この研究は、米海軍医学研究センター、研究作業単位6000.RAD1.J.A0310によってサポートされていました。この記事で表明された見解は執筆者のものであり、必ずしも海軍省、国防省、または米国政府の公式な政策や位置を反映するものではありません。魏メイチンは、米国政府の従業員です。この作品は彼女の公務の一部として用意しました。タイトル17 USC§105」このタイトルの下に著作権保護は、米国政府のいずれかの作業には使用できません。」ことを提供しますタイトル17 USC§101は、その人の公務の一環として、米国政府の兵役のメンバーや従業員によって調製作品として米国政府の仕事を定義します。

1. LAMP反応のための標準としてプラスミドDNA希釈液を調製<ol><li> （C.バーネッティ RSA 493の標的遺伝子IS1111aを含む）プラスミドを決定するために、260nmの光学密度（OD）を測定し、DNA濃度を分光光度計を使用します。 1のOD 260は、DNAを50μg/μlに相当します。 </li><li>コピー数にプラスミドDNAの量に変換します。プラスミドのサイズは6,330 bpであることから、このプラスミドの分子量は4.1×10 6グラム（6,330 bpのX 649グラム/ BP）です。プラスミドDNAの濃度は、34.2 ngの/μL（ステップ1.1で決定される）です。このDNA試料の1μlのDNAの5×10 9コピー（34.2×10 -9グラム/ 4.1×10 6 GX 6.02×10 23）に等しくする、プラスミドDNAの34.2 ngのが含まれています。 </li><li> 5×10 8コピー/μlにDNAサンプルを作るために10倍希釈するための水90μlにプラスミドDNAを10μl（5×10 9コピー/μl）を追加します。。 </li><li>繰り返しステップ1.3は、5×10 7のDNAサンプルを調製した5×10 6、5×10 5、5×10 4、5×10 3、5×10 2、5×10 1、および5×10 0コピー/ μlの。 </li></ol> 2. LAMP反応のための試料からDNAテンプレートを準備します<ol><li>製造業者のプロトコルに従って市販のDNAミニキットを使用して、ヒト血漿試料からのDNAの抽出を行います。抽出のための200μlのサンプルの合計を使用して、20μl容量で抽出したDNAを溶出します。 </li></ol> 3. 2倍LAMP反応緩衝液を準備します<ol><li> 2倍LAMP緩衝液1mlを作るために10倍ポル・バッファ、5 Mトリメチルグリシン、1MのMgSO 4、のdNTP混合物（各10mM）の280μlの水188μlに12μlの320μlの200μlのを混ぜます。 10秒間パルスボルテックスして混和します。 </li></ol> 4.標準LAMP反応を行います<ol><li> 2倍LA12.5μlのミックスMPバッファ（ステップ3で調製した、40mMのトリス-HCl（pHは8.8）、20 mMの塩化カリウム、16mMのMgSO 4を、20mMの（NH 4）2 SO 4、0.2％トリトンX-100、1.6 Mトリメチルグリシン、1.4 mMののdNTP混合物）、プライマーミックスの1.2μlを、 のBst DNAポリメラーゼ（8 U /μl）を1μlの、水5.3μlを0.2ミリリットルのPCRチューブ内で反応混合物（20μl）を作ります。 </li><li> （ステップ1または2から）20μlの反応混合物にDNAの5μl加え、よく混ぜます。 </li><li>近い管および水浴または加熱ブロック中で60分間60℃でインキュベートします。これは、以前に決定され、最適な反応温度です。 </li><li>反応を停止し、10倍ゲルローディング緩衝液5μLを加えます。 </li><li>核酸染色を挿入で染色した2％アガロースゲルに負荷反応生成物5μLを。 </li><li> 1×TBE緩衝液中で35分間、100Vでゲルを実行します。 </li><li> UV光によって結果を可視化します。 </li></ol>前記再構成でLAMP反応を行います試薬<ol><li>再構成緩衝液1mlを作るために10倍ポル・バッファ、5 Mトリメチルグリシン、1 M MgSO 4を、水の667.5μlに7.5μlの200μlを125μlのを混ぜます。 10秒間パルスボルテックスして混和します。 </li><li>密封されたアルミホイル袋から凍結乾燥試薬を含むチューブを外します。それが破損している場合アルミ箔袋が密閉されているかどうかのチューブを使用しないでください。 </li><li>凍結乾燥試薬を再懸濁するために、各チューブに再構成緩衝液20μlを追加します。 </li><li>バッファ上下に5回ピペッティングして混ぜます。凍結乾燥試薬が完全に再懸濁されていることを確認するために見てみましょう。白色粉末をチューブに消えるはずです。 </li><li> （ステップ1または2からの）再構成試薬にDNAテンプレートの5μl加え、よく混ぜます。 </li><li>近い管および水浴または加熱ブロック中で60分間60℃でインキュベートします。 </li><li>反応を停止し、10倍ゲルローディング緩衝液5μLを加えます。 </li><li>負荷反応液5μl核酸染色を挿入で染色した2％アガロースゲルに製品。 </li><li> 1×TBE緩衝液中で35分間、100Vでゲルを実行します。 </li><li> UV光によって結果を可視化します。 </li></ol> 6. HNBまたは蛍光インターカレート色素を含む再構成緩衝液でLAMP反応を行います<ol><li>への水の10倍ポルBufferを125μlの、5 ​​Mトリメチルグリシン、1MのMgSO 4、20mMのHNB（または100倍の蛍光挿入色素の12.5μl）を7.5μlに7.5μlの660μlの（または655マイクロリットル）の200μLをミックス再構成緩衝液1mlを作ります。 10秒間パルスボルテックスして混和します。 </li><li> 5.6 - 手順5.2を繰り返すセクション6で調製した再構成されたバッファを使用してください。 </li><li>肉眼での結果（HNBを含む反応）またはUV光（蛍光挿入色素を含む反応）を視覚化します。 </li></ol> 7.チューブスキャナによるリアルタイムLAMP反応を行います<ol><li> reconstitutに5μlのDNAを追加します。ED試薬は、蛍光挿入色素、近いチューブを含む、チューブスキャナにそれを挿入します。 </li><li> 60℃に設定したインキュベーション温度。 60分間の520nmでの蛍光を測定します。 </li></ol>

