、各温度での洗浄液中の蛍光シグナルの量から、洗浄温度に対する各プローブの遊離曲線を求めた。該遊離曲線を用いて前述の通りに求めた至適洗浄温度は、Alcanivorax borkumensisの検出用プローブでは47℃、Cycloclasticus pugetiiの検出用プローブでは41℃と決定された。
Alcanivorax borkumensis検出用プローブ（Albo222）について得られた遊離曲線を図１、またCycloclasticus pugetii検出用プローブ（Cypug829）について得られた遊離曲線を図２として示した。
【００７５】
６）蛍光シグナルの視覚化と定量解析
上記のCy 5標識DNAプローブとのハイブリダイゼーション、および洗浄処理を行った試料について、蛍光イメージアナライザーSTORM（Amersham Pharmacia Biotech社製）で、フィルターに固定化された核酸にハイブリダイズしているDNAプローブの蛍光シグナルをレッドレーザーダイオード（出力５mW、波長635nm）により励起し、それに対応する蛍光フィルター（650nmロングパスフィルター）を用いて検出し、その蛍光シグナルをドットイメージ画像として視覚化した。さらに、IPLab（Scanalytics社製）を用い、その蛍光画像を解析し定量値を求めた。すなわち、測定する試料のシグナルのイメージ画像からバックグラウンドのシグナルを差し引き、かつ同一メンブレン上にブロットしておいたE.coliの16SrRNA標準試料の段階希釈試料のイメージ画像から求められた検量線を用いて、試料に由来する蛍光シグナルの真の定量値を求めた。
【００７６】
７）特定微生物の優占度解析
全生物ならびにバクテリアドメインに対する標的微生物の優占度は、RI法で提唱されている上記式１によって算出した。
（測定値の補正）
ここで、各種ドメインの優占度算出に用いた補正値は、下記の標準菌株を用いて求めた。
【００７７】
バクテリアドメイン（Bacteria domain）
Escherichia coli DSM30083
Psychrobacter pacificensis IFO16279
Cycloclasticus pugetii ATCC51542
Alcanivorax borkumensis DSM11573
Arcobacter nitrofigilis DSM7299
Methylobacterium extorquens DSM1053
Methylophilus methylotrophus DSM46235
アーキアドメイン（Archaea domain）
Aeropyrum pernix JCM9820
Methanococcoides methylutens DSM2657
ユーカリアドメイン（Eucarya domain）
Saccharomyces cerevisiae IFO10217
これら補正に用いた標準菌株からの核酸の抽出には、RNeasy Maxi kit（Qiagen社製）を用い、各々200 μlずつ小分けして-80℃で保存した。
【００７８】
次に、上述した手法により、全RNA定量、ハイブリダイゼーション、洗浄処理、ドットイメージ画像の取得、画像の定量解析等を実施した。
ドメインレベルでのハイブリダイゼーションを行うプローブとしては、Ammanら（R. I. Amann et al.: 1995、前出）に記載されているEuba338（バクテリアドメイン）、Euka 502（ユーカリアドメイン）、Arch 915（アーキアドメイン）を用いた。また、全生物共通領域に対するDNAプローブには、Univ 1390（D. A. E. Zheng et al.: 1996、前出）を用いた。
【００７９】
そして、バクテリアドメイン中の標的とする特定機能微生物Cycloclasticus pugetiiおよびAlcanivorax borkumensisに対しては、上述した各々の種特異的DNAプローブCypug 829およびAlbo 222を用いた。
測定結果を表１に示す。表１において、試料１は三国町安島漁港内（Stn.A）で採取した試料、また、試料２は同町安島北部海岸船首部着底付近の岩礁域（Stn.B）で採取した試料である。
【００８０】
【表１】

【００８１】
表１に示したように、試料１中にはバクテリアドメインが約４５．２%（全体を１００%とした場合６１．４%）、ユーカリアドメインが約１９．９％、アーキアドメインが約８．５％（合計は、７３．６％）見出された。また、試料２中にはバクテリアドメインが約５３．３%（全体を１００%とした場合５９%）、ユーカリアドメインが約２４．０％、アーキアドメインが約１３．３％（合計は、９０．６％）見出された。
【００８２】
表１において、バクテリアドメインの括弧内の数値は、該ドメイン全体の優占度を表すEuba338に対するCypug829及びAlbo222の割合を示す。すなわち、試料１のバクテリアドメイン中、このドメインに属する標的微生物である芳香族炭化水素分解菌Cycloclasticus pugetiiは２３．６%、もう一つの標的微生物である脂肪族炭化水素分解菌Alcanivorax borkumensisは４．４%の優占度を示す。同様に、試料２のバクテリアドメイン中、このドメインに属する標的微生物である芳香族炭化水素分解菌Cycloclasticus pugetiiは２４．５%、もう一つの標的微生物である脂肪族炭化水素分解菌Alcanivorax borkumensisは６．５%の優占度を示す。
【００８３】
以上、本手法に示したように汚染環境中の微生物の核酸抽出試料を対象とし、Non-RI法で特定機能微生物の優占度を定量化することにより、実際の汚染環境を分子遺伝学的に解析、評価、診断することが可能であることを明らかにした。
【００８４】
【発明の効果】
本発明により、自然環境や人工的な環境中（例えば、石油汚染環境など）に出現する特定機能微生物（例えば、石油分解菌、有用物質生産微生物、病原微生物、硫酸還元菌など）の優占度を、従来の多大な労力と時間を要する培養計数法を用いずに、しかもNon-RI及び（RIを用いない方法）による分子遺伝学的に定量的に解析する手法が提供された。 このような優占度の解析は現在のところPCR法によって実施することはできず、本発明によるNon-RI法による優占度解析は、環境中に含まれる微生物群集の解析・評価にとって極めて有効な手法である。
【００８５】
本発明の手法により、新たに開発された石油分解菌Alcanivorax borkumensisに特異的な核酸プローブの塩基配列は、広く沿岸域の油濁環境で優占する能力を有する天然の石油分解微生物に由来するものである。従って、このプローブの塩基配列は、この石油分解微生物の自然界や油濁環境のバイオ修復過程などの石油処理条件下、および屋外でのバイオ環境修復技術の実証試験や研究室での実験条件下における挙動を解明する上で、また油濁環境の診断ツールや油濁環境のバイオメディエーションによる修復効果を評価するための診断ツールとして、きわめて有益である。
【００８６】
また、本発明により、広く沿岸域に分布することが知られる代表的な脂肪族炭化水素分解菌および/または代表的な芳香族炭化水素分解菌の優占度解析が、分子遺伝学的な手法で実現した。したがって、本発明は、石油汚染海域での石油成分の残存状況や分解過程を生物学的にモニターする上で有益であり、栄養塩等の散布、および栄養塩などとともにこれらの微生物を油濁海域に散布することにより、現場で石油の微生物分解を促進させる環境修復技術（バイオレメディエーション技術）の開発にも有効で、かつ極めて大きな利便性がある。さらに、本発明は、有用物質生産微生物および病原微生物等の有害微生物などの特定機能微生物を検出・定量、スクリーニングするために、ならびに、かかる特定機能微生物を含有する環境試料の解析・評価においても極めて有益である。
【００８７】
【配列表】

【図面の簡単な説明】
【図１】 Alcanivorax borkumensis検出用プローブ（Albo222）について得られた遊離曲線を示す。
【図２】 Cycloclasticus pugetii検出用プローブ（Cypug829）について得られた遊離曲線を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates generally to a method for analyzing and evaluating a polluted environment. Specifically, molecular genetics is used for diagnosis of a hazardous chemical substance polluted environment, particularly a petroleum polluted environment, and a bioenvironmental repair (bioremediation) process by microorganisms in the polluted environment. Analysis and evaluation methods, methods for detecting and quantifying and analyzing harmful microorganisms such as useful substance-producing microorganisms or pathogenic microorganisms, and analysis of the environment and environmental samples containing these microorganisms -It relates to the evaluation method.
[0002]
[Prior art]
Until now, as a method for evaluating the contaminated environment, an analytical instrument combining various fractionation methods such as gas chromatography and high performance liquid chromatography with various elemental analysis methods, mass spectrometry methods, optical detection methods, etc. has been used. Methods for qualitative and quantitative analysis of harmful chemical substances are widely used. There are also COD and BOD methods for measuring the oxygen demand of contaminated environmental samples. However, simple and reliable molecular genetic evaluation methods for specific microorganisms (groups) present in environmental samples as well as contaminated environments are under development, and analysis of dominance of specific microorganisms (groups) A method that enables this has not been established yet. For some environmental pollutants such as petroleum components, various treatment techniques and treatment techniques have been developed through the elucidation of physicochemical factors that influence natural purification.
[0003]
However, there is no simple and highly accurate method for analyzing and evaluating pollutant-degrading bacteria that are dominant in the contaminated environment, including the composition of the natural microbial community that is responsible for natural purification, fluctuations in the community, and the effectiveness of treatment technology. It is difficult to evaluate sex and its universality.
