Name: automating citrus budwood processing for downstream pathogen detection through instrument engineering
Uploaded: 2023-04-21T13:10:00
Description: Watch this Scientific Journal Video about Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering at JoVE.com

作者承认Cahuilla人是完成实验工作的土地的传统监护人。我们感谢加州大学河滨分校的Norman Ellstrand教授提供实验室空间，以便在UCR加州农业和食品企业（CAFÉ）倡议下为该项目开展研究活动。这项研究得到了CDFA-特种作物整笔拨款计划（拨款号18-0001-055-SC）的支持。CRB项目6100也提供了额外的支持;美国农业部国家食品和农业研究所，哈奇项目1020106;以及国家清洁植物网络-美国农业部动植物卫生检验局（AP17PPQS&T00C118，AP18PPQS&T00C107，AP19PPQS&T00C148和AP20PPQS&T00C049授予乔治斯·维达拉基斯。

1. 收集柑橘接穗样品进行运输
<ol><li>向技术演进解决方案发送树信息电子表格，以便他们加载到他们的 Web 服务器中（最终，用户将创建新树）。</li>
	<li>使用手机应用程序 TES 跟踪器 选择一棵树，并将近场通信 （NFC） 项圈标签握在手机上，以将树信息加载到标签中。</li>
	<li>将三到四个柑橘接穗样品插入兼容 BTE 的塑料袋托架中，并盖上盖子。<ol><li>确保接穗的长度不?...

<ol>
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随着HLB柑橘病的出现，为了减少损失，柑橘行业、监管机构和诊断实验室被敦促依靠高通量核酸提取方法与低通量手动样品处理和病原体检测分析相结合，如qPCR34 ，结合疾病管理实践35对单个树木进行测试35.加州的HLB阳性率从2012年的0.01%上升到2020年的1.2%。尽管qPCR是一种强大而可靠的病原体检测工具，但目前可用的技术不允许对足够数量的植物组织进行采样和?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65159">Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering</a>. The Authors section was updated from:
Deborah Pagliaccia1,6 
Douglas Hill2 
Emily Dang1 
Gerardo Uribe1 
Agustina De Francesco1 
Ryan Milton2 
Anthony De La Torre2 
Axel Mounkam2 
Tyler Dang1 
Sohrab Botaghi1 
Irene Lavagi-Craddock1 
Alexandra Syed1 
William Grover3 
Adriann Okamba4,5 
Georgios Vidalakis2 
1Department of Microbiology and Plant Pathology, University of California Riverside 
2Technology Evolving Solutions (TES) 
3Department of Bioengineering, University of California Riverside 
4Ecole Sup&#233;rieure d&#39;Ing&#233;nieurs L&#233;onard de Vinci ESILV 
5University of California Riverside 
6California Agriculture and Food Enterprise (CAF&#201;), University of California Riverside
to:
Deborah Pagliaccia1,6 
Douglas Hill2 
Emily Dang1 
Gerardo Uribe1 
Agustina De Francesco1 
Ryan Milton2 
Anthony De La Torre2 
Axel Mounkam2 
Tyler Dang1 
Sohrab Bodaghi1 
Irene Lavagi-Craddock1 
Alexandra Syed1 
William Grover3 
Adriann Okamba4,5 
Georgios Vidalakis1 
1Department of Microbiology and Plant Pathology, University of California Riverside 
2Technology Evolving Solutions (TES) 
3Department of Bioengineering, University of California Riverside 
4Ecole Sup&#233;rieure d&#39;Ing&#233;nieurs L&#233;onard de Vinci ESILV 
5University of California Riverside 
6California Agriculture and Food Enterprise (CAF&#201;), University of California Riverside

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65159">Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering</a>. The Authors section was updated.

