Name: field-deployable candidatus liberibacter asiaticus detection using recombinase polymerase amplification combined with crispr-cas12a
Uploaded: 2022-12-23T12:45:00
Description: Watch this Scientific Journal Video about Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a at JoVE.com

这项工作得到了国家重点研发计划（2021YFD1400805）、江西省重大科技研发计划（20194ABC28007）、江西省教育厅项目（GJJ201449）、江西省现代农业科学研究协同创新（JXXTCX2015002（3+2）-003）的资助。

1. CLas-DETECTR的构建
注意：CLas-DETECTR的构建是一个四步过程：溶液制备，柑橘总DNA分离，等温DNA扩增和结果可视化。CLas-DETECTR测定的示意图如图 1A所示。
<ol><li>溶液制备<ol><li>制备缓冲液A：20mM NaOH在6%PEG 200中。对于 100 mL，将 113 mg NaOH 和 6 g PEG 200 加入瓶中的 80 mL H2O 中。将瓶子在60°C的水浴中孵育，直到所有PEG 200溶解。然后，将 H2O 加入?...

<ol>
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本研究提出了一种快速便携的CLas检测方法，称为CLas-DETECTR，该方法结合了RPA和CRISPR-Cas12a系统。工作流程如图 1 所示。CLas-DETECTR 检测 CLas 具有特异性和灵敏度（图 2 和图 3）。此外，使用纽霍尔叶样品，CLas-DETECTR以与qPCR相同的灵敏度检测CLas（图4）。值得注意的是，检测结果可以通过独立于实验...

黄龙病（HLB）是全球最成问题的柑橘病害之一1。HLB 由韧皮部定植和挑剔细菌 Liberibacter 属引起，包括亚洲自由念珠菌 （CLas）、非洲乳杆菌和美洲乳杆菌 2。在中国和美国，最普遍的HLB相关物种是CLas，它通过亚洲柑橘木虱（Diaphorina citri）或通过嫁接传播3。柑橘树被CLas感染后，生长下降直至死亡2。柑橘叶感染CLas的常见症状是斑点斑驳，绿岛（小圆形深绿色点），较厚和革质叶子上凸起的软木脉，以及芽不均匀的黄芽2。此外，感染CLas的水果显得小而不平衡2。
由于没有柑橘品种对HLB有抗性，也没有治疗HLB的方法，因此预防HLB需要检疫和分离CLas阳性的柑橘树2，3。因此，早期发现对于监测和检疫至关重要，以防止CLas的传播并最大限度地减少经济损失3。此外，由于感染早期植物中CLas的滴度较低，因此需要进行灵敏的CLas检测3。在中国，CLas检测通常由某些经过认证的测试中心进行。但是，检测过程通常至少需要1周，并且检测费用昂贵。因此，为了帮助监测HLB发病率
已经应用了各种技术来诊断HLB4，5，6，7，8，9。聚合酶链反应 （PCR） 和定量 PCR （qPCR） 因其高灵敏度和特异性而成为最常用的 CLas 检测工具4，5。然而，这些技术严重依赖昂贵的仪器和高技能人员。此外，几种等温扩增方法，如环介导等温扩增（LAMP），由于其简单、快速和低成本，已被开发为传统PCR方法的有吸引力的替代方案8，9，10。然而，由于非特异性扩增信号，应用它们来准确检测CLas具有挑战性，这可能会导致假阳性结果。
RNA引导的CRISPR/Cas（成簇规则间隔短回文重复序列/CRISPR相关）基于核酸内切酶的核酸检测因其高灵敏度、特异性和可靠性而发展成为下一代分子诊断技术11，12，13，14。这些 CRISPR/Cas诊断技术依靠Cas蛋白的附带核酸酶活性来切割用荧光报告基因和寡核苷酸两端的荧光淬灭剂修饰的单链DNA（ssDNA），以及用于捕获释放的荧光报告基因的荧光检测装置11，12.由CRISPR RNA（crRNA）靶标双链体激活的几种Cas效应子的核酸酶活性可以不加选择地切割周围的非靶点ssDNA11。CRISPR-Cas12a（也称为Cpf 1）是一种2类V-A CRISPR / Cas系统，与Cas9相比具有多种优势，例如更低的失配耐受性和更高的特异性13。Cas12a/crRNA系统已被应用于人类病原体和植物病原体14，15，16，17，18的核酸的灵敏和特异性检测。因此，利用Cas12a/crRNA系统应能够准确灵敏地检测CLas的核酸。
单独的Cas12a在理论上不足以检测低水平的核酸。因此，为了提高其检测灵敏度，CRISPR-Cas12a检测通常与等温扩增步骤14、15相结合。重组酶聚合酶扩增 （RPA） 可在 37 °C 至 42 °C 的温度范围内实现灵敏、快速的等温 DNA 扩增19。
最近设计了一种名为DETECTR（DNA核酸内切酶靶向CRISPR 反式 报告基因）的检测平台，该平台将Cas12a的DNA酶活性与RPA和荧光读数相结合12 ，并已被证明可以检测具有更高灵敏度的核酸20。此外，阳性样品发出的荧光信号可以通过现场的手持式荧光检测设备进行观察。
由于我们用RPA扩增DNA，设计了针对CLas 21特异性的五拷贝nrdB（核糖核苷酸还原酶β-亚基）基因的crRNA，并采用了Cas12a蛋白的DNase活性，因此我们称之为CLas检测方法CLas-DETECTR。与现有的CLas检测方法相比，CLas-DETECTR具有快速、准确、灵敏、可部署的特点。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AxyPrep DNA Gel Extraction Kit</td>
			<td>Corning</td>
			<td>09319KE1</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Genomic DNA Extraction Kit</td>
			<td>Solarbio</td>
			<td>D1600</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>EnGen LbCas12a&#160;</td>
			<td>TOLOBIO</td>
			<td>32104-01</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>Ex Taq Version 2.0 plus dye</td>
			<td>TaKaRa</td>
			<td>RR902A</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld fluorescent detection device&#160;</td>
			<td>LUYOR</td>
			<td>3415RG</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole puncher&#160;</td>
			<td>Deli</td>
			<td>114</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate, MgOAc</td>
			<td>TwistDx</td>
			<td>TABAS03KIT</td>
			<td>UK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SCR</td>
			<td>10019718</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB buffer 3.1&#160;</td>
			<td>NEB</td>
			<td>B7203</td>
			<td>USA</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR strip tubes</td>
			<td>LABSELECT</td>
			<td>PST-0208-FT-C</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 200&#160;</td>
			<td>Sigma</td>
			<td>P3015</td>
			<td>USA</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR Max DNA Polymeras</td>
			<td>TaKaRa</td>
			<td>R045A</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-Load Purple 1 kb Plus DNA Ladder&#160;</td>
			<td>New England Biolabs</td>
			<td>N0550S</td>
			<td>USA</td>
			<td></td>
		</tr>
		<tr>
			<td>TB Green Premix Ex Taq II (Tli RNaseH Plus)</td>
			<td>TaKaRa</td>
			<td>RR820B</td>
			<td>China</td>
			<td></td>
		</tr>
		<tr>
			<td>TwistAmp Basic</td>
			<td>TwistDx</td>
			<td>TABAS03KIT</td>
			<td>UK</td>
			<td></td>
		</tr>
	</tbody>
</table>

