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?...

<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>

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?...

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</s...

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 ...

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

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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

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 ...