Erratum: Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

柑橘的嫁接传播限制韧皮部病原体，如类病毒、病毒和细菌，在世界每个柑橘产区都造成了毁灭性的流行病和严重的经济损失。柑橘类病毒限制了生产因素，因为它们在经济上重要的柑橘类型中引起外皮质炎和恶病质疾病，例如三叶、三叶杂交种、柑橘、柑橘和橘子 1,2,3。在加利福尼亚州，这些对类病毒敏感的柑橘类型是“易去皮者”市场增长和盈利的基础，跟随消费者对易于去皮、分段和无籽水果的偏好变化趋势4,5,6。因此，柑橘类病毒受到加州食品和农业部（CDFA）“柑橘苗圃害虫清洁计划-参议院法案140”的监管，CDFA植物害虫诊断处的实验室每年进行数千次柑橘类病毒测试7,8,9,10.自 1930 年代全球流行病开始以来，柑橘 tristeza 病毒 （CTV） 已造成超过 1 亿棵柑橘树死亡 3,9,10,11。在加利福尼亚州，病毒的茎点蚀和三叶断裂抗性对价值 36 亿美元的加州柑橘产业构成严重威胁12,13,14。因此，CDFA将CTV归类为受管制的A类植物害虫，中加州Tristeza根除局（CCTEA）的实验室每年进行广泛的实地调查和数千次病毒测试15,16。据估计，细菌“亚洲念珠菌”（CLas）和黄龙冰病（HLB）对佛罗里达州造成了近90亿美元的经济损失，原因是柑橘种植面积减少了40%，柑橘业务减少了57%，并损失了近8,000个工作岗位17,18。在加利福尼亚州，假设HLB导致的柑橘种植面积减少20%，预计将导致8,200多个工作岗位流失，该州国内生产总值减少超过五亿美元。因此，柑橘病虫害预防计划每年花费超过 4000 万美元进行调查，以测试、检测和根除来自加利福尼亚州14、17、19、20 的 CLas。
管理柑橘类病毒、病毒和细菌的一个关键要素是使用经过病原体测试的繁殖材料（即接穗）进行树木生产。经过病原体测试的柑橘接穗是在采用先进病原体消除和检测技术的综合检疫计划中生产和维护的10,21。加州大学河滨分校的柑橘克隆保护计划 （CCPP） 每年测试来自新进口到该州和美国的柑橘品种以及柑橘接穗树的数千个接穗样品，以保护加州的柑橘并支持国家柑橘清洁植物网络的功能10,17,22.为了处理大量的柑橘检测，高通量、可靠且具有成本效益的病原体检测检测是CCPP 7,10,22等项目成功的基本组成部分。
虽然聚合酶链反应（PCR）等基于分子的病原体检测分析已显著提高植物诊断实验室的通量，但根据我们的经验，实施高通量方案的最关键瓶颈之一是植物组织样品处理步骤。对于柑橘来说尤其如此，因为目前可用于处理富含韧皮部的组织（如叶柄和接穗树皮）的方案是劳动密集型的，耗时的，并且需要昂贵且专业的实验室设备。这些方案需要在低温下手工切碎、称重、冷冻干燥、研磨和离心，以避免核酸降解8,23,24。例如，在CCPP诊断实验室，样品处理包括（i）手工切碎（6-9个样品/小时/操作员），（ii）冷冻干燥（16-24小时），（iii）粉碎（30-60秒）和（iv）离心（1-2小时）。该过程还需要专门的用品（例如，重型安全锁定管，不锈钢研磨球，适配器，刀片，手套）和多件昂贵的实验室设备（例如，超低温冰箱，冷冻干燥机，组织粉碎机，液氮低温，冷冻离心机）。
与任何行业一样，设备工程和流程自动化是降低成本、提高产量以及提供高质量、统一的产品和服务的关键。柑橘行业需要低成本的组织处理仪器，这些仪器需要最少的操作技能，因此易于转移到诊断实验室和现场操作，以实现快速下游病原体检测的高样品处理能力。技术演进解决方案（TES）和CCPP开发（即设计和制造）并验证（即用柑橘样品进行测试并与标准实验室程序进行比较）一种低成本（即消除了对专业实验室设备的需求）仪器，用于快速处理富含韧皮部的柑橘组织（即接穗），称为接穗组织提取器（BTE）。 如图1所示，BTE包括一个用于电源和控制的基本组件，以及一个用于处理柑橘接穗木的可拆卸腔室。BTE腔室由一个砂轮组成，专门设计用于从柑橘接穗中剥离富含韧皮部的树皮组织。切碎的树皮组织通过滑动端口快速喷射到含有提取缓冲液的注射器中，过滤，并准备好进行核酸提取和纯化，无需任何额外的处理或制备（图1）。BTE系统还包括无纸化样品跟踪应用程序和集成称重应用程序，可将样品处理信息实时记录在在线数据库中。
BTE系统将CCPP的实验室诊断能力提高了100%以上，并始终如一地生产出适合纯化高质量核酸和使用PCR测定法检测柑橘移植传播病原体的柑橘组织提取物。更具体地说，BTE将组织处理时间从每个样品的24小时减少到~3分钟，取代了成本超过60,000美元的实验室仪器（图2，步骤2-4），并允许处理更大的样品量。
本文介绍了BTE高通量柑橘树皮组织处理、核酸提取和病原体检测验证数据，包括分别来自CCPP Rubidoux检疫设施和Lindcove基金会设施的所有适当的阳性和阴性对照。我们还介绍了与当前实验室程序相比的吞吐量和处理时间变化（图2）。此外，这项工作为柑橘病原体检测实验室提供了详细的分步协议，并展示了BTE如何支持病原体清洁苗圃，调查和根除计划的功能。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65159/65159fig01.jpg"> 图 1：接穗组织提取器。 BTE包括一个用于电源和控制的基本组件，以及一个用于加工柑橘接穗木的可拆卸室。BTE腔室由一个砂轮组成，专门设计用于从柑橘接穗中剥离富含韧皮部的树皮组织。切碎的树皮组织通过滑口快速喷射到注射器中，过滤，并准备好进行核酸提取和纯化，无需任何额外的处理或准备。缩写：BTE = 接穗组织提取器。 <a href="https://www.jove.com/files/ftp_upload/65159/65159fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65159/65159fig02.jpg"> 图 2：传统手工切碎实验室程序与 BTE 处理之间的分步比较。 BTE 处理涉及高通量柑橘树皮组织处理、核酸提取和病原体检测。每个步骤的时间在括号中表示。 <a href="https://www.jove.com/files/ftp_upload/65159/65159fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.08&#34; Hex Trimmer line</td>
			<td>PowerCare</td>
