我们感谢 Gudmestad 博士在北达科他州立大学提供subterranea质粒 DNA, 华盛顿州立大学 Hanu 帕普博士提供华盛顿州立大学 RNA, 并在 PMTV 博士 Inglis提供 R . 华盛顿州立大学病菌。特别感谢杰里米朱厄尔博士在华盛顿州立大学提供评论的手稿和华盛顿州立大学 CAHNRS 通讯录像。这项研究得到了西北马铃薯研究财团和华盛顿州农业部的支持-专业作物块赠款计划 (批准不。K1764)。PPNS 0741 号, 农业、人力和自然资源科学学院植物病理学系, 农业研究中心, 孵化工程号。WNP00833, 华盛顿州立大学, 铂尔曼, 华盛顿州, 美国。

1. 利用便携式实时 PCR 系统对病原体进行现场分子检测
注意:请参见图 1。
<ol><li>基于磁性珠的 DNA 提取 注意:根据制造商的说明, 使用了一种基于磁性珠的 DNA 提取试剂盒 (从 Primerdesign 的例如)。所有试剂应贮存在室温下 (18-25 摄氏度)。一旦冻干蛋白酶 K (1 瓶) 暂停 (使用1瓶), 存储在-20 摄氏度。<ol>
<li>在微细中混合20-50 毫克的土壤样品和500µL 的样品准备溶液。 注意:土壤的比率: 准备解决方案是重要的, 因为混合在其他比率可能导致下游实验失败 (例如,由 PCR 抑制剂污染)。</li>
		<li>用一根小的不育杵研磨管子底部的泥土, 直到溶液多云。进一步暂停土壤微粒在解答通过摇晃微细并且让它站立, 不受干扰地, 让土壤微粒完全地安定 (典型地在5到10分钟之间)。</li>
		<li>将200µL 的清液转移到新鲜的微细中, 加入200µL 的溶解缓冲 (2 瓶: 盐酸胍溶液) 和20µL 蛋白酶 K (瓶 1)。</li>
		<li>通过反转管并在室温下孵化15分钟, 彻底混合裂解。 注意:如果在微细盖子上发现了裂解液, 请轻敲管子或使用离心机, 如果有的话可以从盖子上取下。</li>
		<li>将500µL 的结合缓冲/磁珠混合 (3 瓶) 添加到裂解样品中。混合吹打上下, 在室温下孵化5分钟远离磁性管架。 注意:在使用前一定要把珠子的溶液搅拌好, 以确保珠子从贮瓶中 aliquoted 均匀。</li>
		<li>将微细放在磁性管架上。等待至少2分钟或直到微细中的所有珠子附着在磁侧壁上。然后, 除去并丢弃所有的上清吹打。 注意:去除和吸上清液时请勿干扰磁化珠。DNA 已经被磁性珠子捕捉到了。</li>
		<li>将微细从磁管机架中取出, 加入500µL 洗涤 Buffer-1 (瓶 4: 高氯酸钠/乙醇溶液), 重复吹打重新悬浮珠, 直到珠子均匀分散。执行此洗涤步骤, 从样品中除去蛋白质和盐。让混合物坐三十年代。</li>
		<li>重复步骤1.1.6。</li>
		<li>将微细从磁管机架中取出, 加入500µL 洗涤 Buffer-2 (瓶 5: 高氯酸钠/乙醇溶液), 重复吹打重新悬浮珠, 直到珠子均匀分散。让混合物坐三十年代。</li>
		<li>重复步骤1.1.6。</li>
		<li>将微细从磁管机架中取出, 然后加入500µL 80% 乙醇 (瓶 6)。 注意:这一步骤是必要的去除残留盐的样品。<ol>
<li>重复吹打重新悬浮珠, 直到珠子均匀分散。让这个立场为10分钟, 偶尔混合反转。</li>
		</ol>
</li>
		<li>重复步骤1.1.6。</li>
		<li>空气干燥磁珠颗粒在室温下与微细盖打开10分钟。 注意:珠子应该从任何可见的残留乙醇中解脱出来, 但不能完全干涸。</li>
		<li>将微细从磁管机架上取出, 加入50-200 µL 洗脱缓冲器 (7 瓶), 重复吹打重新悬浮珠, 直到珠子均匀分散, 并让其站立三十年代。 注意:在上述步骤中, 将纯化的 DNA 从磁珠中释放到洗脱缓冲器中。</li>
		<li>将微细放在磁性管架上。等待至少2分钟或直到微细中的所有珠子附着在磁侧壁上。</li>
		<li>将现在包含纯化 DNA/RNA 的上清液转移到0.5 毫升微细中, 用于下游步骤。</li>
	</ol></li>
	<li>便携式实时 PCR 技术 注意:根据制造商的说明使用便携式 thermocycler 和 PCR 检测试剂盒 (请参阅材料表)。<ol>
<li>打开并运行与 thermocycler 相关的软件, 选择目标检测测试, 并将所有描述信息输入名称 & 详细信息、备注、示例、和测试数据输入字段。 注意:井 #1 和 #2 由软件为消极控制和正面控制分别指定。</li>
		<li>使用前准备 PCR 试剂。将主混合再悬浮缓冲器的500µL 转移到含有冻干母料的管中, 并通过反演混合好。将整个主混合转换为标记为引物/探针的褐色微细 (表 2)。<ol>
<li>帽子和摇动微细混合。必须进行彻底的混合, 以确保所有部件完全重新悬浮。在使用之前, 让这个混合物坐5分钟。 注意:使用后, 将反应混合在-20 摄氏度。</li>
		</ol>
</li>
		<li>通过将所制备的反应混合物的10µL 从上一步转化为0.2 毫升 PCR 管, 然后加入10µL 无菌核酸酶的去离子水, 准备一个负控制。</li>
		<li>通过将10µL 的准备好的反应组合从步骤1.2.2 转化为0.2 毫升的 PCR 管, 然后加入10µL 阳性控制模板, 准备一个正控制。</li>
		<li>对于每个样品, 将准备好的反应混合的10µL 从上一步转化为0.2 毫升 PCR 管, 然后添加10µL 从步骤1.1.16 中制备的样本 DNA。</li>
		<li>在步骤2.1.1 中所述, 将 thermocycler 的水井与其各自的 PCR 管中的内容进行装载。</li>
		<li>输入并确认所有必需的信息后, 选择开始运行, 然后选择以太网连接的仪器或 USB 驱动器。 注意:如果选择了 USB 驱动器选项, 则必须将运行文件保存在要与 thermocycler (例如F:\genesig) 一起使用的驱动器上。在将驱动器插入 thermocycler 后, 将立即开始运行。</li>
	</ol></li>
	<li>便携式实时 PCR 数据分析。
	<ol>
<li>运行完成后, 请使用与 thermocycler 相关的软件从 usb 驱动器打开运行文件 (. usb), 或通过单击结果直接查看软件中的运行结果。</li>
		<li>在分析结果之前, 保存已完成的运行以避免丢失数据。</li>
		<li>在结果选项卡中, 查看按示例分类的运行状态。 注意:可以在此选项卡中获取的数据是在示例中检测到的结果和复制编号的状态。</li>
		<li>单击详细信息选项卡以查看放大曲线。成功检测到目标后, 将显示目标和内部控件的 "量化周期" 值。 注意:这些值是在最终报告中计算出来的, 用于确定样本对目标是否为正数, 以及反应或 DNA 样本是否存在问题。</li>
	</ol></li>
</ol>2. 其他议定书
<ol><li>
基于实验室的替代 DNA 提取方法
	<ol>
<li>
CTAB-苯酚-氯仿基方法
		<ol>
<li>根据道尔方法18和 Dellaporta 方法19 (如前所述), 执行 CTAB 苯酚-氯仿基方法。</li>
		</ol>
</li>
		<li>
DNA 微型制备方法 注意:爱德华兹方法20的执行方式如下所示。<ol>
<li>添加500毫克的土壤, 其次是五1.4 毫米陶瓷珠和750µL 的爱德华兹缓冲 (200 毫米三, pH 8.0, 200 毫米氯化钠, 25 毫米 EDTA, 0.5% SDS) 到一个微细和混合良好。</li>
			<li>孵化的微细在65°c 5 分钟。</li>
			<li>六十年代 (或使用灰浆和杵) 融汇样品与一个珠子搅拌器均质机。</li>
			<li>离心样品在 1.4万 x g 5 分钟。</li>
			<li>将500µL 的清液转移到新鲜的微细, 然后与500µL 的冷冻异丙醇混合。混合通过反转管10次。</li>
			<li>离心样品在 1.4万 x g 15 分钟 pelletize DNA。</li>
			<li>醒酒上清, 让 DNA 颗粒在室温下干燥, 直到剩余的乙醇蒸发。</li>
			<li>用750µL 冷冻70% 乙醇清洗 DNA 颗粒。</li>
			<li>在50-100 µL 的 TE 缓冲器 (10 毫米三 HCl, pH 8.0 和1毫米 EDTA) 重新悬浮之前, 空气干燥颗粒。</li>
		</ol>
</li>
		<li>
其他替代方法
		<ol>
<li>根据制造商的指示, 使用试剂盒 #1 (MP 生物快速 dna 自旋) 和试剂盒 #2 (Zymo BIOMICS dna Miniprep 套件) 进行二氧化硅基 DNA 提取。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
传统的基于实验室的实时 PCR 技术。 注意:传统的 thermocycler 与 mastermix 用于探针 PCR, 各种引物和寡核苷酸探针 (表 2)。<ol>
<li>使用非透明的底 pcr 管或 pcr 板, 准备20µL 反应的所有 DNA 样品进行分析, 以及负控制 (核酸酶无离子水) 和一个积极的模板控制内部准备。</li>
		<li>对于每一个 PCR 管或好, 准备一个混合物, 包括10µL 的 mastermix, 7 µL 无核酸酶去离子水, 2 µL 2 µM 底漆/探针, 1 µL DNA 样本 (从步骤 1.1.16, 或 2.1) 或控制模板, 每样。</li>
		<li>通过选择合适的 pcr 程序, 关闭 pcr 管或板, 开始反应。</li>
	</ol>
</li>
	<li>
基于实验室的常规实时 PCR 数据分析
	<ol>
<li>使用 thermocycler 相关软件分析常规 thermocycler 的结果。要开始数据分析, 请将运行文件从 thermocycler 传输到 usb 驱动器, 插入 usb 驱动器并选择导出。</li>
		<li>从 thermocycler 关联的软件中导出的运行中打开数据文件 (. 梁伯韬)。</li>
		<li>通过在仪器上定位其相应的井来突出样品的井。放大曲线和标准曲线 (如果适用) 将自动生成。如果未输入示例信息, 请单击 "板安装" 或类似函数, 在分析数据之前开始输入数据。</li>
		<li>查看 "量化" 选项卡上的数据;这可以通过第三方软件 (如 CSV、XML 或 HTML 文件读取器) 导出以进行数据分析。</li>
		<li>根据确定的阈值获取重庆数据, 并将其与正负控制进行比较。 注意:如果将靶的 DNA 标准用于检测, 则将样本的数据与标准的样品进行比较, 确定其截止</li>
	</ol>
</li>
</ol>

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作者声明没有竞争的财政利益。

如表 1所示, 最近在病原体分子鉴定方面的技术进展提高了诊断的有效性、准确性和速度, 这有助于检测症状性感染的27。对于现场诊断, 灯和侧流方法经常使用, 因为它们是便携的, 并以较低的成本提供立竿见影的结果。然而, 就血清学方法而言, 特定物种的检测很难实现。这偶尔地导致 misdetection 的非目标微生物例如普通土壤居民。例如, 在疫霉和禾草的血清学检测之间可能存在交叉反应性. 在马铃薯病原体28的情况下, 表明有时难以检测目标植物病原菌.
在本研究中, 我们通过与传统的基于实验室的实时 pcr 系统的性能比较, 开发了一种利用便携式实时 pcr 系统对土壤传马铃薯病原体进行现场分子检测的优化协议。我们发现, 现场方法专门检测到土壤样品中的马铃薯病原体, 尽管灵敏度比实验室化验的结果低10倍。同样值得考虑的是, 在这种情况下, 实验室和田间试验都没有使用生物相关的样本大小。在常规筛选田间土壤时, 需要大量的样本大小, 如前所述的29、30, 其中的样本大小介于250克到1公斤之间, 虽然这些方法需要熟练的操作员和复杂的提取 DNA 的设备。通常, 一个大规模的土壤 DNA 提取物取自一个单一的骨料土壤样本代表的众多标本超过1至4公顷6,29,30。然而, 在这里开发的协议是快速的, 易于使用的用户没有任何经验的分子诊断, 可以在实验室以外使用。与大规模土壤萃取相比, 该方法速度快, 相对便宜, 可用于筛查从相似取样区采集的许多小样本样品到大型骨料样品。这可以克服一些小样本大小的缺陷, 并确定有关该领域病原体空间分布的补充信息。此外, 该方法的可移植性和速度意味着它也可以用于示范活动, 以供教育和参与的目的种植者。
另一项考虑是, 许多实时 PCR 检测已经发布了广泛的植物病原体5。该系统可以利用这些现有的测试, 而无需设计新的灯引物, 以使现场试验。对灯具检测的经常批评是, 它们可能很难设计31。因此, 便携式 PCR 技术允许相对容易地实施多种现成的病原体测试, 供现场测试使用。
传统的方法往往成本高昂、费力、不准确和耗时。我们开发的现场方法的简单性使种植者和工业工人能够自己进行病原体检测, 而且可能产生的结果比送往诊断实验室的速度要快得多。便携式 PCR 方法的及时性和灵敏度可以帮助种植者避免潜在的继发感染, 从而进一步增加病原体的数量和无意传播 (通过设备或人类)。总之, 本研究开发的现场方法能够准确、相对敏感地检测出田间重要的土壤传播病原体。我们的希望是, 本研究开发的现场方法将在当前的诊断管道 (图 6) 中作出贡献, 不仅通过对有关田间植物病害的流行病学问题提供快速准确的答案, 而且还提供加深对植物病原体生物学和流行病学的了解。

准确快速地鉴定致病病原体对植物病害管理的决策有重大影响。土壤传播疾病的诊断特别困难, 因为土壤环境非常大, 相对于植物质量和复杂, 使它成为一个挑战, 了解所有方面的土壤传播疾病。此外, 土壤传播的疾病可以症状在早期感染阶段, 依赖于环境压力, 和一些长期潜伏期, 导致延迟诊断1。许多土壤传播的病原体已经形成了生存结构, 如专门的孢子或 melanized 菌丝, 即使在主人缺席的情况下, 在土壤中也能存活多年。应用于土壤传播疾病管理的方法包括: 避免已知的出没领域, 使用无菌鉴定的种子和幼苗, 保持设备的卫生, 并在可能时限制土壤和水的运动。通过分子检测策略了解病原体的存在也可以发挥有益的作用, 及时作出有关早期治疗的决定或对田间植物进行前期评估。现场测试提供了额外的优势, 提供了一个快速的结果, 而不发送样本到一个诊断实验室, 也许有些距离, 也可以接触种植者, 如果这样的诊断是在他们的存在 "现场"。
对于基于分子检测的现场诊断, 灵敏度、特异性、鲁棒性 (重复性和再现能力) 和效率 (i. e, 简单性和成本性能) 是考虑的关键因素。侧向流动装置 (LFDs), 如 Immunostrip 和 PocketDiagnostic, 是现场病原体检测的常用方法, 因为它们的简单性为一步测定。然而, LFDs 可能不是正确的诊断工具在所有情况下, 因为他们缺乏敏感性和特异性, 并且偶尔地提供模棱两可的结果, 如果目标病原体是在低浓度并且能与相似的种类或属的交叉反应2. 环介导的等温放大 (灯管) 也适用于现场病原体检测, 由于低成本试剂、反应条件保持不变和简单的色度直观分析, 尤其便宜。然而, 无论是 LFDs 还是灯都是定性使用的, 尽管这两种方法都可以用更昂贵的设备3来定量使用。聚合酶链反应 (PCR) 提供了高特异性, 高灵敏度, 和定量能力相比, 上述检测方法。然而, 传统的基于实验室的 PCR 技术需要昂贵的实验室设备和熟练的人员, 这在采用这种技术作为现场检测方法方面是一个主要的劣势。
在该协议中, 演示了一种使用便携式实时 PCR 仪的现场诊断方法。实时 PCR 技术比其他方法在定量准确性、灵敏度和通用性方面具有优势, 并已广泛用于检测各种植物病原体4,5, 包括各种土壤中的马铃薯病原体6。由于最近发展迅速、竞争激烈的市场趋势, PCR 技术所需的设备继续发展得更紧凑、成本更低7。该协议由以下步骤组成: 磁性珠基核酸提取、便携式实时 PCR (荧光探针法) 和定量数据分析, 可以用膝上型计算机远程完成 (图 1)。
利用本发明的便携式 PCR 协议, 对土壤样本进行了分析, 以检测土壤源性致病菌, Spongospora subterranea。选择了Spongospora , 因为它是一种重要的马铃薯病原体, 是粉状痂的致病剂8。近几十年来, 这种疾病的存在被认为已经扩散到许多地方, 在这些地区, 马铃薯被种植9,10。粉状的痂, 通过存在的粉刺如在块茎上的病变可能会造成相当大的质量损失的马铃薯种植者。此外, subterranea还可以传染马铃薯拖把顶病毒 (PMTV), 这可能会导致块茎中的内部病变症状 (称为 spraing)11,12。因此, 了解在种植6之前字段中是否存在subterranea是很重要的。我们还演示了该协议用于检测纹枯病菌吻合组 3 (AG3) 和 PMTV 的有用性。虽然一些吻合组的纹枯病菌导致马铃薯疾病, AG3 可以说是世界上最重要的13, 导致茎溃疡和黑色屑导致可销售的产量损失高达 30%14。PMTV 导致块茎内坏死性病变, 通常称为 spraing。最近在太平洋西北部的几个州首次报告了这种病毒15、16、17, 并对这个重要的马铃薯种植区的种植者越来越关注。本研究除了确定便携式 PCR 对这些重要疾病的有效性外, 还对最佳 DNA 提取方法和土壤样本大小进行了调查。
结果表明, 该方法具有通用性, 适用于不同病原体的检测。我们开发的现场检测方法可以让农业前线工人 (例如,种植者) 及早作出有关疾病管理的决定, 如品种选择或轮作, 并能量化样本中植物病原体。在实地调查期间, 在种植之前, 避免潜在的疾病暴发。