field-deployable candidatus liberibacter asiaticus detection using recombinase polymerase amplification combined with crispr-cas12a

柑橘种植者早期发现亚洲 自由念珠 菌（CLas）有助于早期干预并防止疾病传播。本文介绍了一种用于快速便携的黄龙冰（HLB）诊断的简单方法，该方法结合了重组酶聚合酶扩增和荧光报告基因，该方法利用成簇规则间隔短回文重复序列/CRISPR相关12a（CRISPR-Cas12a）系统的核酸酶活性。该技术的灵敏度远高于PCR。此外，当使用叶样品时，该方法显示出与qPCR相似的结果。与传统的CLas检测方法相比，这里介绍的检测方法可以在90 min内完成，并且在不需要使用PCR机的等温条件下工作。此外，结果可以通过现场的手持式荧光检测设备可视化。

在这里，我们描述了一个便携式平台CLas-DETECTR，它结合了RPA和CRISPR-Cas12a系统，以在现场诊断HLB。CLas-DETECTR 的模式如图 1A 所示。
当来自HLB感染和未感染HLB的Newhall树的叶样品（图1B）通过PCR确认CLas的存在（图1C）时，在HLB感染的样品中观察到绿色荧光信号，但在HLB未感染的样品和阴性对照中没有（图1D）。<stron...

Watch this Scientific Journal Video about Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a at JoVE.com

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

本工作建立了一种基于重组酶聚合酶扩增结合CRISPR-Cas12a的快速、灵敏、便携的亚洲 自由念珠 菌检测方法。

使用重组酶聚合酶扩增结合CRISPR-Cas12a进行现场部署的亚洲 自由念珠 菌检测

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A ...

