Name: virwatest, a point-of-use method for the detection of viruses in water samples
Uploaded: 2019-05-11T14:00:00
Description: Watch this Scientific Journal Video about VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples at JoVE.com

VirWaTest是一个研究项目,由ELHRA(加强人道主义援助的学习和研究)HIF(人道主义创新基金)方案资助。作者感谢WASH团队在这项研究中进行了友好合作。厄瓜多尔的样品分析由厄瓜多尔乐施会和美洲大学调查局资助。BRT.17.01)S.Bofill-Mas是巴塞罗那大学的塞拉-亨特研究员。

1. 准备和包装
注:要包装的材料/设备列于表1。使用手套处理过程控制所需的试剂、浓缩试剂、核酸提取试剂和检测试剂。戴防护眼镜处理核酸提取所需的试剂。
<ol><li>过程控制
	<ol>
<li>按照 ISO 程序 10705-1:199514,在实验室中制备 MS2 噬菌体培养(美国类型文化收藏 [ATCC] 15597-B1)包含 1 x 1010 PFU/mL。</li>
		<li>然后,将每管含有1 x 10<su...

<ol>
	<li>The Sphere Project. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Sphere Project: Humanitarian Charter and Minimum Standards in Humanitarian Response. , (2011).</li><li>Bartram, 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=.">.</a> Water Safety Plan Manual:Step-by-step risk management for drinking-water suppliers. , (2009).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for Drinking-water Quality. , (2011).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 25 Years Progress on Sanitation and Drinking Water. , (2015).</li><li>Girones, 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=Molecular+detection+of+pathogens+in+water+-+The+pros+and+cons+of+molecular+techniques.">Molecular detection of pathogens in water - The pros and cons of molecular techniques.</a> Water Research. 44 (15), 4325-4339 (2010).</li><li>Rodriguez-Manzano, 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=Standard+and+new+fecal+indicators+and+pathogens+in+sewage+treatment+plants,+microbiological+parameters+for+improving+the+control+of+reclaimed+water.">Standard and new fecal indicators and pathogens in sewage treatment plants, microbiological parameters for improving the control of reclaimed water.</a> Water Science and Technology. 66 (12), 2517-2523 (2012).</li><li>Puig, 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=Detection+of+adenoviruses+and+enteroviruses+in+polluted+waters+by+nested+PCR+amplification.">Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification.</a> Applied and Environmental Microbiology. 60 (8), 2963-2970 (1994).</li><li>Carter, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterically+infecting+viruses:+Pathogenicity,+transmission+and+significance+for+food+and+waterborne+infection.">Enterically infecting viruses: Pathogenicity, transmission and significance for food and waterborne infection.</a> Journal of Applied Microbiology. 98 (6), 1354-1380 (2005).</li><li>Bofill-Mas, S., Pina, S., Girones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Documenting+the+epidemiologic+patterns+of+polyomaviruses+in+human+populations+by+studying+their+presence+in+urban+sewage.">Documenting the epidemiologic patterns of polyomaviruses in human populations by studying their presence in urban sewage.</a> Applied and Environmental Microbiology. 66 (1), 238-245 (2000).</li><li>Bofill-Mas, S., 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=Quantification+and+stability+of+human+adenoviruses+and+polyomavirus+JCPyV+in+wastewater+matrices.">Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices.</a> Applied and Environmental Microbiology. 72 (12), 7894-7896 (2006).</li><li>Rames, E., Roiko, A., Stratton, H., Macdonald, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+aspects+of+using+human+adenovirus+as+a+viral+water+quality+indicator.">Technical aspects of using human adenovirus as a viral water quality indicator.</a> Water Research. 96, 308-326 (2016).</li><li>Calgua, 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=Development+and+application+of+a+one-step+low+cost+procedure+to+concentrate+viruses+from+seawater+samples.">Development and application of a one-step low cost procedure to concentrate viruses from seawater samples.</a> Journal of Virological Methods. 153 (2), 79-83 (2008).</li><li>Bofill-Mas, S., 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=Cost-effective+method+for+microbial+source+tracking+using+specific+human+and+animal+viruses.">Cost-effective method for microbial source tracking using specific human and animal viruses.</a> Journal of Visualized Experiments. 58, 5-9 (2011).</li><li>International Organization for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ISO 10705-1:1995: Water quality - Detection and enumeration of bacteriophages - Part 1: Enumeration of F-specific RNA bacteriophages. , (1995).</li><li>Hernroth, B. E., Conden-Hansson, A. C., Rehnstam-Holm, A. S., Girones, R., Allard, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+factors+influencing+human+viral+pathogens+and+their+potential+indicator+organisms+in+the+blue+mussel,+Mytilus+edulis:+the+first+Scandinavian+report.">Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report.</a> Applied and Environmental Microbiology. 68 (9), 4523-4533 (2002).</li><li>Pecson, B. M., Martin, L. V., Kohn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+PCR+for+determining+the+infectivity+of+bacteriophage+MS2+upon+inactivation+by+heat,+UV-B+radiation,+and+singlet+oxygen:+Advantages+and+limitations+of+an+enzymatic+treatment+to+reduce+false-positive+results.">Quantitative PCR for determining the infectivity of bacteriophage MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: Advantages and limitations of an enzymatic treatment to reduce false-positive results.</a> Applied and Environmental Microbiology. 75 (17), 5544-5554 (2009).</li><li>Calgua, B., Barardi, C. R. M., Bofill-Mas, S., Rodriguez-Manzano, J., Girones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantitation+of+infectious+human+adenoviruses+and+JC+polyomaviruses+in+water+by+immunofluorescence+assay.">Detection and quantitation of infectious human adenoviruses and JC polyomaviruses in water by immunofluorescence assay.</a> Journal of Virological Methods. 171 (1), 1-7 (2011).</li><li>Bofill-Mas, S., 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=Cost-effective+method+for+microbial+source+tracking+using+specific+human+and+animal+viruses.">Cost-effective method for microbial source tracking using specific human and animal viruses.</a> Journal of Visualized Experiments. (58), e2820 (2011).</li><li>Gonzales-Gustavson, E., 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=Characterization+of+the+efficiency+and+uncertainty+of+skimmed+milk+flocculation+for+the+simultaneous+concentration+and+quantification+of+water-borne+viruses,+bacteria+and+protozoa.">Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa.</a> Journal of Microbiological Methods. 134, 46-53 (2017).</li></ol>

