The authors have no acknowledgements.

フィールド1.スワブサンプリング<ol><li>手袋のきれいなペアを着用してください。 </li><li>測定テープや定規を用いて表面に触れることなくサンプリング領域のサイズを測定します。できるだけ正確に面積を推定し、報告書（補足表1）を記入してみてください。 </li><li>可能な漏れやラベルサンプル輸送バッグや綿棒キット用綿棒キットを確認してください。 </li><li>水平方向に1ストローク、垂直方向に1ストローク、および斜め方向の1ストロークを次のようにサンプリングエリア全体に綿棒を移動します。 700センチメートル2よりも大きな表面積を綿棒ないでください。 </li><li>チューブに各綿棒を置き、しっかりとキャップを締めます。 </li></ol>研究室への綿棒の2貯蔵および輸送<ol><li>最大48時間、4℃で綿棒を保管してください。長期間保管が必要な場合は、-20℃でスワブを格納する（または-70°C）。 </li><li> すなわち、（0-4℃でチューブを保ちます。 、コレクションの24〜48時間以内に研究室や船への輸送の間に断熱容器に）コールドパックを使用します。 </li></ol> 3.ウイルス濃度、ウイルスRNA抽出および精製注：特に明記しない限り、すべての遠心分離工程は、室温で5分間、5000×gでテーブルトップ遠心機を使用しています。ユニバーサル核酸抽出（UNEX）緩衝液を用いて作業する場合、余分な注意が必要です。ゴーグルまたはフェイスシールドを着用してください。 <ol><li> 1 15 mLチューブと各サンプルに対して1つのRNA Midiカラムにラベルを付けます。各実験における負の抽出制御が含まれます。 </li><li> 10スワブのための溶解液を調製し、PBSTの25ミリリットル（PBS pH7.2の0.02％のTween-80を含む）でUNEXバッファの25ミリリットルを混ぜます。 </li><li>溶解液の50 mLに大腸菌ファージMS2懸濁液（10 6 PFU /μL）の50μLを追加します。 </li><li> 15 mLチューブに綿棒を置き、5ミリリットル溶解液を追加します。混合し、室温で10分間インキュベートします。 </li><li>10秒のための各チューブ、ボルテックスに5ミリリットルの100％エタノールを追加します。 </li><li>慎重に余分な液体を除去した後、綿棒を破棄するために、チューブの側面に軽く押す（これはUNEXのバッファが含まれている）15 mLチューブから綿棒を削除します。残りのボリュームは9-10 mLの間であるべきです。 </li></ol> 4. Midiカラムウイルス核酸の抽出<ol><li>慎重ミディカラム、遠心分離器の上にステップ3.6から4.5 mLのUNEX /エタノール混合物を転送し、ろ液を捨てます。 </li><li>同じ混合物から別の4.5 mLを同じ列、遠心分離機にロードし、ろ液を捨てます。 </li><li>最初の洗浄のための、ミディカラムに70％エタノールの3.5ミリリットルを追加し、遠心分離し、ろ液を捨てます。 </li><li>第二の洗浄のために、ミディカラムに70％エタノールの別の3.5ミリリットルを追加します。ろ液を遠心分離し、廃棄します。 </li><li>ドライスピンのために、（マイナスのウイルスRNAの回収率に影響を与える可能性がある）エタノールのすべての痕跡を除去するために、ミディカラムを遠心してください。 </li><li>新しい15ミリリットルの遠心分離管にMidiカラムを配置します。 Midiカラムに250μlの溶出バッファーを追加します。最高のウイルスRNAの回収率を得るために、遠心分離前に1分間待ちます。 </li><li> -70℃で抽出された核酸を保存するか、手順5に即座に進みます。 </li></ol> RNAクリーンとコンセントレータキットを使用したウイルス核酸の5濃度<ol><li>上記のステップ4.7で溶出したRNAと10秒間渦の250μLに結合バッファーRNAの500μLを追加します。 </li><li> 750μLを10秒間100％エタノールと渦を追加します。 </li><li>各サンプルのスピンカラムにラベルを付けます。スピンカラムに750μLのサンプルをロードします。 </li><li> 1分間12,000×gでスピンカラムを遠心します。フロースルーを捨てます。 </li><li> 1分間12,000×gでスピンカラムと遠心上に残りの750μLをロードします。 </li><li>予備洗浄のために、1分間12,000×gで各スピンカラムや遠心に400μLRNAプレップバッファを追加します。フロースルーを捨てます。 </li><li>最初の洗浄のために、1分間12,000×gで各スピンカラムや遠心に800μLのRNA洗浄バッファーを追加します。フロースルーを捨てます。 </li><li>第二の洗浄のために、1分間12,000×gで列や遠心分離機を回転するように400μLRNA洗浄バッファーを追加します。フロースルーを捨てます。 </li><li>ドライスピンのために、2分間12,000×gで、洗浄緩衝液のすべての痕跡を除去するために、スピンカラムを遠心してください。 </li><li>注意深く新しい1.5 mlのマイクロ遠心チューブにスピンカラムを移します。 </li><li>スピンカラムに25μlの溶出緩衝液を加え、室温で1分間インキュベートします。これは、カラムからのウイルスRNAの回収率を増加させます。 </li><li> 1分間万×gでスピンカラムを遠心分離することにより、RNAを収集します。 </li><li>どちらか-70℃でRT-qPCRを（ステップ6）、またはストアRNAに直接進みます。 </li></ol> 6. Genogroup IのMutiplex RT-qPCRの検出、およびIIノロウイルス、およびコリファージMS 2（補足表2） <ol><li>クリーンな作業面、pipettES、および汚染の可能性を低減するためのRNase汚染除去溶液で遠心分離機。 </li><li>氷の上でRT-qPCRの試薬を解凍します。 （別の領域/客室内に）氷上で解凍RNA。 </li><li>反応の数を決定し、それらの少なくとも10％以上を追加（10の反応が必要な場合など 、11のためのマスターミックスを作ります）。 </li><li> 5秒間の25倍RT-qPCRの酵素を除き、渦個々のマスターミックスコンポーネント、。慎重に指でチューブをフリックすることにより酵素を混ぜます。 </li><li> 5秒間簡単に言えば遠心マスターミックスコンポーネント。 </li><li>キットの説明書に従って、ノロウイルスGIとGIIのリアルタイムRT-PCR検出のためのマスターミックスを調製し、1.5 mlのマイクロ遠心チューブ（補足表3）でノロウイルス特異的オリゴヌクレオチドプライマーおよびプローブを追加します。 </li><li>上下に5〜10回ピペッティングすることによりマスターミックスをミックス（ボルテックスは推奨されません）。リアルタイムPCR 96ウェルプレートの各ウェル内のマスターミックスのアリコートを22μL。 </li><li>渦サンプルRNA5秒間、および簡単な（5秒）遠心分離によって収集します。 </li><li> RT-qPCRのプレート（ 表2からテンプレートに従ってください）に試料RNAとGIとGII陽性対照の3μLを追加します。無テンプレート対照（NTC）をウェルにヌクレアーゼ-Freewaterのの3μLを追加します。 </li><li>光学接着フィルム付きリアルタイムプレートをシール。 </li><li>注意深くウェル中に存在し得る気泡又は液滴を除去するために1分間、1300×gでリアルタイムプレートを遠心。 </li><li> 10分間1）RTステップ：リアルタイムPCR装置とセットアップ以下の熱サイクル条件を設定します。 Taqポリメラーゼの45℃（2）活性化、10分間。 95℃、（3）95℃で15秒の45サイクル、および60℃で60秒で。 </li></ol>スワブ試料中のノロウイルスの7.定量<ol><li> RT-qPCRの結果（ 補足表3）を検証するために、正と負の対照の結果を確認します。 </li><li>各標準曲線は満たしているかどうかを判断許容可能な値は、補足表2に与えられ、式1を使用して標準曲線の全体的な標準偏差を計算します。 （式1）％効率= 100×10（平均CT値-切片）/傾き 。 </li><li> PCRサーマルサイクラーソフトウェアは各標準曲線の傾きを計算していない場合は、（Excel など 、SPSS、またはSAS）統計ソフトウェアを使用して回帰により傾きとR 2の値を決定します。また、私は次式の標準曲線について％の効率を計算します</li><li> 補足表4で指定された基準を満たす標準曲線に基づいて、すべてのテストサンプル用のサーマルサイクラーソフトウェアによって計算されたRNAコピー数を記録します。基準を満たすか、偽陽性コントロールを持っていない標準曲線tの帽子を持つ任意のサンプルを再実行します。大腸菌ファージMS2のCt値をチェックし、PCR阻害を監視するための内部対照として加えました。 </li><li> RNAコピーpの合計数を決定RT-qPCRの反応に使用されるRNA（3μL5）の総RNA溶出液の量（25〜50μL）の比で段階7.2で計算されたRNAコピー数を乗算することにより、ERのサンプル。代替的に、分析対象物の表面積（cm 2）を、総RNAのコピー数を割ることによってRNA濃度を算出します。 </li></ol>ヘミネストされた従来のPCR増幅によるリアルタイムRT-PCR陽性サンプルの8ジェノタイピング<ol><li> RT-PCRアッセイの各プライマーセットのためにマスターミックスの最初のラウンドを準備します（ 補足表2、および5）。 </li><li>マスターミックスの20μLにノロウイルス陽性のRNAの5μLを追加します。 </li><li>以下の条件でRT-PCRを実行する：（1）RTが95℃で、Taqポリメラーゼの42℃（2）活性化、15分で30分間工程、及び（3）95℃で30秒の40サイクル、50℃で30秒、および72℃で60秒。 40サイクル後、72℃でさらに10分間インキュベートします。 </li><li>RT-PCRアッセイの各プライマーセット（ 補足表2および5）のための母校ミックスの第二ラウンドを準備します。 </li><li> 23μLマスターミックスを追加し、（理想的には、第一ラウンドの製品は、RNaseフリー水で1/10に希釈してください）を各チューブに（最初のラウンドから）2μL第一ラウンドのRT-PCRの製品を追加。 </li><li>繰り返し手順8.3。 </li><li> 100ミリリットル1×トリス - 酢酸EDTA（40 mMトリス、20mMの酢酸、およびpH 8.3で1 mMのEDTA）中の2％アガロースゲルを準備します。 60〜70℃に冷却した後、混合物を調製したゲルの100ミリリットルあたりの核酸染色の10μLを追加し、電子レンジで混合物を加熱することにより、アガロース溶解した後、ゲルを注ぎ、コームを挿入します。ゲルは、少なくとも30分間沈降してみましょう。 </li><li> 6×ローディング色素の3μLで各RT-PCR産物の15μlのを混ぜます。負荷の各サンプル15μL、1時間、100Vでアガロースゲルを電気泳動します。 </li><li>ゲルANから適切なサイズの物品税の対象RT-PCR断片（GIとGIIのための341 bpのための330 bp）の商業ゲル抽出キットを用いてRNAを精製dは。精製したPCR産物は、現在サンガー配列決定のために使用することができます。 </li></ol>

