Name: genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)
Uploaded: 2016-11-04T13:00:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

The author thanks R. Boss, A. Cosandey, C. Fournier, I. Ivanovic, and J. Naskova for their excellent work.

1. 黄色ブドウ球菌分離株<ol><li> S.10μlのスプレッド黄色ブドウ球菌株培養または1つのコロニーを5％ヒツジ血液（BA）を含むコロンビア寒天プレート上で選択培地上で増殖させました。 </li><li> 24時間〜18時間、37℃で好気的に培養します。 </li></ol> 2. DNA抽出<ol><li> 10 mMトリス-塩酸および10mMのNa 2 EDTA、pHが8.5を含むTELバッファを準備します。 15分間、121℃でアリコートし、オートクレーブを準備します。 </li><li> 100μlのTELに滅菌プラスチックループまたは滅菌木製の歯のピックと再懸濁を使用して、BAから1コロニーを収集します。スクリューキャップで1.5ミリリットルチューブを使用してください。 </li><li> 10分間95℃でインキュベートします。その後、濡れた氷の上にすぐにサンプルを置きます。 </li><li> PCR（PCR用=鋳型DNA）に適したH 2 Oで100：サンプル1を希釈します。非希釈し、希釈した試料を1ヶ月間-20℃で保存することができます。 </li></ol> 3. RS-PCR <ol><l各サンプルピペットiについて&gt; PCRチューブに1.5μlのH 2 O、12.5のTaqマスターミックス、2μlのG1プライマーμlの10μM、2μlのL1プライマー、10μM、および純粋の約30 ngのに対応するテンプレートDNAの7μlのDNA 2。 </li><li>標準的なPCRサイクラーで以下のサイクリングプログラムを実行します。7分間55℃で2分間ランプアニールに続いて1分間94°Cを含有する27サイクル、続いて15分95°C、。さらに2分勾配後、2分間72℃で延びています。 4℃に冷却し、続いて72℃で10分間の最終伸長によってPCRを終了します。 注：PCR産物は、5日間20℃で保存することができます。 </li><li> RS-PCRの各実行のために知られて遺伝子型（通常はGTB）とポジティブコントロール、 すなわち、サンプル、およびネガティブコントロール（H 2 O）が含まれます。 </li></ol> 4.電気泳動<ol><li>一緒にDNAのための市販の小型化電気泳動キット（MEDキット）を使用しますPCおよびインストールされたソフトウェアに接続されているバイオアナライザと。慎重に、メーカーの対応マニュアルを研究しています。 </li><li>チッププライミングステーションを設定し、製造業者の説明書に従ってシリンジクリップを調整します。 </li><li>製造業者の説明書に従ってゲル - 色素ミックスを調製します。 </li><li> （製造業者の説明書に従って）ゲル - 色素ミックスをロードします。 <ol><li>使用前に30分間RTにゲル染料ミックスを平衡化します。 </li><li>チッププライミングステーション上の新たなDNAチップを置きます。よくマークされG.中のゲル - 色素のピペット9μlの</li><li>プランジャが1ミリリットルに配置されていることを確認した後、チッププライミングステーションを閉じます。それは、シリンジクリップで固定されるまで押しプランジャ。 30秒後、シリンジクリップを解放するために正確に待ちます。 </li><li> 5秒間待ってください。ゆっくりと1ミリリットルの位置にプランジャーを引き戻します。 </li><li>両方のウェル中のゲル色素のオープンチッププライミングステーションとピペット9μlを「G」をマークしました。 </li></ol></li><li> Mのロードarker <ol><li>全12サンプルウェル中、よくはしご中のマーカーのピペットを5μl。空のこれらの13のウェルのいずれかのままにしないでください。 </li></ol></li><li>ラダーとサンプルをロードします。 <ol><li>ラダー記号をよくマークさにDNAラダーのピペット1μlの。 </li><li>サンプルのピペット1μlの（使用のウェル）または脱イオン水（未使用井戸）の1μlの。 </li><li> 2400 rpmで1分間アダプタと渦に水平にチップを置きます。 </li><li> 5分以内にバイオアナライザーでチップを実行します。 </li></ol></li><li>分析の実行<ol><li>バイオアナライザーと接続されたコンピュータを起動します。ソフトウェアを開きます。 </li><li>バイオアナライザのふたを開け、（チップは一方向にしかフィット）チップホルダーにチップを置きます。慎重に蓋を閉じます。 </li><li>ソフトウェアのインストゥルメントの文脈では、「電気泳動&quot;&gt;&quot;のdsDNA &quot;&gt;&quot; DNA 7500 &quot;を選択します。ソフトウェアの現在のファイルの接頭辞を受け入れるか、またはそれを変更します。</li><li>ソフトウェアに存在するスタートボタンをクリックします。ソフトウェアでサンプル名を入力します。チップの実行が終了すると（後に約30分） を実行し 、メッセージの終了は、バイオアナライザーソフトウェアに表示されます。 </li><li>製造業者の指示に従ってチップを取り外し、バイオアナライザーの電極を清掃してください。 </li></ol></li></ol> 5.電気泳動プロファイルから遺伝子型の推定<ol><li> （ソフトウェアは作者から自由に利用可能です）・マハルのソフトウェアをロードします。 </li><li>マハルソフトにインポートする参照データ：「ファイル」&gt;「インポート参考データ」 </li><li>バイオアナライザソフトウェアのデータコンテキストで実行電気泳動を開きます。 1サンプルの電気泳動プロファイルを選択します。 </li><li>関連するピークを確認してください。 <ol><li> 「ツール」&gt;「平均蛍光」：関連するピークを確認するためにマハルソフトウェアを使用してください。 「蛍光」ボックスに挿入します蛍光値は、それらがバイオアナライザーソフトウェアから読み出すことができるように蛍光単位（FU）として示されます。昇順で最も高い蛍光を有する4（3）ピークを挿入し、カンマで区切られています。例：126130132137。 注：すべてのこれらのピークのFUのは、バイオアナライザーソフトウェアによって示されている場合、それらはプロットからそれらを読み出すことによって推定される必要があります。 </li></ol></li><li> 「計算する」ボタンを押してください。観察された蛍光は、ボックス「最小ピーク高さ」で指定された値を超えた場合にピークが関連しています。 </li><li>遺伝子型を推論します。 <ol><li>昇順にマハルのテキストボックス「入力ピークス（bp）の「すべての関連のピークの塩基対での大きさに挿入し、カンマで区切られています。例：440,462,519,579,637。 注：これらのピークの大きさの全てではないが、バイオアナライザーソフトウェアによって示されている場合、それらはプロットからそれらを読み出すことによって推定される必要があります。 </li></ol></li><li> 「計算する」ボタンを押してください。 </li><li>リストでボックスには、最小限の二乗マハラノビス距離 &quot;D2M」を探してください。それは≥0.05ある右に対応するP値は、その後確率は帰無仮説H 0が拒否されたこと≥5％である場合には（H 0：観察された基準プロファイルは同一である）が観察されたプロファイルが同じであることが認められていることを意味します指示された遺伝子型の基準プロファイル。 </li><li> D2Mが最小であるが、対応するP値のフィールドが空の場合、確率&lt;5％H 0を棄却。 注：これはD2Mは最小限ですが、プロファイルが等しくないことを意味します。このような結果は、非平均電気泳動条件に起因し得ます。 <ol><li>これらの偏差を補正するために、値395「ピーク（bp）の観測」テキストボックスに挿入し、「計算」ボタンを押してください。最小限のD2Mと対応するP値≥0.05のために再度確認してください。成功しない場合、値は405、415、および425を試してみてください。 注：時々、小さなステップでもあります示されています。 、観察されたプロファイルが参照データには存在しない成功していないまだ場合、それは新たな遺伝子型または変異体であってもよいです。 </li></ol></li><li>様々なプロファイル<ol><li>繰り返しますが、別途、各電気泳動プロファイルに対して5.6〜5.9を繰り返します。 </li></ol></li></ol>