VirWaTest 方法使未经经验的用户在使用时能够从水样中浓度病毒和核酸。这是一个经济实惠,快速,简单的协议。浓度基于使用脱脂牛奶的有机絮凝原理,通过低pH值和高电导率条件使脱脂牛奶蛋白聚合成絮状物,病毒吸附到。当浮游沉积物时,很容易收集它们,从而有可能浓缩10 L的水,而传统的超离心不能处理大量的水量。
该方法经过修改,在采样点可行,无需使用任何电气设备,电池供...

在任何人道主义紧急情况的第一阶段,获得清洁水供应、环境卫生和个人卫生对于受影响者的生存至关重要。因此,监测水质是预防水传播疫情的优先事项。众所周知,受污染的水往往是疾病的根源,但往往很难确定病毒爆发的来源,如E型肝炎病毒(HEV),即使有传统的实验室方法。水质控制以FIB 1、2、3、4的量化为基础。然而,据广泛记载,FIB的缺乏与病毒性水传播病原体(如轮状病毒(RoV)、诺如病毒(NoV)或HEV5、6)的存在之间没有相关性。因此,使用基于FIB的水质标准可能导致低估与水传播病毒病原体存在相关的风险。监测指标病毒,如人类腺病毒(HAdV),或特定的病原体将有助于确定对病毒病原体的接触和确定人类感染的潜在来源7,8 9、10和在验证卫生措施的功效11。
到目前为止,在这些场景中检测病毒依赖于熟练的员工和复杂的物流。VirWaTest (virwatest.org) 旨在开发一种简单、经济、便携的方法,用于在使用时从水样中浓度和随后检测病毒。
病毒浓度基于10L水样的有机絮凝原理,通过该原理,病毒在较小的体积12、13中回收。在室温下储存不超过2周时,将絮状物收集并添加到缓冲液中,以感染病毒并防止核酸降解。
核酸提取方法基于利用磁粒子,核酸被吸附到这些颗粒上。它们可以通过粒子所连接的磁移液器从一个洗涤缓冲器转移到另一个洗涤缓冲液,最后转移到洗脱缓冲液中。获得病毒核酸悬浮液可运送到参考实验室,在那里可以使用基于PCR的分子方法进行检测。对于每个核酸萃取,测试两种不同的数量,以排除样品产生的酶抑制。或者,在设备可用性最低时,PCR 测试可以在使用点运行。整个过程设计为独立于电源执行(图1)。
一种定量PCR测定,用于检测HAdV,由人类排泄,并在高浓度的废水样品中发现,现已适应在使用点运行。HAdV被用作人类粪便病毒指标。由于 MS2 在 VirWaTest 中用作过程控制,因此还采用了用于 MS2 噬菌体定量的 PCR。该方法可以自定义,以检测任何感兴趣的病毒。
开发后,在中部非洲共和国和厄瓜多尔的两种不同环境中,用户应用了VirWaTest方法,就议定书在实际情况中的应用提供了反馈。
据我们所知,这是第一个允许在使用点集中和检测病毒的程序,独立于任何电源、大型设备和冷冻/冷却条件。建议收集每个水样本的两个副本,以获得可靠的结果。