[0004]
In carrying out environmental evaluation for microorganisms in this contaminated environment, there are a plate culture / counting method using an agar plate medium and an MPN (most probable number) culture / counting method using a liquid medium. However, the above-described conventional culture / counting method has a problem that it takes a lot of labor and labor, and it takes a long culture time to detect a specific degrading microorganism.
[0005]
In addition, the number of microorganisms that can be detected by these conventional separation and culture methods is extremely small among the microorganisms that inhabit the natural world. That is, direct microscopic counting methods such as JE Hobbie, RJ Daley, and S. Jasper: Appl. Environ. Microbiol. 33: 1225-1228, 1977 and KG Porter and YS Feig: Limnol. Oceanogr. 25: 943-948, 1980), the percentage of microorganisms that can be separated and cultured is considered to be 1% or less (RI Amann, W. Ludwig and KH. Schleifer: Phylogenetic Identification and In Situ Detection of Individual Microbial Cell without Cultivation, Microbial. Rev., 59, 143-169, 1995). Therefore, there has been a major drawback in that it is difficult to analyze the composition of the entire microbial community inhabiting there, community fluctuation, behavior of specific microorganisms, and the like.
[0006]
Therefore, in recent years, attempts have been made to detect and quantify molecular genetics independent of conventional separation and culture methods using DNA probes specific to all microorganisms or specific microorganisms. For example, detection and quantification methods at the molecular level by blot hybridization using RI-labeled oligonucleotide probes have been reported (SJ Giovannoni, TB Britschgi, CL Moyer, KG Field: Genitic diversity in Srgasso Sea bacterioplankton. Nature, 345: 60-63, 1990). In addition, a probe release curve analysis technique required for hybridization has been reported using an RI-labeled oligonucleotide probe (DAE Zheng, DA Stahl, and L. Raskin: Characterization of Universal Samll-Subunit). rRNA Hybridization Probes for Quantitative Molecular Microbial Ecology Studies. Applied and Environmental Microbiology, 62: 4504-4513, 1996).
[0007]
However, since conventional methods are premised on the use of RI-labeled probes, the use of special RI handling facilities is indispensable for detection and quantification, and can therefore be monitored in a normal laboratory. Therefore, it is difficult to develop a kit for this monitoring method and to develop a technology for its automation.
[0008]
Another molecular genetic detection and quantification method is a quantitative PCR method that uses a DNA primer specific to a specific microorganism to amplify and quantitatively analyze the target gene region of the specific microorganism. (For example, LG Lee, CR Connell and W. Bloch: Nucleic Acids Research, 21, 3761-3766, 1993). However, with this method, it is difficult to obtain a quantitative value of the total microbial community in the environment, and there is a great technical limitation that the ratio (dominance) of a specific microorganism to the total microorganisms cannot be analyzed.
[0009]
In addition, the FISH method is used to detect specific microorganisms at the cellular level regardless of the separation / culture method (R. I. Amann et al .: 1995, supra). Furthermore, the FISH-DC method is effective when analyzing the proportion of specific microorganisms in the total population at the cellular level for water environmental samples (A. Maruyama and M. Sunamura: Simultaneous direct counting of total and specific microbial cells in seawater, using a deep-sea microbe as biomarker. Applied and Environmental Microbiology, 66: 2211-2215, 2000). However, in order to detect and identify microbial cells with low metabolic activity that occupy the majority in the environment, it is necessary to use some kind of signal amplification means at the same time, and there are still significant technical limitations (A. Maruyama and M.). Sunamura: 2000, supra).
[0010]
The diversity of microorganisms and the microbial community structure in the natural environment include PCR, cloning, sequencing and molecular phylogenetic analysis for direct nucleic acid extraction of environmental microorganism samples, in addition to conventional separation and culture methods. It is gradually elucidated using a series of methods (e.g. TM Schmidt, EF DeLong and NR Pace: Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing, J. Bacteriol., 14, 4371-4378, 1991 ).
[0011]
From the analysis using these methods, it is becoming clear that special microorganism groups that have been difficult to separate and culture by conventional methods appear in an environment contaminated with petroleum or the like. For example, Cycloclasticus pugetii, an aromatic hydrocarbon-degrading bacterium, is released from the US West Coast (SE Dyksterhouse, JP Gray, RP Herwig, JC Lara and JT Staley, Cycloclasticus pugetii gen. Nov., Sp. Nov., An aromatic hydrocarbon-degrading bacterium from marine sediments, Int. J. Syst. Bacteriol., 45, 116-123, 1995) and on the coast of the Sea of Japan, and the aliphatic hydrocarbon-degrading bacterium Alcanivorax borkumensis is found in the North Sea (MM Yakimov, PN Golyshin, S Lang, ER Moore, WR Abraham, H. Lunsdorf and KN Timmis: Alcanivorax borkumensis gen. Nov., Sp. Nov., A new, hydrocarbon-degrading and surfactant-producing marine bacterium, Int. J. Syst. Bacteriol., 48, 339-348, 1998), the Seto Inland Sea coast, the Japan Sea coast, etc. (Kishi and Harayama: Biotechnology Society, Abstracts of lectures P.291, 1998). Among these, Cycloclasticus pugetii's dominance analysis for the total number of bacteria has already been carried out using a method that further improves the accuracy by combining the total cell count method and the MPN culture counting method as described above. (Japanese Patent Application No. 11-237818). However, there is no case where the above-described dominance analysis of the microbial group is performed by molecular genetic techniques regardless of whether or not RI is used.
[0012]
[Problems to be solved by the invention]
The present invention is directed to both all microorganisms inhabiting the natural environment and specific microorganisms that grow on them, and its molecular genetic detection and quantification can be performed without using PCR methods and radioisotopes. Regarding new methods that can be performed by the RI method (methods that do not use radioisotopes) and methods for analyzing and evaluating contaminated environments using such methods, monitoring of hazardous chemical polluted environments, especially petroleum polluted environments, and The purpose is to provide a method for molecular genetic analysis and evaluation of bioenvironmental repair (bioremediation) process by microorganisms. Another object of the present invention is to provide a method for molecular genetic detection and quantification of harmful microorganisms such as useful substance-producing microorganisms and pathogenic microorganisms in natural or artificial environments. Furthermore, molecular genetic analysis of the predominance of specific microorganisms that produce useful substances such as enzymes and specific functional microorganisms such as pathogenic microorganisms in natural or artificial environments, and the environment collected from the environment It is also included in the object of the present invention to provide a method for analyzing and evaluating the usefulness and harmfulness of a sample. Further, the nucleic acid probe specific to the newly developed petroleum-degrading bacterium Alcanivorax borkumensis disclosed in the present specification is used for detection and quantification of the petroleum-degrading bacterium, and analysis and evaluation of the oily environment and environmental samples thereby. As a useful tool for this, it is provided by the present invention.
[0013]
[Means for Solving the Problems]
Environmental pollutants such as oil that have flowed out of an artificially controlled environment are gradually decomposed by natural microbial activity and are naturally purified over time. If it is a hard-to-decompose substance such as PCB, it will take a long time to decompose, but if the pollution affects the growth of general microorganisms, microorganisms specific to the contaminated environment ( Group) is thought to dominate.
[0014]
For example, in the ocean, hydrocarbon-degrading bacteria are said to be widely distributed at a level of 1% or less of the in-situ microbial community, but in oil-polluted areas, the ratio is often said to be 10% or more. (RM Atlas: Petroleum biodegradation and oil spill bioremediation, Marine Pollution Bulltetin, 31, 178-182, 1995, RM Atlas and R. Bartha: Hydrocarbon Biodegradation and Oil Spill Bioremediation. In: Advances in Microbiolbial Ecology, Ed. KC Marshall, PlenumPress, New York, 12, 287-338, 1992), and the proportion of degrading bacteria in the total microbial community is thought to reflect the extent of petroleum contamination (RM Atlas and R. Bartha: 1992, supra).
[0015]
In addition, the number of PAHs-degrading bacteria in sediments from harbors contaminated with creosote (containing about 85% polycyclic aromatic hydrocarbons [PAHs]) discharged from wood treatment facilities has been investigated (AD Geiselbrecht et al: Enumeration and Phylogenetic Analysis of Polycyclic Aromatic Hydrocarbon-Degrading Mairne Bacteria from Puget Sound Sediment, Appl. Environ. Microbiol., 62, 3344-3349, 1996). According to it, the number of PAHs degrading bacteria counted by MPN culture counting method is 10 at the contaminated site.^Four~Ten⁷  MPN / g (dry weight), 10 at non-contaminated site^Three~Ten^Four  MPN / g (dry weight). On the other hand, the total number of bacteria in the sediment is 10⁹  About cells / g (dry weight), there was almost no difference between contaminated and non-contaminated sites. That is, at the creosote-contaminated site, not only the PAHs-degrading bacteria are 10 to 1000 times as many as the non-contaminated site, but the proportion of the total microbial community has increased to about 1%. Thus, it has been found that the proportion of pollutant-degrading bacteria in the microbial community has increased reflecting the pollution of the environment. It will be a good indicator.
