Name: rapid, safe, and simple manual bedside nucleic acid extraction for the detection of virus in whole blood samples
Uploaded: 2018-06-30T15:00:00.000+00:00
Duration: 6 min 59 s
Description: 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

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><tbody><tr><td>Vacutainer K2 EDTA tubes (4 mL)</td><td>Becton Dickinson</td><td>368861</td><td></td></tr><tr><td>Plus Blood Collection</td><td>Becton Dickinson</td><td>362725</td><td></td></tr><tr><td>23G x 1 needle</td><td>Becton Dickinson</td><td>300800</td><td></td></tr><tr><td>LUER LOK syringe (3 mL)</td><td>Becton Dickinson</td><td>309658</td><td></td></tr><tr><td>Butterfly needle with small-bore extension tubing</td><td>J&amp;oslash;rgen Kruuse A/S</td><td>121714</td><td></td></tr><tr><td>1% Virkon</td><td>Nomeco A/S</td><td>849265</td><td></td></tr><tr><td>MagNA Pure LC DNA Isolation Kit I - Lysis/Binding Buffer - Refill</td><td>Roche Diagnostics A/S</td><td>03246752001</td><td></td></tr><tr><td>MagNA Pure LC Total Nucleic Acid Isolation Kit</td><td>Roche Diagnostics A/S</td><td>3038505001</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (1,8 mL)</td><td>Thermo Fisher Scientific</td><td>375418</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (3,6 mL)</td><td>Thermo Fisher Scientific</td><td>379189</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (4,5 mL)</td><td>Thermo Fisher Scientific</td><td>379146</td><td></td></tr><tr><td>DynaMag&amp;trade;-5 Magnet</td><td>Thermo Fisher Scientific</td><td>12303D</td><td></td></tr><tr><td>Transfer pipette (3.5 mL)</td><td>Sarstedt</td><td>86.1171.010</td><td></td></tr><tr><td>Thermo Scientific&amp;trade; Samco&amp;trade; Fine Tip Transfer Pipettes (1.5 mL)</td><td>Thermo Fisher Scientific</td><td>231</td><td></td></tr><tr><td>Thermo Scientific&amp;trade; Nunc&amp;trade; 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>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/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/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/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/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)。

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

rapid-safe-simple-manual-bedside-nucleic-acid-extraction-for

Nanyang Technological University

Research

JoVE Journal

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

科研

环境科学

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

生物工程

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

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 ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

病毒测试,水样中检测病毒的一种使用点方法

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

免疫与感染

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

生物学

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

城市环境 -环境水库SARS-CoV-2采样和检测管道

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.

We present a protocol that combines recombinase polymerase amplification with a CRISPR/Cas12a system for trace detection of DNA viruses and builds portable smartphone microscopy with an artificial intelligence-assisted classification for point-of-care DNA virus detection.

基于RPA-CRISPR/Cas12a-SPM和深度学习的DNA病毒检测系统

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase ...