我们感谢 Susanne. 拉斯穆森和 Solvej Kolbjørn 詹森为他们的技术援助和处理临床样品。该项目是 EbolaMoDRAD 财团的一部分, 该集团已从创新医学倡议2根据赠款协议 N°115843的联合承诺获得资金。这项联合承诺得到欧洲联盟2020地平线研究和创新计划和 EFPIA 的支持。

资本区域生物医学研究伦理学委员会已给予知情同意, 此处描述的所有方法均获豁免, 不受道德委员会制度的审查, 这是根据丹麦关于化验发展项目的法律。
1. 用于病毒灭活和快速 NA 提取的采血真空管的研制
注意: 此协议使用的缓冲区包含胍硫氰酸盐 (GITC) 和非离子表面活性剂, 这是刺激物。采取适当的实验室安全措施, 使用流动罩, 并在处理时戴上手套。避免皮肤和眼睛接触。如果缓冲区溢出, 污染的表面绝不能直接与胺或次氯酸钠 ("漂白剂" 中的活性成分) 进行消毒, 因为这种混合物可能导致有毒氰化物的形成。首先, 用吸收性组织擦拭溢出的缓冲器。接下来, 用70% 乙醇和水清洗表面, 最后使用胺或次氯酸钠。
<ol><li>要准备采血真空管, 将1.6 毫升的特定商业裂解缓冲液注入4毫升 EDTA 真空管, 用25克 x 1 针和3毫升注射器刺穿管盖。 注: 请勿拆卸真空管盖。必须保持真空。</li>
	<li>将含有缓冲液的真空管贮存在室温下, 直至使用。 注: 该管在制备后至少1年稳定。</li>
</ol>2. 制备 NA 萃取缓冲器
<ol><li>要准备用于 NA 提取的缓冲器, 请在机架中放置两个1.8 毫升、两个4.5 毫升和一个3.6 毫升管。</li>
	<li>添加, 由吹打, 960 µL 磁性玻璃微粒 (MGPs) 到一个干净的1.8 毫升管和标签它与 MGPs. 并用重悬赛车暂停完全之前吹打它。 注: MGPs 往往迅速收集在底部的管。</li>
	<li>添加, 由吹打, 4 毫升的洗涤缓冲器 I 到一个干净的4.5 毫升管和标签它为 WB-1。 注意: 洗涤缓冲器我含有胍氯, 这是刺激性的。采取适当的实验室安全措施, 使用流动罩, 并在处理时戴上手套。避免皮肤和眼睛接触。</li>
	<li>添加, 由吹打, 1.5 毫升洗涤缓冲 II 到一个干净的3.6 毫升管和标签它为 WB-2。</li>
	<li>添加, 由吹打, 3 毫升洗涤缓冲 III 到一个干净的4.5 毫升管和标签它为 WB-3。</li>
	<li>添加, 由吹打, 100 µL 洗脱缓冲器到一个干净的1.8 毫升管和标签为 EB。</li>
	<li>将 aliquoted 缓冲区存储在室温下, 直到使用。 注: 该管在制备后至少1月内稳定。</li>
</ol>3. 病毒感染体征和症状患者的血液收集
注意: 在收集病人的全血时, 采取适当的实验室安全措施。戴上手套和眼镜。如果病人是孤立的, 请遵循生物安全4级程序。
<ol><li>要收集病人的静脉全血, 使用蝶针与小孔延长管和血液收集真空管含有裂解/捆绑缓冲。把病人的手臂放在向下的位置, 把收集管放在比蝶针低的位置。将蝶形针插入病人的静脉内, 并将采血真空管连接至小孔延伸。 注: 这将防止回流。</li>
	<li>血液收集后, 通过翻转管 5-10 倍, 混合的管的内容。 注: 由于血液收集管中含有1.6 毫升溶解/结合缓冲液的残余真空, 采集的样品体积将自动为1.6 毫升。</li>
	<li>用70% 乙醇消毒管外。</li>
	<li>室温下将试管孵化20分钟。 注: 该协议可以暂停在这里, 完整的采血管可以储存在-20 °c, 5 °c, 25 °c 或37°c 至少1月7。</li>