<table><tbody><tr><td>White shell PCR plate</td><td>Bio-Rad</td><td>HSP9601</td><td></td></tr><tr><td>CFX Manager</td><td>Bio-Rad</td><td>1845000</td><td></td></tr><tr><td>SsoAdvanced Universal Probes Supermix&amp;nbsp;</td><td>Bio-Rad</td><td>1725280</td><td></td></tr><tr><td>CFX96 Touch qPCR instrument</td><td>Bio-Rad&amp;nbsp;</td><td>1855195</td><td></td></tr><tr><td>Ethylalcohol, pure 200 proof</td><td>Decon Laboratories</td><td>04-355-222</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid (EDTA)</td><td>Fisher BioReagents</td><td>BP118-500</td><td></td></tr><tr><td>Phenol, Saturated&amp;nbsp;</td><td>Fisher BioReagents</td><td>BP1750I-400</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Tris)</td><td>Fisher BioReagents</td><td>BP152-5</td><td></td></tr><tr><td>q16 real-time PCR machine</td><td>genesig</td><td>MBS486001</td><td></td></tr><tr><td>Ultrapure Sodium Dodecyl Sulfate (SDS)</td><td>Invitrogen</td><td>15525-017</td><td></td></tr><tr><td>2-Propanol (Isopropanol)</td><td>JT Baker</td><td>67-63-0</td><td></td></tr><tr><td>Chloroform</td><td>JT Baker</td><td>9180-03</td><td></td></tr><tr><td>Hydrochloric Acid, 36.5-38.0% (HCl)</td><td>JT Baker</td><td>9535-03</td><td></td></tr><tr><td>iso-Amyl Alcohol</td><td>JT Baker</td><td>9038-01</td><td></td></tr><tr><td>FastDNA Spin kit&amp;nbsp;</td><td>MP Biomedicals</td><td>6560-200</td><td>Silica-based DNA extraction kit #1</td></tr><tr><td>Luna Universal One-Step RT-qPCR Kit</td><td>New England Biolabs</td><td>E3005S</td><td></td></tr><tr><td>0.1ml Low Profile Individual PCR Tubes</td><td>Phenix Research</td><td>MPC-100LP</td><td></td></tr><tr><td>Easy DNA/RNA Extraction Kit&amp;nbsp;</td><td>Primerdesign</td><td>genesigEASY-EK</td><td></td></tr><tr><td>genesig Easy 1.5 mL tubes</td><td>Primerdesign</td><td>genesigEASY-1.5</td><td></td></tr><tr><td>Magnetic tube rack</td><td>Primerdesign</td><td>genesigEASY-MR</td><td></td></tr><tr><td>Spongospora subterranea f. sp. subterranea genesig Easy Kit</td><td>Primerdesign</td><td>Path-S.subterranea-EASY</td><td></td></tr><tr><td>Hexadecyltrimethylammonium bromide (CTAB)</td><td>Sigma-Aldrich</td><td>H6269-100G</td><td></td></tr><tr><td>Pellet pestles</td><td>Thermo Fisher Scientific</td><td>12-141-364</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Thermo Fisher Scientific</td><td>7647-14-5</td><td></td></tr><tr><td>DNA Miniprep kit&amp;nbsp;</td><td>ZymoBIOMICS</td><td>D4300</td><td>Silica-based DNA extraction kit #2</td></tr></tbody></table>

on-site molecular detection of soil-borne phytopathogens using a portable real-time pcr system

对植物病害进行现场诊断可以成为种植者及时作出决定的有用工具, 以便及早实施疾病管理战略, 减少疾病的影响。目前在许多诊断实验室中, 聚合酶链反应 (pcr) 特别是实时 pcr 技术被认为是植物病原体检测最敏感、最准确的方法。然而, 基于实验室的 PCRs 通常需要昂贵的实验室设备和熟练的人员。在本研究中, 利用马铃薯的土壤传播病原体, 证明了现场分子检测的潜力。这是使用快速和简单的协议, 包括磁性珠基核酸提取, 便携式实时 PCR (荧光探针为基础的检测) 实现。便携式实时 PCR 方法与基于实验室的系统相比比较有利,从Spongospora subterranea中检测到的 DNA 只有100份。在这里开发的便携式实时 PCR 方法可以替代实验室的方法和一个有用的现场工具的病原体诊断。