<table border="0" cellpadding="0" cellspacing="0" style="width:1677px;" width="1677">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">5x HOT FIREPol Probe qPCR Mix Plus (ROX)</td>
			<td style="width:195px;">Solis BioDyne</td>
			<td style="width:123px;">08-14-00001</td>
			<td style="width:989px;">Includes Solis Biodyne&#39;s 5x HOT FIREPol Probe qPCR Mix Plus (qPCR Mix), 50 Reactions</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">8-Microtube Strips with Caps</td>
			<td style="width:195px;">dD Biolab</td>
			<td style="width:123px;">840637</td>
			<td>Low Profile, Thin Walls, Adapted for Quantitative and Qualitative PCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Aquagenx CBT E. coli Kit</td>
			<td style="width:195px;">Aquagenx, LLC</td>
			<td style="width:123px;">ECCBT10</td>
			<td>10 Tests per Kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Batteries and Power Adapters for Magnetic Stirrer</td>
			<td style="width:195px;">GenIUL</td>
			<td style="width:123px;">900011674</td>
			<td>Includes 12V car power adapter</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Bucket Support</td>
			<td style="width:195px;">GenIUL</td>
			<td style="width:123px;">900011648</td>
			<td>Aluminium support</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Bucket, 10 L</td>
			<td style="width:195px;">Cater4You</td>
			<td style="width:123px;">10LTR</td>
			<td>Polypropilene, Tamperproof, Clear color</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Centrifuge Tube, 50 mL</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">CTSP-E50-050</td>
			<td>Polypropylene, Sterile, Graduated, With Skirt</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Citric Acid 1-Hydrate, 500 g</td>
			<td style="width:195px;">PanReac AppliChem</td>
			<td style="width:123px;">1410181211</td>
			<td>Pure, Pharma Grade, 1 Kilogram</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Clear PET Bottle</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">FPET-500-088</td>
			<td style="width:989px;">Clear Color, PET, Cap Not Included</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Difco Skim Milk, 500 g</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:123px;">232100</td>
			<td>Dehydrated</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">DNA/RNA Shield, 250 mL</td>
			<td style="width:195px;">Zymo Research</td>
			<td style="width:123px;">R1100-250</td>
			<td>DNA/RNA Preservation Medium, 250 mL</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Easy9 Pipette Controller</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">EAS9-001-001</td>
			<td>0.3 &#956;m filter, Pipettes from 0.1 to 100 mL, Autoclavable silicone pipette holder</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Eppendorf Tube, 0.5 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:123px;">0030121023</td>
			<td>Polypropilene, Safe-Lock</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Eppendorf Tube, 2 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:123px;">0030120094</td>
			<td>Polypropilene, Safe-Lock</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Eppendorf Tube, 5 mL</td>
			<td style="width:195px;">dD Biolab</td>
			<td style="width:123px;">999542</td>
			<td>Polypropylene, Sterile, Graduated</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Ethanol 96% V/V, 1 L</td>
			<td style="width:195px;">Panreac AppliChem</td>
			<td style="width:123px;">361085-1611</td>
			<td>For UV, IR&#160; and HPLC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Laboratory Tweezers</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">FORS-007-002</td>
			<td>Thin, Curved End, L= 120 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Magnetic Stirrer</td>
			<td style="width:195px;">GenIUL</td>
			<td style="width:123px;">900017000</td>
			<td>Battery-powered</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Marker</td>
			<td style="width:195px;">dD Biolab</td>
			<td style="width:123px;">929203</td>
			<td style="width:989px;">Black, Extra fine Tip, Water Resistant, Fast Drying, For Plastic and Glassware</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Micro Rota-Rack for Microtubes</td>
			<td style="width:195px;">dD Biolab</td>
			<td style="width:123px;">37782</td>
			<td>4 Modules, L x W x H= 208 x 100 x 100 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Mini8 Real-Time PCR Cycler</td>
			<td style="width:195px;">Coyote Biosciences, China</td>
			<td style="width:123px;">Mini-8</td>
			<td>Portable, Works with 12V Power Supplies or External Batteries, Two channels, Capacity for 8 Tubes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">NucliSens Lysis Buffer</td>
			<td style="width:195px;">Biomerieux</td>
			<td style="width:123px;">200292</td>
			<td>Reagents for up to 48 Isolations, Store at Ambient Temperature</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Open Tip Serological Pipette, 10 mL</td>
			<td style="width:195px;">Deltalab</td>
			<td style="width:123px;">900136N</td>
			<td>Sterile, Individually Wrapped (Paper/Plastic)</td>
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		<tr height="22">
			<td height="22" style="height:23px;width:371px;">PE Screw Cap PP28</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">TP28-004-020</td>