[0016]
However, the numbers of petroleum-degrading bacteria and PAHs-degrading bacteria reported above were detected and counted by the plate culture method and the culture method using the MPN method, which require a great amount of labor and time. In addition, since the number of microorganisms that can be separated and cultured as described above is usually 1% or less of the total microbial community, there is a drawback that the number of degrading microorganisms concentrated in the environmental microbial community is not sufficiently reflected in the counting by the culture method. is there. Further, as in the method according to the present invention, the counting or quantification of specific degrading bacteria (microorganisms of a specific genus species or microorganisms having genetic information that can be specified by molecular phylogenetic taxonomy) concentrated in an environmental microbial community has not yet been made. Absent.
[0017]
Therefore, if it becomes possible to easily and quickly monitor the behavior of specific microorganisms that serve as an indicator of the environment including those degrading bacteria, and the proportion of them (dominance) in the total microbial community, It is considered that diagnosis of the contaminated environment can be performed with high accuracy and at an early stage, such as the degree of repair and the degree of restoration and recovery of the contaminated environment. For this purpose, it is indispensable to develop detection and quantification methods for specific microorganisms in environmental samples, instead of the conventional separation and culture methods, which have great limitations in analyzing total microbial communities and specific function microorganisms. is there. Furthermore, these methods must be applicable to diagnosis of various polluted environments and can be used easily. Therefore, the following steps were taken to develop a new method that meets these needs.
[0018]
First, two types of petroleum-degrading bacteria with different characteristics of decomposing petroleum components that are becoming dominant in actual oil-sparing environments, namely, aliphatic hydrocarbon-degrading bacteria and aromatic hydrocarbon-degrading bacteria are selected. Then, a nucleic acid probe specific to each of them was developed.
[0019]
The selected aliphatic hydrocarbon-degrading bacterium decomposes aliphatic hydrocarbons, but does not decompose carbohydrates such as aromatic hydrocarbons and glucose. On the other hand, aromatic hydrocarbon-degrading bacteria degrade persistent polycyclic aromatic hydrocarbons, but do not use carbohydrates such as aliphatic hydrocarbons and glucose. Therefore, the dominance of these two types of petroleum-degrading bacteria is considered to vary depending on the hydrocarbon components and concentration in the spilled oil, and the nucleic acids of these two degrading bacteria existing in the oil-spilled environment. Quantitatively grasping and analyzing the microflora at the nucleic acid level makes it possible to analyze and evaluate the natural purification process of pollutants and bioenvironmental restoration processes, such as the ratio, concentration and fate of hydrocarbon components in the environment It is considered to be.
[0020]
Next, the present inventors have succeeded in developing a Non-RI method for molecular genetic detection and quantification of all microorganisms and specific microorganisms in the environment for the first time. In other words, the hybridization and washing process conditions, which are indispensable when using a new probe, are examined using a technique that does not use radioisotopes, and the most important hybridization process is performed using fluorescent or chemiluminescent labels. The detection process can also be performed by fluorescence detection method or chemiluminescence detection method. As a result, we succeeded in developing a relative quantification method for microbial nucleic acids in the environment using the Non-RI method without using radioisotopes throughout the entire process.
[0021]
Environmental samples contain various contaminants, and it is difficult to set internal standards. Nucleic acids, especially RNA, are components that are susceptible to enzymatic degradation. It is difficult to accurately estimate efficiency. Therefore, even if a highly purified nucleic acid sample can be quantified, there is a great limitation in accurately quantifying (absolute quantification) the total nucleic acid content present in the used environmental sample. Therefore, the target sequence and the reference sequence are selected from nucleic acid molecules (the same molecule or multiple molecules) that exhibit the same behavior during extraction / purification, and the abundance ratio is obtained from these abundances to calculate the relative amount. This is considered to be most effective for evaluating the abundance of microorganisms in environmental samples. This analysis method is called a relative molecular quantification method.
[0022]
The principle of this relative molecular quantification method is based on the premise of hybridization with multiple probes in the same sample, and the quantitative value of hybridization of a specific microbial probe (detecting a target sequence) is a universal probe (reference sequence) common to all organisms. Is detected with a quantitative value of hybridization, and an accurate relative value is obtained. Actually, standardization can be performed by using an average value of values obtained for various standard strains as reference data. it can. In this standardization, the technique presented by the existing RI method can be used, but the novelty of the present invention is that all processes are realized by the Non-RI method for the first time. This analysis method also has a great feature and advantage in that it does not use the PCR method at all.
[0023]
An on-site microbial sample that had been concentrated, collected, and cryopreserved immediately after the oil spill in 1997 was analyzed using this newly developed method. As a result, it was possible to estimate the degree of dominance of the aliphatic hydrocarbon-degrading bacteria and the aromatic hydrocarbon-degrading bacteria that occupy the entire microbial group concentration. Moreover, the nucleic acid probe newly developed for the aliphatic hydrocarbon-degrading bacterium enables detection and quantification at the cell level by the fluorescence in situ hybridization (FISH) method.
[0024]
As described above, the Non-RI analysis method according to the present invention is effective for molecular genetic diagnosis of an actual petroleum-contaminated environment and analysis / evaluation of a bio-environment repair process. In addition, the analysis method is highly convenient for kits and automation, and furthermore, this series of methods is used to evaluate environments contaminated with hazardous chemicals other than petroleum, environmental samples, and contaminated environments. It has advantages such as being extremely useful for analyzing and evaluating the bioenvironmental restoration process. Furthermore, it is extremely useful for detecting and quantifying, analyzing and evaluating harmful microorganisms such as useful substance-producing microorganisms and pathogenic microorganisms without depending on the separation and culture method.
[0025]
Based on these findings, the present invention has been completed.
That is, the gist of the present invention is as follows.
1. Detects and quantifies nucleic acids of specific functional microorganisms in samples obtained from natural and artificial environments by the Non-RI method (a method that does not use radioisotopes), and the degree of dominance of specific functional microorganisms in the environment A method for analyzing the following steps:
1) A process for extracting, purifying and storing a target nucleic acid from a microorganism-containing sample collected from a natural environment or an artificial environment,
2) Select a nucleic acid sequence specific to the specific function microorganism from the nucleic acid base sequence information targeted by the specific function microorganism, synthesize an oligonucleotide having the sequence, and label it with a Non-RI label. A step of producing a Non-RI-labeled nucleic acid probe specific to the specific functional microorganism,
3) After immobilizing the target nucleic acid extracted and purified in 1) on a hybridization substrate and bringing it into contact with a non-RI-labeled nucleic acid probe specific for a specific functional microorganism, a hybridization treatment is performed. A process of performing a cleaning process,
4) a step of visualizing and analyzing a signal derived from the hybridized Non-RI labeled nucleic acid probe and obtaining a quantitative value thereof;
5) a step of calculating the dominance of the specific functional microorganism by using the obtained quantitative value and comparing it with the quantitative value measured for each domain of all living organisms and organisms;
A method as described above.
[0026]
2. 3. The method according to 1 above, wherein the washing treatment of 3) is performed at an optimum washing temperature determined from a release curve of a probe prepared using a Non-RI-labeled nucleic acid probe specific to a specific functional microorganism. .
3. 3. The method according to 1 or 2 above, wherein the non-RI labeled nucleic acid probe is fluorescently or chemiluminescently labeled.
4). 4. The method according to any one of 1 to 3, wherein the specific function microorganism is a microorganism that decomposes a specific chemical substance.
5). 5. The method according to 4 above, wherein the specific chemical substance is a harmful chemical substance.
[0027]
6). 5. The method according to 4 above, wherein the specific chemical substance is petroleum and petroleum components.
7. 4. The method according to any one of 1 to 3 above, wherein the specific function microorganism is a specific useful substance-producing microorganism.
8). 4. The method according to any one of 1 to 3 above, wherein the specific function microorganism is a specific harmful microorganism including a pathogenic microorganism.
9. A method for analyzing and evaluating a microbial community function of an environment by analyzing the dominance of a specific function microorganism in a natural environment or an artificial environment using the method described in any one of 1 to 8 above.
10. A method for analyzing and evaluating a contaminated environment using the method according to any one of 1 to 8 above.
[0028]
11. A method for analyzing and evaluating a hazardous chemical substance-contaminated environment using the method described in 4 or 5 above.
12 A method for analyzing and evaluating an oily environment using the method described in 6 above.
13. RNA having a base length of 10-50 bp having a part of the base sequence of SEQ ID NO: 5 or a part of the corresponding riboxynucleotide sequence and capable of specifically hybridizing with a nucleic acid derived from the Alcanivorax family or genus petroleum-degrading bacteria Or a DNA probe.
14 DNA or RNA having a base sequence set forth in SEQ ID NO: 1 or a riboxynucleotide sequence corresponding to the base sequence set forth in SEQ ID NO: 1 that specifically hybridizes with a nucleic acid derived from the Alcanivorax family or genus petroleum-degrading bacteria, and can detect or quantify it probe.
15. 15. The DNA or RNA probe according to 13 or 14 above, wherein the petroleum-degrading bacterium of the Alcanivorax family or genus is Alcanivorax borkumensis or a related species thereof.
