概念化、h. c. および Z. Q. F;Z. Q. F ・ j ・ C、h. c. 調査リソース、Z. Q. F.;書く - 原案、I. A. P.;ライティング-レビューと編集、Z. Q. F.;、s. l. t. h. c. j. c. i. a. p.可視化, H. C.;監督、Z. Q. F.;資金獲得、Z. Q. F と f. q. l.
サウスカロライナ改善先生品質高等教育国家助成金タイトルによるプロジェクト、強化中間等級科学教師の工場プロセスの知識、構造、および植物科学研究 (工場の機能を通じて婚約科学)
ユナイテッド立ち会える教育学科 (CFDA 番号 84.367B) 資金
著者らはゲノム DNA のサンプルを提供するためにフロリダ大学の博士インディアン王を感謝したいから感染シトラスシネンシスバレンシア。

1. ゲノム抽出キットを使用して植物組織からのゲノム DNA を分離します。
<ol><li>切り出して全体の新鮮なきれいなはさみを使用して柑橘類の木から葉し、葉がきれいなプラスチック サンドイッチ バッグ、柑橘類の葉の組織を取得します。 注記: 葉のティッシュの入った袋を配置する 4 ° C の冷却装置でまたは断熱容器で氷の腐敗を防ぐため、コレクション後できるだけ早く。</li>
	<li>乳鉢と乳棒を寒さに液体窒素の少量を追加します。彼らが冷やしている間葉組織、サイズ約 1 平方インチの小片をカットするはさみを使用します。それを瞬時に凍結するモルタルに切断組織を配置します。必要な場合より多くの液体窒素を追加します。 注: 絶縁手袋は、液体窒素を処理しながら常に着用する必要があります。</li>
	<li>すぐに、モルタルを用いた微粉末に植物組織を粉砕します。液体窒素が完全に蒸発して緑の粉のままなるまで研削を続けます。</li>
	<li>簡単に、ヘラを使って 1.5 mL 遠心チューブにモルタル内部地組織をスクープして 10-15 s、液体窒素中での金属のヘラを冷やします。</li>
	<li>核溶解液の 600 μ L を追加 (材料の表を参照) を使用して 1000 μ L ピペットと渦 1-3 s のサンプルは、15 分の 65 ° C の水浴中孵化させなさい。</li>
	<li>逆転管 2 - 5 回、それを混合し、37 ° c 15 分間キャビネット インキュベーターで孵化して、ライセート 10µL ピペットを使用する RNase 溶液 3 μ L を追加します。 注: 続行する前に室温に冷却する混合物を許可します。</li>
	<li>しっかりしたペレットを形成する 13,000 x g でタンパク質沈殿物溶液、20 秒の高速、3 分間遠心する渦の 200 μ L を追加します。サンプルを遠心分離されているが、新鮮な 1.5 mL 遠心チューブに室温イソプロパノールの 600 μ L を追加します。</li>
	<li>1000 μ L ピペットを使用して、慎重にイソプロパノールを含む新鮮な 1.5 mL 遠心管に移し、遠心サンプルから上澄みを取り外します。ペレットを破棄今可能性があります。 注: を使用すること、ペレット上清の非常に小さい量を残すことによって蛋白質の上澄みが汚れない。</li>
	<li>DNA が糸状の繊維の塊として目に見えるようになるまで、チューブを反転し、室温で 1 分 13,000 × g で遠心分離します。</li>
	<li>上清をデカント、ペレットを 70% のエタノールの 600 μ L を追加、DNA を洗浄し、1 分間 13,000 × g で遠心チューブを数回反転します。</li>
	<li>慎重に緩やかな DNA の餌を残して、エタノールを吸引し、開き、吸収紙の上に休んで、チューブを反転します。15 分間室温で乾燥した空気。</li>
	<li>乾燥 DNA の餌に DNA 補水液 100 μ L を追加し、チューブをタップして定期的に混合しながら、1 時間 65 ° C の水浴の DNA を孵化させなさい。</li>
	<li>精製水 1 μ L を microcuvette に置き、空白として使用する分光光度計に挿入します。Microcuvette に 1 μ L のサンプルを配置し、分光光度計を挿入して、現在の DNA の濃度を計算します。DNA 濃度 (ng/μ L) (260-320) として計算される × 希釈係数 × 50 ng/μ L をすることができます。 注: プロトコルはここで一時停止することができます。サンプルは 4 ° C で保存できます。</li>
</ol>2. ゲノム DNA のサンプルの PCR を実行します。
<ol><li>結合 10 μ L 2 x PCR マスター ミックス (材料の表を参照)、1 μ L 前方プライマー (10 pM/μ L)、1 μ L 逆プライマー (10 pM/μ L) (プライマー シーケンスについて表 2を参照)、100 ng の DNA のサンプルから抽出され、精製水 (最大 20 μ L 最終巻) で50 μ L の PCR チューブ、ミックス、フリックで簡単に遠心分離機。</li>
	<li>たちに PCR チューブを挿入し、それに応じて実行 (表 1参照)。 注: プロトコルはここで一時停止することができます。必要に応じて、反応が完了した後、4 ° C でサンプルを格納します。</li>
</ol>3. Agarose のゲルに増幅された DNA を electrophorese します。
<ol><li>アガロース パウダー 0.4 g の重量を量る、きれいな 200 mL 三角フラスコ、50 mL を充填する 1 x TAE バッファーと共にそれを追加します。</li>
	<li>マイクロ波高 1.5 分、すべての 30 の揺れの s、アガロースは完全に溶解するまで。 注: 火傷を防ぐために液体のアガロースを処理しながら絶縁手袋を着用ください。</li>
	<li>液体アガロースとミックスする手ふれに臭化エチジウム 10 mg/mL の 2.5 μ L を追加し、レベル ゲル鋳型に液体 agarose ゲルを注ぎなさい。注入後標準 12 歯ゲル櫛をすばやく挿入します。</li>
	<li>ゲルを許可、室温で 20 分間冷却し、ゲルの櫛を削除して、ジェルを 1 x TAE バッファーでいっぱいレベル ジェル ボックスに。</li>
	<li>ピペットを使用すると、増幅された DNA サンプルの合計を井戸に読み込みます。あなたのサンプルから別のよく 5 μ L の DNA の梯子 (材料の表を参照) を追加します。</li>
	<li>定数 90 V、35 分間のサンプルを electrophorese し、ゲルを取り外し、UV transilluminator に置きます。</li>
	<li>写真し、302 の紫外線透過を使用しながらパターンをバンディング ゲル分析 nm。</li>
</ol>