			<td style="width:989px;">For PET Bottles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">pH Indicator Strip</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">WSPH-001-001</td>
			<td>Range pH 2.8 to pH 4.4, 50 Strips per Pack</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Plastic Test Tube</td>
			<td style="width:195px;">Quimikals</td>
			<td style="width:123px;">300913</td>
			<td>Includes Cap</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Polyethylene Pasteur Pipette</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">PIPP-003-500</td>
			<td>Graduated, 7 mL Overall Volume, Non-Sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Polypropylene Screw Flask With Screw Cap, 150 mL</td>
			<td style="width:195px;">Deltalab</td>
			<td style="width:123px;">409726</td>
			<td>Screw cap, Sterile, graduated up to 100 mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Polypropylene Screw Flask With Screw Cap, 60 mL</td>
			<td style="width:195px;">Deltalab</td>
			<td style="width:123px;">409526G</td>
			<td>Screw cap, Sterile, Graduated up to 50 mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Powder Powder Detergent</td>
			<td style="width:195px;">-</td>
			<td style="width:123px;">-</td>
			<td>Regular Powder Soap for washing clothes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Power Cables for Magnetic Stirrer</td>
			<td style="width:195px;">GenIUL</td>
			<td style="width:123px;">900011692</td>
			<td>Connection between batteries and magnetic stirrers</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">QuickPick Magnetic Tool</td>
			<td style="width:195px;">BioNobile</td>
			<td style="width:123px;">24001</td>
			<td>Hand-held tool for magnetic particles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">QuickPick Tips in Box</td>
			<td style="width:195px;">BioNobile</td>
			<td style="width:123px;">24296</td>
			<td>RNase-Free, Autoclaved, 96 Units</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">QuickPick XL gDNA Magnetic Particles</td>
			<td style="width:195px;">BioNobile</td>
			<td style="width:123px;">SN51100</td>
			<td>3.2 mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Sea Salts</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:123px;">S9883-500G</td>
			<td>An artificial salt mixture closely resembling the composition of the dissolved salts of ocean water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Silicone Tubing</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">SILT-006-005</td>
			<td>Roll of 5 Meters, Inner&#160; &#248; x Outer&#160; &#248;= 6 x 10 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Sodium Hydroxide Pellets, 98.5 - 100.5%</td>
			<td style="width:195px;">VWR Chemicals</td>
			<td style="width:123px;">28244295</td>
			<td>Pellets, 1 Kg</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Solar Rotary Platform</td>
			<td style="width:195px;">SOL-EXPERT Group</td>
			<td style="width:123px;">70020</td>
			<td>Acrylic Plate, 10 RPM, Supports up to 300 Grams</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">SOLIScript 1-step Probe Kit</td>
			<td style="width:195px;">Solis BioDyne</td>
			<td style="width:123px;">08-57-00250</td>
			<td>Includes Solis Biodyne&#39;s 5x One-Step Probe Mix (qPCR Mix) and 40x One-Step SOLIScript Mix (Reverse Transcriptase Enzyme), 250 Reactions</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">SPEEDTOOLS RNA Virus Extraction Kit</td>
			<td style="width:195px;">BioTools</td>
			<td style="width:123px;">21.141-4197</td>
			<td>Includes BioTools&#39;s BAW Buffer (Washing Buffer 1), BAV3 Buffer (Washing Buffer 2 and 3) and BRE Buffer (Elution Buffer).</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">SpinBar Octhaedral Stirring Magnet</td>
			<td style="width:195px;">dD Biolab</td>
			<td style="width:123px;">045926</td>
			<td>Pivot Ring, L x&#160; &#248; = 38 x 8 mm, Blue</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Tape-End Serological Pipette, 10 mL</td>
			<td style="width:195px;">Deltalab</td>
			<td style="width:123px;">PN10E1</td>
			<td>Sterile, Individually Wrapped (Paper/Plastic)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Tape-End Serological Pipette, 50 mL</td>
			<td style="width:195px;">Deltalab</td>
			<td style="width:123px;">900043</td>
			<td>Sterile, Individually Wrapped (Paper/Plastic)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Termi-DNA-Tor - Nucleic Acid Remover</td>
			<td style="width:195px;">BioTools</td>
			<td style="width:123px;">22001-4291</td>
			<td>Remover of nucleic acids, bacteria, fungi and mycoplasma from material and surfaces, 450 mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Water Molecular Biology Reagent, 1L</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:123px;">W4502-1L</td>
			<td>Nuclease and Protease Free, 0.1 &#956;m Filtered</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Whirl-Pak Bag, 540 mL</td>
			<td style="width:195px;">Deltalab</td>
			<td style="width:123px;">200361</td>
			<td>Stable bottom</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:371px;">Zip Lock Plain Bag</td>
			<td style="width:195px;">LabBox</td>
			<td style="width:123px;">BZIP-080-100</td>
			<td>Polyethylene, L x W= 120 x 80 mm</td>
		</tr>
	</tbody>
</table>