			<td>FPRO07065</td>
			<td>Needed to replace blades.</td>
		</tr>
		<tr>
			<td>1 Hp, 8 gal air compressor</td>
			<td>California Air Tools</td>
			<td>8010</td>
			<td>Quickly dry chambers after rinsed</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Globe Scientific</td>
			<td>111558B</td>
			<td>Store sample in after swishing with syinges</td>
		</tr>
		<tr>
			<td>10 mL Syringe Set</td>
			<td>Technology Evolving Solutions</td>
			<td>TE006-F1-10A-G1000-E1</td>
			<td>Syringe material is cut into. 1 L bottle with guanidine thiocyanate buffer. WARNING - contains guanidine thiocyanate, hazardous waste service required - do not mix with bleach</td>
		</tr>
		<tr>
			<td>12&#34; Ruler</td>
			<td>Westcott</td>
			<td>&#8206;16012</td>
			<td>To measure trimmer line before cutting</td>
		</tr>
		<tr>
			<td>12% Sodium Hypochlorite</td>
			<td>Hasa</td>
			<td>1041</td>
			<td>Disinfects chambers after processing</td>
		</tr>
		<tr>
			<td>-20 C Freezer</td>
			<td>Insignia</td>
			<td>NS-CZ70WH0</td>
			<td>Store sample after processing</td>
		</tr>
		<tr>
			<td>4&#34; x 12&#34; plastic bags</td>
			<td>Plymor</td>
			<td>FP20-4x12-10</td>
			<td>Bags to hold branches during shipping. O-rings attach bag to BTE chamber to seal</td>
		</tr>
		<tr>
			<td>6&#34; Cotton Swab</td>
			<td>Puritan</td>
			<td>806-PCL</td>
			<td>Swab to remove clogs</td>
		</tr>
		<tr>
			<td>7 Gallon Storage Tote</td>
			<td>HDX</td>
			<td>206152</td>
			<td>Holds sodium hypochlorite solution to disinfect chambers and water to rinse chambers</td>
		</tr>
		<tr>
			<td>Air blow gun</td>
			<td>JASTIND</td>
			<td>&#8206;JTABG103A</td>
			<td>Directs air into the chambers at high pressure</td>
		</tr>
		<tr>
			<td>Black Sharpie</td>
			<td>Sharpie</td>
			<td>&#160;S-19421</td>
			<td>Mark 1.5 mL tubes so you can identify sample later</td>
		</tr>
		<tr>
			<td>Bottle Top Dispensor</td>
			<td>Brand</td>
			<td>Z627569</td>
			<td>Adjustable bottle top dispensor to dispense guandine into syringe</td>
		</tr>
		<tr>
			<td>BTE Chamber</td>
			<td>Technology Evolving Solutions</td>
			<td>TE002BB-A05-E1</td>
			<td>Used to process budwood. Includes O-rings, BTE Slide, slide plunger, drain valve, lid, blade set, and blade set removal tool</td>
		</tr>
		<tr>
			<td>Dish Soap</td>
			<td>Dawn</td>
			<td>57445CT</td>
			<td>Surfectant to improve sodium hypochlorite penetration into chamber</td>
		</tr>
		<tr>
			<td>Fume hood with hepa filter</td>
			<td>Air Science</td>
			<td>P5-36XT-A</td>
			<td>Fume hood with hepa filter (ASTS-030)&#160; to limit possible contamination and protect against chemical spills</td>
		</tr>
		<tr>
			<td>Insulated foam shipping container</td>
			<td>PolarTech</td>
			<td>261/J50C</td>