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著者らは、開示することは何もありません。

以前、我々は、挿入要素IS1111a 14をターゲットに高感度で特異的なLAMPアッセイを開発しました。それは非常にC.の異なる株の間で保存されているため、IS1111要素が選択されましたバーネッティと細菌中のコピーのその高い番号（110から7）（クレー2006）。我々の結果は、LAMPはコクシエラ DNAのわずか1染色体コピーに相関し得るIS1111要素の...

小さ ​​いグラム陰性菌コクシエラバーネッティは、世界中の人獣共通感染症であり、Q熱の原因物質です。 Q熱の世界的な分布とCの高い感染へ海外展開バーネッティ 、米軍と文民要員が流行地域に感染しているの危険にさらされています。 急性および慢性の感染症：Q熱は、2つの形で現れます。急性Q熱は、インフルエンザ様症状、肝炎、または肺炎で自身を提示し、通常、低い死亡率と自己制限疾患です。慢性Q熱は、あまり普及しているが、多くの場合、1,2-放置すれば、はるかに高い死亡率を有している心内膜炎、になります。したがって、適切な治療を導くために早期診断は患者のケアのために重要です。ポリメラーゼ連鎖反応（PCR）および定量的リアルタイムPCR（定量PCR）アッセイは、Cを検出するために開発されています細胞培養物および臨床サンプル3-5 &lt;中バーネッティ DNA/ SUP&gt;。 PCRおよび定量PCRの両方がルーチンワークのリソースに制約のある地域でのコストがかかり、多くの場合、容易に入手できません。 もともと納富6で説明した、LAMP法（LAMP）は、代替のDNA増幅法を提供しています。 LAMPは、標的遺伝子の少なくとも6つの独立した領域を認識する特別に設計されたプライマーセットと共に、鎖置換型DNA合成のためのBst DNAポリメラーゼを使用します。 LAMPの最も重要な利点は、増幅が等温条件下で起こることです。したがって、唯一の水浴、加熱ブロックまたはインキュベータが必要です。この方法は、いくつかのリケッチア病原体7-9を検出するために使用されてきました。ゲル電気泳動により増幅されたDNA産物の可視化は、非特異的な増幅に起因する偽陽性から真陽性を区別することができる最も正確な方法です。しかし、ゲル電気泳動に関わる手順は、リソースが制限されたARのための実用的ではありませんEAS。いくつかの代替方法は、反応生成物を検出するために開発されました。このようなピロリン酸マグネシウムの形成10またはUV光11,12の下で可視化する蛍光挿入色素を使用してから派生した濁度としてこれらの代替は、ゲルを実行するよりも有利で ​​す。 Q熱が流行し13である熱帯および亜熱帯の国では、周囲温度である25℃、37℃、で保存した場合LAMP試薬は、1ヶ月間安定でした。 LAMPアッセイはCの存在を検出するために我々の研究室で開発されました血漿試料中のバーネッティ 14。ここでは、凍結乾燥試薬からLAMP反応液を調製するためのシンプルなプロトコルを記述します。室温で保存した場合、凍結乾燥試薬は、1ヶ月間安定です。簡単に可視化手法と組み合わせると、これはCを検出するために使用するのに理想的な方法であり、リソース制限の設定においてバーネッティ 。

<table><tbody><tr><td>LAMP primers</td><td>Eurofins MWG operon</td><td>10 nmol, salt free</td><td>Sequence designed by customer</td></tr><tr><td>Bst DNA polymerase</td><td>New England Biolabs</td><td>M0275L</td><td>8 units/ml</td></tr><tr><td>10x Thermo pol buffer</td><td>New England Biolabs</td><td>B9004S</td><td></td></tr><tr><td>dNTP mixture</td><td>New England Biolabs</td><td>N0447L</td><td>10 mM each</td></tr><tr><td>Betaine</td><td>Sigma-Aldrich</td><td>B0300</td><td>5 M, Trimethylglycine</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M3409-1ML</td><td>1 M</td></tr><tr><td>10x Bluejuice</td><td>Invitrogen</td><td>10816-015</td><td>gel loading buffer</td></tr><tr><td>SYBR green</td><td>Invitrogen</td><td>S7585</td><td>10,000x, visualize products in tubes</td></tr><tr><td>GelRed</td><td>Phenix Research Products</td><td>RGB-4103</td><td>10,000x, visualize products in gels</td></tr><tr><td>Lyophilized reagents</td><td>Gene Reach</td><td></td><td></td></tr><tr><td>Hydroxynaphthol blue</td><td>Fluka</td><td>33936-10G</td><td></td></tr><tr><td>ESEQuant tube scanner</td><td>Qiagen</td><td></td><td>real-time detection</td></tr></tbody></table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

Q熱リケッチア 、Q熱を引き起こす因子は、偏性細胞内細菌です。 PCRに基づく診断アッセイは、Cを検出するために開発されています細胞培養物および臨床試料中バーネッティ DNA。 PCRは、特殊な装置や大規模なエンドユーザのトレーニングを必要とし、したがって、それは、特に、リソ​​ースに制約のある領域にルーチン作業には適していません。我々は、Cの存在を検出するためのループ媒介等温増幅（LAMP）アッセイを開発しました患者サンプル中バーネッティ 。この方法は、水浴または加熱ブロック中60℃付近の単一の温度で行われます。このLAMPアッセイの感度は、反応当たり約25コピーの検出限界を用いたPCRに非常に類似しています。このレポートでは、凍結乾燥試薬およびhydroxynaphtholブルー（HNB）または反応中の蛍光挿入色素とUVランプを使用して結果の可視化を用いて反応の調製を記載します。 LAMP試薬はlyophiliました検出感度を損なうことなくZED 1ヶ月室温（RT）で保存しました。反応混合物の調製は、複雑な工程を必要としないので、このLAMPアッセイは、特に頑強です。この方法は、Q熱が流行しているリソースが制限された設定での使用に最適です。