virwatest, a point-of-use method for the detection of viruses in water samples

当这种水用于饮用、食物灌溉、洗涤等时,人类和动物排出的病毒可能会污染水源,对人类健康构成威胁。传统的粪便细菌指标并不总是检查病毒病原体是否存在,因此病毒病原体和病毒指标的检测是相关的,以便采取降低风险的措施,特别是在人道主义情况和领域水传播病毒爆发频繁。
目前,在使用点可以进行若干允许粪便指示细菌(FIB)定量的商业测试。但是,此类商业测试不适用于病毒检测。在环境水样品中检测病毒需要将几升浓缩成更小的体积。此外,病毒的检测一旦集中,就依赖于病毒基因组的核酸提取和分子检测(例如,基于PCR的聚合酶链反应[PCR]测定)等方法。
此处描述的方法允许从 10 L 水样中浓度病毒,以及在使用时使用简单和便携式设备提取病毒核酸。这允许在使用点测试水样几种病毒,在人道主义情景以及没有设备化实验室的任何情况下都很有用。或者,该方法允许浓缩水样中的病毒,并将浓缩物运送到室温下的实验室进行进一步分析。

方法开发
这一程序是在GenIUL和乐施会的实验室开发的。它包括三个不同的步骤。第一种是病毒颗粒浓度,是以前描述的脱脂牛奶絮凝法的改编方法。原始方法被修改为独立于电源,更简单,并且没有离心步骤。
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Watch this Scientific Journal Video about VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples at JoVE.com

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

在这里,我们介绍VirWaTest,这是一种简单、经济、便携的方法,用于在使用时从水样中集中和检测病毒。

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 ...

