病毒测试,水样中检测病毒的一种使用点方法
1Laboratory of Viruses Contaminants of Water and Food (VIRCONT), Department of Genetics, Microbiology and Statistics, Section of Microbiology, Virology and Biotechnology,University of Barcelona, 2Grupo de Investigación Biodiversidad, Medio Ambiente y Salud (BIOMAS), Facultad de Ingenierías y Ciencias Agropecuarias (FICA), Ingeniería en Biotecnología,Universidad de las Américas, 3Municipal Laboratory – Waters of Mataró, 4GenIUL
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 |
References
- The Sphere Project. . The Sphere Project: Humanitarian Charter and Minimum Standards in Humanitarian Response. , (2011).
- Bartram, J., et al. . Water Safety Plan Manual:Step-by-step risk management for drinking-water suppliers. , (2009).
- World Health Organization. . Guidelines for Drinking-water Quality. , (2011).
- World Health Organization. . 25 Years Progress on Sanitation and Drinking Water. , (2015).
- Girones, R., et al. Molecular detection of pathogens in water – The pros and cons of molecular techniques. Water Research. 44 (15), 4325-4339 (2010).
- Rodriguez-Manzano, J., et al. Standard and new fecal indicators and pathogens in sewage treatment plants, microbiological parameters for improving the control of reclaimed water. Water Science and Technology. 66 (12), 2517-2523 (2012).
- Puig, M., et al. Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Applied and Environmental Microbiology. 60 (8), 2963-2970 (1994).
- Carter, M. J. Enterically infecting viruses: Pathogenicity, transmission and significance for food and waterborne infection. Journal of Applied Microbiology. 98 (6), 1354-1380 (2005).
- Bofill-Mas, S., Pina, S., Girones, R. Documenting the epidemiologic patterns of polyomaviruses in human populations by studying their presence in urban sewage. Applied and Environmental Microbiology. 66 (1), 238-245 (2000).
- Bofill-Mas, S., et al. Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices. Applied and Environmental Microbiology. 72 (12), 7894-7896 (2006).
- Rames, E., Roiko, A., Stratton, H., Macdonald, J. Technical aspects of using human adenovirus as a viral water quality indicator. Water Research. 96, 308-326 (2016).
- Calgua, B., et al. Development and application of a one-step low cost procedure to concentrate viruses from seawater samples. Journal of Virological Methods. 153 (2), 79-83 (2008).
- Bofill-Mas, S., et al. Cost-effective method for microbial source tracking using specific human and animal viruses. Journal of Visualized Experiments. 58, 5-9 (2011).
- International Organization for Standardization. . ISO 10705-1:1995: Water quality – Detection and enumeration of bacteriophages – Part 1: Enumeration of F-specific RNA bacteriophages. , (1995).
- Hernroth, B. E., Conden-Hansson, A. C., Rehnstam-Holm, A. S., Girones, R., Allard, A. K. Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report. Applied and Environmental Microbiology. 68 (9), 4523-4533 (2002).
- Pecson, B. M., Martin, L. V., Kohn, T. 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. Applied and Environmental Microbiology. 75 (17), 5544-5554 (2009).
- Calgua, B., Barardi, C. R. M., Bofill-Mas, S., Rodriguez-Manzano, J., Girones, R. Detection and quantitation of infectious human adenoviruses and JC polyomaviruses in water by immunofluorescence assay. Journal of Virological Methods. 171 (1), 1-7 (2011).
- Bofill-Mas, S., et al. Cost-effective method for microbial source tracking using specific human and animal viruses. Journal of Visualized Experiments. (58), e2820 (2011).
- Gonzales-Gustavson, E., et al. Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa. Journal of Microbiological Methods. 134, 46-53 (2017).
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).