人类诺沃克病毒的表面上检测拭子采样方法
Summary
A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.
Abstract
诺如病毒的人是流行性和世界各地的零星胃肠炎的主要原因。因为大多数感染或者直接经由人对人的路线通过环境表面或食物传播或间接地受污染的非生物媒介和无生命的表面是用于病毒的期间诺罗病毒爆发的传播重要的车辆。
我们开发和评估使用大泡拭子用于检测和从硬表面人类诺如病毒的打字的协议。纤维拭子或防静电抹布相比，大泡拭子允许从高达700 cm 2的马桶座面恢复病毒（范围1.2-33.6％）。该协议包括用于病毒从拭子提取和使用旋转柱的病毒RNA的进一步浓缩的步骤。总共127已经从在游船和长期护理设施的表面，其中诺罗病毒性胃肠炎已经收集到217拭子样品（58.5％）报道药检呈阳性被RT-qPCR的诺如病毒GII。这些29（22.8％）的可以成功基因分型。总之，检测使用我们开发可以帮助确定环境污染的暴发期间水平以及检测病毒的当临床样品是不可用的协议环境表面上诺罗病毒的;它也可能有利于整治策略的有效性进行监测。
Introduction
诺如病毒的人是流行性和散发性急性胃肠炎全世界1，2，3的主要原因。该病毒是极具传染性并通过直接的人发生互动的人或间接通过受污染的食物，水或环境表面接触传播。诺如病毒可棚长时间和长期的环境表面上的病毒的生存已记载1，2，3。在高峰脱落，十亿病毒颗粒被每克释放粪便和呕吐物还含有足够数量的病毒颗粒中，以引起感染4，5，6，7，8，EF“> 9,10，另外，可以很容易地发生无生命的表面和人的皮肤之间的病毒传输2，11，12，因此，环境污染的监测可有助于爆发调查和评估的清理的有效性和消毒程序。
几个环境取样协议已经用于检测轮状病毒了描述，大肠杆菌噬菌体MS2，猫杯状病毒（FCV），和噬菌体P22 13，14，15，16。然而，在这些研究中，包括快速干燥（<1小时）和小的表面区域中所述的验证条件（25×100厘米2）时，可能不能充分代表字段设置。此外，预期ENVIRO的低的污染水平nmental表面需要是能够检测很少的病毒颗粒的协议。
我们开发了诺如病毒的检测和分型基于大泡，表面取样方法。这种方法已经在几次爆发诺沃克病毒被证实。该协议包括1）如何收集环境表面拭子样本（2）如何收集和运输到实验室过程中最好保持样本的完整性，以及3）诺如病毒的实验室测试和打字。
Protocol
1.棉签取样的场穿一双干净的手套。 测量取样区域的大小，而无需使用卷尺或直尺接触到表面。尝试尽可能准确地估计该地区并填写报告表（补充表1）。 检查可能的泄漏和标签样本运输袋，包棉签拭子套件。 移动拭子穿过取样面积如下：在水平方向的行程，在垂直方向的一个行程，并在对角方向的一个笔划。不要擦拭表面面积超过700 平方厘米大…
Representative Results
图1呈现拭子采样协议的流程图。这个协议包括四个主要步骤; 1）样品的采集，2）样品存储和运输，3）病毒RNA纯化和浓缩和4），RT-qPCR分析和基因分型。 图1：流量为诺如病毒的环境表面取样最终协议的图表 <a href="http://ecsource.jove.com/files/ftp_upload/55205…
Discussion
诺如病毒有18和10 3病毒颗粒20之间的50％的人感染剂量。因此，即使表面的低水平的污染可能对公共健康危险。拭子采样协议的若干方面进行了评价，包括：1）不同拭子的材料，2）在运输过程中贮存条件拭子，3）病毒RNA的浓度，和4）噬菌体MS2作为内提取的控制。
直到最近，才由棉，涤纶，锦纶和抗静电擦拭）制成拭子的性能进行了评估，并提出适?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| Generic name for kits | ||||
| Macrofoam swab | Premoistened EnviroMax Swab kit | Puritan | 2588060PFUW | |
| RNA Lysis buffer | CDC UNEX buffer | Microbiologics | Cat No MR0501 | |
| RNA extraction spin column | Midi column | Omega Biotek | Cat No R6664-02 | |
| RNA purification spin column | Zymol RNA Clean and Concentrator kit | Zymo Research | Cat No R1016 | |
| Real time RT-PCR kit | AgPath kit One-Step RT-PCR Kit | Life Technologies | Cat No 4387391 | |
| Conventional RT-PCR kit | Qiagen one step RT-PCR kit | Qiagen kit | Cat No 210212 | |
| Gel extraction kit | Qiagen QIAquick gel extraction kit | Qiagen kit | Cat No 28704 or 28706 | |
| Coliphage MS2 | ATCC | Cat No 15597-B1 | ||
| RNA run-off transcripts | Bacteriophage MS2 (ATCC No. 15597-B1) can be cultivated using Escherichia coli (E.coli) Famp (ATCC No. 700891). | |||
| Realtime PCR platform | Applied Biosystems | Model ABI 7500 | GI and GII RNA run off transcripts were quantified spectrophotometrically at A260, diluted in diethyl pyrocarbonate-treated water to 1 × 106 copies/ μl, and stored at −80°C with 1.0 U /μl RNasin (Promega, Madison, WI). | |
| Optical 96-well reaction plate | Thermo Scientific | Cat No 4316813 | ||
| MicroAmp Clear Adhesive Film | Thermo Scientific | Cat No 4306311 |
Riferimenti
- Isakbaeva, E. T., et al. Norovirus transmission on cruise ship. Emerg. Infect. Dis. 11, 154-158 (2005).