[0029]
16. The non-RI-labeled nucleic acid probe specific to a specific functional microorganism, the DNA or RNA probe according to the above 13, which can specifically hybridize with a nucleic acid derived from the Alcanivorax family or genus petroleum-degrading bacteria, or the sequence according to SEQ ID NO: 1 The nucleotide sequence of SEQ ID NO: 6 or the like, which can specifically hybridize with a DNA or RNA probe having a nucleotide sequence or a corresponding riboxynucleotide sequence, and / or a nucleic acid derived from a cycloclasticus genus petroleum-degrading bacterium Selected from RNA or DNA probes having a base length of 10-50 bp having a part of the roxynucleotide sequence, or DNA or RNA probes having the nucleotide sequences described in SEQ ID NOs: 2 to 4 or ribonucleotide sequences corresponding thereto The Alcanivorax strain concentrated in the environmental microbial community according to the method described in 1 above. A method to analyze the oil spill environment by analyzing the dominance of petroleum-degrading bacteria of the genus or genus and / or Cycloclasticus.
[0030]
17. 16. Analyzing the dominance of an aliphatic hydrocarbon-degrading bacterium Alcanivorax borkumensis or related species thereof and / or an aromatic hydrocarbon-degrading bacterium Cycloclasticus pugetii or related species thereof The method described in 1.
[0031]
18. The DNA or RNA probe according to the above 13, or a part of the nucleotide sequence of SEQ ID NO: 6 capable of specifically hybridizing with a nucleic acid derived from an oil-degrading bacterium of the genus Cycloclasticus or a nucleotide length of 10- One or more nucleic acid probes selected from a 50 bp DNA or RNA probe and / or a DNA or RNA probe having a nucleotide sequence described in SEQ ID NOs: 1 to 4 or a ribonucleotide sequence corresponding thereto are used as Non-RI. A kit for molecular genetic analysis / evaluation of contaminated environment and environmental samples, characterized by containing a non-RI labeled probe obtained by labeling.
[0032]
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes 1) a step of extracting, purifying, and storing a target nucleic acid such as 16S rRNA from a microorganism-containing sample collected from a natural environment or an artificial environment, and 2) a 16S rDNA that is a target of a specific functional microorganism. A sequence specific to the specific function microorganism is selected from the nucleotide sequence information of the above, and an oligonucleotide having the sequence is synthesized, labeled with a Non-RI label, and specific to the specific function microorganism. Non-RI labeled nucleic acid probe production process, 3) Specific target microorganisms produced in 2) by immobilizing the target nucleic acid such as 16S rRNA extracted and purified in 1) on the substrate for hybridization. A step of performing a hybridization treatment after contacting with a non-RI-labeled nucleic acid probe specific to, and a washing step. 4) Visualizing and analyzing a signal derived from the hybridized Non-RI-labeled nucleic acid probe. A step of obtaining the quantitative value, and 5) a step of calculating the dominance of the specific functional microorganism by using the obtained quantitative value and comparing it with the quantitative value of the reference sequence measured for each organism and each domain of the living world. A method for detecting and quantifying nucleic acids of specific function microorganisms from natural and artificial environments using the Non-RI method (a method that does not use radioactive isotopes) and analyzing the predominance of specific function microorganisms in the environment provide.
[0033]
In this method, measuring the abundance of all organisms and the communities of each domain of the living world is indispensable to know the size of the entire microbial community (the size of the population) in the target environmental sample. And has a very important meaning in the process of estimating the degree of dominance of specific functional microorganisms concentrated in the group.
[0034]
The “dominance” refers to the ratio of the specific microorganism of the subject to the total microorganisms in the sample or the microorganisms belonging to each domain.
Here, the “natural environment” means the biosphere on the earth, hydrospheres such as oceans, lakes and rivers, geospheres such as soil, land underground, submarine underground, aerosphere such as the Earth's surface, animals and plants, It means all the environments on the earth where microorganisms such as dead bodies live.
[0035]
“Artificial environment” means an environment that is artificially controlled to some extent. For example, the environment in laboratory flasks and demonstration test equipment, chemical and chemicals such as food and brewing, pharmaceuticals, etc. Artificial production, processing, storage, and transportation of the environment in production tanks such as fermenters used in the bio field, or water such as tap water, domestic and industrial water, cooling water, circulating water, waste water, and sewage This refers to the environment related to use and disposal, and refers to all artificially controlled environments where microorganisms may exist or be mixed.
[0036]
“Specific functional microorganisms” refers to microorganisms whose functions and characteristics are specified by culture, genetic analysis, etc. among microorganisms inhabiting the natural environment. For example, specific chemicals such as petroleum and petroleum components Microorganisms that decompose substances, trichlorethylene, and organic chlorine compounds such as PCB and dioxin, microorganisms that decompose harmful chemical substances such as environmental hormone substances (such as alkylphenols, bisphenol A, and phthalates), organic mercury cyanide compounds, and organic tin compounds Etc. can be illustrated. Also, microorganisms that produce useful enzymes such as chitinase, lipase, cellulase, xylanase, lignin degrading enzyme and various antibiotics, pathogenic microorganisms such as Escherichia coli, Escherichia coli O157, Escherichia coli, and Bacillus anthracis, and Examples of specific functional microorganisms include, but are not limited to, sulfate-reducing bacteria that cause hydrogen corrosion to cause tank corrosion and malodor.
[0037]
Judging from the phenotypic traits and homology of gene sequences, it is not possible to assign it to a known species, but it is not possible to establish a new species by distinguishing it from the properties of the known species. This species refers to species that have very similar phenotypic traits and very close phylogenetic positions among certain species or known species. When used in the present specification, judging from the homology of 16S rDNA widely used as an indicator of organism phylogeny, its homology is 90% or more, preferably 94% or more, more preferably Means 96% or more. Examples of closely related species of Alcanivorax borkumensis include Alcanivoraxsp.ST-T1, Alcanivorax sp.WF-1, Alcanivorax sp.Shm-2, Alcanivorax sp.K3-3, Alcanivorax sp.K2-1, Alcanivorax sp.TE- 9, fFundlbacter jadensis and so on. Cycloclasticus pugetii related species include Cycloclasticus sp.M4-6, Cycloclasticus sp.E16-S, Cycloclasticus sp.W, Cycloclasticus sp.G, Cycloclasticus sp.N3-PA321, Cycloclasticus sp. (Cycloclasticus oligotrophus) (These rDNA sequences are registered in the Japan DNA Data Bank (DDBJ)). However, it is not limited to the examples listed here.
[0038]
“Microorganism-containing samples collected from natural and artificial environments” means natural environment samples such as seawater, lake water, river water, bottom mud, sediment, soil, minerals, groundwater, pore water, animals and plants, or the above In the case of the artificial environmental sample itself, and in the case where there are few microorganisms in the environment, it means a sample in which microorganisms in the environment are concentrated by filtration or centrifugation. For example, using a filter such as a flat membrane filter or hollow fiber membrane filter having a pore size of 0.2 micrometers smaller than the size of most common microorganisms, the microorganisms in the environmental water are concentrated on the filter by filtration, and the concentrate Can be used as a sample. In addition, using a flat membrane filter with a pore size of 0.2 micrometers, for example, using a tangential flow filter manufactured by Millipore, flow the liquid sample in the horizontal direction instead of the vertical direction and concentrate by filtration, and use the concentrated liquid as the sample. Can do. In addition, by directly subjecting the sample to high-speed centrifugation operation, for example, centrifugation for about 10 minutes or more with a centrifugal force of about 8,000 × g or more, microorganisms are precipitated to obtain a concentrated sample, which is used as a sample for nucleic acid extraction. Can do.
[0039]
A known method (for example, a method described in M. G. Murray and W. F. Thompson, Nucleic Acids Research 8: 4321-4325, 1980) can be used for nucleic acid extraction from the above microorganism-containing sample. For soil and sediment samples, purification methods using hydroxyapatite (for example, KJ Purdy, DB Nedwell, TM Embley and S. Takii: Use of 16S rRNA-targeted oligonucleotide probes to Investigate the occurrence and selection of sulfate -reducing bacteria In response to nutrient addition to sediment slurry microcosms from aJapanese estuary, FEMS Microbiol. Ecol., 24, 221-234, 1997). In particular, when the analysis target is RNA such as 16S rRNA, commercially available RNA extraction kits such as Qiagen RNeasy kit, Stratagene RNA RT-PCR Miniprep kit, Clontech NucleoSpin RNA kit, Ambion RNAqueous kit and the like can be used. When a sample contains a large amount of impurities, several nucleic acid extraction techniques can be used in combination to improve the purity of the nucleic acid extract. This degree of purification can be easily confirmed, for example, by measuring an absorption spectrum in the vicinity of a wavelength of 220 to 400 nm using a spectrophotometer and comparing it with the absorption spectrum of a pure RNA or DNA standard sample. In any case, careful attention is required during the extraction and purification operations in order to prevent contamination with microorganisms, nucleolytic enzymes and contaminants.