<ol>
	<li>Field Office, F. l. o. r. i. d. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Citrus October Forecast Maturity Test Results and Fruit Size. , (2016).</li><li>Singerman, A., Useche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+citrus+greening+on+citrus+operations+in+Florida.">Impact of citrus greening on citrus operations in Florida.</a> Food and Resource Economics Department, UF/IFAS Extension. , (2015).</li><li>Spreen, T. H., Hodges, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The economic impact of HLB on the florida citrus industry. , (2012).</li><li>UICEP. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology. , (2013).</li><li>Jagoueix, S., Bove, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phloem-limited+bacterium+of+greening+disease+of+citrus+is+a+member+of+the+alpha+subdivision+of+the+Proteobacteria.">The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria.</a> Int J Syst Bacteriol. 44 (3), 379-386 (1994).</li><li>Fujikawa, T., Iwanami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+detection+of+citrus+greening+(huanglongbing)+bacterium+&amp;#34;Candidatus+Liberibacter+asiaticus&amp;#34;+by+DNA+amplification+with+new+16S+rDNA-specific+primers.">Sensitive and robust detection of citrus greening (huanglongbing) bacterium &amp;#34;Candidatus Liberibacter asiaticus&amp;#34; by DNA amplification with new 16S rDNA-specific primers.</a> Mol. Cell. Probes. 26 (5), 194-197 (2012).</li><li>Davis, M. J., Mondal, S. N., Chen, H., Rogers, M. E., Brlansky, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-cultivation+of+'Candidatus+Liberibacter+asiaticus'+with+actinobacteria+from+citrus+with+huanglongbing.">Co-cultivation of 'Candidatus Liberibacter asiaticus' with actinobacteria from citrus with huanglongbing.</a> Plant Dis. 92 (11), 1547-1550 (2008).</li><li>Department of Agriculture &amp; National Agriculture Statistics Service. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Citrus Fruits 2015 Summary (September 2015). , (2015).</li><li>Hocquellet, A., Toorawa, P., Bov&#233;, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+the+two+Candidatus+Liberobacter+species+associated+with+citrus+huanglongbing+by+PCR+amplification+of+ribosomal+protein+genes+of+the+&#946;+operon.">Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the &#946; operon.</a> Mol. Cell. Probes. 13 (5), 373-379 (1999).</li><li>Mafra, V., 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=Reference+genes+for+accurate+transcript+normalization+in+citrus+genotypes+under+different+experimental+conditions.">Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.</a> PLoS One. 7 (2), e31263 (2012).</li><li>Li, W., Hartung, J. S., Levy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+PCR+for+detection+and+identification+of+Candidatus+Liberibacter+species+associated+with+citrus+huanglongbing.">Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing.</a> J. Microbiol. Meth. 66 (1), 104-115 (2006).</li></ol>

還陳、イアン ・ アーサー ・ パーマー、Jian 陳、明チャン、スティーブン ・ トンプソン、Fengquan リュー、そして鄭清 Fu 利害の対立を宣言しません。

まさに最適な実験条件を達成するためにすべての手順が続いていることが重要です。全体の葉の組織がこのデモ中に使用されました。しかし、理論的には植物組織の任意の種類を含めるにプロトコルを変更できます。以前は、研究者は葉静脈の組織ではなく、全体の葉組織からのゲノム DNA を抽出して同様の結果11を達成しました。シトラスFBOX遺伝子に対応するバン?...