	<li>继续直接到简化 NA 提取方法。</li>
</ol>4. 全血的简化 NA 提取
<ol><li>用手 5-10x 翻转真空管, 将钠从裂解/捆绑缓冲液中纯化出来, 并将该管的内容混合在一起。</li>
	<li>小心拆卸管盖, 并排出盖子。</li>
	<li>将准备好的整除 MGPs (1 毫升) 直接倒入采血管中。</li>
	<li>在含有该样本的管上放置一个未使用的采血管的新盖子。</li>
	<li>把手指放在盖子上, 以确保管子紧紧地闭合, 并用手翻转采血管 5-10 倍来混合管的内容。</li>
	<li>把管子放在磁性支架上, 把手指放在盖子上, 以确保管子紧闭。</li>
	<li>用手轻轻翻转磁性持有者几次, 以确保所有的 MGPs 都收集在管的一侧与磁铁。 注: 磁性支架可以用细长磁铁和橡皮筋代替。</li>
	<li>去掉管子的盖子, 用一次性吸管或简单地将内容倒入50毫升的收集管中, 丢弃管子的内容。 注: 用盖子关闭管, 避免50毫升收集管中的气溶胶。</li>
	<li>将准备好的整除 WB-1 (4 毫升) 直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把手指放在盖子上, 以确保管子紧闭。</li>
	<li>把管子从磁支架上取下, 用手指把盖子牢牢地拧紧。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>并用重悬 MGPs 通过手翻转采血管 5-10 倍。</li>
	<li>重复步骤 4.6-4.8。</li>
	<li>将准备好的整除 WB-2 (1.5 毫升) 直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把管子从磁支架上取下。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>把一根手指放在盖子上, 以确保管子紧闭。</li>
	<li>并用重悬 MGPs 通过手动翻转血液收集管几秒钟。</li>
	<li>重复步骤 4.6-4.8。</li>
	<li>将准备好的整除 WB-3 (3 毫升) 直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把管子从磁支架上取下。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>把一根手指放在盖子上, 以确保管子紧闭。</li>
	<li>并用重悬 MGPs 通过手翻转采血管 5-10 倍。</li>
	<li>重复步骤 4.6-4.8。</li>
	<li>将 EB (100 µL) 的制备整除直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把管子从磁支架上取下。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>并用重悬用手指敲击采血管 5-10 次, 在 EB 中 MGPs。 注意: 该协议可以在这里暂停, 管可以存储在-20 摄氏度。</li>
	<li>将悬浮 MGPs 的一滴 (5-8 µL) 转移到下游 NA 放大反应混合使用1.5 毫升一次性吸管 (任何下游诊断 NA 放大试验, 如灯/rt 灯或 qPCR/rt-qPCR 化验可以使用)。 注: NAs 将坚持 MGPs, 所以一定要使用 MGPs 在下游 NA 放大反应。使用前混合赛车悬浮液。混合后, MGPs 将收集在管的底部。</li>
</ol>