肠病毒大多存在于不同的水生水库，包括地下水、静止水、溪流、河流和 r 水。它们与健康风险有关，并对人类感染负责。肠病毒通常引起肠胃炎，并且大多通过粪便-口腔途径传播。其他水传播疾病由甲型肝炎病毒和E型肝炎病毒引起急性肝炎，其相关死亡率显著，特别是孕妇的E型肝炎。为人道主义情况提议的水质监测实际协议旨在将E.Coli等细菌作为粪便污染的指标。但众所周知，与其他微生物（如对许多激活过程更具抵抗力的病毒）没有相关性。到目前为止，在人道主义危机环境中已经进行了病毒检测，使现有协议适应这些情况，并运送样品到参考实验室进行病毒分子检测。需要从观点中检测病毒病原体和病毒指标的方法，以减少公共卫生风险，防止疫情爆发，提高人道主义干预的效率。提出了一种10升水样病毒检测方法，分为病毒浓度、核酸提取和病毒检测三个步骤。所有这些步骤都根据我们实验室目前使用的方法进行了调整，并且可以通过使用便携式试剂和设备，生产出电源和冰柜的受抚养者，在观点上执行。此外，在方法中添加了防腐剂溶液，以保证核酸的稳定性，如果需要，可运往参考实验室。我们提出的方法叫做VirWaTest，是经过GenIUL和乐施会的合作开发的。在开始之前，应考虑要测试的样品数量，以制备试剂和材料。建议收集每个水样两个副本，以获得有代表性的结果。如果有适当数量的材料可用，可以同时测试多个样品。在移动或运到需要的方法的地方之前，应准备和包装试剂和材料。表一描述了应包装的材料和小型设备清单。准备一个噬菌体MS2库存，按照相应的ISO方法作为过程控制添加到每个水样中。然后分配10微升的悬浮液，其中含有10至10个平台装置，预制作成10毫升管，让水干燥。每个水样需要一管。此外，每采集的样品制备一个含有10mL无菌蒸馏水的管子。对于预絮状脱脂牛奶溶液，准备一个管子，里面装着脱脂牛奶，一个拉链塑料袋，里面装着海盐，还有一个装有蒸馏水的塑料瓶。对于 pH 调整，准备一个含有柠檬酸的管子、一个含有氢氧化钠的管子以及两个含有 25 和 50 mL 蒸馏水的塑料容器。对于要测试的每个水样，通过将海盐和柠檬酸加入拉链塑料袋来准备一个调理袋，通过将解解缓冲液和核酸防腐剂添加到 5 mL 管中来制备保鲜溶液。最后，准备四个中和小袋，在四个拉链塑料袋中加入强力洗涤剂。对于要提取的每个水样，准备一个含有磁性颗粒和乙醇的五毫升管，以及三个含有500、600和200微升洗涤缓冲液的2mL管，以及一个含有洗涤缓冲液的管。如果使用纽厄尔热循环器，则每个 PCR 测定中可同时分析两种不同的核酸提取。对于所有PCR测定，准备分子生物学水。根据协议中描述的体积和浓度，准备包含两个底向和一个特定目标病毒的混合物。对于每种病毒，将指示的卷添加到管内一至八个管托盘。切条，分成两条。管子一到五，管子六到八。对于每种病毒，含有10至5、10至7和10至9的风干DNA悬浮液，以及预制作成管6、7和8的已知拷贝。这种浓度协议基于有机絮凝原理，低pH和高导电性条件驱动脱脂牛奶蛋白组织成病毒吸收的絮凝。一旦絮凝，可以很容易地收集。在带盖子的平底桶中收集 10 升水样。如果从管道中收集，让水在收集水之前运行几秒钟。使用透明铲斗来可视化颗粒非常重要。在远离阳光直射的新鲜地方寻找一个平坦的空间，将磁力搅拌器连接到桶支撑下的电池，并在每个支撑上放置包含水样的水桶。重新考虑柠檬酸和氢氧化钠，并摇动，直到它们完全溶解。将 500 mL 的蒸馏水倒入立式袋中，然后将袋子放在磁性搅拌器上。加入一个海盐袋的内容和一个脱脂奶管的内容，以中等速度搅拌五分钟。使用柠檬酸和氢氧化钠将絮状脱脂牛奶的 pH 值调整为 3.4 和 3.6 之间的 pH 值。关闭磁力搅拌器，确保浮子可见。手电筒可能有助于可视化絮凝。将搅拌磁铁扔进水中。将磁力搅拌器设置为最大速度并打开。将 10 mL 蒸馏水加入过程控制管。倒置几次，把溶液倒入水中。将一个样品调理袋的内容倒入水中，搅拌五分钟。确保水样（应介于 3.4 和 3.6 之间）的 pH 值。如果需要，调整 pH。加入 100 mL 预絮状脱脂牛奶溶液，用盖子密封水桶，以最低速度将水搅拌 8 小时。八小时后，关闭磁力搅拌器，让絮凝器至少多等五个小时。使用基于移液器控制器、塑料管和几个移液器的系统，丢弃上清液，注意不要打扰铲斗底部的絮状物。