DNA 提取方法的比较
通过检测土壤样本中的S. subterranea dna 的数量, 从病原体上的田间标本中测定了磁性珠基 dna 提取方法与实时 PCR 的相容性。如补充图 1所示, 将磁珠法与其他方法进行比较, 其中包括基于 CTAB 苯酚-氯仿的方法18、快速 DNA 小制备方法19、20和其他标准硅基 DNA 提取试剂盒。采用六种不同方法提取的 DNA 样本, 均接受常规的基于实验室的实时 PCR。结果表明, 磁珠基方法与其它方法相比较, 尽管硅基 DNA 萃取试剂盒在我们测试的方法中表现出最佳的性能。所有套件含有胍硫氰酸盐或盐酸胍: 两者都是强大的 chaotropic 剂, 变性大多数的细胞蛋白, 包括 RNases 和 DNases。因此, 使用该方法既适用于 DNA, 也适合 RNA 提取。
便携式实时 pcr 与常规实验室实时 pcr 的比较研究
为了将便携式 pcr 的灵敏度和特异性与常规的实验室 pcr 方法进行比较, 使用不同数量的subterranea对病原体 DNA 进行绝对量化, 该基因由 pGEM 向量21进行..使用 SsTQ 引物/探针集0分析了其基因的一系列10倍稀释 (106到 1022拷贝)。结果表明, 该方法检测了靶向病原体 DNA (100 份), 但灵敏度比传统的基于实验室的 pcr 方法低10倍, 检测到至少10份 (图 2)。
为了进一步验证, 试验了人工侵染的土壤。subterranea sporosori 是从马铃薯根部的粉状赤霉病根瘿获得的。土壤 sporosori 悬浮在最后浓度 105 sporosori/克土壤干重。利用磁性珠基方法, 从受侵土样品中提取 DNA, 并制备10倍的串行稀释, 以获得相当于 105、104、103、102、101和 100 的浓度。sporosori 土壤干重。DNA 样本用于 PCR, 使用的是 "23"。结果表明, 便携式 pcr 方法对传统的基于实验室的 pcr 方法具有比较强的分析能力, 但同样, 灵敏度降低了10的因子 (图 3)。
最后, 我们从一个自然污染了subterranea的字段中测试了一个土壤样本。以磁性珠为基础, 对不同数量的土壤 (10、20、50和100毫克每500µL 的萃取缓冲液) 进行了 DNA 提取。结果表明, 土壤作为 DNA 提取起始材料的最佳重量为50-100 毫克 (图 4)。土壤量超出范围导致下游 PCR 步骤失败。这种效应可能是因为当过量的土壤用作起始材料时, 污染物 (例如, 酚醛化合物) 会干扰 PCR24。在土壤体积较小的情况下, 提取的 DNA 量可能低于 PCR 检测的限度 (例如, 10-20 毫克土壤中提取的总 dna 的产量变化)。便携式 pcr 和常规 pcr 方法的灵敏度相当。不同提取方法在 DNA 样品中得到相似的结果 (补充图 2)
用便携式实时 PCR 技术检测其他病原体的现场检测系统
我们测试了便携式 PCR 方法检测其他重要的土壤传播的马铃薯病原体, R. 病菌AG3 和 PMTV。在本研究中, 我们使用 RsTq primers/RQP1 探针集25对R. 病菌AG3 检测与纯文化中的 DNA 进行实时 PCR。我们还进行了实时 PCR 使用 PMTV 底漆/探针集26 PMTV 检测与 RNA 从一个 spraing 症状块茎样本使用。如图 5所示, 便携式 PCR 方法成功地检测到这两种病原体。结果可与便携式和常规仪器相比较, 表明便携式 pcr 方法具有通用性, 适用于其他病原体检测, 如果为实时 PCR 设计的底漆序列可用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56891/56891fig1.jpg"> 图 1。一种便携式实时 PCR 系统在现场病原体检测中的程序。该协议由以下顺序组成: 用简单均匀化 (A)、磁性珠基核酸萃取 (B)、便携式实时 PCR (C) 以及使用便携式计算机 (D) 进行定量数据分析的裂解制备方法。请注意, 所有步骤都可以在站点上完成。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56891/56891fig2v2.jpg"> 图 2.便携式 pcr 与常规实验室 pcr 方法的灵敏度比较.利用 SsTQ 引物/探针集, 使用不同数量的subterranea其基因 (106到 100拷贝) 对病原体 DNA 进行量化。在常规 thermocycler (A) 和便携式 thermocycler (B) 中, subterranea质粒 DNA 的对数值与重庆的倒数对数值之间的线性回归。<a href="/files/ftp_upload/56891/56891fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56891/56891fig3v2.jpg"> 图 3.人工侵染土壤中检测性能的比较与 subterranea.土壤人为地被侵扰了 (105到 100 sporosori 土壤的干重) 与S. subterranea sporosori 悬浮物。利用磁珠基方法, 从受侵染土壤样品中提取 DNA。用 PCRs 的土壤样品进行了试验。sporosori 每克土壤起始量的测井值与传统 thermocycler (A) 和便携式 thermocycler (B) 的倒数对数值之间的线性回归。<a href="/files/ftp_upload/56891/56891fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56891/56891fig4v2.jpg"> 图 4.DNA 提取中土壤样品起始量的比较.采用磁珠法对10、20、50、100毫克土壤样品进行 DNA 提取。使用便携式 thermocycler 进行实时 PCRs。标准曲线代表了从土壤样品 (x 轴) 中提取的总 DNA 总量与由该荧光底漆/探针集放大的 PCR 产物 (y 轴) 数量之间的关系。<a href="/files/ftp_upload/56891/56891fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56891/56891fig5.jpg"> 图 5.检测其他马铃薯病原体, R. 病菌和 PMTV.使用便携式 thermocycler 和常规 thermocycler 进行实时 PCRs。R. 病菌AG3 是通过 RsTq 引物从纯培养中提取的总 DNA 中检测到的, 而 RQP1 探针 (A) PMTV 是用 PMTV-D 底漆/探针集 (B) 从 PMTV 感染的块茎样品中提取的总 RNA 中检测到的。<a href="/files/ftp_upload/56891/56891fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56891/56891fig6.jpg"> 图 6.用于 phytopathogens 的诊断管道.流程图显示了病原诊断的一般工作流。请注意, 如果使用现场分子检测, 则可以省略传统的步骤例如视觉识别, 这使得整个诊断过程简单而快速。<a href="/files/ftp_upload/56891/56891fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>便携式实时 PCR 技术</td>
			<td>实时 PCR</td>
			<td>灯</td>
