ミカエラ·ホール、ラリッサビスポおよび技術支援のためのグスタボ·タバレスに感謝します。

生で食べたり、軽く調理される食品は、ヒトの健康のために最も懸念されます。例としては、サラダ、野菜、果物、甲殻類や甲殻類が含まれます。 1.サンプルの採取<ol><li>汚染を最小限に抑える条件下でサンプルを採取し、輸送中に独立した、クリーンバッグに格納されています。採取した後、サンプルを4℃で保存し、24時間以内に処理されるべきです。 </li></ol> 2.エンリッチド文化の準備<ol><li>果物と野菜： <ol><li>丈夫なビニール袋に材料の約10グラムを配置します。大きな材料を加工する場合は、果物や野菜の皮に集中しています。 90ミリリットルの0.1Mリン酸ナトリウム緩衝液、pH7.0の処理は30秒間パドルブレンダーに加えます。パドルブレンダーが使用できない場合は別の方法として、材料を手動で撹拌することができます。 </li><li> 2 50ミリリットルファルコンチューブ内に液体を収集します。ペレット細菌に7分間4,000×gで遠心分離管。 </lI&gt; <li>上清を慎重に除去し、無菌ルリアベルターニ（LB）ブロス30ml中のペレット化した細菌を中断。 </li><li> 37℃で25℃で1チューブを保持し、他の、シェーカーO / Nでインキュベートします。培養物は、毎分200振動（OPM）で振盪されるべきです。 4℃で保存培養必要になるまで。 </li></ol></li><li>シーフード： <ol><li>滅菌ピンセットとピンセットを用いて、胃（カキ）または消化管（エビ）を解剖し、0.1Mリン酸ナトリウム緩衝液、pH 7.0の200μLを含む滅菌1.5mlチューブに入れます。ホモジネートを作成するためのサンプルを柔らかく。 </li><li> 2滅菌5ミリリットルチューブにLBブロス5mlのを分配し、シーフードホモジネートとそれぞれに接種。 </li><li> 25℃、他の37℃で1チューブを保持する、シェーカーO / Nでインキュベートします。培養物を200 OPMで振とうすべきです。 4℃で保存培養必要になるまで。 </li></ol></li></ol>インテグロン3.スクリーニング文化2濃度で提供される緩衝液を用いて、この方法で使用されています。必要に応じて、ローレンツ（2011）24は、この雑誌の以前の問題で、PCRの最適化とトラブルシューティングの方法を概説しました。 <ol><li>ゆでたDNAの調製： <ol><li>滅菌0.5ミリリットルのPCRチューブに濃縮された培養物100μlを分注します。水浴またはヒートブロックを用いて、10分間99℃にサンプルを加熱します。 2分間氷上で寒さをスナップ。 </li><li> 5分間、14,000×gでマイクロ遠心は、細胞破片をペレット化します。氷に戻ります。 DNAサンプルは、必要になるまで-20℃で凍結することができますが、氷上で解凍する必要があります。 </li></ol></li><li> PCRを使用して、クラス1インテグロンのスクリーニング： 注：O / Nインキュベーションからの混合培養物をHS463a / HS464 PCRプロトコル（ 表1）を使用してクラス1インテグロンについてスクリーニングされます。 <ol><li>任意の潜在的SOUから離れて、氷の上のすべてのPCR試薬を解凍DNAの混入のRCE。陰性対照（ 表2）を含む、サンプルあたり48μlのPCRマスターミックスを構成します。バリアのヒントを使用して、各チューブにPCRマスターミックスの48μLを分注します。 </li><li>バリアのヒントを使用して、個々のPCRチューブに解凍したDNAの2μlを添加します。 PCRサーモに適切なプログラムを実行します。 </li><li>増幅の結果を評価するために、注入し、TBE緩衝液（90 mMトリス - ホウ酸、2mMのEDTA）で実行する2％アガロースゲル上でPCR産物の7μLを電気泳動。 100bpラダーなどの分子量マーカーを含みます。 </li><li>ポストは、DNA染色でゲルを染色し、UVトランスイルミネーターを用いて可視化します。陰性コントロールには増幅がなされていないことを確認してください。任意の特定のサンプルのための周りの470塩基対での強いバンドが混合培養がクラス1インテグロンを運ぶ細菌を含んでいることを示します。純粋なコロニーは今PCR陽性を返された混合培養物から単離されます。 </li></ol></li></ol><ol><li>連続希釈と拡散板： 注：PCR陽性の混合培養物からの単一コロニーを単離するために、混合培養物を連続リン酸ナトリウム緩衝液中で10倍に希釈し、その後、単一コロニーを回収するためにメッキされます。 <ol><li> 0.1Mリン酸ナトリウム緩衝液、pH 7.0の9ミリリットルに混合培養の1ミリリットルを加え、反転により混合します。リン酸緩衝液をさらに9ミリリットルに希釈の1ミリリットルを追加し、10 -8の連続希釈に達するまで繰り返します。 </li><li>二重に、LB寒天上に10 -4〜10 -8希釈液100μLを広げます。最初の混合培養に用いたのと同じ温度でプレートO / Nをインキュベートします。次いで、プレートを、しかし、それは非常に迅速に、単一のコロニーを処理するのが最善である、必要になるまで4℃で保存することができます。 </li></ol></li><li>単一コロニーの選択と煮DNA調製： <ol><li>連続希釈プレートから単一コロニーを選び、SEこのようなコロニーの大きさ、形状、色などの基準を使用して、できるだけ多くの異なるタイプのコロニーをlecting。スプレッドプレートから単一コロニーを選択するために、滅菌爪楊枝を使用してください。 </li><li>コロニーにつまようじをタッチし、次に100μlの滅菌水を含むPCRチューブに移します。多数のコロニーをスクリーニングする場合は、その後コロニーをマイクロタイタートレイ中で調製する​​ことができます。水への細胞の一部を除去するために指の間つまようじをスピン。 </li><li>同じつまようじを使用して、コロニーのストリークを含むLBプレートに接種。各縞は約1cmの長さである場合、多数のコロニーは、標識されたプレート上に記憶することができます。あなたが分離株の適切な数を選択するまで繰り返し、1〜3を繰り返します。 </li><li>最初の富化培養に用いたものと同じ温度で、LBプレートO / Nインキュベートします。必要になるまで、培養物を、次いで、4℃で保存することができます。 </li><li>細菌懸濁液からDNAを調製するために、OUなどの手順に従ってください3.1節でtlined。純粋培養溶解物のスクリーニングのために、3.2節で概説されるようにHS463a / HS464 PCRを行います。 PCR陽性を返すの分離株は、ゲノムDNA（第5節）の製造のための、およびさらなる分析のために使用されます。 </li></ol></li></ol> 25を打つビーズを使用したインテグロン陽性単一コロニーの5のゲノムDNA抽出<ol><li> 4.2節で単離された純粋培養のサンプルとLBブロス5mlの接種。最初の富化培養に使用したのと同じ温度で振とう、O / Nをインキュベートします。 </li><li>ペレット細胞は7分間、4,000×gで、その後、上清を除去します。ライジング行列Eの高速取管で1ミリリットルCLS-TC溶液中でペレット化した細胞を再懸濁します。ビーズ叩解機を用いて、30秒間5.5メートル/秒でサンプルを処理します。 </li><li> 5分間、14,000×gで遠心分離管や滅菌1.5mlチューブに上清700μlのを回復します。 </li><li> M関6と5：1に希釈した結合マトリックスの700μLを追加5分間室温でidiniumチオシアンミックス。 1分間14,000×gで遠心し、上清を捨てます。 </li><li>ペレットを完全に再懸濁されるまで、800μlの塩エタノール洗浄（70％エタノール、0.1 M酢酸ナトリウム）、ボルテックスを追加。 1分間14,000×gで室温で5分間、遠心分離のために洗浄し、上清を捨てます。すべての上清を必要に応じて、マイクロピペットを使用して、削除されたことを確認し、その後5分間空気乾燥させ。 </li><li>ピペッティングにより200μlのTE（10mMのトリス-HCl、1mMのEDTA、pHは7.6）でペレットマトリックスを再懸濁します。 3分間室温でインキュベートします。 3分、転送滅菌チューブに溶出したDNAの160μlのための14,000×gで遠心します。 DNA抽出の収量および完全性をチェックするために2％W / Vアガロースゲル上のゲノムDNA抽出の一部を実行します。精製ゲノムDNAは-20℃で保存し、PCR試験のために必要な場合に、氷上で解凍されます。 </li></ol> 6.診断のPCRおよびDNAシークエンシング注意：セクション5で調製したゲノムDNAは、すべての診断のPCRのために使用され、クラス1インテグロンのための陽性を確認します。 <ol><li>簡単に20秒間14,000×gでDNAサンプルとパルススピンをボルテックス、氷の上でゲノムDNAを解凍します。分離株は、471塩基対のアンプリコン（3.2節）について陽性であることを確認し、HS463a / HS464を使用して、クラス1インテグロンインテグラーゼ遺伝子（ 表1）のためのPCRを繰り返します。 </li><li>種レベルの陽性培養の同定小サブユニットrRNA遺伝子（16S rDNAの）を分析することによって達成されます。プライマーF27 / r1492（ 表1）を用いて16S遺伝子を増幅します。電気泳動を用いてPCR産物を確認してください。すべての細菌のターゲットは、約1450塩基対の16Sアンプリコンを生成する必要があります。 </li><li>種同一性を決定するためのシーケンス16S rRNA遺伝子アンプリコンが、そこステップ6.2からの複数のPCRポジティブである可能性があり、これらの陽性の多くは、同じ細菌種になるので、我々は最初usin異なる種を区別します16S PCR産物gの制限消化。 <ol><li>制限酵素関数hinf 1と16S PCRの一部を消化。任意の4塩基対切断部位致します認識する酵素が、 関数hinf 1は、16Sのターゲットからの良好な多様性を生成し、信頼性の高い、安価な酵素です。 </li></ol></li><li> 30μlの制限は、滅菌チューブにマスターミックス（ 表2）を消化し ​​、未purfied 16S PCR産物の20μlを添加設定します。 37℃の水浴O / Nでインキュベートします。 2％アガロースゲル上でダイジェストを確認してください。 </li><li> 関数hinf 1によって生成される消化パターンをスコア。同じ16S制限プロフィールと分離株は、同じ種である可能性が高いです。むしろ、すべての16S PCR産物は、各関数hinf 1制限パターンの最も3代表での配列を配列決定するよりも。 </li><li>市販のキットを用いて16S PCR産物を精製します。一本鎖エキソヌクレアーゼキットを使用することができるが、列ベースのまたは沈殿法もuninc除去するためにうまく機能しますプライマーおよび一本鎖DNAをorporated。シーケンスプライマーR910（ 表1）を用いてシークエンシング機能によって指定されたような反応をセットアップ16Sアンプリコン。 </li><li> BLASTN機能（NCBIでのDNAデータベースを調べるために、得られたDNA配列を使用し<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> ）。 97％以上のヌクレオチド同一性は、通常、種のアイデンティティとします。 </li></ol> 7.マッピングとインテグロンとカセット配列の特徴注：人間が支配生態系から発するクラス1インテグロンは、すべてのサンプルの中で最も一般的なインテグロンである可能性が高いです。これらのインテグロンすべては、最近の単一起源を持っているため、高度に保存されたDNA配列5を持っています。 <ol><li>クラスの間に1人のソースから発するインテグロンと行うことにより、環境試料中に天然に存在するものを区別プライマーを用いたPCRはintI1F165 / intI1R476 10（ 表1）を設定します。 注：環境クラス1インテグロンが任意の製品を生成しませんしながら、臨床的または共生細菌からのクラス1インテグロンは、〜300 bpの産物を生成します。 </li><li>プライマーセットHS458 / HS459とカセットの配列を増幅することにより、臨床インテグロンを特徴づけます。このPCRは、ほとんどのカセットの配列の末端にintI1および3 &#39;保存されたセグメント間の領域を増幅します。カセットの数および同一性は可変であるので、PCR産物の大きさも変化するであろう。 </li><li>アンプリコンを精製し、増幅プライマーのそれぞれを用いて、DNAを配列決定します。 BLASTN機能（使用して、DNAデータベースを調べるために配列を使用<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgiを</a> ）。 </li><li>カセットの命名はインテグロン遺伝子カセットのための多くのデータ·堆積するので、標準化された命名法9に従うことを確認Sは矛盾やあいまいな名前を使用します。これらは伝達、病原性や毒性8,21を増加与える遺伝子を含む、新たな表現型をコードすることができるので、新規遺伝子カセットに特に注意を与えます。 注：クラス1インテグロンの前臨床形態は、まだ自然環境に循環します。特に、アクティブテネシー402トランスポゾンにリンクされているものは、ほとんど関心26のものです。これらのインテグロンのカセット配列はプライマーセットHS458 / HS459で増幅されませんが、カセットコンテンツに依存して可変サイズのアンプリコンを生成し、MRG284 / MRG285プライマーセット（ 表1）を用いて増幅することができます。 7.2に概説するようにPCR産物は精製されるべきであり、DNAを配列決定しました。 </li></ol>