			<td>Insulated shipping container to ship samples on ice after they are collected</td>
		</tr>
		<tr>
			<td>Lab coat</td>
			<td>Red Kap</td>
			<td>KP14WH LN 46</td>
			<td>Lab coat to limit possible contamination and protect against chemical spills</td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>Microsoft</td>
			<td>Surface</td>
			<td>Wifi capable laptop to run TES GUI. Needed for initial setup and provides more indepth information about the tissue processing base</td>
		</tr>
		<tr>
			<td>NFC Capable Phone</td>
			<td>Samsung</td>
			<td>Galaxy S9</td>
			<td>Phone to download and use TES phone app</td>
		</tr>
		<tr>
			<td>NFC clip tag</td>
			<td>Technology Evolving Solutions</td>
			<td>TE005-Clip-E1</td>
			<td>Sample tag that can be linked with trees. Made to function with TES phone app</td>
		</tr>
		<tr>
			<td>NFC Collar Tag</td>
			<td>Technology Evolving Solutions</td>
			<td>TE005-Collar-E1</td>
			<td>Tag that is attached to a tree. Made to function with TES phone app</td>
		</tr>
		<tr>
			<td>Nitrile Gloves</td>
			<td>Usa Scientific</td>
			<td>3915-4400</td>
			<td>Gloves to limit possible contamination and protect against chemical spills</td>
		</tr>
		<tr>
			<td>Noise-Reducing Earmuff</td>
			<td>3M</td>
			<td>90565-4DC-PS</td>
			<td>Protect ears while operating air compressor and tissue processing base</td>
		</tr>
		<tr>
			<td>Polyurethane Recoil Air Hose</td>
			<td>FYPower</td>
			<td>&#8206;510019</td>
			<td>Attaches air gun to compressor</td>
		</tr>
		<tr>
			<td>Saftey glasses</td>
			<td>Solidwork</td>
			<td>SW8329-US</td>
			<td>Protect eyes for chemical and physical hazards</td>
		</tr>
		<tr>
			<td>Spray bottle</td>
			<td>JohnBee</td>
			<td>B08QM81BJV</td>
			<td>Spray bleach to deconatinate surfaces</td>
		</tr>
		<tr>
			<td>Tissue Extractor Base</td>
			<td>Technology Evolving Solutions</td>
			<td>TE001-A-E1</td>
			<td>System to process plant tissue. Needs BTE or LTE chambers to function. Includes power cable, blade adapter, and 8/32&#34; allen wrench</td>
		</tr>
		<tr>
			<td>Tissue Processing Base Weight Scale</td>
			<td>Technology Evolving Solutions</td>
			<td>TE003-A05-200g-01-E1</td>
			<td>200 g, 0.01 resolution weight scale that connects to tissue processing base to enforce weight ranges and/or link weights with sample. Includes scale, power cable, connection cable, 5ml syringe holder, tower air shield&#160;</td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td>EasyGoProducts</td>
			<td>B07WQDZGRP</td>
			<td>Needed to transport hazardous waste (guanidine thiocyanate) using a hazardous waste disposal service</td>
		</tr>
		<tr>
			<td>Wire Cutter</td>
			<td>Boenfu</td>
			<td>&#8206;BOWC-06002-US</td>
			<td>Wire cutters to cut trimmer line</td>
		</tr>
	</tbody>
</table>