			<td>elisa</td>
			<td>横向流动</td>
		</tr>
<tr>
<td>每个目标反应的成本</td>
			<td>$ 0.60-$ 8.47</td>
			<td>0.60 元</td>
			<td>0.75 元</td>
			<td>0.60 元</td>
			<td>4.74 元</td>
		</tr>
<tr>
<td>敏感性</td>
			<td>100份</td>
			<td>10份</td>
			<td>10份</td>
			<td>1-10 sporosori33 1-10 ng/毫升 (蛋白质)33
</td>
			<td>1-10 sporosori34 5x105CFU/mL35
</td>
		</tr>
<tr>
<td>时间费用</td>
			<td>90分钟</td>
			<td>80-240 分钟</td>
			<td>50-90 分钟32
</td>
			<td>3-24 小时</td>
			<td>10-15 分钟</td>
		</tr>
<tr>
<td>需要准备</td>
			<td>核酸提取 底漆设计</td>
			<td>核酸提取 底漆/探头设计</td>
			<td>Nucelic 酸提取物 底漆设计</td>
			<td>蛋白质提取 抗体</td>
			<td>N/A</td>
		</tr>
<tr>
<td>所需材料</td>
			<td>便携 thermocycler</td>
			<td>常规 thermocycler</td>
			<td>改良染色 孵化器</td>
			<td>平板读卡器 洗涤设备</td>
			<td>N/A</td>
		</tr>
</tbody></table>表1。phytopathogens 分子和血清学检测方法比较图
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆</td>
			<td colspan="2">序列 (5′–3′)a
</td>
			<td>目标b
</td>
		</tr>
<tr>
<td>SsTQ13
</td>
			<td colspan="2">CCGGCAGACCCAAAACC</td>
			<td>subterranea中的 ITS1-ITS2
</td>
		</tr>
<tr>
<td>SsTQ13
</td>
			<td colspan="2">CGGGCGTCACCCTTCA</td>
			<td>subterranea中的 ITS1-ITS2
</td>
		</tr>
<tr>
<td>SsTQ13
</td>
			<td colspan="2">fam]CAGACAATCGCACCCAGGTTCTCATG [谭]</td>
			<td>subterranea中的 ITS1-ITS2
</td>
		</tr>
<tr>
<td>Genesig subterranea底漆/探头</td>
			<td colspan="2">N/A</td>
			<td>subterranea中的肌动蛋白
</td>
		</tr>
<tr>
<td>SPO1014
</td>
			<td colspan="2">GGTCGGTCCATGGCTTGA</td>
			<td>它在subterranea中
</td>
		</tr>
<tr>
<td>SPO1114
</td>
			<td colspan="2">GGCACGCCAATGGTTAGAGA</td>
			<td>它在subterranea中
</td>
		</tr>
<tr>
<td>SPOPRO114
</td>
			<td colspan="2">fam]CCGGTGCGCGTCTCTGGCTT [谭]</td>
			<td>它在subterranea中
</td>
		</tr>
<tr>
<td>RsTqF119
</td>
			<td colspan="2">AAGAGTTTGGTTGTAGCTGGTCTATTT</td>
			<td>ITS1-ITS2 R. 病菌中的
</td>
		</tr>
<tr>
<td>RsTqR119
</td>
			<td colspan="2">AATTCCCCAACTGTCTCACAAGTT</td>
			<td>ITS1-ITS2 R. 病菌中的
</td>
		</tr>
<tr>
<td>RQP119
</td>
			<td colspan="2">fam]TTTAGGCATGTGCACACCTCCCTCTTTC [谭]</td>
			<td>ITS1-ITS2 R. 病菌中的
</td>
		</tr>
<tr>
<td>Genesig PMTV 底漆/探头</td>
			<td colspan="2">N/A</td>
			<td>PMTV 中的 CP-RT</td>
		</tr>
<tr>
<td>PMTV20
</td>
			<td colspan="2">AGAATTGRCATCGAAACAGCA</td>
			<td>CP 在 PMTV</td>
		</tr>
<tr>
<td>PMTV20
</td>
			<td colspan="2">GTCGCGCTCCAATTTCGTT</td>
			<td>CP 在 PMTV</td>
		</tr>
<tr>
<td>PMTV20
</td>
			<td colspan="2">fam]CCACAAACAGACAGGTATGGTCCGGAA [谭]</td>
			<td>CP 在 PMTV</td>
		</tr>
<tr>
<td colspan="4">
a用 6-羧基) 或 TAM (5-carboxytetramethylrhodamine) 修饰寡核苷酸 DNA 引物</td>
		</tr>
<tr>
<td colspan="4">
b其: 内转录间隔, CP: 涂层蛋白;CP: 涂层蛋白系通读</td>
		</tr>
</tbody></table>表2。本研究中使用的引物
辅助图1。DNA 提取方法对粉状赤霉病病原菌检测的比较.比较了六种不同的 DNA 提取方法 (A F) 对土壤样品中粉状赤霉病病原体、 subterranea的检测。(B、D、F)。DNA 是用硅基试剂盒 #1 (见所有套件名称的材料表), 硅基套件 #2, 磁性珠基套件, repsectively。采用常规的实验室 pcr thermocycler 进行 pcr。标准曲线代表了从土壤样品中提取的总 DNA 总量与 SsTQ 引物/探针集扩增的 PCR 产物数量之间的关系。<a href="/files/ftp_upload/56891/Supplementary_Figure_1.pdf" target="_blank">请单击此处下载此图.</a>
辅助图2。便携式 pcr 与常规实验室 pcr 检测的界限比较。总 DNA 是用三种不同的提取方法从土壤样品中分离出来的: (a、B) 道尔法、(C、D) 硅基试剂盒 #2 和 (E、F) 磁珠基套件。左边显示的图是使用便携式 thermocycler 的数据, 其中的图形是用该方法引物/探针集, 而右边的图表示使用传统的基于实验室的 thermocycler 与 SsTQ 引物/探针集生成的数据。<a href="/files/ftp_upload/56891/Supplementary_Figure_2.pdf" target="_blank">请单击此处下载此图.</a>

