这项工作得到了来自 CDC/NIOSH 高平原山间农业健康与安全中心 (5U54OH008085) 和科罗拉多生物科学发现评估补助金计划 (14 改编-16) 的资助。

1. 气溶胶室的设置 (见图 2)
<ol><li>
预加载液体 impingers 与20毫升0.01 米磷酸盐缓冲盐水, pH 值 7.5 (PBS)。
	<ol>
<li>加入0.5 克阴离子交换树脂, 悬浮在其中一个液体 impingers 的 PBS 内, 另一种液体冲击器用作控制。</li>
	</ol>
</li>
	<li>在气溶胶腔内平行放置液体 impingers, 使用夹持器, 面对喷雾器的气溶胶入口。 注: 有关其他详细信息, 请参见图 2 。</li>
	<li>在液体 impingers 附近共找到一个直接读取的气溶胶监测器, 以测量气溶胶的质量浓度 (mg/米3)。</li>
	<li>在液体 impingers 附近共同定位其他直接读仪器 (室内空气质量监测和热风速计), 以监测气溶胶室内的环境变量, 如温度、相对湿度和风速。</li>
	<li>在气溶胶室的拐角处运行轴流风机, 以方便气溶胶的混合。 注: 轴流风机以固定速度运行, 不可调节。</li>
</ol>2. 模型 Bioaerosolization 实验 (见图 3)
<ol><li>对磷酸盐缓冲盐水 (PBS) 进行连续、10倍稀释的 MS2 噬菌体和流感疫苗的浓度。 注: 在这里演示的方法, 103.5 FFU/毫升的 a 型流感病毒疫苗菌株和 105 PFU/毫升的 MS2 噬菌体使用。为了确定菌的效价, 噬菌体被传播到大肠杆菌HS (pFamp) R, 并列举如前所述的20。该商业疫苗含有已知数量的四只活减毒菌株, 两种流感病毒和两种 B 型流感病毒, 以荧光聚焦单位或 FFU 表示。</li>
	<li>在6射流碰撞雾化器的玻璃容器内制备菌, 在100毫升的 PBS 中创建所需的病毒粒子浓度悬浮液, 并在自定义气溶胶腔内安装。</li>
	<li>启动仪器测量气溶胶腔内的变量, 如温度、相对湿度、二氧化碳浓度和风速 (使用轴流风机产生)。使用直接读取的气溶胶监测仪跟踪气溶胶室内的质量浓度, 并在试验之间建立一致性。</li>
	<li>密封气溶胶室, 用高效过滤空气净化10分钟。</li>
	<li>将经过过滤的干燥空气输送到6.9 帕的雾化器上。</li>
	<li>在每个气溶胶室试验中产生病毒 bioaerosols (通过雾化器) 的靶浓度。在此演示中, 将生成浓度为5毫克/米3的 bioaerosols。</li>
	<li>一旦达到目标质量浓度的气溶胶, 停止雾化。</li>
	<li>对每一个已校准为12.5 升/分流量的液体冲击器操作一个空气取样泵. 在每次取样事件前后使用主流量标准进行校准, 以确保采样量的一致性。使用位于雾化器附近的高效过滤线提供稀释空气, 并在燃烧室内保持恒定的压力。</li>
	<li>积极取样气溶胶室大气40分钟的时间, 以达到每样样品500升的取样量。 注: 在12.5 升/分两泵平行取样40分钟, 可取样气溶胶的1000升。因此, 在实验完成后, 预计气溶胶室中的大部分空气通过一个高效过滤系统被取样并释放到环境中。这是由颗粒浓度下降的证明。后实验, 表面被净化与10% 家用漂白剂和70% 乙醇。在本示范中使用指标;然而, 如果已知的病原体在该系统中使用, 则可根据需要实施额外的生物安全措施。</li>
</ol>3. 核酸提取和 qRT PCR 检测
<ol><li>
将核酸直接从阴离子交换树脂中分离出来, 并为比较起见, 从液体 impingers 中使用的液体隔离。在这个例子中, 我们描述了一个 RNA 隔离过程, 但是类似的协议可以用于 DNA 病毒。
	<ol>
<li>从负离子交换树脂中分离出核酸。<ol>
<li>将液体冲击器的阴离子交换树脂的所有液体转移到无菌50毫升圆锥管。</li>
			<li>允许树脂沉淀, 并慢慢地从负离子交换树脂中醒酒液体样品。使用1毫升吸管尖端, 以去除所有剩余的液体从阴离子交换树脂。</li>
			<li>从病毒 RNA 隔离试剂盒中, 将含有载体 RNA 的560µL 病毒裂解缓冲液直接添加到阴离子交换树脂中, 在室温下进行周期性混合孵化10分钟。</li>
			<li>使用1毫升吸管尖端, 转移病毒裂解物 (病毒裂解缓冲) 到一个无菌1.5 毫升圆锥管, 并进行 rna 隔离使用病毒 rna 隔离套件 (见材料表) 按照制造商的指示, 除了RNA 是洗脱在总容量60µL 洗脱的缓冲。</li>
		</ol>
</li>
		<li>从液体 impingers 中分离出核酸。<ol>
<li>吸管140µL 的液体样品成一个无菌1.5 毫升圆锥管和执行 rna 隔离使用病毒 rna 隔离套件按照制造商的指示, 除洗脱 RNA 的总容量60µL 洗脱缓冲。 注: 140 µL 是最大样本量, 可以处理与特定的病毒 RNA 隔离套件在这里使用的一个单一步骤的准备。</li>
		</ol>
</li>
	</ol>
</li>
	<li>执行 qRT PCR 检测 MS2 和流感 A 和 B 病毒, 如前所述23,24。在这一示范中, 5 µL 核酸 eluates 用于 qRT PCR。</li>
</ol>

