概念化、H.C. 和 z.q.f.;调查, h.c., J, 和 z.q. F;资源, z.q.f.;文字原件草稿, i.a.p.;写作-审阅 & 编辑, i.a.p., h.c., s.l.t. 和 z.q.f.;可视化, h.c.;监督、z.q.f.;资金购置、z.q. F 和 f.q.l。
南卡罗来纳州支持的项目提高教师素质高等教育国家补助金标题,通过参与植物科学研究提高中级理科教师对植物过程、结构和功能的认识 (植物科学)
美国教育部颁发的资金 (CFDA 号 84.367B)
作者要感谢佛罗里达大学的年王博士提供来自受感染的柑橘的瓦伦西亚的基因组 DNA 样本。

1. 使用基因组提取试剂盒分离植物组织中的基因组 DNA
<ol><li>用干净的剪刀从柑橘树上切下一整片新鲜的叶子, 将叶子放在干净的塑料三明治袋中, 获得柑橘叶组织。 注: 在收集后应尽快将含有叶组织的包放在4摄氏度冰箱或保温容器中的冰上, 以避免变质。</li>
	<li>加入少量液氮冷却砂浆和杵。当它们冷却时, 用剪刀剪下一小片叶子组织, 大小大约1平方英寸。将切割组织放在砂浆中立即将其冻结。如果需要, 添加更多的液氮。 注: 在处理液氮时, 应始终佩戴绝缘手套。</li>
	<li>用砂浆将植物组织快速研磨成细粉。继续研磨直至液氮完全蒸发, 并保持良好的绿色粉末。</li>
	<li>在 10-十五年代, 在液态氮气中短暂冷却金属铲, 然后用刮刀将砂浆内的地面组织舀入1.5 毫升的离心管中。</li>
	<li>添加600µL 核溶解溶液 (见材料表) 使用1000µL 吸管和涡流样品 1-3 s, 然后孵化在65°c 水浴15分钟。</li>
	<li>添加3µL RNase 溶液的裂解液使用10µL 吸管, 反转管 2-5 倍, 以混合, 并孵化37°c 在一个橱柜孵化器为15分钟。 注: 允许混合物冷却到室温, 然后再继续。</li>
	<li>添加200µL 的蛋白质沉淀溶液, 在二十年代高速涡旋, 然后离心机3分钟在 1.3万 x g 形成一个坚实的颗粒。当样品被离心, 添加600µL 室温异丙醇到一个新鲜的1.5 毫升离心管。</li>
	<li>使用1000µL 吸管, 小心地从离心样品中取出上清, 将其转移到含有异丙醇的新鲜1.5 毫升离心管。颗粒现在可能被丢弃。 注意: 避免用蛋白质污染上清液, 在颗粒上方留下少量的上清液。</li>
	<li>反转管直到 DNA 变为可见的一大类线程状股, 然后离心机在 1.3万 x g 1 分钟室温。</li>
	<li>醒酒上清液, 将70% 乙醇的600µL 添加到颗粒中, 多次反转管以清洗 DNA, 离心机在 1.3万 x g 处进行1分钟。</li>
	<li>小心地吸干乙醇, 留下松散的 DNA 颗粒, 然后打开并倒置管子, 搁在吸水性纸上。室温空气干燥15分钟。</li>
	<li>将100µL dna 补液溶液添加到干 dna 颗粒中, 然后在65摄氏度的水浴中孵化出1小时的 dna, 同时通过轻敲管进行混合。</li>
	<li>将1µL 的纯净水放在 microcuvette 上, 并将其插入分光光度计中用作空白。通过在 microcuvette 上放置1µL 样品, 并插入分光光度计, 计算出目前 DNA 的浓度。DNA 浓度 (ng/µL) 可以计算为 (260-320) x 稀释因子 x 50 ng/µL。 注意: 协议可以在这里暂停。样品可以储存在4摄氏度。</li>
</ol>2. 对基因组 DNA 样品进行 PCR 处理
<ol><li>结合10µL 2x PCR 大师组合 (参见材料表), 1 µL 前底漆 (下午10点/µL), 1 µL 反向底漆 (下午10点/µL) (见表 2为底漆序列), 100 ng 的基因组 DNA 提取的样本, 和纯净水 (最多20µL 最终体积) 在50µL PCR 管, 轻弹混合, 并简要离心机。</li>
	<li>将 PCR 管插入 thermocycler 并相应运行 (见表 1)。 注意: 协议可以在这里暂停。如有必要, 在反应完成后, 将样品贮存在4摄氏度。</li>
</ol>3. Electrophorese 琼脂糖凝胶中扩增的 DNA
<ol><li>重0.4 克琼脂糖粉, 并添加它连同1x 泰的缓冲器到一个干净的200毫升锥形瓶, 填补多达50毫升。</li>
	<li>微波在高为1.5 分钟, 摇晃每三十年代, 直到琼脂糖完全地被溶化。 注: 在处理液体琼脂糖时应佩戴绝缘手套以防止烫伤。</li>
	<li>添加2.5 µL 10 毫克/毫升溴溴化液琼脂糖和摇动混合, 然后倒入液琼脂糖凝胶成水平凝胶模具。快速插入标准的12牙凝胶梳后浇。</li>
	<li>允许凝胶在室温下冷却20分钟, 然后去除凝胶梳, 并将凝胶放入一个充满1x 泰缓冲器的水平凝胶盒中。</li>
	<li>使用微, 将总扩增的 DNA 样本加载到水井中。添加5µL 的 DNA 阶梯 (见材料表) 在一个单独的井从你的样品。</li>
	<li>Electrophorese 样品35分钟在恒定的 90 V, 然后去除胶凝体和地方在紫外 transilluminator。</li>
	<li>在302纳米紫外线透视的情况下, 对凝胶条纹图案进行照相和分析。</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>

