Name: localization and quantification of begomoviruses in whitefly tissues by immunofluorescence and quantitative pcr
Uploaded: 2020-02-08T12:00:00
Description: Watch this Scientific Journal Video about Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR at JoVE.com

这项工作得到了国家重点研究与发展计划（赠款号：2017YFD0200600）、中国农业研究系统专项资金（赠款号：CARS-23-D07）和比尔和梅林达·盖茨基金会（投资IDOPP1149777）的支持。).我们感谢吴建祥教授提供TYLCV CP抗体。

1. 白蝇、病毒、植物和病毒的获取
<ol><li>棉花上的后白蝇 （MEAM1）在温室的防虫笼中，温度为 26 ° 1 °C，相对湿度为 14：10：黑暗循环，60 ± 10% 相对湿度。</li>
	<li>基于白蝇线粒体细胞色素氧化酶I基因执行常规PCR，以确定白蝇种群的纯度。<ol>
<li>收集20只成年白蝇，并把它们单独转移到含有30μL的发合缓冲液（10 mM Tris，pH = 8.4，50 mM KCl，0.45%[wt/vol] 补间-20，0.2% [wt/vol] 明胶，0.45% [vol/vol] 诺?...

<ol>
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A., 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=A+sensitive+method+for+the+quantification+of+virion-sense+and+complementary-sense+DNA+strands+of+circular+single-stranded+DNA+viruses.">A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses.</a> Scientific Reports. 4, 6438 (2014).</li><li>Gotz, M., 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=Implication+of+Bemisia+tabaci+heat+shock+protein+70+in+Begomovirus-whitefly+interactions.">Implication of Bemisia tabaci heat shock protein 70 in Begomovirus-whitefly interactions.</a> Journal of Virology. 86 (24), 13241-13252 (2012).</li><li>Zhao, J., Chi, Y., Zhang, X. J., Wang, X. W., Liu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+whitefly+vesicle+associated+membrane+protein-associated+protein+B+in+the+transmission+of+Tomato+yellow+leaf+curl+virus.">Implication of whitefly vesicle associated membrane protein-associated protein B in the transmission of Tomato yellow leaf curl virus.</a> Virology. 535, 210-217 (2019).</li><li>Maluta, N. K., Garzo, E., Moreno, A., Lopes, J. R., Fereres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+yellow+leaf+curl+virus+benefits+population+growth+of+the+Q+biotype+of+Bemisia+tabaci+(Gennadius)+(Hemiptera:+Aleyrodidae).">Tomato yellow leaf curl virus benefits population growth of the Q biotype of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae).</a> Neotropical Entomology. 43 (4), 385-392 (2014).</li><li>Czosnek, H., Hariton-Shalev, A., Sobol, I., Gorovits, R., Ghanim, 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+incredible+journey+of+begomoviruses+in+their+whitefly+vector.">The incredible journey of begomoviruses in their whitefly vector.</a> Viruses. 9 (10), 273 (2017).</li><li>Wei, J., 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=Vector+development+and+vitellogenin+determine+the+transovarial+transmission+of+begomoviruses.">Vector development and vitellogenin determine the transovarial transmission of begomoviruses.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (26), 6746-6751 (2017).</li><li>Ghanim, M., Morin, S., Zeidan, M., Czosnek, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+transovarial+transmission+of+tomato+yellow+leaf+curl+virus+by+its+vector,+the+whitefly+Bemisia+tabaci.">Evidence for transovarial transmission of tomato yellow leaf curl virus by its vector, the whitefly Bemisia tabaci.</a> Virology. 240 (2), 295-303 (1998).</li><li>Xie, Y., 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=Highly+sensitive+serological+methods+for+detecting+tomato+yellow+leaf+curl+virus+in+tomato+plants+and+whiteflies.">Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies.</a> Virology Journal. 10, 142 (2013).</li><li>Arya, M., 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=Basic+principles+of+real-time+quantitative+PCR.">Basic principles of real-time quantitative PCR.</a> Expert Review of Molecular Diagnostics. 5 (2), 209-219 (2005).</li><li>Wei, J., 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=Specific+cells+in+the+primary+salivary+glands+of+the+whitefly+Bemisia+tabaci+control+retention+and+transmission+of+begomoviruses.">Specific cells in the primary salivary glands of the whitefly Bemisia tabaci control retention and transmission of begomoviruses.</a> Journal of Virology. 88 (22), 13460-13468 (2014).</li><li>Wang, L. 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在这里，我们描述了一种方案，用于通过免疫荧光和qPCR在其白蝇载体的组织中定位和定量果蝇病毒。解剖是白蝇组织中对病毒进行本地化和量化的第一步。白蝇的身体长约1毫米，这意味着组织非常小，很难解剖它们。此外，组织之间有很强的联系。例如，卵巢与细菌紧密结合，使其难以分离。在这里，考虑到不同组织的特点和位置，我们描述了不同的解剖方法。然而，需要大量的实践来快速准?...