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作者没有什么可透露的。

本协议概述了一种使用改进的液体 impingers 对 bioaerosols 进行敏感病毒捕获的方法。该方法对 bioaerosols 中病毒载量的检测和定量进行了优化。这里所展示的具体修改包括在普通液体冲击器中加入负离子交换树脂。该方法是为简化下游样品加工而开发的, 而其他样品处理技术, 如离心、过滤和沉淀法等, 并没有提供这样的优势。核酸的提取是直接从树脂, 不需要高容量 elutions 和多样品准备步骤, 最终有?...

该方法的目的是提供一个高度敏感的气溶胶取样平台, 以促进分子检测的负电荷病毒从 bioaerosols。微生物, 包括病毒微粒, 可以在 bioaerosols 中存活1长时间。Bioaerosols 可以旅行较远的距离和保持生存能力和传染性, 这证明了军团的疾病爆发的起源于距离6公里的工业冷却塔从受影响的人和导致18人死亡2。通过 bioaerosols 介导的病毒向人类间接传播可能发生在多种环境中, 并已证明诺如病毒在学校和餐馆3、4爆发。同样, 气溶胶传播病毒可能发生在农业环境中, 如在猪和家禽养殖场, 这一传输路线被认为是一个主要因素在病毒的移动之间的生产设施5,6,7,8,9。
对病毒携带的 bioaerosols 的有效取样可以改善快速诊断和预防疫情的准备, 如在中国活动物市场 bioaerosols 检测到 H5 流感病毒的示威活动中所示, 以及美国10,11。目前的气溶胶取样技术涉及许多不同的粒子捕获原理, 可大致分为 impingers、旋风、撞击和过滤器12。在本议定书范围之外, 详尽地涵盖了这些平台对 bioaerosols 病毒取样的所有利弊;但是, 可以说, 大多数这些取样装置尚未针对病毒和噬菌体13的收集进行优化。此外, 病毒微粒的传染性往往受到负面影响, 液体 impingers 认为更有效地保持病毒传染性比取样设备, 如固体撞击或过滤器14。然而, 液体撞击的一个缺点是靶稀释效应, 这是因为病毒在收集容器中相对较大的体积 (通常是≥20毫升) 收集的。另一个重要的缺点是液体 impingers 的次优效率, 浓缩颗粒 < 0.5 µM 大小15。然而, 这些装置的捕获效率可以通过固定在固体基质上来改善, 因为固定化可以增强病毒核酸的保存和病毒传染性16,17。
我们以前已经证明, 阴离子交换树脂是一种有效的工具, 从液体基质中捕获和浓缩病毒, 包括 F RNA 噬菌体, 甲型肝炎病毒, 人腺病毒, 和轮状病毒18,19 ,20。根据制造商的定义, 在这项工作中使用的阴离子交换树脂是一种吸附聚苯乙烯强碱性阴离子交换树脂, 功能性季胺基团介导在液体介质中对阴离子的吸引和捕获21.因此, 阴离子交换树脂有望捕获病毒的净负表面电荷, 包括许多肠道病毒, 流感病毒和其他与公共和动物健康有关的病毒。
目前的协议涉及增加负离子交换树脂的液体冲击器。在该系统中, 树脂作为冲击器液中捕获的病毒微粒的二次浓缩步骤。核酸可以直接洗脱在小体积, 提供了一个浓缩样本的分子分析。因此, 该方法的主要优点是提高了病毒检测灵敏度, 主要是通过减少样本量。另外, 由于对负电荷病毒的固有非特定捕获, 该方法可能适用于检测大量感兴趣的病毒。对 A 型和 B 型流感病毒的疫苗株和 FRNA 噬菌体 MS2 (MS2) 进行了验证。这些病毒随后使用标准的 qRT PCR 方法检测, 如前所述22。终端用户不应期望在执行此方法时遇到困难, 因为对当前现有设备的修改不构成对常规气溶胶取样和分析流程的重大干扰。