<ol>
	<li>Lee, D., Shin, Y., Chung, S., Hwang, K. S., Yoon, D. S., Lee, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+Highly+Sensitive+Molecular+Diagnosis+of+Zika+Virus+by+Lateral+Flow+Assays.">Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays.</a> Analytical Chemistry. 88, 12272-12278 (2016).</li><li>Benzine, J. W., Brown, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diagnostic+Field+Test+for+Point-of-Care+Detection+of+Ebola+Virus+Directly+from+Blood.">Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly from Blood.</a> Journal of Infectious Diseases. 214 (Suppl 3), S234-S242 (2016).</li><li>Faye, O., Faye, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+deployment+of+a+rapid+recombinase+polymerase+amplification+Ebola+virus+detection+assay+in+Guinea+in+2015.">Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015.</a> Euro Surveillance. 20 (44), 10-18 (2015).</li><li>Yang, M., Ke, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Evaluation+of+a+Rapid+and+Sensitive+EBOV-RPA+Test+for+Rapid+Diagnosis+of+Ebola+Virus+Disease.">Development and Evaluation of a Rapid and Sensitive EBOV-RPA Test for Rapid Diagnosis of Ebola Virus Disease.</a> Scientific Reports. 6, (2016).</li><li>Smither, S. J., Weller, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buffer+AVL+alone+does+not+inactivate+Ebola+virus+in+a+representative+clinical+sample+type.">Buffer AVL alone does not inactivate Ebola virus in a representative clinical sample type.</a> Journal of Clinical Microbiology. 53 (10), 3148-3154 (2015).</li><li>Byrnes, S., Fan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Portable,+Pressure+Driven,+Room+Temperature+Nucleic+Acid+Extraction+and+Storage+System+for+Point+of+Care+Molecular+Diagnostics.">A Portable, Pressure Driven, Room Temperature Nucleic Acid Extraction and Storage System for Point of Care Molecular Diagnostics.</a> Analytical Methods. 5 (13), 3177-3184 (2013).</li><li>Rosenstierne, M. W., Karlberg, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+bedside+inactivation+of+Ebola+virus+for+safe+nucleic+acid+tests.">Rapid bedside inactivation of Ebola virus for safe nucleic acid tests.</a> Journal of Clinical Microbiology. 54 (10), 2521-2529 (2016).</li><li>Vinner, L., Fomsgaard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+orthopoxvirus+for+diagnostic+PCR+analysis.">Inactivation of orthopoxvirus for diagnostic PCR analysis.</a> Journal of Virological Methods. 146, 401-404 (2007).</li><li>Schmitz, H., K&#246;hler, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+clinical+and+laboratory+data+in+two+cases+of+imported+Lassa+fever.">Monitoring of clinical and laboratory data in two cases of imported Lassa fever.</a> Microbes and Infection. 4 (1), 43-50 (2002).</li><li>De La Vega, M. A., Caleo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ebola+viral+load+at+diagnosis+associates+with+patient+outcome+and+outbreak+evolution.">Ebola viral load at diagnosis associates with patient outcome and outbreak evolution.</a> Journal of Clinical Investigation. 125 (12), 4421-4428 (2015).</li><li>Hasanoglu, I., Guner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+viral+load+in+Crimean+Congo+hemorrhagic+fever.">Dynamics of viral load in Crimean Congo hemorrhagic fever.</a> Journal of Medical Virology. 90 (4), 639-643 (2017).</li><li>Biava, M., Colavita, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+rapid+and+sensitive+RT-qPCR+assay+for+the+detection+of+Ebola+Virus.">Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus.</a> Journal of Virological Methods. 252, 70-74 (2018).</li><li>Fern&#225;ndez-Carballo, B. L., McBeth, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous-flow,+microfluidic,+qRT-PCR+system+for+RNA+virus+detection.">Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection.</a> Analytical and Bioanalytical Chemistry. 410 (1), 33-43 (2017).</li><li>Weller, S. A., Bailey, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Biofire+FilmArray+Biothreat+E-test+(v2.5)+for+rapid+identification+of+Ebola+virus+disease+in+heat-treated+blood+samples+obtained+in+Sierra+Leone+and+United+Kingdom.">Evaluation of the Biofire FilmArray Biothreat E-test (v2.5) for rapid identification of Ebola virus disease in heat-treated blood samples obtained in Sierra Leone and United Kingdom.</a> Journal of Clinical Microbiology. 54 (1), 114-119 (2015).</li><li>Semper, A. E., Broadhurst, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+GeneXpert+Ebola+Assay+for+Diagnosis+of+Ebola+Virus+Disease+in+Sierra+Leone:+A+Field+Evaluation+Study.">Performance of the GeneXpert Ebola Assay for Diagnosis of Ebola Virus Disease in Sierra Leone: A Field Evaluation Study.</a> PLoS Medicine. 13 (3), e1001980 (2016).</li><li>Biocartis. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Instructions for Use: Idylla TM Ebola Virus Triage Test. , (2016).</li><li>MPLC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Total Nucleic Acid Isolation Kit. Safety data sheet version 1.7. , (2015).</li></ol>