在过去的几十年里，贝戈莫病毒（贝戈莫病毒属，家族双子病毒）已经给全世界许多蔬菜、纤维和观赏作物的生产造成了严重损害。贝戈莫病毒由白蝇贝米西亚·塔巴奇（赫米特拉：阿莱罗迪达）持续传播，这是一种复杂的物种，包含超过35个神秘的物种2，3。白蝇病毒可能直接或间接地影响白蝇的生理和行为，如粪便4、长寿4和宿主偏好5、6。此外，即使在相同的实验条件下，在相同的实验条件下，特定白蝇病毒物种/菌株的传播效率也因不同的白蝇神秘物种而异，这表明白蝇与白蝇之间存在复杂的相互作用。为了更好地了解白蝇-白蝇病毒相互作用背后的机制，在白蝇组织中对病毒进行定位和定量至关重要。
番茄黄叶卷曲病毒（TYLCV）是一种在以色列首次报告的果子病毒，但如今对全世界番茄生产造成了严重破坏。由于其经济重要性，它是研究最好的贝戈病毒13之一。像其他单部分体细胞病毒一样，TYLCV是一种单链圆形DNA病毒，其基因组大小约为2，800个核苷酸14。虽然仍在争论中，几行证据支持在白蝇15，16，17复制TYLCV。此外，TYLCV粒子和白蝇蛋白的相互作用已经报告6，18，19，20。对于病毒传播，白蝇通过喂食受病毒感染的植物获得TYLCV，病毒通过食物管到达食道，穿透中肠道壁到达溶血区，然后转移到主要的唾液腺（PSG）。最后，病毒通过唾液管沿着唾液管道进入植物噬菌体21。此外，几项研究表明，TYLCV能够从雌性白蝇的转传传给它们的后代22，23。换句话说，为了实现生产性传播，病毒必须克服白蝇体内的细胞屏障，才能从一个组织转移到另一个组织。在跨越这些障碍期间，白蝇和病毒蛋白之间很可能发生相互作用，这可能决定病毒的传播效率。
免疫荧光是蛋白质分布分析常用技术。抗体与其抗原结合的特异性是免疫荧光的基础。由于TYLCV的经济意义，针对TYLCV涂层蛋白的单克隆抗体已经开发出来，为病毒24的局部化提供了一种高度敏感的方法。定量PCR（qPCR）允许对核酸进行敏感和具体的定量。该技术最常基于水解探针（例如，TaqMan）或荧光染料（如SYBR绿色）检测。对于基于水解探针的qPCR，需要特定的探头，从而增加了成本。荧光染料qPCR更简单，更具成本效益，因为标记的放大里昂特异性杂交探头不需要25。到目前为止，一些研究已经使用免疫荧光和qPCR以及其他方法来研究复杂的果子病毒-白蝇相互作用。例如，Pan等人对白蝇组织中的病毒进行了qPCR和免疫荧光分析，发现白蝇物种亚洲II 1和东亚小1（MEAM1）之间传播烟草卷曲病毒的能力差异是由于该病毒能够有效地穿过亚洲的中肠道壁，而不是MEAM18。同样，虽然地中海（MED）白蝇可以轻易传播TYLCV，但它们不能传播番茄黄叶卷曲中国病毒（TYLCCNV）。使用PSG中病毒的免疫荧光检测对选择性传播进行了研究，结果表明TYLCCNV不容易穿过MED白蝇26的PSG。TYLCV CP和白蝇中古的自噬标记蛋白ATG8-II的免疫荧光共定位表明，自噬在抑制白蝇27的TYLCV感染中起着关键作用。
在这里，我们以TYLCV为例，描述了一种通过免疫荧光技术在白蝇中肠道、PSG和卵巢中定位的果子病毒的协议。该技术包括用原抗体和染料标记的二级抗体进行解剖、固定和孵育。荧光信号显示病毒蛋白在白蝇组织中的位置，然后可以在共聚焦显微镜下检测到。更重要的是，该协议可用于共同地化果病毒和白蝇蛋白。我们进一步描述了在白蝇中古、PSG、淋巴和卵巢中使用SYBR绿色qPCR对TYLCV进行定量的协议，该协议可用于比较不同白蝇组织样本中的病毒量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>MultiSciences</td>
			<td>F0001</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-ph&#233;nylindole (DAPI)</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>MultiSciences</td>
			<td>A3828</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System</td>
			<td>Bio-RAD</td>
			<td>185-5201</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 549-goat anti-mouse</td>
			<td>Earthox</td>
			<td>E032310-02</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Monoclonal antibody (MAb 1C4)</td>
			<td></td>
			<td></td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sangon Biotech</td>
			<td>B548119-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>TB green premix Ex Taq (Tli RNase H Plus)</td>
			<td>TaKaRa</td>
			<td>RR820A</td>
			<td>qPCR master mix</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Thermofisher</td>
			<td>A41182</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissuelyzer</td>
			<td>Shaghai jingxin</td>
			<td>Tissuelyser-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>BBI life sciences</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BBI life sciences</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization and quantification of begomoviruses in whitefly tissues by immunofluorescence and quantitative pcr

