小体积的方法从水中病毒浓度
Summary
An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.
Abstract
Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.
Introduction
人类肠道病毒是水源性疾病1-3中重要病原体，但一般存在于被污染的环境水低的数字，使得它们的检测困难而不浓度。用于浓缩病毒程序通常包括一个过滤步骤中的过滤器的洗脱液中，接着过滤洗脱，和仲浓度。一个常见的过滤方法依赖于使用荷电膜，如正电的过滤器（最近在4,5审查）的。这些过滤器依赖于捕捉的病毒悬浮在水中使用的过滤表面（正电荷）和靶向的病毒颗粒之间的静电相互作用（带负电）。两个正电的过滤器是商业上可用的依赖于该技术中，玻璃/纤维素和纳米氧化铝/玻璃纤维过滤器。该玻璃/纤维素滤器成本高达10倍，该纳米氧化铝/玻璃纤维的，其限制使用的玻璃/ CE的llulose过滤器进行例行病毒监控。最近的研究已经得出结论差异是名义在从室温水中6,7肠道病毒的恢复这两个滤波器之间，证明使用一种更便宜的过滤器的替代。其他过滤选项，例如带负电和玻璃毛过滤器进行了研究，然而，他们要么需要源水（电负性过滤器）的预处理或没有市售的（玻璃毛过滤器）。的病毒浓度程序的发展主要集中在以提高病毒回收率从水优化初级浓缩技术（过滤器）。然而，次级浓度程序，其中降低洗脱液的体积通常为1升至毫升卷，也可对病毒回收率8显著影响。
的肠道病毒次要浓度通常依赖于絮凝剂如一些类型的牛肉提取物（有机floccuLATION）或脱脂奶絮凝9-12从过滤器表面除去病毒颗粒。最近，利用再加上另外硅藻土（细颗粒）的牛肉膏另一二次浓度过程已表明承诺回收腺病毒，肠道病毒，诺如病毒和8,13,14。根据相似的原理，以在该病毒颗粒中的有机絮凝方法的硅藻土的浓度的作品附加到并通过改变悬浮液的pH释放从粒子（絮凝物或硅藻土）。这两个次级浓缩技术之间的比较中尖刺腺病毒（ADV）类型40和41 8的恢复已被评估。该研究的结论是，两个次级浓缩技术人腺病毒的回收统计学相似。然而，有机絮凝方法需要30分钟。孵育在pH 3.5，而硅藻土技术需要一个较短的温育（10分钟），在pH 4.0。有机flocculat离子也需要使用昂贵的实验室设备（离心机）叔浓缩过程中收集的絮凝物颗粒，与此相反的硅藻土技术仅使用基本的实验室设备（真空过滤），以硅藻土颗粒从悬浮液中分离出来。
过滤器和二次洗脱技术的某些组合也可影响病毒的回收率。一项研究的结论是，初级（正电的过滤器）的某些组合和次级浓缩技术（硅藻土或有机絮凝）的 腺病毒13的回收的显著影响。这些结果表明，优化使用这些技术时，需要以最佳地从给定的水基质回收靶病毒。优化是一个耗时的，艰苦的过程许多研究者积极避免，因为无数的变量进行评估（过滤器类型/品牌，pH值洗脱液，硅藻土/有机絮凝）。
对于这种Study，一个步骤，开发了使用加标人腺病毒株40和41可以推测，以确定病毒浓度的最佳条件，从水，由于每个病毒类型显示了独特的衣壳形态和特定的衣壳电荷浓度的协议可能需要对每个病毒进行优化目标，以达到最佳的病毒回收。本研究为腺病毒40和41浓度的方法：1）评估病毒回收率自来水使用阳电过滤盘之后2）建立有机絮凝法对硅藻土技术作为辅助的浓度评价，以及3）评估洗脱缓冲液为大专浓度。
Protocol
Representative Results
Discussion
阳电过滤器是集中在水的病毒有效;然而，这些过滤器可以在它们的结构和组合物，其可以反过来改变其效力不同。私了这个问题，衣壳的结构和收费病毒株需要浓缩技术进行定制，以确保最佳恢复15之间变化。通过对现有的浓度的技术（ 例如，正电性的过滤器，牛肉提取物洗脱），靶病毒更有效浓度可以达到16,17简单的修改。
研究迄今为止通常集中于?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Adenovirus 40 stock | ATCC | VR-931 | |
| Adenovirus 41 stock | ATCC | VR-930 | |
| Sodium Thiosulfate | Fluka Chemical Co. | 72051 | |
| Celites #577 | Fluka Chemical Co. | 22142 | |
| NanoCeram 47mm | Argonide | N/A | |
| 1MDS 47mm | 3M | 6408502 | |
| AP-20 Prefilter 47mm | Millipore Corp. | AP2004700 | |
| Glycine | Sigma | 50046-1KG | |
| Sodium Polyphosphate | Acros Organics | 390930010 | |
| Trypsin | Gibco | 25200 | |
| PBS | Sigma | P5368 | |
| Hydrochloric Acid | Fisher | A481-212 | |
| BBL Beef Extract | BD Biosciences | 212303 | |
| Difco Beef Extract | BD Biosciences | 211520 | |
| ABI 7900 Real-time PCR system | ABI | N/A | |
| Stainless Steel Filter Housing | Millipore Corp. | XX2004720 | |
| Blood DNA Extraction Kit | Qiagen | 51104 | |
| EPA MPN Calculator | http://www.epa.gov/nerlcwww/online.html |
References
- Sinclair, R. G., Jones, E. L., Gerba, C. P. Viruses in recreational water-borne disease outbreaks: a review. Journal of Applied Microbiology. 107, 1769-1780 (2009).