automating citrus budwood processing for downstream pathogen detection through instrument engineering

移植传播、韧皮部限制的柑橘病原体，如病毒、类病毒和细菌，是造成全球毁灭性流行病和严重经济损失的原因。例如，柑橘tristeza病毒杀死了全球超过1亿棵柑橘树，而“念珠菌亚洲自由杆菌”使佛罗里达州损失了90亿美元。使用经过病原体测试的柑橘接穗进行树木繁殖是管理此类病原体的关键。加州大学河滨分校的柑橘克隆保护计划 （CCPP） 使用聚合酶链反应 （PCR） 测定每年测试来自柑橘接穗树的数千个样品，以保护加州的柑橘并为国家清洁植物网络提供清洁繁殖装置。柑橘病毒和类病毒的高通量分子检测的一个严重瓶颈是植物组织处理步骤。
正确的组织制备对于提取优质核酸和PCR测定的下游使用至关重要。植物组织在低温下切碎、称重、冷冻干燥、研磨和离心以避免核酸降解是时间和劳动密集型的，需要昂贵且专业的实验室设备。本文介绍了一种专门用于快速处理柑橘接穗木富含韧皮部的树皮组织的仪器的验证，称为接穗组织提取器 （BTE）。与现有方法相比，BTE 可将样品通量提高 100%。此外，它还减少了劳动力和设备成本。在这项工作中，BTE样品的DNA产量（80.25 ng / μL）与CCPP的手工切碎方案（77.84 ng / μL）相当。该仪器和快速植物组织处理方案可以使加利福尼亚州的几个柑橘诊断实验室和项目受益，并成为全球其他木本多年生作物组织处理的模型系统。

使用 BTE 加工的接穗柑橘组织进行 RNA 提取、纯化和质量，并评估组织处理时间 我们使用来自255棵代表性柑橘树的接穗样品进行此测试，以比较BTE与标准程序的RNA质量。样品由接穗组织提取器（BTE）（方案步骤4.1-4.6和图2，右侧，步骤1，步骤5和步骤6）处理或按照法规批准的柑橘接穗组织处理方法制备，该方法利用手工去皮和切碎，冷冻干燥，粉碎和树皮组织的离心，如Dang等人所述...