作者没有什么可透露的。

在本报告中, 我们描述了一种安全、快速、简单的人工床边 NA 提取方法, 用于在裂解/捆绑缓冲-灭活全血中对病毒进行下游分子检测。所述的 NA 提取方法是直接对含有裂解/捆绑缓冲 (材料表) 的真空采血管收集的全血标本进行的.7。此特定缓冲区钝化 EBOV7 , 是该方法中的关键组件。血液收集后, 我们建议孵化管至少20分钟7 , 以确保完全失活的样本。床边 EBOV 灭活消除了在严格的包容条件下处理任何 EBOV 阳性样品的需要, 如 BSL-4 条件, 并允许在正常 BSL-2 条件下处理样品。其他风险组四病原体, 如拉沙病毒、马尔堡病毒和克里米亚刚果出血热病毒也可能被此缓冲区灭活, 但仍有待证明。
所述的 NA 提取方法是在医院隔离病房或野战医院的病人旁边进行的, 因此不需要实验室设备, 如离心机、加热块或电。NA 萃取方法所需要的是含有固定容积的 MGPs 或洗涤缓冲器、磁持器和一次性吸管的管子。预准备的包含缓冲区的管简化了协议, 因为它消除了对吸管的需要, 而相反, aliquoted 缓冲器直接倒入样品管。磁性持有者抓住含有 NA 的 MGPs, 调解洗涤步骤的简单处理。磁保持架可以用普通磁铁和橡皮筋代替, 以防磁支架不可用;然而, 磁性持有者减少了在应用磁铁和橡皮筋时, 降低管子和溢出内容的动手风险。由于溶解/捆绑缓冲-灭活全血样品的粘度, 一次性吸管被用来去除从管上的上清/液/洗缓冲。一次性吸管可替代直接浇注的内容从管成废物收集管, 但请注意不要创建任何水滴外管。如果产生水滴, 污染的表面绝不能直接与胺或次氯酸钠 ("漂白剂" 中的活性成分) 进行消毒, 因为这种混合物可能导致有毒氰化物的形成。因此, 如果首先溢出, 用吸收性组织擦拭溢出。接下来, 用70% 乙醇清洁表面, 然后用大量的水, 最后, 使用胺或次氯酸钠。这包括所有与溶解/粘结缓冲液 (包括废物收集管和一次性吸管) 接触的废物。相反, 收集所有的废物在一个单独的废物容器, 只用70% 乙醇消毒容器的外部。
NAs 通常在加热步骤中从 MGPs 洗脱, 但在简化的 NA 提取协议中删除了该加热步骤, 以消除用电和设备的使用。因此, NAs 倾向于坚持 MGPs, 因此, 必须添加到下游 na 检测化验, 以进行阳性 na 检测。MGPs 被转移到下游反应混合使用一滴从1.5 毫升一次性吸管。这加法不象正常加法使用翅片尖端吸管一样精确;然而, unprecise 添加 MGPs 的灯, rt 灯, qPCR, 或 rt qPCR 反应组合不抑制反应或影响荧光信号在我们的内部诊断化验。然而, 这可能不同于化验和化验, 我们建议每一个下游 NA 测试验证, 并使用简化 NA 提取方法测试的灵敏度。NA 提取方法相对敏感, 在下游分子试验中, 如 qPCR, 可以很容易地提取和检测到含有 DNA 或 RNA 病毒低至3000拷贝/毫升的全血样本。临床标本中的病毒载量通常在感染的急性期很高, 出血热病毒, 如 EBOV、拉沙病毒和克里米亚刚果出血热病毒, 通常含有超过 105拷贝/毫升9 ,10,11。因此, 这些样本中的病毒可以使用此 NA 提取方法很容易地检测到。然而, 除 EBOV7、痘苗和牛痘病毒8以外的病毒的 MPLB 灭活仍有待证明。
这种简化的 NA 提取方法可以在医院隔离病房或现场设置中进行, 是治疗点分子诊断的理想选择。然而, 该方法不适用于高通量 NA 提取, 由于操作操作的开放管。如果需要高通量 na 提取, 则用 NA 萃取机器人7可以很容易地纯化灭活全血标本。另一个缺点是处理有害试剂, 如裂解/捆绑缓冲 [20-30% 聚乙二醇 p-(11, 33-tetramethylbutyl)-苯基醚和 30-50% 胍硫氰酸盐 (GITC)]17和洗涤缓冲器 I (30-50% 胍氯化物)17在一个开放管外面一个流动罩。浓缩形式中的溶解/束缚缓冲器包含在真空管内, 血液收集 1:1 (v/v), 浓度在管打开前被减少。真空管的开启和 MGPs 或洗涤缓冲液的添加/清除我是在几秒钟内完成的, 因此, 吸入气溶胶的风险极小。清洗后的洗涤缓冲器-I, 从管, 没有其他有害试剂使用的协议。
自 2013年 EBOV 爆发以来, 许多快速的护理点分子测试和装置已经发展到3、6、12、13、14、15、16.然而, 其中许多测试仍需要对传染性物质、BSL-3 生物安全柜和最低限度的实验室设备 (如吸管、离心机、加热块或电) 进行上游处理。生物安全柜的使用复杂化并延长了快速诊断的速度。描述的床边病毒灭活与直接收集血液进入病毒灭活管结合简化 NA 提取方法, 消除了对生物安全柜, 电力和实验室设施的需要。另外, 由于简化的 NA 提取方法可以在病人旁边进行, 并结合一个便携式电池驱动的等温装置中的灯反应, 可以在40分钟内获得安全的床边分子诊断。

