Summary

病毒测试,水样中检测病毒的一种使用点方法

Published: May 11, 2019
doi:

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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。 过程控制 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。 然后,将每管含有1 x 10<su…

Representative Results

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。 <p class="jove_content" fo:keep-together.with…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

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Cite This Article
Aguado, D., Fores, E., Guerrero-Latorre, L., Rusiñol, M., Martínez-Puchol, S., Codony, F., Girones, R., Bofill-Mas, S. VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples. J. Vis. Exp. (147), e59463, doi:10.3791/59463 (2019).

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