カンキツグリーニング、カンキツグリーニング病とも呼ばれる、柑橘類の木々 の重要な損失を引き起こした。1 つの代表格は、フロリダ州 2016-2017 シーズン1現在 7000 万箱に 2 億 4400 万ボックスから 1997-1998 年シーズンのフロリダ オレンジ ボックスの生産の 70% 削減を引き起こす病気を予想する場所です。以上フロリダの柑橘類の木の 90% が感染した2、およびフロリダの柑橘類工業は、前記カンキツグリーニングは3主要な役割を果たしている柑橘類の病気が毎年約 10 億ドルを失うこと推定されています。カンキツグリーニング、侵襲ミカンキジラミミカンキジラミ、によって主に広がる、.の感染組織の接木によって広げることができるも病気の静脈、非対称的な黄化斑点、早期落葉、枝枯れ、根面齲蝕、植物の死の黄変が発生します。重要なことは、疾患の原因となりフルーツ、ほとんど、季節外れの花を作り出す柑橘類の植物。プロデュースは感染したカンキツ果実が未熟な緑、および4の味が苦いです。
このメソッドの目的は、正確し、運動性細菌カンキツグリーニング病病原細菌の属、感染したカンキツ5の師部内に生きてカンキツグリーニング病原を正確に識別。ゲノム DNA は生きている細菌を含む全体の葉組織から抽出されます。この抽出したゲノム DNA は細菌の 16S rdna に相補的なオリゴヌクレオチドは、このシーケンスを増幅するため、従来の PCR のテンプレートとして使用されます。シトラスFBOX遺伝子相補的なオリゴヌクレオチドは、内部増幅制御として使用されます。以前の研究6で成功するために実証されているため、このメソッドを使用にしました。
この方法には、簡単な比較的安価な任意の通常の整った生化学のラボで実施することができるの明確な利点があります。さらに、PCR のままこの病原体の7と病原体の能力を生き残るため、無症候性ホスト8年の再現を養殖の難しさのため、この病原体を検出するため最も正確かつ正確な方法。多くの異なる専門 PCR の試金はこの病原体; を正常に検出に使用されています。しかし、従来の PCR は、無症候性ホスト8の内で特に、正確かつ特定の検出のための最も簡単なアッセイを残ります。

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Wizard Genomic DNA Purification Kit</td>
			<td style="width:175px;">Promega</td>
			<td style="width:111px;">A1120</td>
			<td style="width:111px;">Source of Nuclei Lysis, RNase, Protein Precipitation, and DNA Rehydration solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td>Air Products</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mastercycler pro with control panel</td>
			<td>Eppendorf</td>
			<td>6321000019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1250 W Microwave</td>
			<td>Panasonic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5424 table top centrifuge</td>
			<td>Eppendorf</td>
			<td>05-403-93</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isotemp 215 digital water bath</td>
			<td>Fisher scientific</td>
			<td>15-462-15Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal spatula</td>
			<td>Sigma-Aldrich</td>
			<td>Z283274</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mortar and pestle</td>
			<td>Sigma-Aldrich</td>
			<td>Z247464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSE Vortex Mixer</td>
			<td>Corning</td>
			<td>6775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 10 &#956;L tips</td>
			<td>TipOne</td>
			<td>1161-3730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 200 &#956;L tips</td>
			<td>TipOne</td>
			<td>1163-1730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 1250 &#956;L tips</td>
			<td>TipOne</td>
			<td>1161-1750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Variable Volume Pipettor Kit</td>
			<td>VWR</td>
			<td>75788-460</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethidium bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E1510</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>IBI Scientific</td>
			<td>IB70041</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>17892</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Fisher Chemical</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#956;cuvette</td>
			<td>Eppendorf</td>
			<td>6138000018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stackable casting tray and combs</td>
			<td>Carolina</td>
			<td>213655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini-Sub Cell GT System</td>
			<td>Bio-Rad</td>
			<td>1704487EDU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biospectrometer basic</td>
			<td>Eppendorf</td>
			<td>6135000009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 mL PCR tubes</td>
			<td>Fisher scientific</td>
			<td>AB-0620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL microcentrifuge tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3439</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2X Taq Green PCR Master Mix</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forward Primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reverse Primer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water, PCR grade</td>
			<td>Sigma-Aldrich</td>
			<td>3315953001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel Doc XR+</td>
			<td>Bio-Rad</td>
			<td>1708195</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 KB+ DNA ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>10787018</td>
			<td></td>
		</tr>
	</tbody>
</table>

specific and accurate detection of the citrus greening pathogen candidatus liberibacter spp. using conventional pcr on citrus leaf tissue samples