在病毒暴发的情况下, 当病人被限制在医院隔离病房或需要快速诊断时, 安全、简单、准确的点对诊的分子诊断是必要的病人护理和风险遏制。最近两次病毒暴发, 在西非的埃博拉病毒 (EBOV) 和南美的 Zika 病毒 (2015), 增加了对改进的护理点分子诊断测试的兴趣, 如反向转录回路介导的等温放大 (RT 灯)1,2和 recombinase 聚合酶放大 (RPA)3,4。RT 灯和 RPA 都是快速、敏感和特定的分子测试, 可以在简化的样品制备中进行。对 Zika 病毒, 将 RT 灯与侧流法 (LFA) 相结合, 在30分钟内检测非纯化全血标本中的 Zika 病毒;然而, 对于被归类为风险组4病原体并具有高度传染性的 EBOV, 需要在生物安全等级 4 (BSL-4) 条件下处理和灭活, 然后才能进行任何安全诊断程序。
EBOV 的简化失活方法, 例如在2、3、4、5的样品中加入裂解缓冲剂, 在暴发期间使用;然而, 这些方法需要在 BSL-4 条件下处理与实验室设备, 如 BSL-3 生物安全柜, 离心机, 暖气块, 吸管, 至少。这种设备通常不存在于隔离病房或外地医院。为了克服这一挑战, 已尝试在手提箱3中进行诊断, 并开发了一些便携设备和机器 (例如, 一种用于核酸 (NA) 提取的便携设备)6。然而, 在使用这些设备之前, EBOV 阳性样品仍需进行灭活。
我们以前报告了一个快速的床边病毒灭活方法为 EBOV7, 痘苗病毒和牛痘病毒8通过增加一个商业裂解/捆绑缓冲到普通的真空采血管, 允许直接血液从患者转移到灭活缓冲器7。这种直接和直接的钝化在一个封闭的系统, 消除了处理样品使用任何严格的遏制, 如 BSL-4 条件7, 并在正常 BSL-2 条件下处理样品的需要。这种灭活方法与几种 NA 萃取系统兼容, 如机器人和手净化套件7;然而, 这些方法需要实验室设备, 如机器人, 离心机, 和电力, 这并不总是存在于现场设置或在医院隔离病房。
在本报告中, 我们描述了一种安全、快速和简化的人工 NA 提取方法, 用于随后在裂解/捆绑缓冲-灭活全血中的病毒分子检测。NA 萃取方法不需要任何设备, 除了磁铁/磁性持有人。NA 萃取不需要离心机、加热块或电。因此, 这种方法不依赖于实验室设施, 而且可以很容易地用于任何地方 (如在野战医院、医院隔离病房或低资源环境下)。na 萃取方法快速简便, 可直接用于任何下游 NA 试验, 如 qPCR、rt qPCR、灯或 rt 灯。当该 NA 提取方法与灯和便携式电池驱动的等温仪相结合时, 可以在40分钟内采集到血液中的床边诊断。