[0040]
“Non-RI labeled nucleic acid probe preparation” means detection without using RI such as fluorescence detection, chemiluminescence detection, chemiluminescence detection, etc. for oligonucleotides of about 10-50 bases in length, which have sequences selected as probes This means that a non-RI labeled nucleic acid probe is prepared by performing labeling that enables analysis by a technique. Here, the oligonucleotide can be synthesized by a known method, for example, a phosphoramide method or a triester method. Alternatively, it may be synthesized by an automatic DNA synthesizer. It is well known to those skilled in the art that oligonucleotides may be directly labeled with a fluorescent dye, or labeled with a peptide substance such as digoxigenin and indirectly labeled via a substance that binds to the peptide substance. is there. Fluorescent dyes include rhodamine, fluorescein-isothiocyanate (FITC), Lucifer Yellow CH (LuciferYellow CH), rhodamine 123 (Rhodamine 123), pyronin Y (Pyronin Y), propidium iodide, ethidium homodimer (Ethidium) homodimer), carboxyfluorescein diacetate (CFDA), fluorescein diacetate (FDA), carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), 5-cyano-2,30 ditolyltetrazolium (CTC), tetramethylrhodamine isothioti Anate (TRITC), Sulforhodamine 101 acid chloride (Texas Red), Cy3, Cy5, Cy7, 2-hydroxy-3-naphtholic acid-2′-phenylanilide phosphate ( 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate (HNPP) These without limitation, may be used any fluorescent substance known to those skilled in the art. In addition, a technique can be used in which an oligonucleotide is labeled with an antigenic substance and chemiluminescence or fluorescence (also referred to as chemical fluorescence) generated by enzymatic degradation of a substrate is detected using an enzyme immunological technique. As a label for using indirect labeling and enzyme immunological technique, biotin / avidin, digoxigenin / anti-digoxigenin system, and the like can be used, but are not limited thereto.
[0041]
In any of the above cases, there is no or weak background fluorescence or luminescence derived from a substrate such as a membrane filter used for hybridization and a glass slide treated with an organic coating, and there is no non-specific binding to the substrate. Preferably, very few fluorescent dyes or protein-protein binding or enzyme-substrate reaction systems are used. For example, when fluorescent detection is performed using a membrane filter as a base for hybridization, it is preferable to use a fluorescent dye such as Cy 5, and when chemiluminescence detection is performed similarly, for example, labeling with digoxigenin (DIG) or the like A chemiluminescence system (for example, a kit manufactured by Roche) using CDP-Star as a substrate for alkaline phosphatase may be used using an alkaline phosphatase labeled anti-digoxigenin antibody.
[0042]
To obtain the probe release curve using the Non-RI method, a nucleic acid probe labeled with an antigenic substance or peptide substance such as the fluorescent dye or digoxigenin prepared above is used, and the rRNA sequence complementary to the probe sequence is used. Hybridization is carried out on a substrate for hybridization such as a positively charged nylon membrane filter or a glass slide treated with an organic coating such as poly-L-lysine. Next, the amount of fluorescence and the amount of chemiluminescence are measured according to the method performed by Zheng et al. (D.A.E.Zheng et al .: 1996, supra) using an RI-labeled nucleic acid probe. That is, the temperature is increased by several degrees C., and the cleaning solution is changed every time the temperature is increased, and the cleaning process is performed. The non-RI-labeled nucleic acid probe is released in the cleaning solution every time the cleaning process is performed. The amount of fluorescence or chemiluminescence is measured, and the measured value is plotted against the washing temperature to obtain a release curve. Finally, in the obtained S-shaped free curve, the temperature near the inflection point seen on the low temperature region side is set as the optimum cleaning temperature of the probe.
[0043]
In order to “immobilize nucleic acid on the substrate for hybridization”, first, for example, a positively charged nylon membrane filter or a slide glass treated with an organic coating such as poly-L-lysine is used for hybridization. A predetermined amount of a nucleic acid sample is dropped on the substrate to adhere the nucleic acid. Next, when the filter is used as a base, the nucleic acid is placed on the filter by performing UV irradiation using a UV irradiation device such as a UV crosslinker (Stratagene) or by performing alkaline solution treatment. By strongly adsorbing or binding, the nucleic acid can be immobilized on a hybridization substrate. When a slide glass or the like is used as a substrate, a film is formed on the glass surface with a substance that mediates adsorption of nucleic acid between the substrate and the nucleic acid (for example, poly-L-lysine), and then a nucleic acid sample is placed thereon. The nucleic acid can be immobilized on the substrate for hybridization by dripping and adsorbing to the substrate.
[0044]
In order to “perform hybridization treatment and washing treatment by the Non-RI method”, the following steps are performed. First, a nucleic acid sample immobilized on a substrate by the above-described method is first used in a hybridization solution (for example, Raskin L, SJ, Rittmann BE, Stahl DA: Group-Specific 16S rRNA Hybridization Probes to Describe Natural Communities of Methanogens. Applied and Environmental Microbiology, 60: 1232-1240, 1994) and an arbitrary temperature, for example, 35 ° C. for 30 minutes (prehybridization process). Next, the hybridization solution to which the Non-RI labeled nucleic acid probe is added is contacted overnight at an arbitrary temperature, for example, 35 ° C. (hybridization process). In these steps, the latter treatment is essential. Finally, the immobilized nucleic acid sample after hybridization is washed with the washing solution (for example, the solution described in DAE Zheng et al .: 1996 [supra]) and the optimum washing temperature obtained by the above method for the probe used. In contact with each other for 30 minutes (cleaning process).
[0045]
“Visualizing and analyzing a fluorescent or chemiluminescent signal signal derived from a hybridized Non-RI-labeled nucleic acid probe and determining its quantitative value” is performed according to the following procedure. After performing the above hybridization and washing treatment, the fluorescent or chemiluminescent signal derived from the DNA probe hybridized to the nucleic acid sample immobilized on the substrate by complementary binding of the nucleic acid is derived from the labeled probe. Use equipment and conditions according to the type of signal, that is, in the case of fluorescent signals, fluorescent image analyzers such as FluoroImager, Storm, Typhoon (above Amersham Pharmacia Biotech), FX Pro (BioRad) and cooled CCD In the case of a chemiluminescence signal such as a camera, the signal intensity is obtained as a quantitative value by visualizing it as an image using an apparatus such as a photomultiplier, a cooled CCD camera, or a photon counting camera and analyzing it.
[0046]
“Whole organism” means the sum of all three domains of the biological kingdom, such as Archea, Bacteria, and Eucarya. “Calculate the degree” means that the sum of the quantitative values measured for all three domains or the quantitative value measured for the domain to which the target specific functional microorganism belongs is used as the denominator and the target is measured for the specific functional microorganism. The determined quantitative value is calculated as a molecule. However, in practice, it is necessary to make some corrections as follows.
[0047]
For example, when 16S rRNA is targeted, even if the target nucleotide sequence regions of two types of probes are present in a ratio of 1: 1 in one rRNA molecule, their quantitative values are affected by the three-dimensional structure of rRNA. Not necessarily the same. In such a case, it is necessary to correct so that a quantitative value proportional to the abundance ratio of the target base sequence region can be obtained.
[0048]
Moreover, unless the extraction / purification efficiency is accurately estimated, absolute quantification of the target base sequence region specific to the specific functional microorganism is impossible. However, as described above, the accurate estimation of the extraction / purification efficiency is currently not possible. Impossible. Therefore, the base sequence region (universal region) common to all three domains is used as a comparative control, and the abundance of the target base sequence region needs to be relatively evaluated with the ratio of the target base sequence region to that.
[0049]
In the case of rRNA, the abundance per cell is considered to vary depending on the cell type in addition to the cell activity. Furthermore, the ratio of the regions specific to the various domains relative to the universal region is also considered to be different depending on the type between the same domains due to the three-dimensional structure of rRNA. Therefore, it is necessary to measure variations in quantitative values based on these types of differences in advance using some typical standard strains, obtain an average value, and perform calculation correction based on the average value.
[0050]
Finally, it is necessary to subtract the quantitative value of the background caused by the hybridization substrate itself or the non-specifically adsorbed nucleic acid probe from the measured value.
When correcting for the dominance of these specific function microorganisms, the following formula 1 (SJ Giovannoni et al .: 1990, supra) already proposed in the RI method can be used. it can.
[0051]
[Expression 1]

[0052]
In this calculation, DNA probes such as Euba338, Arch 915, and Euka 502 described in Amann et al. (RI Amann et al .: 1995, supra) were used for detection and quantification of bacterial, archaic, and eucalyptus domains. Can be used. Further, as a DNA probe targeting the common area of all organisms, Univ 1390 (D.A.E.Zheng et al .: 1996, supra) can be used. For detection and quantification of a specific functional microorganism, a specific nucleotide sequence region specific to that species is selected from gene nucleotide sequence regions unique to the microorganism, such as 16S rDNA, 18S rDNA, 23S rDNA, and gyrB. Select and synthesize a probe containing the sequence. Non-RI labeled nucleic acid probes can be prepared by the method described above.