黄龙兵，简称HLB。这是一种毁灭性的柑橘病害。在大多数柑橘生产国，如中国和美国，HLB是由韧皮部限制性细菌Liberibacter asiaticus引起的，缩写为CLas。CLas的早期检测对于种植者促进早期干预和防止疾病传播非常重要。因此，我们开发了一种快速便携式HLB诊断的新方法，结合了重组酶聚合酶扩增和CRISPR-Cas12a系统。我们称之为CLas-DETECTR。我们平台的灵敏度远高于PCR。此外，当使用叶片样品时，它显示出与qPCR相似的结果。与传统的CLas检测相比，我们的方法可以在90分钟内完成，并且在等温条件下工作。结果也可以通过现场的手持式荧光检测设备进行可视化。CLas-DETECTR 包含四个步骤：溶液制备、柑橘总 DNA 分离、等温 DNA 扩增和可视化结果。此处显示了CLas-DETECTR测定的示意图。缓冲液A，溶液B和溶液C按照协议中所述制备。为了节省时间并使该方法适用于现场CLas检测，采用基于大肠杆菌聚乙二醇的方法获得用于DNA扩增的粗植物提取物。该工具中使用了柑橘叶。从叶子上打出几块叶盘。将叶盘放入 1.5 毫升的 Eppendorf 管中。然后，将200微升缓冲液A加入管中。用塑料棒手动研磨叶盘直至光滑。让管子不受干扰 10 分钟。随后将上清液应用于DNA扩增。对于等温DNA扩增，将步骤2.3中的1微L上清液和1微L乙酸镁加入溶液B中并充分混合。在实验室中，将它们在37度的简单培养箱中孵育15分钟。为了可视化结果，将步骤3.2中的10微升混合物加入溶液C中并充分混合。将它们在 37 度培养箱中孵育 60 分钟。戴上护目镜，通过手持荧光检测装置观察绿色荧光信号。为了测试CLas-DETECTR的特异性，根癌农杆菌GV3101，柑橘黄单胞菌亚种的第一个纯菌落。柑橘溃疡病的病原体柑橘和我们实验室分离的伯克霍尔德氏菌株1440用于提取其基因组DNA。然后将这三种细菌基因组DNA和CLas阳性的Newhall叶子基因组DNA进行CLas-DETECTR测试。为了测试CLas-DETECTR的灵敏度，使用PCR，CLas-DETECTR和qPCR检测一系列CLas DNA片段稀释液，如方案中所述，在特异性和灵敏度测试之后，使用CLas-DETECTR方法检测现场是否存在CLas。从中国江西甘南师范大学校园种质资源苗圃中生长的纽霍尔甜橙树收集的叶子样本。还应用了qPCR，一种通常在实验室中使用的令人信服的CLas检测方法。来自HLB感染和未感染HLB的Newhall树的叶子样本，其中CLas的存在在我们的实验室中得到证实，进行了CLas-DETECTR测试。绿色荧光信号在HLB感染的样本中被捕获，但在HLB未感染和阴性对照中没有被捕获。我们使用从其他细菌中提取的核酸确定了CLas-DETECTR的特异性。在CLas-DETECTR测定中，只有从CLas阳性Newhall叶中提取的gDNA显示出绿色荧光信号。从根癌农杆菌GV3101，柑橘黄单胞菌亚种和伯克霍尔德氏菌株1440中提取的gDNA没有，这意味着CLas-DETECTR可以特异性检测CLas。我们还使用协议中列出的一系列CLas DNA稀释液测试了CLas-DETECTR的灵敏度。PCR每兆升可检测201个拷贝。另一方面，CLas-DETECTR每兆升可以检测2.01个拷贝，这表明它可作为敏感的现场诊断。qPCR每兆升可检测0.201拷贝，Ct值高于36。因此，当使用稀释的CLas特异性扩增子时，CLas-DETECTR的灵敏度比传统PCR高两个数量级，比qPCR低一个数量级。最后，重新考察了CLas-DETECTR在现场样品中的可行性，并比较了其与qPCR的灵敏度。在数字中，我们看到。我们可以看到，当CLas浓度小于每兆升0.2拷贝时，Ct值高于36，这是一个非常低的浓度。15份样本中，qPCR检测的样本1、2、3、4、8、10、13和14的Ct值均小于36。观察到明显的绿色荧光信号。另一方面，当qPCR检测的样品6、7、9、12和15的Ct值未定时，未观察到绿色荧光信号。此外，当qPCR检测到的样品5和11的CT值高于36时，观察到微弱的绿色荧光信号。结果表明，我们的平台是现场HLB诊断的快速，强大和灵敏的工具。

Research

JoVE Journal

Biology

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Principles of Site-Specific Recombinase (SSR) Technology

Principles of Site-Specific Recombinase SSR Technology

特定地点的重组酶（SSR）技术的原则

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Molecular Evolution of the Tre Recombinase

TRE重组酶的分子进化

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

专访：HIV - 1前病毒DNA切除使用一个演进的重组酶

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

从细胞亚群特异性RNA提取使用激光捕获显微切割加上快速的免疫标记

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

一个定量聚合酶重组酶扩增法的发展与内部阳性对照

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

使用重组酶聚合酶扩增结合CRISPR-Cas12a进行现场部署的亚洲 自由念珠 菌检测

使用重组酶聚合酶扩增结合CRISPR-Cas12a进行现场部署的亚洲自由念珠菌检测