<table border="0" cellpadding="0" cellspacing="0" style="width:1080px;" width="1081">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:544px;">Vacutainer K2 EDTA tubes (4 mL)</td>
			<td style="width:251px;">Becton Dickinson</td>
			<td style="width:119px;">368861</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plus Blood Collection</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">362725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23G x 1 needle&#160;</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">300800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LUER LOK syringe (3 mL)</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">309658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butterfly needle with small-bore extension tubing</td>
			<td style="width:251px;">J&#248;rgen Kruuse A/S</td>
			<td style="width:119px;">121714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% Virkon&#160;</td>
			<td>Nomeco A/S</td>
			<td style="width:119px;">849265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Pure LC DNA Isolation Kit I - Lysis/Binding Buffer - Refill</td>
			<td>Roche Diagnostics A/S</td>
			<td style="width:119px;">03246752001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Pure LC Total Nucleic Acid Isolation Kit</td>
			<td>Roche Diagnostics A/S</td>
			<td style="width:119px;">3038505001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (1,8 mL)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">375418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (3,6 mL)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">379189</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (4,5 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">379146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag&#8482;-5 Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">12303D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette (3.5 mL)</td>
			<td>Sarstedt</td>
			<td style="width:119px;">86.1171.010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#8482; Samco&#8482; Fine Tip Transfer Pipettes (1.5 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">231</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermo Scientific&#8482; Nunc&#8482; 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">339652</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid, safe, and simple manual bedside nucleic acid extraction for the detection of virus in whole blood samples

对感染的快速诊断对于暴发管理、风险控制和病人护理至关重要。我们以前已经显示了一种方法, 快速床边的埃博拉病毒在血液取样的安全核酸 (NA) 测试通过添加一个商业裂解/捆绑缓冲直接进入真空采血管。利用这种床边灭活方法, 我们开发了一种安全、快速、简便的床边 NA 提取方法, 用于随后在裂解/捆绑缓冲-灭活全血中检测病毒。钠萃取直接在采血管中进行, 无需设备或电。
将血液收集到溶解/结合缓冲液中后, 通过手工翻转管将其内容混合, 并在室温下孵化出20分钟的混合物。将磁性玻璃微粒 (MGPs) 添加到管中, 通过手工翻转收集管将其内容混合。MGPs, 然后收集在血液收集管的一侧使用磁性持有人或磁铁和橡皮筋。MGPs 清洗三次, 在将洗脱缓冲器直接添加到采集管后, NAs 就可以进行 NA 测试, 如 qPCR 或等温环放大 (灯管), 而不需要从反应中去除 MGPs。NA 提取方法不依赖任何实验室设施, 可以很容易地在任何地方使用 (例如, 在外地医院和医院隔离病房)。当该 NA 提取方法与灯和便携式仪器相结合时, 可在采血40分钟内获得诊断。