<table>
	<tbody>
		<tr>
			<td>Escherichia coli bacteriophage MS2 (ATCC 15597-B1)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15597-B1</td>
			<td></td>
		</tr>
		<tr>
			<td>FluMist Quadrivalent</td>
			<td>AstraZeneca</td>
			<td>Contact manufacturer</td>
			<td>Viral constitutents of this vaccine are subject to change on an annual basis</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855195</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and probes</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;M sterile filter</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L pyrex bottles or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipettor</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL serological pipet</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-aid or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Probe RT-PCR Kit</td>
			<td>Qiagen</td>
			<td>204443</td>
			<td></td>
		</tr>
		<tr>
			<td>Amberlite IRA-900 chloride form</td>
			<td>Sigma-Aldrich</td>
			<td>216585-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (molecular biology grade)</td>
			<td>Sigma-Aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf DNA LoBind Microcentrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>13-698-791</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes&#160;</td>
			<td>ThermoFisher</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>05-538-62</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Platinum One-Step qRT-PCR Kit w/ROX</td>
			<td>ThermoFisher</td>
			<td>11745100</td>
			<td></td>
		</tr>
		<tr>
			<td>SKC Biosampler 20 mL, 3-piece glass set</td>
			<td>SKC Inc.</td>
			<td>225-9593</td>
			<td></td>
		</tr>
		<tr>
			<td>Vac-u-Go sample pumps</td>
			<td>SKC Inc.</td>
			<td>228-9695</td>
			<td></td>
		</tr>
		<tr>
			<td>Collison nebulizer (6-jet)</td>
			<td>BGI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPA capsule</td>
			<td>PALL</td>
			<td>12144</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-TRAK indoor air quality monitor 8554</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Alnor velometer thermal anemometer AVM440-A</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SidePak AM510 personal aerosol monitor</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioaerosol chamber</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of viruses from bioaerosols using anion exchange resin

该协议演示了一种针对病毒的自定义气溶胶采样方法。在该系统中, 负离子交换树脂与液体撞击式空气取样装置相结合, 有效地集中了 bioaerosols 的负电荷病毒。因此, 树脂在气溶胶取样工作流中充当额外的浓度步骤。然后直接从阴离子交换树脂中提取出病毒微粒的核酸, 得到的样品适用于分子分析。此外, 本议定书还描述了一个定制的气溶胶室, 能够在各种环境条件下产生带有病毒的 bioaerosols, 并允许对环境变量 (如温度、湿度) 进行连续监测,风速和气溶胶质量浓度。使用此协议的主要优点是增强了病毒检测的灵敏度, 这是通过与未修改的常规液体冲击器进行直接比较来评估的。其他优势包括可能集中各种负电荷病毒, 低成本的阴离子交换树脂 (每样0.14 美元), 并易于使用。缺点包括本议定书无法评估树脂吸附的病毒微粒的传染性, 并可能需要优化冲击器中使用的液体取样缓冲器。

图 1展示了基于电荷的从 bioaerosols 捕获病毒的原理, 通过将树脂加入液体基 impingers。图 2显示了自定义构建的气溶胶室的设置。图 3描述了设置 aerosolization 实验的步骤和确保质量控制的措施。图 4显示了从 bioaerosols 捕获的负电荷病毒 qRT PCR 检测的放大曲线。表 1显示了用?...

Watch this Scientific Journal Video about Detection of Viruses from Bioaerosols Using Anion Exchange Resin at JoVE.com

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

证明了一种基于液体冲击的气溶胶病毒取样方法。当与下游分子检测相结合时, 该方法允许对 bioaerosols 的病毒进行简便、灵敏的检测。

用阴离子交换树脂检测 Bioaerosols 病毒

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

detection-of-viruses-from-bioaerosols-using-anion-exchange-resin

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

8.1K 次观看.  Colorado State University. 证明了一种基于液体冲击的气溶胶病毒取样方法。当与下游分子检测相结合时, 该方法允许对 bioaerosols 的病毒进行简便、灵敏的检测。

视频: Detection of Viruses from Bioaerosols Using Anion Exchange Resin

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

比色纸为基础的检测<em&gt;大肠杆菌</em&gt;<em&gt;沙门氏菌</em&gt;属，和<em&gt;单核细胞增生李斯特氏菌</em&gt;从农业用水大容量

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

科研

环境科学

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

从农业土壤使用静态钱伯斯温室气体流量测量

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

从个人污秽苍蝇检测食源性细菌病原体

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A multidimensional gas chromatography method for the analysis of dissolved hydrogen sulfide in liquid crude oil samples is presented. A Deans switch is used to heart-cut light sulfur gases for separation on a secondary column and detection on a sulfur chemiluminescence detector.

h的测量<sub&gt; 2</sub&gt; S在原油和原油顶空用多维气相色谱法，院长交换和选择性硫检测

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

Ø的底栖交易所<sub&gt; 2</sub&gt;，N<sub&gt; 2</sub&gt;和营养盐使用小的核心孵化

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

建筑双层疏水阀早期检测翡翠灰螟

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

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

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

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 ...

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

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

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

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

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 ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

一种用于检测汞和镉的厌氧生物传感器检测方法

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

用阴离子交换树脂检测 Bioaerosols 病毒

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此视频中的章节

比色纸为基础的检测

从农业土壤使用静态钱伯斯温室气体流量测量

从个人污秽苍蝇检测食源性细菌病原体

h的测量

Ø的底栖交易所

建筑双层疏水阀早期检测翡翠灰螟

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

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

一种用于检测汞和镉的厌氧生物传感器检测方法

一种精确、自主的昆虫产生模式检测系统