- Lopman, B. A., Gastañaduy, P., Park, G. W., Hall, A. J., Parashar, U. D., Vinjé, P. Environmental transmission of norovirus gastroenteritis. Curr. Opin. Virol. 2 (1), 1-7 (2011).
- Malek, M., et al. Outbreak of norovirus infection among river rafters associated with packaged delicatessen meat, Grand Canyon, 2005. Clin Infect Dis. 48 (1), 31-37 (2009).
- Atmar, R. L., et al. Norwalk virus shedding after experimental human infection. Emerg. Infect. Dis. 14 (10), 1553-1557 (2008).
- Glass, R. I., Parashar, U. D., Estes, M. K. Norovirus gastroenteritis. N. Engl. J. Med. 361 (18), 1776-1785 (2009).
- Park, G. W., et al. Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces. Appl. Environ. Microbiol. 81 (17), 5987-5992 (2015).
- Barker, J., Jones, M. V. The potential spread of infection caused by aerosol contamination of surfaces after flushing a domestic toilet. J. Appl. Microbiol. 99, 339-347 (2005).
- Tung-Thompson, G., Libera, D. A., Koch, K. L., de Los Reyes, F. L., Jaykus, L. A. Aerosolization of a Human Norovirus Surrogate, Bacteriophage MS2, during Simulated Vomiting. PloS one. 10, 0134277 (2015).
- Atmar, R. L., et al. Determination of the 50% human infectious dose for Norwalk virus. J. Infect. Dis. 209 (7), 1016-1022 (2014).
- Petrignani, M., van Beek, J., Borsboom, G., Richardus, J. H., Koopmans, M. Norovirus introduction routes into nursing homes and risk factors for spread: a systematic review and meta-analysis of observational studies. J. Hosp. Infect. 89 (3), 163-178 (2015).
- . Centers for Disease Control Prevention. Norovirus outbreak in an elementary school–District of Columbia, February 2007. MMWR. Morb. Mortal. Wkly. Rep. 56 (51-52), 1340-1343 (2008).
- Cheesbrough, J. S., Barkess-Jones, L., Brown, D. W. Possible prolonged environmental survival of small round structured viruses. J. Hosp. Infect. 35, 325-326 (1997).
- Julian, T. R., Tamayo, F. J., Leckie, J. O., Boehm, A. B. Comparison of surface sampling methods for virus recovery from fomites. Appl. Environ. Microbiol. 77, 6918-6925 (2011).
- Taku, A., et al. Concentration and detection of caliciviruses from food contact surfaces. J. Food. Prot. 65, 999-1004 (2002).
- Scherer, K., Ellerbroek, L., Schulenburg, J., Johne, R., Klein, G. Application of a swab sampling method for the detection of norovirus and rotavirus on artifically contaminated food and environmental surfaces. Food. Environ. Virol. 1 (42), 42-49 (2009).
- Herzog, A. B., et al. Evaluation of sample recovery efficiency for bacteriophage P22 on fomites. Appl. Environ. Microbiol. 78, 7915-7922 (2012).
- Vega, E., et al. CaliciNet: A Novel Surveillance Network for Norovirus Gastroenteritis Outbreaks in the United States. Emerging Infectious Diseases. 17 (8), 1389-1395 (2011).
- Rolfe, K. J., et al. An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping. J Clin Virol. 39 (4), 318-321 (2007).
- Kittigul, L., et al. Norovirus GII-4 2006b variant circulating in patients with acute Thailand during a 2006-2007 study. J. Med. Virol. 82 (5), 854-860 (2010).
- Teunis, P. F., et al. Norwalk virus: how infectious is it. J. Med. Virol. 80 (8), 1468-1476 (2008).
- Wollants, E., et al. Evaluation of a norovirus sampling method using sodium dodecyl sulfate/EDTA-pretreated chromatography paper strips. J. Virol. Methods. 122, 45-48 (2004).
- Weir, M. H., Shibata, T., Masago, Y., Cologgi, D., Rose, J. B. The Effect of Surface Sampling and Recovery of Viruses and Non-Spore Forming Bacteria on a QMRA Model for Fomites. Environ. Sci. Technol. 50 (11), 5945-5952 (2016).
- . Microbiology of food and animal feed-Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR. International Organization for Standardization (ISO). , (2013).
- Huslage, K., Rutala, W. A., Sickbert-Bennett, E., Weber, D. J. A quantitative approach to defining “high-touch” surfaces in hospitals. Infect. Control. Hosp. Epidemiol. 31 (8), 850-853 (2010).
- Wu, H. M., et al. A norovirus outbreak at a long-term-care facility: the role of environmental surface contamination. Infect. Control. Hosp. Epidemiol. 26 (10), 802-810 (2005).
- Ikner, L. A., Gerba, C. P., Bright, K. R. Concentration and recovery of viruses from water: a comprehensive review. Food Environ. Virol. 4 (2), 41-67 (2012).
- Gallimore, C. I., et al. Environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit. J. Clin. Microbiol. 44 (2), 395-399 (2006).
- Ganime, A. C., et al. Dissemination of human adenoviruses and rotavirus species A on fomites of hospital pediatric units. Am J Infect Control. , (2016).
- Verani, M., Bigazzi, R., Carducci, A. Viral contamination of aerosol and surfaces through toilet use in health care and other settings. Am J Infect Control. 42 (7), 758-762 (2014).
Citazione di questo articolo
Park, G. W., Chhabra, P., Vinjé, J. Swab Sampling Method for the Detection of Human Norovirus on Surfaces. J. Vis. Exp. (120), e55205, doi:10.3791/55205 (2017).