贝戈莫病毒（贝戈莫病毒属，家族双子病毒）由贝米西亚塔巴奇复合物的白蝇以持续、循环的方式传播。考虑到果虫病毒对全世界作物生产造成的广泛损害，必须了解果蝇病毒与其白蝇载体之间的相互作用。为此，在病媒组织中定位和定量病毒至关重要。在这里，以番茄黄叶卷曲病毒（TYLCV）为例，我们描述了一个详细的方案，通过免疫荧光在白蝇中肠道、原发唾液腺和卵巢中本地化果子病毒。该方法基于对病毒涂层蛋白、染料标记的二级抗体和共聚焦显微镜使用特定抗体。该协议还可用于共同本地化果病毒和白蝇蛋白。我们进一步描述了通过定量PCR（qPCR）对白蝇中肠、原发性唾液腺、淋巴和卵巢中的TYLCV进行定量的一个方案。使用专为TYLCV设计的引基，定量协议允许比较白蝇不同组织中的TYLCV量。所述协议对于白蝇和受病毒感染植物体内的果子病毒的定量可能有用。这些协议可用于分析白蝇中贝戈病毒的循环途径，或作为研究白蝇-白蝇-白蝇病毒相互作用的其他方法的补充。

此处使用B. tabaci复合体和 TYLCV 的 MEAM1 白蝇作为示例来描述这些过程。本手稿中描述的免疫荧光和病毒定量程序概述如图1所示。图2显示了PSG、中肠和卵巢中TYLCV和DAPI染色免疫荧光检测的代表性结果，表明TYLCV在PSG和中肠道中积累较多，卵巢中积累较少。图3显示了白蝇PSG、中古、淋巴和卵巢中24、48和72小时在TYLCV感染番茄上?...

Watch this Scientific Journal Video about Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR at JoVE.com

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

我们描述了一种免疫荧光和定量PCR方法，用于昆虫组织中果子病毒的定位和定量。免疫荧光方案可用于共定病毒和载体蛋白。定量PCR协议可以扩展，以量化整个白蝇体内和受病毒感染的植物中的病毒。