[0053]
By analyzing the dominance degree of a specific functional microorganism that dominates in the natural environment or an artificial environment using the above method, the microbial community function of the environment can be evaluated. Here, the “microbe community function” refers to a function as a whole operated by a plurality of types of microorganisms that live in the target environment. The type of microorganism referred to here is preferably a species that is a normal classification unit, but may be an experimental functional unit, for example, one functional unit such as a group of petroleum-degrading bacteria or a group of PCB-degrading bacteria. Therefore, the function of only one specific function microorganism is not called a microbial community function.
[0054]
For example, in the process of petroleum decomposition, in addition to microorganisms that decompose aliphatic hydrocarbons and microorganisms that decompose aromatic hydrocarbons, many microorganisms are involved in the process of decomposing these decomposition intermediate metabolites to carbon dioxide. Microorganisms that supply micronutrients such as vitamins to these microorganisms are considered to coexist, and multiple types of microorganisms are involved in the mineralization and detoxification of petroleum in cooperation. Can think. In this case, if a degradation product can become a growth inhibitory substance, it is considered that the degradation efficiency increases by coexisting with the available microorganism, and a high degradation function is expressed as a whole microbial community. Will be.
[0055]
Specifically, one or more types of specific function microorganisms within the microbial community, such as aerobic hydrocarbon-degrading bacteria, PCB-degrading bacteria, proteolytic bacteria, glucose-utilizing bacteria, and the like, and anaerobic By investigating the dominance of harmful microorganisms such as sulfate-reducing bacteria and methanogenic bacteria, pathogenic bacteria, and other useful substances that produce various enzymes or antibiotics, and examining their temporal changes, each specific function Microbial transitions can be analyzed. As a result, for example, in coastal surface water, if the predominance of sulfate-reducing bacteria increases at a certain time, the microbial community at that time can be evaluated as potentially having a high sulfate-reducing function, and the cause is It can be presumed that they have migrated from the normal habitat due to the rolling up of the bottom mud due to typhoons and crustal activities and the inflow of anaerobic drainage before collection.
[0056]
In addition, the contaminated environment can be analyzed and evaluated using the above method. For example, if the target specific microorganism is a heterotrophic bacterium that predominates specifically in the effluent of a pulp mill, if its dominance increases at some point, the environment If it can be judged that there is a high possibility of being contaminated and the change in the degree of dominance is monitored over a long period of time to grasp the periodicity and seasonality of the transition, the change is sudden. It can be evaluated whether there is any and whether the load is artificial. In addition, it is possible to estimate the collection location, time and season of environmental samples containing useful substance-producing microorganisms, and the occurrence location, time and season of harmful microorganisms.
[0057]
Furthermore, using the above method, it is possible to analyze and evaluate an environmental sample that is considered to contain a hazardous chemical substance-contaminated environment and specific microorganisms. For example, if the target specific functional microorganism is a PCB-degrading bacterium, if the degree of dominance rises at a certain time, it can be determined that the environment is likely to be contaminated with PCB, and the microbial community is As a whole, it can be determined that the PCB decomposition function is increasing. Furthermore, if the change in the degree of dominance is monitored over a long period of time and the periodicity and seasonality of the transition are grasped, whether the change is sudden or not, the load is artificial, such as the inflow of factory effluent. It can be estimated.
[0058]
In addition, the oil spill environment can be analyzed and evaluated using the above method. For example, if the target specific functional microorganism is derived from petroleum components, for example, tetradecane or anthracene-degrading bacteria, if the degree of dominance rises at a certain time, the environment is the former case Among the petroleum components, saturated hydrocarbons such as n-alkanes, and in the latter case, aromatic hydrocarbons can be judged as highly likely to be contaminated, and the microbial community as a whole has a function of decomposing the petroleum components. It can be determined that it is increasing. Further, by examining the transition of the degree of dominance of specific function microorganisms, it is possible to analyze and evaluate the degree of petroleum pollution or other pollution and the bioremediation process. Furthermore, if the change in the degree of dominance is monitored over a long period of time and the periodicity and seasonality of the transition are grasped, whether the change is sudden or not, the load may be caused by a ship accident, etc. It can be estimated whether it is caused by artifacts.
[0059]
When focusing on aliphatic hydrocarbon-degrading bacteria that are known to be distributed in many marine areas as specific function microorganisms, the aliphatic hydrocarbon-degrading bacteria GR211-P1 strain isolated purely from Shikoku coastal area through enrichment culture (Patent deposit number FERM BP-7788: transferred to the international deposit on November 5, 2001) part of the base sequence of 16S rDNA (SEQ ID NO: 5), The nucleotide sequence region of SEQ ID NO: 1 that specifically hybridizes with a nucleic acid derived from Alcanivorax borkumensis and its related species, and can detect or quantify it (region of nucleotide numbers 207 to 226 in SEQ ID NO: 5. 16S of Escherichia coli In the numbering system [HF Noller and CR Woese, Science, 212: 403-411, 1981] showing the position from the 5 ′ end in the base sequence of rDNA, the region corresponding to base numbers 222 to 241) and the region Complete base sequence Alternatively, a DNA probe having a base length of 10 to 50 bp including a part, preferably a base length of 15 to 25, or an RNA probe having a ribonucleotide sequence corresponding thereto may be designed. As an example, the following probes can be mentioned.
(1) 5'-CGA CGC GAG CTC ATC CAT CA-3 '(Albo 222, 20 mer) (SEQ ID NO: 1)
3'-GCT GCG CTC GAG TAG GTA GT-5 '(SEQ ID NO: 7)
When these probes are synthesized and labeled with a Non-RI label, that is, with a fluorescent dye or an antigenic substance, the method described above may be used.
[0060]
When paying attention to aromatic hydrocarbon-degrading bacteria whose distribution has been confirmed in a plurality of sea areas as specific function microorganisms, DNA probes and RNA probes having the following base sequences can be used.
(1) 5'-GGAAACCCGCCCAACAGT-3 '(Cypug829-846^*, 18mer: region of base numbers 823 to 840 in SEQ ID NO: 6) (SEQ ID NO: 2)
3'-CCTTTGGGCGGGTTGTCA-5 '(SEQ ID NO: 8)
(2) 5'-TGCACCACTAAGCGGAAACC-3 '(Cypug840-859^*, 20mer: region of base numbers 834 to 853 in SEQ ID NO: 6) (SEQ ID NO: 3)
3'-ACGTGGTGATTCGCCTTTGG-5 '(SEQ ID NO: 9)
(3) 5'-TGCACCACTAAGCGGAAACCCGCCCAACAGT-3 '
(Cypug829-859^*, 31mer: region of base numbers 823 to 853 in SEQ ID NO: 6) (SEQ ID NO: 4)
(Note that^*(Number) indicates the position (numbering system) from the 5 ′ end in the 16S rDNA base sequence of Escherichia coli (Noller H. F. and C. R. Woese, 1981. Science, 212: 403-411). The base sequence of the probe in (3) is the one in which the base sequences of the probes in (1) and (2) are adjacent. In this case, care must be taken because the probe may be self-bonded.
[0061]
After the oil spill in the Sea of Japan by the Russian tanker Nakhodka on January 2, 1997, these probes were installed in the coastal waters of Mikuni-cho, Fukui Prefecture, where the bow bottomed and a large amount of oil spilled. It has a part of the 16S rDNA base sequence (SEQ ID NO: 6) derived from petroleum-degrading bacteria found from the sample collected on the 15th of May, and is an aromatic hydrocarbon-degrading bacterium of the genus Cycloclasticus, especially Cycloclasticus pugetii and It is designed to detect the related species.
[0062]
In addition to these nucleic acid probes for detecting a specific function microorganism, a sample containing a target environment microorganism using a detection nucleic acid probe for a domain to which the specific function microorganism belongs and a universal probe for all living organisms containing the probe. By applying the molecular genetic detection and quantification method (relative molecular quantification method) described above to Alcanivorax genus aliphatic hydrocarbon-degrading bacteria, especially Alcanivorax borkumensis and related species, and Cycloclasticus genus aromatics The molecular level of the degree of dominance of hydrocarbon-degrading bacteria, especially Cycloclasticus pugetii and related species, in the bacterial domain to which the entire microbial community and / or both belong. Can be measured. Furthermore, by analyzing temporal and / or spatial changes in the degree of dominance, the state of natural and man-made environments, especially oily environments contaminated with oil, can be analyzed from a microbiological viewpoint.・ It becomes possible to evaluate.