Watch this Scientific Journal Video about On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System at JoVE.com

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

快速准确地检测植物病原体, 特别是土壤传播病原体, 对于防止田间植物病害进一步接种生产和增殖至关重要。采用便携式实时 PCR 检测系统开发的方法, 能够在野外条件下进行现场诊断。

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 ...

on-site-molecular-detection-soil-borne-phytopathogens-using-portable

Universidad San Sebastian

Research

JoVE Journal

Environment

16.4K 次观看.  Washington State University. 快速准确地检测植物病原体, 特别是土壤传播病原体, 对于防止田间植物病害进一步接种生产和增殖至关重要。采用便携式实时 PCR 检测系统开发的方法, 能够在野外条件下进行现场诊断。

视频: On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

科研

免疫与感染

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

环境科学

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

使用 循环介质等温扩 增检测灌溉水中的植物浮肿

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

生物工程

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale 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 combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

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 ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

基因分型的植物和动物样品，恕不另行DNA的纯化

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

生物学

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

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

摘要

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此视频中的章节

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

循环介导等温扩增 (灯管) 法快速识别烟粉虱

使用循环介质等温扩增检测灌溉水中的植物浮肿

电化学 DNA 生物传感器检测食源性致病菌的研究进展

在动物标本呼吸道病原体的高通量检测通过纳米PCR

基因分型的植物和动物样品，恕不另行DNA的纯化

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

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

摘要

探索更多视频

此视频中的章节

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

循环介导等温扩增 (灯管) 法快速识别烟粉虱

使用 循环介质等温扩 增检测灌溉水中的植物浮肿

电化学 DNA 生物传感器检测食源性致病菌的研究进展

在动物标本呼吸道病原体的高通量检测通过纳米PCR

基因分型的植物和动物样品，恕不另行DNA的纯化

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

使用循环介质等温扩增检测灌溉水中的植物浮肿