当水位即将到达搅拌磁铁时，挤压塑料管以停止水流，然后将移液器从铲斗中移开。摇动水桶重新浇注絮片，然后倒入立式塑料袋中。允许絮凝，多等一小时。使用移液器小心吸水，避免干扰絮状物。使用移液器吸气絮，并将它们转移到锥形管中。记下样品浓缩物的最终体积，将 1 mL 转移到含有防腐剂溶液的管中。悬浮液可用于在田间进行核酸提取，或运到参考实验室作进一步分析。收集絮凝物时丢弃的水必须在处理前处理。将中和剂袋的内容添加到包含废弃水的桶中。混合水，让水静止三十分钟。然后将处理过的水作为一般废物处理。磁性颗粒可以使用磁性移液器从一个管转移到另一个管。在执行提取之前，先习惯磁移液器的操作。将含有提取所需试剂的管子排列在机架中。也就是说，从左到右：绑定缓冲区、洗涤缓冲区和洗脱缓冲区。使用一次性塑料移液器将 1 mL 样品浓缩物转移到含有结合缓冲液的管中。反转管十次以优化解决方案。在室温下孵育溶液十分钟，同时使用单独轨道或手动连续混合。使用磁性移液器和可重复使用的清洁尖端收集磁性颗粒，并释放磁性颗粒到含有洗涤缓冲液的管中。用尖端轻轻摇动溶液三十秒钟，然后收集它们。重复此清洗过程以清洗缓冲液 2 和 3。收集洗涤缓冲液三中的磁性颗粒，并释放到含有洗涤缓冲液的管中。然后丢弃尖端。在室温下孵育溶液五分钟，同时使用独奏轨道连续混合。将磁性移液器上的尖端更换为任何一个，然后从洗脱缓冲液中收集所有磁性颗粒。将附加颗粒的尖端丢弃。核酸留在洗脱缓冲液中，应储存以作进一步分析或装运。使用五管和三管带准备检测人类腺病毒。根据协议，将水、PCR混合物和核酸外加加入到管中。之后，将水和PCR混合到管六至八。将两条带放入热循环器中并选择适当的运行。运行完成后，收集结果并分析获得的数据。只有当病毒测试导致阴性时，才应进行 MS2 检测。使用为 MS2 检测准备的管条，并选择适合 PCR 的热轮廓。VirWaTest磁核酸提取与QIAgen病毒RNA进行比较，通过测试与人类腺病毒和MS2尖峰的水样来模拟它。减少测试 p 值显示，使用 VirWaTest 比 QiAgen 作为两种测试病毒的提取方法，病毒恢复率显著提高。在位于中非共和国班吉和厄瓜多尔佩德纳莱斯的乐施会洗涤小组的合作下，在实地测试了病毒浓度的开发方法，这些小组收集水样，采用开发浓度方法进行浓缩，并把它们送到巴塞罗那的实验室进行核酸提取和病毒定量。在厄瓜多尔，在所有检测的样本中检测到人类腺病毒，而在班吉采集和浓缩的五个样本中，有一个对人腺病毒检测呈阳性。在中非共和国班吉的乐施会际洗涤小组和厄瓜多尔佩德纳莱斯的另一个小组对该方法进行评估时，已证明该方法是有用的。VirWaTest 可能是预警系统的一部分，也可能用于参与水卫生的组织对疫情进行调查。这种设备有助于分析水中的病毒病原体，改善水安全管理，以减少人道主义危机情况下的病毒性疾病事件。

Research

JoVE Journal

Immunology and Infection

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

使用荧光免疫化学检测爱德华ictaluri</em斑点叉尾鮰>（一毛虫）样品

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

科研

免疫与感染

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

为集中的的水性病毒和农业人畜共患病病原体的玻璃纤维过滤器

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性<em&gt;隐</em&gt;和<em&gt;贾第虫属。</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

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

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

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 ...