Watch this Scientific Journal Video about Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering at JoVE.com

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

我们设计、制造并验证了一种快速处理富含韧皮部的树皮柑橘接穗组织的仪器。与现有方法相比，接穗组织提取器（BTE）提高了样品通量，并降低了所需的劳动力和设备成本。

通过仪器工程自动化柑橘接穗加工，用于下游病原体检测

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious ...

所展示的技术使用低成本仪器快速处理富含韧皮部的柑橘组织，无需劳动密集型方案和专门的实验室设备。这样可以快速检测和管理柑橘类病毒、病毒和细菌的下游。这种方法可以提高检测服务的通量，提高质量和统一性，使柑橘产业受益，并可能有助于诊断和治疗苗圃和田间作业中的疾病。柑橘类植物受到病原体的感染，在世界范围内造成重大的经济损失。先进的病原体检测技术，如布穗组织提取器（BTE），对于生产用于大规模树木生产的病原体测试繁殖材料是必要的。虽然该方法经过柑橘样品的测试和验证，但可用于诊断实验室和许多其他作物的田间操作。执行此技术的初学者必须熟悉协议，严格遵循说明并耐心等待。首先，使用手机应用测试跟踪器选择一棵树，并将近场通信 （NFC） 项圈标签固定到手机，以将树信息加载到标签中。将三到四个柑橘接穗样品插入布穗组织提取器或BTE，兼容的塑料袋载体中，并用夹子将其关闭。按照文本中描述的步骤确保样品质量后，使用手机应用程序测试跟踪器扫描树的 NFC 项圈标签，并将其与样品袋上的 NFC 夹标签链接。在经过适当消毒的通风橱内，拨动组织提取器底座背面的开关。确保按下盒子左侧的开关顶部，然后等待，直到闪烁的绿色 LED 表示腔室已准备就绪。要制备BTE室，请使用O形圈将其固定在后喷嘴上，将空样品袋连接到腔室的背面。检查刀片是否有磨损或损坏迹象，例如刀片上的切口延伸到塑料中，或尖端的切口。确保刀片上的箭头与单锁符号对齐，并且腔室底部的空气释放转向 O 符号。将透明盖子放在腔室开口上。使用腔室右侧的轨道，将透明的BTE滑轨滑到腔室上，将腔室底部的锁推入滑轨。确保柱塞盖安装在滑块顶部。要将 BTE 腔室装载到 BTE 底座上，请将准备好的腔室放在底座上，BTE 底座喷嘴突出到腔室的背面。通过将NFC夹标签上的白色贴纸以缓慢的圆周运动移动到盒子右侧的Z轴上，直到黄灯开始闪烁，将样品加载到BTE底座上。将样品连接到底座后，确保样品袋上没有孔。然后将 O 形圈放在样品袋上，将其固定在 BTE 喷嘴的前面。要装载未使用的注射器套件，首先，撕裂注射器套件。然后将未使用的注射器放在秤塔上。当红灯开始闪烁或刻度显示零时，取出注射器组。从注射器上取下带有过滤器的柱塞，同时确保液体留在底部注射器中。将柱塞放在纸巾或塔上，不要接触任何表面。最后，通过将出口口压入注射器并将其旋转90度，将注射器连接到滑动出口。要开始处理接穗样品，请按顶部的黑色按钮启动机器。从袋子里拿一根接穗棒，把它穿过BTE喷嘴的顶部。用另一只手抓住腔室另一侧的接穗棒，慢慢地向下插入刀片。在旋转接穗的同时慢慢来回移动接穗。当听到轻微的嗡嗡声时，将树枝更深地移动到机器中以专注于韧皮部。完成所有分支的相同操作后，按顶部的黑色按钮停止处理，并等待指示灯再次开始闪烁黄色。要验证样品重量，请将注射器旋转 90 度并将其向下拉。将柱塞放回注射器上，然后将注射器放在塔上。秤自动检测样品后，绿色 LED 闪烁表示样品重量在适当范围内。如果红灯闪烁，表示样品重量过低，请重复样品处理。