<ol>
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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+quantitative+approach+to+defining+"high-touch"+surfaces+in+hospitals.">A quantitative approach to defining "high-touch" surfaces in hospitals.</a> Infect. Control. Hosp. Epidemiol. 31 (8), 850-853 (2010).</li><li>Wu, H. 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=A+norovirus+outbreak+at+a+long-term-care+facility:+the+role+of+environmental+surface+contamination.">A norovirus outbreak at a long-term-care facility: the role of environmental surface contamination.</a> Infect. Control. Hosp. Epidemiol. 26 (10), 802-810 (2005).</li><li>Ikner, L. A., Gerba, C. P., Bright, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+recovery+of+viruses+from+water:+a+comprehensive+review.">Concentration and recovery of viruses from water: a comprehensive review.</a> Food Environ. Virol. 4 (2), 41-67 (2012).</li><li>Gallimore, C. I., 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=Environmental+monitoring+for+gastroenteric+viruses+in+a+pediatric+primary+immunodeficiency+unit.">Environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit.</a> J. Clin. Microbiol. 44 (2), 395-399 (2006).</li><li>Ganime, A. C., 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=Dissemination+of+human+adenoviruses+and+rotavirus+species+A+on+fomites+of+hospital+pediatric+units.">Dissemination of human adenoviruses and rotavirus species A on fomites of hospital pediatric units.</a> Am J Infect Control. , (2016).</li><li>Verani, M., Bigazzi, R., Carducci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+contamination+of+aerosol+and+surfaces+through+toilet+use+in+health+care+and+other+settings.">Viral contamination of aerosol and surfaces through toilet use in health care and other settings.</a> Am J Infect Control. 42 (7), 758-762 (2014).</li></ol>