丰泉、伊恩？帕默、建安、明昌、斯蒂芬·汤普森、刘和郑庆府宣告没有利益冲突。

至关重要的是, 所有的步骤都是完全遵循, 以达到最佳的实验条件。全叶组织在本示范期间使用;然而, 该协议可以修改为理论上包括任何类型的植物组织。此前, 研究人员从叶静脉组织中提取基因组 DNA, 而不是全叶组织, 并取得了类似的结果11。如果与柑橘FBOX基因相对应的带不出现, 结果无效, 在操作过程中可能存在一个或多个技术错误。常见的错误包括: 使用受污染或不适当灭菌的水、微小贴士或离心管;在 PCR 组合或基因组 DNA 提取过程中不添加一个或多个必要的成分;未能保持适当的 thermocycler 条件;使用超过其有效期的试剂或其它成分; 或未在琼脂糖凝胶中添加溴溴或另一种 DNA 染色;electrophoresing 放大的 DNA 在太高的电压或太长;使用旧的或不适当的混合泰缓冲器, 或简单地使用水代替泰的缓冲填充凝胶盒。
引物 LAA2_F 和 LAJ5_R 是从以前的研究中选择的, 因为他们成功地确定Candidatus Liberibacter asiaticum 和 africanum。这些引物放大核糖体亚基基因rplA和rplJ,产生一个放大 669 bp 片段从C.l. africanum 或 703 bp 片段从C.l. asiaticum9。本文从前人的研究中选择了引物 LSS_F 和 LSS_R, 其结果为C 的低假阴性率、准确度和特异性。asiaticum 湖LSS 底漆集在 C 中所有三 16S rDNA 基因中放大了500的 bp 序列.l. asiaticum6。FBOX 引物被构造为用作内部放大控制, 并放大400在柑橘植株中发现的 bp 序列。以往的研究表明, FBOX是稳定的表达在柑橘植物, 并将成为一个伟大的候选者作为参考基因, 当适应这一检测为 RT-qPCR10。
这种检测可用于任何类型的柑橘组织, 并可修改为使用其他 PCR 检测, 如 qPCR。这种方法的局限性很少, 大多数是常规 PCR 所固有的。这些限制因素包括: 在 PCR 混合物中存在乙醇或其他抑制性化学物质, 缺乏定量而不执行更先进的化验, 需要在较小的实验室 thermocycler, 以及存在 DNA 污染。
虽然这一检测的特异性并没有直接与更具体的 qPCR 方法进行比较, 但这一分析对于从事Candidatus liberibacter 研究的科学家和寻求诊断可能感染柑橘的农民是有用的。树在他们的领域, 特别是如果更具体的方法是不可用或成本效益。的确, 一个柑橘种植者或研究人员可以在几天内快速、廉价地测定数个样品, 方法是使用多通道吸管和几个96井板, 而不购买昂贵的 RT qPCR thermocycler。