本文提出的协议简单有效, 可广泛应用于用裂解/结合缓冲剂灭活的传染性全血标本进行的任何分子检测。血液失活和 NA 提取工作流程如图 1所示, 包括采血真空管的制备7 (图 1A), 采血7 (图 1B), NA 提取 (图 1C - 1H) 和下游 NA 分析 (图 1I)。
由于简化的 NA 提取协议, MGPs 不会从洗脱步骤中删除, 因此 NAs 倾向于坚持 MGPs。任何下游分子分析, 如 qPCR 或灯分析, 必须直接在 MGPs (图 1I和图 2) 进行, 以确保一个积极的结果。
NA 提取方法是有效的, RNA 或 DNA 病毒阳性全血样本的病毒负载低至 130-3000 拷贝/毫升可以提取和分析使用 qPCR 或 RT qPCR (图 3)。
使用 NA 提取方法, 灯或 RT 灯, 和便携式电池驱动的恒温装置的病毒阳性全血样本, 诊断可以获得40分钟内从血液收集 (图 4)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58001/58001fig1.jpg"> 图 1: 简化 NA 提取全血样品的工作流程.(A) 使用针头和注射器制备裂解/捆绑缓冲真空采血管。(B) 用蝴蝶针收集血液。(C) 将采血管放在支架内, 取下盖子, 将 MGPs 倒入管内。(D) 用新盖子把管子关上, 用手翻转管子来混合内容。(E) 通过在磁支架上翻转管, 收集采血管一侧的 MGPs。(F) 使用一次性吸管去除上清液。(G) 将洗涤或洗脱缓冲器直接倒入采血管内, 并重复在D组中显示的动作。(H) 此面板显示 MGPs 已准备好直接用于 qPCR、rt qPCR、灯管或 rt 灯反应。(I) 使用1.5 毫升一次性吸管将一滴 (5-8 µL) 的 MGPs 转移到 PCR 或灯管反应管中。小组A和B: 这个数字已经被修改了从 Rosenstierne等。7.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58001/58001fig1large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58001/58001fig2.jpg"> 图 2: 用简化的 na 提取方法提取28种病毒阳性全血样品的 na 检测.全血标本 (1.6 毫升) 测试阴性的病毒被用 DNA 病毒 (eb 病毒) (2.0 x 104 -1.0 x 103拷贝/毫升)] 或 RNA 病毒 [丙肝病毒 (HCV) (6.0 x 105 -3.0 x 103拷贝/毫升)]或登革病毒 (DENV) (1.7 x 108 -1.7 x 106拷贝/毫升)。使用上述方法提取 nas, 并通过特定的内部 qPCR、RT PCR、灯管或 rt 灯对提取的 nas 进行了重复分析, 其中有或不 MGPs。x轴 = NA 检测方法, y轴 = qPCR/灯泡中样品阳性率。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58001/58001fig2large.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/58001/58001fig3v2.jpg"> 图 3: 简化 NA 提取方法的灵敏度.为了证明该方法的概念, 对病毒阴性全血进行了不同的 RNA 或 DNA 病毒制剂, 含有定义的病毒负载, 并提取了 NAs 使用简化 NA 提取方法。(A) 负全血 (1.4 毫升) 以200µL 10 倍稀释的 EBOV 标准为诊断目的而准备 (ENIVD), 这是由最近在盖凯杜/几内亚爆发的 (2.0 x 106个拷贝/毫升)。用 MX3005P 热循环仪在内部 EBOV 特定的 RT qPCR7中对提取的 NAs 进行了重复分析。(B) 全血 (1.2 毫升) 以400µL 10 倍稀释的丙型肝炎病毒为例, 标准血清样本 (1.2 x 106英寸/毫升)。用 MX3005P 热循环仪对提取的 NAs 进行了重复的内部 HCV 特异 RT qPCR 分析。(C) 全血 (1.2 毫升) 的400µL 不同浓度的 eb 病毒 (2.0 x 104拷贝/毫升, 8.0 x 103拷贝/毫升, 2.7 x 103拷贝/毫升, 9.3 x 102拷贝/毫升)。通过内部 eb 病毒特异 qPCR 对提取的 NAs 进行了重复分析。(D) 全血 (1.2 毫升) 的400µL 10 倍稀释的浅滩病毒 (1.3 x 104拷贝/毫升)。对提取的 NAs 进行了重复分析, 由内部的浅滩特定 qPCR。x轴 = 病毒在尖刺全血样品中的最终浓度 (拷贝/毫升或内/毫升), y轴 = Ct 值 (平均值 * SD)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58001/58001fig3v2large.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/58001/58001fig4v2.jpg"> 图 4: 用便携式等温装置简化 NA 萃取和 RT 灯或灯.(A) 由于缺乏 EBOV 阳性的全血样本在病毒 & 微生物学特别诊断, 丹麦 Statens 血清研究所, 1.4 毫升的负全血与200µL 不同稀释 EBOV为诊断目的准备的标准 (ENIVD)。用简化的 na 萃取方法进行了 na 提取, 并通过 EBOV 特异性 RT 灯反应进行了分析。作为阴性对照, 含有阴性的全血样本。x轴 = EBOV 的最终浓度在尖刺的整个血液样品 (拷贝或 mL), y轴 = min 到正面。(B)关于概念的证明, 全血 (1.6 毫升) 整除数从7名患者, 确诊为阳性的 eb 病毒 (1.3万-500 拷贝/毫升) 在艾滋病毒 & 微生物学特别诊断, Statens 血清研究所, 丹麦, 接受了 NA用便携式电池驱动的精灵 II 等温装置, 利用简化的 NA 萃取方法提取和分析了 eb 特异灯的反应。作为一个负控制, 包括细小 B19-positive 样本。x轴 = 在患者样本 (拷贝/mL) 中 eb 病毒负载, y轴 = 最小值为正数。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58001/58001fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples at JoVE.com

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

在这里, 我们提出了一个快速病毒核酸提取从病毒灭活全血的协议。萃取是直接在采血管, 不需要设备或电力。该方法不依赖于实验室设施, 可以在任何地方使用 (例如在野战医院)。

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Immunology and Infection

7.3K 次观看.  Statens Serum Institut. 在这里, 我们提出了一个快速病毒核酸提取从病毒灭活全血的协议。萃取是直接在采血管, 不需要设备或电力。该方法不依赖于实验室设施, 可以在任何地方使用 (例如在野战医院)。

视频: Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）:细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...