[0063]
According to the method of the present invention, an environment polluted with harmful chemical substances such as petroleum, microorganisms living in the environment, without using conventional separation and culture techniques, and RI (radioisotope) and PCR methods. Simple, rapid molecular genetic evaluation. In addition, the method of the present invention can be applied to the detection and quantification of specific function microorganisms such as microorganisms that produce useful substances such as useful enzymes and antibiotics, and screening. Furthermore, the method of the present invention can also be applied to detection / quantification and screening of specific function microorganisms such as harmful microorganisms such as pathogens. It is therefore possible and beneficial to provide the reagents and instruments necessary for the method of the present invention as a kit and to automate the method of the present invention. Furthermore, the method of the present invention is extremely useful as a diagnostic tool for evaluating the environment contaminated with harmful chemical substances and analyzing the bioenvironmental repair process (bioremediation) and evaluating the repair effect. In addition, the present invention is useful for detecting / quantifying and screening microorganisms having specific functions such as useful substance-producing microorganisms and harmful microorganisms such as pathogenic microorganisms, which are contained in natural and artificial environments. Therefore, the present invention is extremely useful in the analysis and evaluation of environmental samples containing such specific function microorganisms.
[0064]
In addition, the DNA probe disclosed in the present invention can specifically identify a specific aliphatic hydrocarbon-degrading bacterium that has been reported to appear in many sea areas in recent years, and is extremely useful for the detection and quantification thereof. It is.
[0065]
【Example】
The present invention will be specifically described by the following examples. These examples are for explaining the present invention more specifically, and do not limit the scope of the present invention.
[Example 1]
(1) Development of DNA probe for detection of Alcanivorax borkumensis
Aliphatic hydrocarbon-degrading bacterium GR211-P1 strain successfully isolated from samples collected from the coast of Shikoku through enrichment culture (patent deposit number FERM BP-7788: transferred to international deposit on November 5, 2001) From 16S rDNA nucleotide sequence information (SEQ ID NO: 5), Stahl and Amann (Development and Application of Nucleic Acid Probes. In: Nucleic Acid Techniques in Bacterial Systematics. Ed .: E. Stackebrandt and M. Goodfellow, John Wiley and Sons, Chichester, pp. 205-248, 1991), a sequence specific to this bacterial species was selected from the region considered to be free from damage due to the higher-order structure, and an oligonucleotide having the sequence was selected. The non-RI labeled DNA probe (hereinafter sometimes referred to as Albo 222) having the sequence shown in SEQ ID NO: 1 is finally synthesized by labeling the 5 ′ end with a fluorescent dye of FITC, TRITC or Cy 5 Produced.
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  The sequence of this probe is the Alcanivorax borkumensis SK2 (reference strain; M. M. Yakimov et al .: 1998, supra) on the international nucleotide sequence database.)ofAlcanivorax borkumensi confirmed to show 100% homology only with the sequences inspectionIt was shown to be effective as a DNA probe.
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In using the DNA probe shown in the present invention, Alcanivorax borkumensis DSM11573 (standard strain) is a target microorganism, Pseudomonas aeruginosa IFO12689 (standard strain) and the like are target microorganisms, and Maruyama and Sunamura (Simultaneous direct counting of total and specific microbial cells in The effectiveness was actually confirmed by the FISH method using the apparatus and method described in Seawater, using a deep-sea microbe as biomarker. Applied and Environmental Microbiology, 66: 2211-2215, 2000). Hybridization was performed at 43 ° C. in the presence of 20% formamide, and washing was performed at 44 ° C.
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(2) DNA probe for detection of Cycloclasticus pugeti
As a DNA probe for detecting Cycloclasticus pugetii as another target microorganism, Cyclopug 829-846 (SEQ ID NO: 2, hereinafter sometimes referred to as Cypug829) was used. This probe array is located in the coastal waters of Mikuni-cho, Fukui Prefecture, where the bow struck and a large amount of oil spilled after the oil spill in the Sea of Japan caused by the Russian Tankanahotoka on January 2, 1997. From the sample collected on January 15th, we obtained a growth positive front sample in a serially diluted culture count (MPN) sample of petroleum degrading bacteria using C heavy oil as the only carbon source, and extracted DNA directly from it. It was found as a sequence specific to Cycloclasticus pugetii from Cycloclasticus pugetii 16S rDNA nucleotide sequence information revealed by sequencing.
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[Example 2]
The results of dominance analysis of Cycloclasticus pugetii and Alcanivorax borkumensis using these DNA probes in actual oil-contaminated sea areas are shown below.
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1) Sample collection and storage
In the analysis, in the coastal waters of Mikuni-cho, Fukui Prefecture, where a large amount of spilled oil drifted after the oil spill in the Sea of Japan by the Russian tankana hotoka that occurred on January 2, 1997, A sample collected on January 15, 1997 and stored frozen was used. That is, about 3 liters of oily seawater collected locally was immediately filtered and concentrated on the filter using a hollow fiber membrane filter HF400 (manufactured by Millipore) having a pore diameter of 0.2 micrometers, and the filter was stored frozen.
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2) Cell disruption and RNA extraction and storage
The obtained environmental microorganism concentrated sample was transferred to a 12 ml hard glass tube together with the hollow fiber membrane filter, and RLT buffer contained in RNeasy Midi Kit manufactured by Qiagen was added. After adding glass beads that have been sterilized by dry heat at 180 ° C for 1 hour or more (GLASS BEADS 106 microns and finer manufactured by Sigma) in an amount of 1.5 g with respect to 1.5 ml of the RLT buffer used, cell disruption The cells were disrupted by reciprocal shaking for 5 minutes in a vessel (B. Braun MSK Cell Homogenizer). This treatment was performed while monitoring with an electronic thermometer and spraying liquid carbon dioxide to maintain the temperature of the sample at about 5 ° C. In addition, the treatment time was determined by using Bacillus subtilis IFO13719 cells belonging to Gram-positive bacteria that have a stronger cell wall than Gram-negative bacteria and inhabit a lot of land. The nucleic acid thus analyzed was analyzed by agarose gel electrophoresis, and the time during which the amount of nucleic acid eluted was high and excessive nucleic acid fragmentation was not selected was set as a processing condition. RNA was extracted and purified from the obtained cell disruption sample using RNeasy Midi Kit manufactured by Qiagen, and each 50-100 μl was aliquoted and stored at −80 ° C.
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3) Measurement of total RNA amount
Prior to hybridization analysis, the total RNA amount in the sample was measured using a RiboGreen RNA Quantitation Kit (Molecular Probes) with a fluorescence spectrophotometer RF-5300PC (Shimadzu) with an excitation wavelength of 480 nm and a fluorescence wavelength of 520. The measurement was performed under the setting conditions of nm and a bandwidth of 5 nm.
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4) Hybridization by Non-RI method
Hybridization by the Non-RI method using a fluorescent probe was performed as follows. First, a sample that has been cryopreserved is in a buffer solution (a solution obtained by mixing dimethyl pyrocarbonate (DMPC) -treated water, 20xSSC [0.15 M NaCl and 0.015 M sodium citrate], formaldehyde in a ratio of 5: 3: 2). The nucleic acid in the sample was adsorbed onto a positively charged nylon membrane filter (Roche Diagnostics) and immobilized on the filter using a UV Stratalinker (Stratagene). Next, using a hybridization solution (Raskin L, SJ, Rittmann BE, Stahl DA: Group-Specific 16S rRNA Hybridization Probes to Describe Natural Communities of Applied and Environmental Microbiology, 60: 1232-1240, 1994), 35 ° C. For 30 minutes. Thereafter, overnight hybridization was performed at 35 ° C. using the above hybridization solution to which the Cy 5 labeled DNA probe had been added to a final concentration of 10 pmol / ml, and then the filter was washed with a washing solution (1% Sodium In 1xSSC [0.15 M NaCl and 0.015 M sodium citrate] to which Dodecyl Sulfate was added, the sample was subjected to a washing treatment at the optimum washing temperature of each probe determined by the following method for 30 minutes for a total of 2 times.
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5) Probe release curve measurement by Non-RI method and determination of optimum washing temperature
The optimum washing temperature was determined by the Non-RI method with reference to Zheng et al. (D.A.E. Zheng et al .: 1996, supra). Specifically, nucleic acid extraction samples of the respective standard strains (Cycloclasticus pugetii ATCC51542 and Alcanivorax borkumensis DSM11573) were immobilized on the membrane filter by dodd blotting (adding about 2 μg of RNA per dot), and each by the method described above. And hybridized overnight at 35 ° C. Thereafter, the membrane filter was cut into 8 dot portions, and the fragments were transferred to a 5 ml plastic tube containing 2 ml of washing solution. Next, the cleaning temperature was increased from 35 ° C. to 80 ° C. every 3 ° C., and each time the temperature was increased, the cleaning solution was replaced with a new cleaning solution. The amount of the fluorescence signal of the probe released into each of the obtained washing solutions was measured with a fluorescence spectrophotometer RF-5300PC (manufactured by Shimadzu Corporation) (measurement conditions were excitation wavelength 640 nm, fluorescence wavelength 662) nm and bandwidth 5 nm). Finally, the release curve of each probe with respect to the washing temperature was determined from the amount of fluorescent signal in the washing solution at each temperature. The optimal washing temperature determined as described above using the release curve was determined to be 47 ° C. for the detection probe for Alcanivorax borkumensis and 41 ° C. for the detection probe for Cycloclasticus pugetii.
The release curve obtained for the Alcanivorax borkumensis detection probe (Albo222) is shown in FIG. 1, and the release curve obtained for the Cycloclasticus pugetii detection probe (Cypug829) is shown in FIG.