Authors have no conflicting interest. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

ノロウイルスは18と10 3ウイルス粒子20との間に50％のヒト感染用量を持っています。したがって、表面のも低レベルの汚染は、公衆衛生上のリスクをもたらすことができます。 1）異なるスワブ材料、2）貯蔵条件スワブ輸送の間、3）ウイルスRNAの濃度、及び4）大腸菌ファージMS2内部抽出コントロールとして：スワブサンプリングプロトコルのいくつかの側面を含め?...

<html>人間のノロウイルスは、世界的流行と散発的な急性胃腸炎の主要な原因1、2、3です。ウイルスは非常に伝染性であり、送信は、人との対話にまたは間接的に汚染された食物、水や環境表面との接触を介して直接人を介して行われます。ノロウイルスは、長時間流すことができ、環境表面上のウイルスの生存期間の延長は1、2、3に記載されています。ピークの排出時には、ウイルス粒子の数十億は糞便1g当たり放出される、および嘔吐はまた、感染4を引き起こすウイルス粒子の十分な数が含まれている5、6、7、8、EF &quot;&gt; 9、10。また、無生物表面と人間の皮膚との間のウイルスの移動が容易に発生する可能性があります2、11、12。したがって、環境汚染のモニタリングがアウトブレイク調査を支援し、クリーンアップの有効性を評価する際にすることができ、消毒手順。 いくつかの環境サンプリングプロトコルは、ロタウイルスの検出のために記載されており、大腸菌ファージMS2、ネコカリシウイルス（FCV）、及びバクテリオファージP22 13、14、15、16。しかし、高速乾燥（&lt;1時間）と小さい表面領域（25×100 cm 2）を含む、これらの研究に記載の検証条件は、適切にフィールドの設定を表していない場合があります。また、エンバイロの低い汚染レベルを期待nmental表面は、非常に少数のウイルス粒子を検出することができるプロトコルを必要とします。 私たちは、ノロウイルスの検出とタイピングのためのマクロフォームベースの表面サンプリング法を開発しました。この方法は、いくつかのノロウイルス集団発生時に検証されています。プロトコルは、1）（2）どのように最善の研究室に収集・輸送中のサンプルの完全性を維持するために、およびノロウイルスの3）実験室での試験とタイピング環境表面からスワブサンプルを収集する方法。