0.2ミリリットル管内の凍結乾燥LAMP試薬はのBst DNAポリメラーゼ、プライマー、およびdNTPが含まれています。再構成用緩衝液20μlを、凍結乾燥された試薬を再懸濁するために使用される。 図1は、新たに調製した試薬および凍結乾燥試薬を用いて、アガロースゲル上でLAMP反応結果を示します。凍結乾燥試薬は、その活性を低下させません。両方の試?...

Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

の検出のための凍結乾燥LAMP法試薬の開発<em&gt; Q熱リケッチア</em

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

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Immunology and Infection

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

10.5K ビュー.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

ビデオ: The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

ループ媒介等熱増幅を用いた灌漑水中フィ トフトーラカプシ の検出

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

環境学

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

免疫・感染学

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

トリパノソーマ・クルジまたは他の病原性生物由来のDNAを検出するためのすぐに使用できるqPCR

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...

生化学

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用<em&gt; Q熱リケッチア</em&gt;のsiRNAサイレンシング中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

キャピラリー配列で複数の核酸の視覚的検出

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

生物工学

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

オープンソースのミニチュア蛍光計で、資源制限設定でリアルタイムの等温核酸増幅反応を監視

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

等温増幅法を用いたヒト呼吸器細菌感染症診断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

著者スポットライト:マイクロ流体チップを使用した呼吸器病原体の迅速な検出の進歩 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

動物性食品中の サルモネラ菌 をスクリーニングし、培養分離から サルモネラ菌 を確認するためのループ媒介等温増幅

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

の検出のための凍結乾燥LAMP法試薬の開発

要約

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この動画の章

ループ媒介等熱増幅を用いた灌漑水中フィトフトーラカプシの検出

発生とのマルチ表現型ハイコンテンツスクリーニング

トリパノソーマ・クルジまたは他の病原性生物由来のDNAを検出するためのすぐに使用できるqPCR

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用

キャピラリー配列で複数の核酸の視覚的検出

オープンソースのミニチュア蛍光計で、資源制限設定でリアルタイムの等温核酸増幅反応を監視

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

動物性食品中のサルモネラ菌をスクリーニングし、培養分離からサルモネラ菌を確認するためのループ媒介等温増幅

の検出のための凍結乾燥LAMP法試薬の開発

要約

さらに動画を探す

この動画の章

ループ媒介等熱増幅を用いた灌漑水中フィ トフトーラカプシ の検出

発生とのマルチ表現型ハイコンテンツスクリーニング

トリパノソーマ・クルジまたは他の病原性生物由来のDNAを検出するためのすぐに使用できるqPCR

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用

キャピラリー配列で複数の核酸の視覚的検出

オープンソースのミニチュア蛍光計で、資源制限設定でリアルタイムの等温核酸増幅反応を監視

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

マイクロ流体チップベースのループ媒介等温増幅による下気道感染症を引き起こす細菌性病原体の迅速な検出

動物性食品中の サルモネラ菌 をスクリーニングし、培養分離から サルモネラ菌 を確認するためのループ媒介等温増幅

ループ媒介等熱増幅を用いた灌漑水中フィトフトーラカプシの検出

動物性食品中のサルモネラ菌をスクリーニングし、培養分離からサルモネラ菌を確認するためのループ媒介等温増幅