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Call+of+the+wild:+antibiotic+resistance+genes+in+natural+environments.">Call of the wild: antibiotic resistance genes in natural environments.</a> Nature Rev. Microbiol. 8, 251-259 (2010).</li><li>Wellington, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+natural+environment+in+the+emergence+of+antibiotic+resistance+in+Gram-negative+bacteria.">The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria.</a> Lancet Infect. Dis. 13 (2), 155-165 (2013).</li><li>Gillings, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+a+Tn402-like+class+1+integron+with+a+novel+cassette+array+via+flanking+miniature+inverted-repeat+transposable+element-like+structures.">Mobilization of a Tn402-like class 1 integron with a novel cassette array via flanking miniature inverted-repeat transposable element-like structures.</a> Appl. Env. Microbiol. 75 (18), 6002-6004 (2009).</li><li>Graham, D. W., Collignon, P., Davies, J., Larsson, D. J., Snape, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Underappreciated+role+of+regionally+poor+water+quality+on+globally+increasing+antibiotic+resistance.">Underappreciated role of regionally poor water quality on globally increasing antibiotic resistance.</a> Env. Sci. Technol. 48 (20), 11746-11747 (2014).</li><li>Perry, J. A., Westman, E. L., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistome:+what's+new.">The antibiotic resistome: what's new.</a> Curr. Opinion Microbiol. 21, 45-50 (2014).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J. Vis. Exp. 63, e3998-e3998 (2011).</li><li>Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Extraction+of+PCR-Competent+DNA+from+Recalcitrant+Environmental+Samples.">Rapid Extraction of PCR-Competent DNA from Recalcitrant Environmental Samples.</a> Env. Microbiol. 1096, 17-23 (2014).</li><li>Sajjad, A., Holley, M. P., Labbate, M., Stokes, H., Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+class+1+integron+with+a+complete+Tn402-like+transposition+module.">Preclinical class 1 integron with a complete Tn402-like transposition module.</a> Appl. Env. Microbiol. 77 (1), 335-337 (2011).</li><li>Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+in+the+environment:+A+link+to+the+clinic.">Antibiotic resistance in the environment: A link to the clinic.</a> Curr. Opinion Microbiol. 13 (5), 589-594 (2010).</li><li>Stokes, H., Nesbo, C., Holley, M., Bahl, M., Gillings, M., Boucher, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+1+integrons+predating+the+association+with+Tn402.-like+transposition+genes+are+present+in+a+sediment+microbial+community.">Class 1 integrons predating the association with Tn402.-like transposition genes are present in a sediment microbial community.</a> Journal of Bacteriology. 188, 5722-5730 (2006).</li><li>Lane, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acid Techniques in Bacterial Systematics. , 115-175 (1991).</li><li>Holmes, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+activity+of+a+distinctive+integron-gene+cassette+system+associated+with+stutzeri.+populations+in+soil.">Recombination activity of a distinctive integron-gene cassette system associated with stutzeri. populations in soil.</a> Journal of Bacteriology. 185, 918-928 (2003).</li></ol>