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6) Visualization and quantitative analysis of fluorescent signal
Samples that had been hybridized with the Cy5-labeled DNA probe and washed were analyzed with a fluorescent image analyzer STORM (manufactured by Amersham Pharmacia Biotech) for the DNA probe hybridized to the nucleic acid immobilized on the filter. The fluorescence signal was excited by a red laser diode (output 5 mW, wavelength 635 nm), detected using a corresponding fluorescence filter (650 nm long pass filter), and the fluorescence signal was visualized as a dot image image. Furthermore, using IPLab (manufactured by Scanalytics), the fluorescence image was analyzed to obtain a quantitative value. That is, using a calibration curve obtained from the image of the serially diluted E. coli 16S rRNA standard sample subtracted from the background signal from the image of the sample signal to be measured and blotted on the same membrane. Thus, the true quantitative value of the fluorescent signal derived from the sample was determined.
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7) Dominance analysis of specific microorganisms
The degree of dominance of target microorganisms for all organisms and bacterial domains was calculated by the above formula 1 proposed by the RI method.
(Measurement value correction)
Here, the correction values used for calculating the dominance of various domains were determined using the following standard strains.
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Bacteria domain
Escherichia coli DSM30083
Psychrobacter pacificensis IFO16279
Cycloclasticus pugetii ATCC51542
Alcanivorax borkumensis DSM11573
Arcobacter nitrofigilis DSM7299
Methylobacterium extorquens DSM1053
Methylophilus methylotrophus DSM46235
Archaea domain
Aeropyrum pernix JCM9820
Methanococcoides methylutens DSM2657
Eucarya domain
Saccharomyces cerevisiae IFO10217
For extraction of nucleic acids from the standard strains used for these corrections, RNeasy Maxi kit (manufactured by Qiagen) was used and each 200 μl was aliquoted and stored at −80 ° C.
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Next, total RNA quantification, hybridization, washing treatment, acquisition of dot image images, quantitative analysis of images, and the like were performed by the above-described methods.
Probes that perform hybridization at the domain level include Euba338 (bacterial domain), Euka 502 (eukaria domain), and Arch 915 (archia domain) described in Amman et al. (RI Amann et al .: 1995, supra). ) Was used. Moreover, Univ 1390 (D.A.E.Zheng et al .: 1996, supra) was used as a DNA probe for the common region of all organisms.
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For the specific function microorganisms Cycloclasticus pugetii and Alcanivorax borkumensis targeted in the bacterial domain, the above-mentioned species-specific DNA probes Cypug 829 and Albo 222 were used.
The measurement results are shown in Table 1. In Table 1, sample 1 is a sample collected in the Yasushima fishing port (Stn.A) in Mikuni-cho, and sample 2 is a sample collected in the rocky area (Stn.B) near the bottom of the bow of the north coast of the town. .
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[Table 1]

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As shown in Table 1, in the sample 1, the bacterial domain is about 45.2% (61.4% when the whole is 100%), the Eucalyptus domain is about 19.9%, and the Archa domain is about 8%. 0.5% (total 73.6%) was found. In Sample 2, the bacterial domain is about 53.3% (59% when the whole is 100%), the Eucalyptus domain is about 24.0%, and the Archa domain is about 13.3% (the total is 90%). .6%) was found.
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In Table 1, the numbers in parentheses for bacterial domains indicate the ratio of Cypug829 and Albo222 to Euba338, which represents the dominance of the entire domain. That is, in the bacterial domain of sample 1, 23.6% of the aromatic hydrocarbon-degrading bacterium Cycloclasticus pugetii which is a target microorganism belonging to this domain, and 4.4% of the aliphatic hydrocarbon-degrading bacterium Alcanivorax borkumensis which is another target microorganism. Indicates% dominance. Similarly, in the bacterial domain of sample 2, the target microorganism belonging to this domain, the aromatic hydrocarbon-degrading bacterium Cycloclasticus pugetii, is 24.5%, and the other target microorganism, the aliphatic hydrocarbon-degrading bacterium Alcanivorax borkumensis, is 6. 5% dominance.
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As described above, the nucleic acid extraction sample of microorganisms in the contaminated environment as described in this method is targeted, and the prevalence of specific function microorganisms is quantified by the Non-RI method, so that the actual contaminated environment can be analyzed by molecular genetics. It was revealed that analysis, evaluation, and diagnosis are possible.
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【The invention's effect】
According to the present invention, the degree of dominance of specific functional microorganisms (for example, petroleum-degrading bacteria, useful substance-producing microorganisms, pathogenic microorganisms, sulfate-reducing bacteria, etc.) that appear in natural or artificial environments (for example, petroleum-contaminated environments) Thus, there has been provided a method for quantitatively analyzing molecular genetics by Non-RI and (method not using RI) without using a conventional culture counting method which requires a lot of labor and time. Such dominance analysis cannot be performed by PCR at present, and dominance analysis by Non-RI method according to the present invention is extremely effective for analysis and evaluation of microbial communities contained in the environment. It is a technique.
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The nucleotide sequence of the nucleic acid probe specific to the newly developed petroleum-degrading bacterium Alcanivorax borkumensis by the method of the present invention is derived from natural petroleum-degrading microorganisms that have the ability to dominate widely in coastal oily environments. It is. Therefore, the base sequence of this probe is under the conditions of petroleum treatment, such as the bioremediation process of the natural environment and oily environment of this oil-degrading microorganism, as well as in the field of experimental verification of bioenvironmental remediation technology and laboratory experimental conditions. It is extremely useful as a diagnostic tool for elucidating the behavior, and as a diagnostic tool for diagnosing oil spill environments and for evaluating the repair effect of bio-mediation of oil spill environments.
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In addition, according to the present invention, analysis of the dominance of typical aliphatic hydrocarbon-degrading bacteria and / or typical aromatic hydrocarbon-degrading bacteria known to be widely distributed in coastal areas is a molecular genetic technique. Realized. Therefore, the present invention is useful for biologically monitoring the residual state and decomposition process of petroleum components in oil-contaminated sea areas, and disperses nutrient salts and the like, and removes these microorganisms together with nutrient salts. It is effective for the development of environmental remediation technology (bioremediation technology) that promotes the microbial degradation of petroleum on site, and is extremely convenient. Furthermore, the present invention is extremely useful for detecting, quantifying and screening specific function microorganisms such as useful substance-producing microorganisms and harmful microorganisms such as pathogenic microorganisms, as well as for analyzing and evaluating environmental samples containing such specific function microorganisms. It is beneficial.
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[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a release curve obtained for an Alcanivorax borkumensis detection probe (Albo222).
FIG. 2 shows a release curve obtained for a probe for detection of Cycloclasticus pugetii (Cypug829).

Claims (11)

Alcanivorax borkumensis and - derived nucleic acids that specifically hybridize, can detect or quantify it is Non-RI label, DNA consisting of ribonucleotide carboxymethyl nucleotide sequence corresponding nucleotide sequence or to set forth in SEQ ID NO: 1 or RNA probe. Detection and quantification of Alcanivorax borkumensi s nucleic acid in samples obtained from natural and artificial environments by Non-RI method (method not using radioisotope), and the predominance level of Alcanivorax borkumensi s In which the following steps are performed:
1) A process for extracting and purifying a target nucleic acid from a microorganism-containing sample collected from a natural environment or an artificial environment,
2 ) A step of immobilizing the target nucleic acid extracted and purified in 1) on a hybridization substrate, bringing it into contact with the probe according to claim 1, performing a hybridization treatment, and then performing a washing treatment ,
3 ) a step of visualizing and analyzing a signal derived from the hybridized probe according to claim 1 and obtaining a quantitative value thereof;
4 ) A step of calculating the degree of dominance of Alcanivorax borkumensi s by using the obtained quantitative value and comparing it with the quantitative value measured using the probe according to claim 1 for each domain of the whole organism and organism kingdom. Including said method. The cleaning process of 2), and performs at optimum washing temperature obtained from the free curve of the probe created using the specific Non-RI-labeled nucleic acid probe Alcanivorax borkumensi s, according to claim 2 the method of. The method according to claim 2 or 3 , wherein the non-RI-labeled nucleic acid probe is fluorescently or chemiluminescently labeled. The method according to any one of claims 2 to 4 , wherein Alcanivorax borkumensi s is a microorganism that degrades a specific chemical substance. The method according to claim 5 , wherein the specific chemical substance is a harmful chemical substance. 6. A method according to claim 5 , characterized in that the specific chemicals are petroleum and petroleum components. A method for analyzing and evaluating Alcanivorax borkumensi s in an environment by analyzing the dominance of the Alcanivorax borkumensi s in a natural environment or an artificial environment using the method according to any one of claims 2 to 7. . Using a method according to any one of claims 2-7, a method for analyzing and evaluating the contaminated environment. A method for analyzing and evaluating a hazardous chemical substance-contaminated environment using the method according to claim 5 or 6 . A method for analyzing and evaluating an oily environment using the method according to claim 7 .

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