如果重量太高，请取出一些样品，黄色LED的闪烁会变慢。要使样品均质化，请从装有样品的注射器中取出柱塞。将液体推入注射器并将柱塞插入回去。推动柱塞通过缓冲液，植物汁液通过网状过滤器通过橡胶管进入空注射器。通过将缓冲液和植物汁液从一个注射器来回推到另一个注射器三到四次来混合样品，直到样品变成均匀的绿色液体。一旦充分均质化，将植物汁液样品推入没有网状过滤器的注射器中，并从中分离橡胶管和带有过滤器的注射器。然后将植物汁液样品从注射器中排出到两毫升无菌微量离心管中。使用永久性记号笔用袋号标记样品后，将它们储存在零下 20 摄氏度直至进一步使用。处理第 10 个样品后，对 BTE 可拆卸腔室进行消毒，绿色 LED 继续闪烁而不是变为蓝色。要拆卸腔室并清洁所有污染物，请拆下透明塑料盖、透明腔室滑块和腔室滑轨上的堵塞端口。然后将腔室底部的空气释放阀转到打开的挂锁标记。将腔室组件放入设置超声波清洗机后，将腔室放入浴槽中，盖子在其下方，以防止盖子漂浮。运行超声波清洗机15分钟。在将腔室组件移动到干燥站之前，在水浴中冲洗腔室组件至少 30 秒。要开始干燥，请将气枪与腔室上部开口的凹槽对齐，然后按下喷枪的扳机以分配空气约30秒。将气枪转向腔室的顶部喷嘴，按下扳机后，缓慢地跟踪每个喷嘴入口的三个完整圆圈。将气枪放在BTE的中心，从最内侧开始按住扳机并移动，直到气枪指向滑块。快速将空气流过整个腔室前后，以去除外部的地表水。BTE和柑橘组织处理常规实验室方案的比较表明，在两种情况下，通过测量260纳米吸光度确定的提取核酸浓度是相当的。BTE提取的核酸纯度为260和280纳米处的吸光度比，蛋白质污染低。通过RT-qPCR分析的靶向柑橘NADH脱氢酶基因mRNA的核酸完整性对于BTE和标准手动方案非常相似。传统的实验室程序需要将近 7 到 10 分钟的时间手工切碎每个样品，而组织处理需要更多时间，包括冷冻干燥、研磨和离心。然而，BTE在三分钟内处理了每个样本以进行核酸提取，包括制备、称量和清洁步骤。来自第一次BTE样品处理的72个健康样品中没有一个对测试的柑橘病原体产生扩增曲线，从而验证了柑橘组织的BTE处理。在第二次BTE样品处理中，使用两个引入混合感染样品，提取的核酸在第1批和第5批中显示出不同的柑橘病毒和类病毒的存在。然而，没有检测到头部之间的交叉污染，或假阳性或阴性结果。在加工过程中，将树枝放在一个地方太久可能会切入树枝太深，从而增加堵塞的机会，并收集无细胞纤维材料。一旦处理，该材料就可以用于大多数DNA和RNA提取和纯化方案，并且可以使用任何植物病原体的分子检测测定进行测试。开发布穗组织提取器为高通量区域和树林范围的筛选打开了大门。与叶组织提取器一起，它允许大规模处理以前被认为难以处理的植物组织，例如根和花梗。

Research

JoVE Journal

Bioengineering

Procedure for Lung Engineering

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

科研

生物工程

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

在180°弧形动脉试验段的二次流动结构下游的型号IV型支架失败的实验研究

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

蛋白质的MS检测快速酶法处理与流通型微反应器

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

破碎散装水凝胶并加工成颗粒状水凝胶，用于生物医学应用

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...