柑橘的绿化, 也被称为黄龙, 已经造成了大量的柑橘树的损失。一个典型的案例是佛罗里达州, 预计该疾病将导致佛罗里达州橙色盒的生产减少 70%, 从1997至1998季的2亿4400万箱, 7000万盒 2016 2017 赛季1。佛罗里达州90% 以上的柑橘类植物感染了2, 据估计, 由于柑橘类疾病, 佛罗里达柑橘业每年损失约10亿美元, 其中柑橘绿化在3起主要作用。柑橘的绿化主要由侵袭性的亚洲柑橘木虱Diaphorina 桔传播,但也可以通过嫁接感染的组织传播.该病引起静脉发黄, 黄叶斑驳不对称, 过早落叶, 枝复生, 根腐, 植物死亡。重要的是, 这种疾病使柑橘类植物生产出的季节开花, 很少产生水果。被感染的柑橘类植物生产的水果是不成熟的, 绿色的和苦涩的品尝4。
该方法的目的是准确和准确地识别Candidatus liberibacter 菌的运动细菌, 其致病剂为柑橘的绿化, 生活在被感染的柑桔树的韧皮部内5。基因组 DNA 从整个叶组织中提取, 其中含有活细菌。这种提取的基因组 DNA 被用来作为常规 PCR 的模板, 其中寡核苷酸补充细菌的 16S rDNA 序列被用来放大这个序列。寡核苷酸互补的柑橘FBOX基因作为内部放大控制。我们选择使用这个方法, 因为它已经被证明是成功的在以前的研究6。
这种方法有明显的优势, 简单, 相对便宜, 并能够在任何设备正常的生物化学实验室进行。此外, PCR 仍然是最准确和最精确的方法, 以检测这种病原体, 由于难以培养这个病原体7, 和病原体生存和繁殖的能力在无症状的主机8年。许多不同的特异性 PCR 检测方法已经成功地发现了这种病原体;然而, 常规 PCR 仍然是最简单的检测, 特别是在无症状宿主8。

<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

柑橘类绿化, 又称黄龙, 是一种破坏性的柑橘病, 在全球范围内肆虐柑橘养殖场。这种疾病导致不对称的黄叶斑驳, 静脉变黄, 落叶, 根腐, 最终死亡的柑橘植株。在感染时, 柑橘类植物生长发育迟缓, 并在季节内产生花朵。这些花很少产生果实, 而那些产生小的, 苦涩的, 不规则形状的柑橘类水果是不可取的。这种疾病是由亚洲柑橘木虱、 Diaphorina 桔和被感染的柑橘组织嫁接而传播的。病原体在柑橘植株内有长而多变的潜伏期--有时在症状出现前数年。在体外培养这种病菌的尝试是不成功的, 可能是由于感染的柑橘组织中的病原体浓度低和不均匀, 或者因为很难复制有利于生长的环境条件。病原体。由于其潜伏期长, 研究人员无法培养病原体, 很难在病毒传播前发现疾病。因此, 这种疾病只在突然破坏柑橘农民的全部产量之后才变得明显。本文介绍了一种准确、特异地检测柑桔绿化病原体、 Candidatus liberibacter 的方法. 使用基因组 DNA 提取试剂盒和 PCR 技术。该方法简单、高效、成本效益高, 适用于定量分析。该方法可用于任何柑橘组织;然而, 它有可能受组织中的病原体数量的限制。然而, 这种方法将使柑橘养殖者能够及早识别受感染的柑橘类植物, 并遏制这种破坏性疾病的蔓延, 然后才能进一步扩散。

一个正面结果产生一个不同的波段对应于 500 bp 为Candidatus Liberibacter 胭脂Las606/Lss6和/或一个独特的带对应于 700 bp 为Laa2/Laj5,插入在16S 核糖体β操纵9。内部放大控制带也必须出现在 400 bp。这个波段对应于柑橘FBOX基因10的放大, 并且必须出现一个结果被认为是有效的 (图 1和 图 2)。500 bp 和 700 bp 波段的缺乏, 而 400 bp 波段存在代表一个负面的结果。在健康样品上进行 PCR 可产生约 700 bp 和 400 bp 的非特异带型;然而, 只有一个与这些分子量对应的加粗带表明了一个积极的结果 (图 1)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>操作</td>
			<td>临时。</td>
			<td>时间</td>
			<td>周期</td>
		</tr>
<tr>
<td>初始变性</td>
			<td>95°c</td>
			<td>3-5 分钟</td>
			<td>1</td>
		</tr>
<tr>
<td>变性</td>
			<td>95°c</td>
			<td>30秒</td>
			<td rowspan="3">35</td>
		</tr>
<tr>
<td>退火</td>
			<td>55°c</td>
			<td>30秒</td>
		</tr>
<tr>
<td>伸长</td>
			<td>72°c</td>
			<td>40秒</td>
		</tr>
<tr>
<td>最终伸长率</td>
			<td>72°c</td>
			<td>10分钟</td>
			<td>1</td>
		</tr>
</tbody></table>表1。Thermocycler 设置用于放大目标 16S rDNA 基因和柑橘 FBOX。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆名称</td>
			<td colspan="2">序列</td>
			<td>参考</td>
		</tr>
<tr>
<td>LAA2_F</td>
			<td colspan="2">5 '-达 AAA GGT TGA CCT TTT-3 '</td>
			<td>Hocquellet, et, 1999</td>
		</tr>
<tr>
<td>LAJ5_R</td>
			<td colspan="2">5 '-aca AAA 级 GAA ATA A-3 '</td>
			<td>Hocquellet, et, 1999</td>
		</tr>
<tr>
<td>FBOX_F</td>
			<td colspan="2">5 '-GAA 行动铁通 TCG 共同国家评估 CT-3 '</td>
			<td>这种化验</td>
		</tr>
<tr>
<td>FBOX_R</td>
			<td colspan="2">5 '-AGC GGC 达达动漫法案 G-3 '</td>
			<td>这种化验</td>
		</tr>
<tr>
<td>LSS_F</td>
			<td colspan="2">5 '-GGA GTG AGT GGA CCG A-3 '</td>
			<td>Fujikawa, et, 2012</td>
		</tr>
<tr>
<td>LSS_R</td>
			<td colspan="2">5 '-ACC 民航局 GGT AAA 瑞声 C-3 '</td>
			<td>Fujikawa, et, 2012</td>
		</tr>
</tbody></table>表2。引物的名称, 他们的 DNA 序列, 和参考。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57240/57240fig1.jpg"> 图1。四例感染柑桔叶组织样品和两种健康样本作为阴性对照的常规 PCR 结果分析。"gDNA" 表示从柑橘叶组织提取的基因组 DNA 样本。样品 gDNA 1 和 gDNA 4 是来自未感染的柑橘叶片样本;gDNA 2、3、5和6是Candidatus liberibacter asiaticum (课室) 感染的样本。DNA 是按照协议中的文字提取的。PCR 是使用提取的基因组 DNA 作为模板, 根据协议 (见表 1)。LAA2/LAJ5、LAS606/LSS和FBOX是指使用的底漆集 (见表 2)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57240/57240fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57240/57240fig2.jpg"> 图2。完整的凝胶照片三放大 gDNA 样品.样品1是健康的柑橘组织;样本2和3是被感染的组织样本。这些示例对应于 gDNA 1、2和 3, 如图 1所示。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57240/57240fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

柑橘类绿化是影响全球柑橘作物的一种特殊破坏性疾病。本文介绍了一种利用 PCR 和基因组 DNA 提取柑桔叶组织的简便方法, 对柑桔绿化病原体、 Candidatus liberibacter 进行准确、准确的鉴定。

利用常规 PCR 技术对柑桔叶片组织样品Candidatus liberibacter的特异和准确检测

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

Nanyang Technological University

Research

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 次观看.  University of South Carolina. 柑橘类绿化是影响全球柑橘作物的一种特殊破坏性疾病。本文介绍了一种利用 PCR 和基因组 DNA 提取柑桔叶组织的简便方法, 对柑桔绿化病原体、 Candidatus liberibacter 进行准确、准确的鉴定。

视频: 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.

比色纸为基础的检测<em&gt;大肠杆菌</em&gt;<em&gt;沙门氏菌</em&gt;属，和<em&gt;单核细胞增生李斯特氏菌</em&gt;从农业用水大容量

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

科研

环境科学

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.

双工数字PCR方法的同时定量<em&gt;肠球菌。</em&gt;和人的粪便相关HF183标记的水域

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.

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

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.

用便携式实时 PCR 系统进行土壤载体 Phytopathogens 的现场分子检测

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.

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

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.

用阴离子交换树脂检测 Bioaerosols 病毒

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.

CD8+ T 细胞活化的各向异性聚合物人工抗原呈现细胞的制备

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.

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

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.

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

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.

零售食品样品中丁酸二氮的浓缩和检测

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