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

澄清白蝇病媒中病毒的空间性和数量有助于理解白蝇病毒与白蝇的相互作用，并有助于制定控制严重白蝇病毒病的新策略。这项技术的优点是，我们可以快速分离白蝇组织，联合本地化病毒和白蝇蛋白，并量化白蝇全身和受病毒感染的植物中的病毒。在适当的实验时间点，在获得病毒后，将冰麻醉雌性白蝇放入显微镜幻灯片上的一滴PBS中，并使用精细的针灸针在立体显微镜下打开白蝇的腹部。使用针头提取中针，将中针放入PBS的收集盘中。要收集 PSG，请将胸部部位的主体从头部附近的后侧分离，用针头固定白蝇穿过胸部。使用第二根针摇动白蝇，直到两个唾液腺从身体分离，并转移腺体到PBS的新菜。要收集卵巢，完全打开腹部，用针将卵巢与其他组织分离。使用移液器去除多余的组织，将卵巢转移到PBS的新盘中。要收集血与升样品，请将 10 微升 PBS 添加到新的显微镜幻灯片上，并将新的白蝇放入液滴中。使用细针灸针轻轻打孔在腹部。然后对腹部施加轻微压力，将血氧释放到缓冲液中，并收集液体的八微升样品。要在收获的白蝇PSG、中毛病或卵巢组织中可视化TYLCV定位，请将15至20个标本转移到一个玻璃3.5厘米的培养皿中，并吸进多余的缓冲液。将组织在一毫升4%甲醛中固定两小时，在室温下，每次洗涤一毫升新鲜TBS，然后仔细清洗样品三次，两分钟。上次洗涤后，用 0.5%Triton X-100 的 0.5% 的一毫升渗透样品 30 分钟，然后洗涤三次，每次洗涤用一毫升 TBST 进行证明。上次洗涤后，在室温下用一毫升1%牛血清白蛋白进行两个小时的非特异性结合，随后在TBST中进行三次洗涤，如证明。上次洗涤后，在四摄氏度下将细胞与感兴趣的主要抗体标记在四摄氏度下，防止光线。第二天早上，用TBST清洗样品三次，然后加入适当的二级抗体，在室温下保持两小时，防止光线。在孵育结束时，洗涤中组三次，将试样转移到干净的显微镜幻灯片中。在安装盖玻片和通过共和显微镜检查标记组织之前，尽可能多地取出缓冲液，用一滴 DAPI 染色核。对于 TYLCV 定量，在新鲜 PBS 中将收获的白蝇组织样本洗涤 2 到 3 次，并在 5 微升 PBS 中将 20 个中结、20 个 PSG 或 1 个卵巢转移到含有 25 微升解解的单个 PCR 管中。在每个管中加入五到七个直径两毫米的陶瓷珠子，并将样品研磨在均质器中。将一个八微升的血升样品加入新的PCR管中，并加入10微升的解菌，并进行彻底的混合。在65摄氏度下孵育所有样品一小时，随后在100摄氏度下孵育10分钟，并短暂离心。接下来，在定量PCR条管的小瓶中加入18微升主混合物，并将每个DNA样本的两微升添加到至少三个小瓶中。当所有样品都装好后，关闭管子并离心机将溶液沉淀在每瓶的底部。将小瓶加载到用于定量 PCR 的实时热循环器上，选择网络作为荧光染料，选择未知作为样品类型。然后将定量数据导出到电子表格中，以分析每个样品中病毒DNA的相对数量。在这里，显示了在白蝇 PSG、中古和卵巢中，DAPI 染色中的 TYLCV 的代表性图像。正如观察到的，TVLCV表明在PSG和中古体内比在卵巢中积累更多。在喂养受TYLCV感染的番茄24、48和72小时后，白蝇PSG、中古、血红和卵巢中病毒的定量表明，不同白蝇组织中病毒的数量增加，与采集获取期增加有直接关系。最好使用新鲜的白蝇，因为死昆虫会增加解剖的难度。按照这个程序，其他分析，如西方印迹和共免疫沉淀，可以进行，以回答有关病毒蛋白表达和病毒和载体蛋白相互作用的其他问题。

Research

JoVE Journal

Immunology and Infection

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

科研

免疫与感染

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

为马科动物疱疹病毒2诊断的呼吸道液体定量PCR检测方法的开发和验证

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

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

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

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

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

代谢图谱: 定量酶化学和组织化学法测定脱氢酶在细胞和器官中的活性

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Quantitative PCR of T7 Bacteriophage from Biopanning

以此 T7 噬菌体的定量 PCR 研究

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

在异种性鼠模型中的移植-无源疾病的诱导和评分,以及使用数字PCR对小鼠组织中人类T细胞的定量

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...