著者らは、開示することは、関連は何もありません。

インテグロンおよびそれらに関連する遺伝子カセットの同定は、潜在的に、新たな日和見病原体の出現を予測する人間の食物連鎖の中に病原体のための経路を追跡し、新たな耐性を特定する際に重要なステップであり、毒性は8,21,26を決定因子。本稿の目的は、そのカセットの配列を特徴付けるし、それらが存在する細菌種を識別し、クラス1インテグロンのためのサンプルをスクリー...

抗生物質の発見は20 世紀の最も偉大な科学的成果の一つでした。しかし、抗生物質の使用や乱用は、抗生物質耐性菌の急速な進化につながっている、これらは現在、21世紀の公衆衛生に深刻な脅威をもたらします。ほとんどの治療の選択肢に耐性細菌株の上昇は、我々は抗菌薬がもはや1,2に効果的である時代に入っている可能性を高めます。 抗生物質耐性を付与する遺伝子機構は年間3百万のすることによって、ヒトおよび抗生物質選択圧をより以前、古代のシステムです。このようなプラスミド、トランスポゾン、ゲノムの島々 、統合的な接合要素とインテグロンなどのモバイル遺伝的要素は、内および細菌種4との間の両方の抗生物質耐性遺伝子（ARG）を広めることができます。これらのうち、インテグロンはもかかわらず、ARGの普及において中心的な役割を果たしてきました彼らは細菌ゲノム5への動員および挿入するためのプラスミドおよびトランスポゾンに依存しているという事実。インテグロンはインテグロンインテグラーゼを用いた遺伝子カセットをキャプチャして、[エンコードされインテグロンプロモーター6,7（ 図1）を使用して、カセットを発現します。 インテグロン遺伝子カセットは、その製品の抗生物質や消毒剤8に対する抵抗性を付与することができる単一のオープンリーディングフレーム（ORF）からなる小型のモバイル要素です。クラス1インテグロンは、最も一般的に、それらをまとめて130の異なる抗生物質耐性遺伝子カセット9を介して取得した臨床分離株5から回収インテグロンです。 人間関連共生や病原性細菌へのクラス1インテグロンの広がりは、これらの遺伝子エレメント10の多くが含まれている人間の廃棄物の流れを生成します。クラス1インテグロンが含まれている推定10 19細菌が毎年下水汚泥を経てリリースされた私nはイギリス11。これは、抗生物質耐性を付与するクラス1インテグロンは今野生の鳥類、魚類、および他の野生生物の12-14の微生物叢中に検出されていることは驚くべきことではありません。新しい遺伝子カセットなどのモバイル要素と複雑な再配列の取得は、特に下水処理場や他の水域15-18で、引き続き発生するので、戻って環境にインテグロンを解放すると、重大な公衆衛生上の脅威をもたらします。自然環境は、新しい耐性決定と日和見病原体19,20のための肥沃な募集の地面になります。新規インテグロン含有細菌や新しいargsが汚染された水や食物21,22を通じて人間社会に戻って一周することができます。環境ARGSのサーベイランスは、理解と将来23に抗生物質耐性を管理するための重要な戦略です。具体的には、注意が生で食べたりしている食品に支払われるべきですこれらは、新しいモバイル要素と病原体の伝播のための最大の脅威を提示するので軽く、調理。 食品中のクラス1インテグロンおよびそれらに関連する遺伝子カセットが概説されて、検出、同定および特徴づけるためのこのプロトコルでは、効率的なアプローチ（ 図2）。培養およびポリメラーゼ連鎖反応（PCR）の組み合わせを使用して、インテグロンは急速に複雑な細菌群集及び個々の分離株で検出することができます。細菌およびインテグロン関連遺伝子カセットの立体構造と同一の種を同定するための方法が挙げられます。この方法は、植物及び動物の食品の広い範囲に適しており、典型的なワークフローの例は、これらの食品の種類ごとに与えられています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>GoTaq Colourless Mastermix</td>
			<td>Promega</td>
			<td>M7132</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>RNAse (Ribonuclease A from bovine pancreas)</td>
			<td>Sigma</td>
			<td>R6513-10MG</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>HinFI restriction enzyme</td>
			<td>Promega</td>
			<td>R6201</td>
			<td>Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA</td>
		</tr>
		<tr>
			<td>100 bp ladder</td>
			<td>GE Healthcare</td>
			<td>27400701</td>
			<td>Used as a size standard on all agarose gels</td>
		</tr>
		<tr>
			<td>GelRed DNA stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>Guanidinium Thiocyanate</td>
			<td>Life Technologies</td>
			<td>AM9422</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>CLS-TC Solution</td>
			<td>MP Biomedicals</td>
			<td>6540409</td>
			<td>Resuspension solution used at the begining of the genomic DNA extraction</td>
		</tr>
		<tr>
			<td>Lysing Matrix E FastPrep tubes</td>
			<td>MP Biomedicals</td>
			<td>116914500</td>
			<td>Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available.</td>
		</tr>
		<tr>
			<td>binding matrix</td>
			<td>MP Biomedicals</td>
			<td>116540408</td>
			<td>Diluted 1:5 with 6M guanidinium thiocyanate and used in the genomic DNA extraction method.</td>
		</tr>
		<tr>
			<td>Fast Prep machine</td>
			<td>MP Biomedicals</td>
			<td>Number of options available</td>
			<td>MP Biomedicals has a number of FastPrep machines available to purchase. Visit http://www.mpbio.com for more information</td>
		</tr>
	</tbody>
</table>