<table><tbody><tr><td>&lt;strong&gt;Generic name for kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Macrofoam swab&lt;/strong&gt;</td><td>Premoistened EnviroMax Swab kit&amp;nbsp;</td><td>Puritan</td><td>2588060PFUW</td></tr><tr><td>&lt;strong&gt;RNA Lysis buffer&amp;nbsp;&lt;/strong&gt;</td><td>CDC UNEX buffer</td><td>Microbiologics</td><td>Cat No MR0501</td></tr><tr><td>&lt;strong&gt;RNA extraction spin column&lt;/strong&gt;</td><td>Midi column</td><td>Omega Biotek</td><td>Cat No R6664-02</td></tr><tr><td>&lt;strong&gt;RNA purification spin column&lt;/strong&gt;</td><td>Zymol RNA Clean and Concentrator kit&amp;nbsp;</td><td>Zymo Research</td><td>Cat No R1016</td></tr><tr><td>&lt;strong&gt;Real time RT-PCR kit&lt;/strong&gt;</td><td>AgPath kit One-Step RT-PCR Kit</td><td>Life Technologies</td><td>Cat No 4387391</td></tr><tr><td>&lt;strong&gt;Conventional RT-PCR kit&lt;/strong&gt;</td><td>Qiagen one step RT-PCR kit</td><td>Qiagen kit</td><td>Cat No 210212</td></tr><tr><td>&lt;strong&gt;Gel extraction kit&lt;/strong&gt;</td><td>Qiagen QIAquick gel extraction kit</td><td>Qiagen kit</td><td>Cat No 28704 or 28706</td></tr><tr><td></td><td>Coliphage MS2</td><td>ATCC</td><td>Cat No 15597-B1</td></tr><tr><td></td><td>RNA run-off transcripts</td><td></td><td></td></tr><tr><td></td><td>Realtime PCR platform</td><td>Applied Biosystems</td><td>Model ABI 7500</td></tr><tr><td></td><td>Optical 96-well reaction plate</td><td>Thermo Scientific</td><td>Cat No 4316813</td></tr><tr><td></td><td>&lt;a href=&quot;http://www.thermofisher.com/order/catalog/en/US/adirect/%20https:/www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catProductDetail&amp;amp;showAddButton=true&amp;amp;productID=4306311&amp;amp;_bcs_=H4sIAAAAAAAAAI1RTWvDMAz9Nb4srDg2yXpNW3rZVyHbuRhHSQx2HWwnJf9%2BUkO6scMYGEmWnqX3%0A5Mec8e0p%2BGbUKWZMlFkNYTIa4h%2F5PqWByYqJI57r9bpJPQTnWxPRb7R3mB4jGrig6b0DdD40WOyT%0As9iBCUmHb1MYge78iYi06ZznPEd0exacF6LkD5h%2BNTr4yg1II0DH5C6rmh6imSA7GuuInsIq5TUy%0AjD6Yf%2FKU1Zav3NBrlZT13cr8s0ajGhNAJ4xsYvKoXcPkAYEHEwer5jrNFuWVdF1ieajNpbPwAhPY%0AvUrQ%2BTAjAt88w4zlm8Ly%2FYRha2yiyeUSUJM35ajHt%2BZiUS0KuWNityrHkLSjuyHUDbkgfuxgbfwx%0AD9Q0vydo9rrs4te675%2FTKhvpd74AhB0C9yUCAAA%3D&amp;amp;returnURL=http%3A%2F%2Fwww.thermofisher.com%3A80%2Forder%2Fcatalog%2Fen%2FUS%2Fadirect%2Flt%3Fcmd%3DcatDisplayStyle%26catKey%3D101%26filterType%3D1%26OP%3Dfilter%26filter%3Dft_1101%252Ff_2005260*&quot;&gt;MicroAmp Clear Adhesive Film&amp;nbsp;&lt;/a&gt;</td><td>Thermo Scientific</td><td>Cat No 4306311</td></tr></tbody></table>

swab sampling method for the detection of human norovirus on surfaces

人間のノロウイルスは、世界的流行と散発的な胃腸炎の主要な原因です。ほとんどの感染はいずれかの環境表面や食品を介して間接的に個人対個人の経路を介して直接広がったりしているため、汚染された媒介物と無生物表面は、ノロウイルスの集団感染時のウイルスの拡散のための重要な媒体です。 私たちは、硬質表面からのヒトノロウイルスの検出およびタイピングのためのマクロフォームスワブを使用してプロトコルを開発し、評価しました。繊維が先端に付いた綿棒や帯電防止ワイプと比較すると、マクロフォームスワブは、最大700 cm 2の便座表面からのウイルス回収（範囲1.2から33.6パーセント）を許可します。プロトコルは、スピンカラムを用いてウイルスRNAのスワブからウイルスを抽出し、さらに濃縮するためのステップを含みます。ノロウイルス胃腸炎がされていたクルーズ船や介護施設での表面から収集された217スワブサンプルの合計で、127（58.5パーセント）RT-qPCRによりGIIのノロウイルス検査で陽性と報告しました。これらの29（22.8％）のうち、正常遺伝子型を決定することができました。結論として、私たちが開発したプロトコルを使用して、環境面でのノロウイルスの検出は、臨床試料が入手できないときに大流行中の環境汚染のレベルだけでなく、ウイルスの検出を判断する際に役立つかもしれ。それはまた、改善戦略の有効性の監視を容易にすることができます。