- WHO. . Guidelines for Safe Recreational Water Environments. , (2003).
- Westrell, T., et al. Norovirus outbreaks linked to oyster consumption in the United Kingdom. Euro Surveillance : Bulletin Europeen Sur les Maladies Transmissibles = European Communicable Disease Bulletin. 15, (2010).
- Wong, K., Fong, T. T., Bibby, K., Molina, M. Application of enteric viruses for fecal pollution source tracking in environmental waters. Environment International. 45, 151-164 (2012).
- Cashdollar, J. L., Wymer, L. Methods for primary concentration of viruses from water samples: a review and meta-analysis of recent studies. Journal of Applied Microbiology. 115, 1-11 (2013).
- Ikner, L. A., Soto-Beltran, M., Bright, K. R. New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water. Applied and Environmental Microbiology. 77, 3500-3506 (2011).
- Karim, M. R., Rhodes, E. R., Brinkman, N., Wymer, L., Fout, G. S. New electropositive filter for concentrating enteroviruses and noroviruses from large volumes of water. Applied and Environmental Microbiology. 75, 2393-2399 (2009).
- McMinn, B. R., Cashdollar, J. L., Grimm, A. C., Fout, G. S. Evaluation of the celite secondary concentration procedure and an alternate elution buffer for the recovery of enteric adenoviruses 40 and 41. Journal of Virological Methods. 179, 423-428 (2012).
- Katzenelson, E., Fattal, B., Hostovesky, T. Organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water. Applied and Environmental Microbiology. 32, 638-639 (1976).
- Fout, G. S., Schaefer, F. W., Messer, J. W., Dahling, D. R. . ICR microbial laboratory manual. 178, (1996).
- 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, 79-83 (2008).
- Calgua, B., et al. Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas. Water Research. 47, 2797-2810 (2013).
- McMinn, B. R. Optimization of adenovirus 40 and 41 recovery from tap water using small disk filters. Journal of Virological Methods. 193 (2), 284-290 (2013).
- Brinkman, N. E., Haffler, T. D., Cashdollar, J. L., Rhodes, E. R. Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater. Journal of Virological Methods. 193, 140-146 (2013).
- Albinsson, B., Kidd, A. H. Adenovirus type 41 lacks an RGD alpha(v)-integrin binding motif on the penton base and undergoes delayed uptake in A549 cells. Virus Research. 64, 125-136 (1999).
- He, C., Lian, J. S., Jiang, Q. Electronic structures and hydrogen bond network of high-density and very high-density amorphous ices. The Journal of Physical Chemistry. B. 109, 19893-19896 (2005).
- Sedmak, G., Bina, D., Macdonald, J., Couillard, L. Nine-year study of the occurrence of culturable viruses in source water for two drinking water treatment plants and the influent and effluent of a Wastewater Treatment Plant in Milwaukee, Wisconsin. 71, 1042-1050 (2005).
- Soto-Beltran, M., Ikner, L. A., Bright, K. R. Effectiveness of poliovirus concentration and recovery from treated wastewater by two electropositive filter methods. Food and Environmental Virology. 5, 91-96 (2013).
- Haramoto, E., Katayama, H. Application of acidic elution to virus concentration using electropositive filters. Food and Environmental Virology. 5, 77-80 (2013).
- Lee, H., et al. Evaluation of electropositive filtration for recovering norovirus in water. Journal of Water and Health. 9, 27-36 (2011).
- Gibbons, C. D., Rodriguez, R. A., Tallon, L., Sobsey, M. D. Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses, adenoviruses and male-specific coliphages from seawater. Journal of Applied Microbiology. 109, 635-641 (2010).
- Hill, V. R., et al. Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants. Applied and Environmental Microbiology. 71, 6878-6884 (2005).
- Polaczyk, A. L., Roberts, J. M., Hill, V. R. Evaluation of 1MDS electropositive microfilters for simultaneous recovery of multiple microbe classes from tap water. Journal of Microbiological Methods. 68, 260-266 (2007).
- Rhodes, E. R., Hamilton, D. W., See, M. J., Wymer, L. Evaluation of hollow-fiber ultrafiltration primary concentration of pathogens and secondary concentration of viruses from water. Journal of Virological Methods. 176, 38-45 (2011).
- Melnick, J. L., et al. Round robin investigation of methods for the recovery of poliovirus from drinking water. Applied and Environmental Microbiology. 47, 144-150 (1984).
- Schwab, K. J., De Leon, R., Sobsey, M. D. Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR. Applied and Environmental Microbiology. 61, 531-537 (1995).