screening foodstuffs for class 1 integrons and gene cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

intI1用混合培養し、細菌分離株のスクリーニングプライマーセットHS463a / HS464 PCRは、クラス1インテグロン、インテグラーゼ遺伝子、intI1（ 図1）の存在を検出することができます。このプライマーセットは、混合培養でintI1を検出するためにうまく機能し、また、拡散板（ 図2）から回収した細菌コロニーをスクリーニングするために?...

Watch this Scientific Journal Video about Screening Foodstuffs for Class 1 Integrons and Gene Cassettes at JoVE.com

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

クラス1インテグロンおよび遺伝子カセットのための食品のスクリーニング

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

screening-foodstuffs-for-class-1-integrons-and-gene-cassettes

Research

JoVE Journal

Environment

9.0K ビュー.  Macquarie University. This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs. 

ビデオ: Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

ギブソンアセンブリと遺伝子銃を用いてタバコの一過性の遺伝子発現

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

環境学

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

レタスの潜在的な干ばつ耐性のためのセミハイスループットスクリーニング（<em&gt;レタス</em&gt;）遺伝資源のコレクション

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

リグノセルロース系バイオマス中の反抗バリエーションのハイスループットスクリーニング：総リグニン、リグニンモノマー、および酵素シュガーリリース

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

で捕食摂食行動を分析します<em&gt; Pristionchus</em&gt;およびその他の線虫

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

水中の内分泌活性のスクリーニング市販の使い方<em&gt;インビトロ</em&gt;トランスバイオアッセイ

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

海面とサンプル分析にMicroplasticsサンプリングのためのプロトコル

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

プラスチックペレットからの有機塩素系農薬の抽出と塑性解析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

大気エアロゾルからの抽出および界面活性剤の特性評価

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

海洋モデルとしてフジツボタテジマフジツボ improvisus培養と遺伝子発現

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

アグロバクテリウムとサツマイモネコブセンチュウ-ジャガイモの変換を介したと GUS 染色によってスベリン遺伝子のプロモーター活性

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...