図1は、スワブサンプリングプロトコルを示すフローチャートです。このプロトコルは、4つの主要なステップで構成されています。 1）サンプル収集、2）サンプルの保管および輸送、3）ウイルスRNA精製および濃縮し、4）RT-qPCRのアッセイおよび遺伝子型決定。 <img alt="図1" src="/files/ftp_upload/55205/55205fig1.jpg" /><br ...

Watch this Scientific Journal Video about Swab Sampling Method for the Detection of Human Norovirus on Surfaces at JoVE.com

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

表面上のヒトノロウイルスの検出のためのスワブサンプリング方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

swab-sampling-method-for-the-detection-of-human-norovirus-on-surfaces

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

19.5K ビュー.  Centers for Disease Control and Prevention. A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces. 

ビデオ: Swab Sampling Method for the Detection of Human Norovirus on Surfaces

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

エンテロウイルスと文化およびRT-QPCRによる水中のノロウイルス発生のEPAメソッド1615測定。ウイルスサンプルのI.コレクション

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

環境学

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫・感染学

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

エンテロウイルスと文化とRT-qPCRにより水中のノロウイルス発生のEPAメソッド1615計測。 II。総培養可能なウイルスアッセイ

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

エンテロウイルスと文化およびRT-qPCRにより水中のノロウイルス発生のEPAメソッド1615測定。パートIII。 RT-qPCRによりウイルス検出

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

運動誘発性リンパ球のアポトーシスの決定のための指先穿刺の採血方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

のTaqManワンステップRT-PCR法によるキッズスツールからノロウイルスの検出とGenogrouping

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

医学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

代替体外ウイルスのカプシド構造健全性の定量方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

フローサイトメトリーを用いた経月細菌に結合するヒトノロウイルスウイルス様粒子の定量化

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

都市環境のスワッピング - 環境貯留層からのSARS-CoV-2のサンプリングと検出のためのパイプライン

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

生物学

An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL II biosafety cabinet.
Virus-like particles for human norovirus are listed as BSL II pathogens, and all work performed using VLPs should be conducted in a certified biosafety cabinet.
Add 10 micrograms of human norovirus VLPs to each tube of bacteria, and mix thoroughly by pipetting. Incubate the tubes for 1 hour at 37 degrees Celsius with constant rotation. After incubation, centrifuge the tubes at 10,000 times g for 5 minutes.
Aspirate and discard the supernatant and resuspend the bacterial pellet in 1 milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10,000 times g for 5 minutes. Discard the supernatant and resuspend the VLP bacteria pellet in 150 microliters of 5% blocking buffer.
A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment, and add one treatment all at once to the tubes.
For each bacterial sample, prepare 50 microliters of diluted human norovirus G2 antibody. Using 5% blocking buffer, dilute the antibody 1 to 125 for E. coli case samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios. Divide each attachment assay sample into 350-microliter aliquots by transferring them into clean 1.5-milliliter centrifuge tubes.
To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the G2 antibody dilution to the second set of aliquots. Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots.
Incubate all the samples on ice and in the dark for 30 minutes. Then, centrifuge all the samples at 10,000 times g for 5 minutes. Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10,000 times g for 5 minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB.
After centrifuging the samples one final time and discarding the supernatant, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degrees Celsius until they are analyzed by flow cytometry.

表面上のヒトノロウイルスの検出のためのスワブサンプリング方法

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