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The authors have nothing to disclose.

RS-PCR（&gt; 30純粋なDNA /アッセイをNG）におけるDNAの過剰がある場合手順全体の最も重要なステップがある2。そのすべてのピークがベースラインから始まり、最後ならば一般的には、プロファイルの解像度が最適です。 2つのピークが一緒に近すぎる場合は、解像度が不完全です。手動識別が必要とされるように、これらの条件下では、バイオアナライザーソフトウェアが不適切なピ?...

ブドウ （ 黄色ブドウ球菌）は、牛1における乳房内感染（IMI）のための世界的な責任を最も重要な病原体として知られています。欧州12カ国というの牛でCosandey ら 3及びボスら 4によりフルニエら 2スイス牛で実証・研究による研究S.ウシIMIから分離された黄色ブドウ球菌は、遺伝的に不均一な群です。 16S-23S rRNAの遺伝子間スペーサー領域のPCR増幅（RS-PCR）により、17遺伝子型の合計は、最初に101疫学的に独立した単離物2で検出されました。他の15の遺伝子型（GTS）は、各すべての分離株の1〜4％を作り、まれに発生していないのに対し、遺伝子型Bと遺伝子型C（GTC）は、（80％）が最も一般的でした。欧州レベル3では、GTBは、GTCは、GTF、GTI、およびGTRは、すべての分析株（N = 456）の76％を含む、最も重要な遺伝子型でした。 GTBは、ワット中央ヨーロッパに位置していましたhereas他の遺伝子型は、広く普及しました。株の残りの24％は41の遺伝子型が含まれ、それらの多くは、しかし、一度のみ観察されました。 フルニエ者らの研究ら 2、Cosandey ら 3、及びグラバーら 5は、さらに、遺伝子型が非常に欧州レベルでのように、スイスで、ならびに、それらの病原性遺伝子のパターンと関連していたことを実証しました。研究はさらにS.間のIMIの有病率の大幅な違いを明らかにしました球菌 GTB、GTCおよび他の遺伝子型2,5：GTB考慮して、群れでの牛の87％までは、この遺伝子型に感染していました。 GTCのと他の遺伝子型の場合には、しかし、IMIは唯一の群れの1〜2頭の牛に認められました。 IMI S.によって引き起こされます黄色ブドウ球菌は 、通常、対応する乳腺の炎症および乳中の炎症細胞の増加をもたらします。その結果、体細胞の共同乳中のunts（SCC）、ほとんどの国で乳質の重要な指標は、減少した牛乳の価格、あるいは配信停止につながる増加されます。 フルニエら 2のRS-PCRに加えて、他のタイプの方法は、Sのウシ株サブタイプについて記載されています球菌 8-11。 2つの一般的なものは、 スパタイピング 12と多座配列タイピング（MLST）13が含まれます。後者の1が7ハウスキーピング遺伝子の塩基配列決定を必要とするのに対し、前者は、プロテインAのブドウ球菌スパコードする遺伝子の可変スペーサー領域の配列を決定するDNAに基づいています。これは、1つの12および7のPCR 13は 、それぞれ、特定の装置を使用してアンプリコンの精製、配列決定反応及び分析を行い、実行する必要があることを意味します。 RS-PCRと比較すると、これらの方法は、低いサンプルスループットと高いコストが生じ、実験室にかなりの付加的な努力を必要とします。ザスループットは、PFGEのために特に低いです。 S.のウシの株について、これらの方法の解像度を考慮すると黄色ブドウ球菌 、RS-PCRは、少なくともMLST 4またはPFGE 15より4、より良いと入力し、スパと同じくらい良いです。 バイナリタイピング13を含むすべてのこれらの方法は、Sによって引き起こされるウシIMIの病因にさらなる洞察を得ることにその有効性を実証しました黄色ブドウ球菌 、しかし理由述べた制限の彼らは、大規模な臨床研究にはあまり適しています。また、RS-PCRによる遺伝子型決定フルニエらによって記載されているように2は 、疫学的及びS.の病原性の可能性のある信頼性の高い予測を可能にしますウシIMIに関与球菌 、臨床獣医学における2のキー値。

<table><tbody><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Merck</td><td>1.08382.0500</td><td>Mw = 121.14 g/mol</td></tr><tr><td>Titriplex III</td><td>Merck</td><td>1.08418.0250</td><td>Mw = 372.24 g/mol; Substance is equivalent to Na&lt;sub&gt;2&lt;/sub&gt;EDTA&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hydrochloric acid 5 mol/L</td><td>Merck</td><td>1.09911.0001</td><td></td></tr><tr><td>Columbia agar+5% sheep blood</td><td>bioM&amp;eacute;rieux</td><td>43049</td><td></td></tr><tr><td>Agilent DNA7500 Kit</td><td>Agilent Technologies</td><td>5067-1504</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>Agilent 2100 expert sofware</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>HotStarTaq master mix&amp;nbsp;</td><td>Qiagen</td><td>203445</td><td></td></tr><tr><td>Primer L1</td><td>Mycrosinth</td><td></td><td>5&#039;CAA GGC ATC CAC CGT3&#039;</td></tr><tr><td>Primer G1</td><td>Mycrosinth</td><td></td><td>5&#039;GAA GTC GTA ACA AGG3&#039;</td></tr></tbody></table>

genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

遺伝子型とその変種の定義すべての識別された遺伝子型から複数の帯域が異なる電気泳動プロファイルが新たな遺伝子型であると考えられます。新しい遺伝子型に名前を付けて拡張フルニエらによると。5 GTZにGTAの遺伝子型につながる、GTBZに遺伝子型GTAZにGTAA、およびGTBAが続き、現在ではGTCCにGTCAさ?...

Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

のジェノタイピング<em&gt;黄色ブドウ球菌</em&gt;によってリボソームスペーサーPCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

genotyping-of-staphylococcus-aureus-by-ribosomal-spacer-pcr-rs-pcr

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

9.6K ビュー.  Agroscope. Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

ビデオ: Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

遺伝子破壊ミュータンス連鎖球菌緊張せず遺伝子クローニングの生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

遺伝学

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

血液培養から細菌性病原体の検出および抗菌剤耐性テストのための一日のワークフロースキーム

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

免疫・感染学

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

抗生物質耐性ブドウ球菌の細菌の検出のためのバイオセンサー

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

生物工学

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

グラム陽性菌におけるRNAシーケンシングと結合したMS2-アフィニティ精製

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

生化学

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNAの遺伝子配列決定およびMALDI-TOF MSによるレア細菌性病原体の同定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

ゲノムワイドな遺伝子欠失で<em&gt;ストレプトコッカス血統</em&gt;ハイスループットPCRによる

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

生物学

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

16SリボソームRNA遺伝子アンプリコンの次世代シーケンシング

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

細菌群集の特徴付けのための効率的な核酸抽出および16S rRNAの遺伝子配列決定

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

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