カンキツグリーニング、カンキツグリーニング病として知られているは、世界的に柑橘類の農場を荒らす破壊的な柑橘類疾患です。この病気には、非対称の黄色葉の斑点、葉脈黄、摘葉、根面齲蝕、最終的には、柑橘類の植物の死が発生します。感染、柑橘類の植物は成長の発育を妨げているし、季節はずれの花を作り出します。これらの花はほとんど果物をもたらすし、小さく、苦い、不規則な形の柑橘系の果物を降伏行うことが望ましい。この病気は、感染したカンキツ組織からの移植、ミカンキジラミ、ミカンキジラミ、広がっています。病原体は柑橘類の植物の中で長いと変数の潜伏期間-時々 年、症状が現れる前に。この病原体の in vitro培養の試みが成功すると、可能性が低いのためにされている、内の病原体の凹凸濃度感染カンキツ組織や環境を複製することは困難だから条件の成長を助長病原体。前にそれが広がっており、その長い潜伏期間と、病原体を培養する研究者の無力のために病気を識別する非常に困難です。その結果、病気のみ明らかになります突然柑橘類の農夫の全体収穫を破壊した後。ここに提示のカンキツグリーニング病原体、ゲノム DNA 抽出キットや PCR を用いたカンキツグリーニング病病原細菌属の正確かつ具体的検出法です。このメソッドは、シンプルで効率的、費用対効果、かつ定量化するための適応です。このメソッドは、カンキツ組織で使用するために合わせることができます。しかし、それは可能性のある病原体の組織内に存在の量によって制限されます。それにもかかわらず、このメソッドは以前、感染した柑橘系植物を識別するために柑橘農家を許可し、それがさらに広がることができる前に、この破壊的な病気の広がりを抑制します。

肯定的な結果が 500 に対応する明瞭なバンドを得られる bpカンキツグリーニング病病原細菌うわさの花姫Las606/Lss6,および/または 700 に対応する異なるバンドのLaa2/Laj5、 16S リボソーム β への挿入のための bpオペロン9。内部増幅制御帯域 400 に現れる場合も bp。このバンドは柑橘類FBOX遺伝子の<sup class...

Watch this Scientific Journal Video about Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples at JoVE.com

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

カンキツグリーニングは柑橘類の作物に影響を与える世界的に特に破壊的な病気です。ここで紹介は単純なメソッドを使用して PCR およびカンキツ葉組織のゲノム DNA 抽出カンキツグリーニング病原体、カンキツグリーニング病病原細菌属の正確かつ正確な同定のため

特定病原体緑化シトラスカンキツグリーニング病病原細菌属カンキツ葉組織サンプルで使用した従来の PCR の正確な検出

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

specific-accurate-detection-citrus-greening-pathogen-candidatus

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JoVE Journal

Environment

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

7.6K ビュー.  University of South Carolina. カンキツグリーニングは柑橘類の作物に影響を与える世界的に特に破壊的な病気です。ここで紹介は単純なメソッドを使用して PCR およびカンキツ葉組織のゲノム DNA 抽出カンキツグリーニング病原体、カンキツグリーニング病病原細菌属の正確かつ正確な同定のため

ビデオ: Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

比色検出の紙ベース<em&gt;エシェリヒア·コリ</em&gt;<em&gt;サルモネラ</em&gt;属、および<em&gt;リステリア菌</em&gt;農業大量の水から

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

環境学

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

の同時定量のための二重デジタルPCRアッセイ<em&gt;エンテロコッカス属。</em&gt;およびWatersにおけるヒトの糞便関連HF183マーカー

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

DMMTAV DMDTAV環境用 DMAVを使用しての作製: 合成、精製、および確認

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

ポータブル リアルタイム PCR を用いた土壌伝染性植物の敷地内の分子の検出

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

植物感染試験: スプレーと傷を介した植物病原体イネいもち病菌接種

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

陰イオン交換樹脂を用いるバイオエアロゾルからのウイルスの検出

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

異方性高分子人工抗原提示細胞 cd 8 + T 細胞活性化のための加工

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

ティラピア湖ウイルスを使用して従来 RT-PCR および SYBR の検出グリーン Rt-qpcr

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

純粋な落葉スタンドにおける3つの異なる方法を用いてリーフエリア指数推定

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 &#176;C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a &#34;sandwiched&#34;, anaerobic environment. Agar plates are incubated overnight at 37 &#176;C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 &#176;C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15&#8211;20% of samples test positive for C. perfringens.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

小売食品サンプルにおけるクロストリジウム・パーフリンゲントキシノタイプの濃縮と検出

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized ...