Name: a small volume procedure for viral concentration from water
Uploaded: 2015-02-03T13:00:00
Description: Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

1. Preparação de material de vidro e Carcaças de filtro <ol style="margin-left: 40px;"><li> Salvo disposição em contrário, esterilizar todos os copos, caixas de filtro e soluções a 121 ° C por 15 min. Para garantir a esterilidade, cobrir todas as aberturas ou as superfícies expostas, quer com a folha de alumínio ou papel fixada com fita antes da esterilização. </li><li> Montar aparelho de filtração, anexando carcaça do filtro (47 mm de diâmetro) para 1 L Erlenmeyer braço lateral. Recolha filtros nece...

<ol>
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Filtros electropositiva são úteis na concentração vírus de água; no entanto, estes filtros podem diferir na sua estrutura e composição, que por sua vez poderia alterar a sua eficácia. Para agravar este problema, as estruturas do capsídeo e encargos variam entre cepas de vírus que requerem ser adaptados técnicas de concentração para garantir a recuperação óptima 15. Através de modificações simples das técnicas de concentração existentes (por exemplo, filtros electropositivos, ex...

Vírus entéricos humanos são importantes causadoras doenças transmitidas pela água 1-3, mas são geralmente presentes em números baixos nas águas ambientais contaminados, tornando difícil a sua detecção, sem concentração. Os procedimentos usados ​​para concentrar os vírus incluem, tipicamente, uma etapa de filtração, seguido por eluição do filtro, e concentração secundária do eluato filtro. Um procedimento de filtração comum se baseia em uso de membranas carregadas, tais como filtros electropositivos (recentemente revisados ​​em 4,5). Estes filtros dependem de captura de vírus em suspensão em água usando interacções electrostáticas entre a superfície de filtro (carregado positivamente) e partículas de vírus segmentados (carregada negativamente). Dois filtros electropositivos que estão disponíveis comercialmente confiam nesta tecnologia, os filtros de vidro / celulose e nano-alumina / fibra de vidro. Os custos de filtro de vidro / celulose são até 10 vezes maior do que a nano-alumina / fibra de vidro, o que limita o uso do vidro / cefiltros llulose para monitoramento de vírus de rotina. Estudos recentes concluíram que as diferenças são nominais entre estes dois filtros em recuperação de enterovírus de 6,7 água do ambiente, o que justifica o uso de um filtro alternativa mais barata. Outras opções de filtros, como filtros electronegativos e de fibra de vidro foram estudadas, no entanto, que quer impor ao pré-tratamento da água de nascente (filtros electronegativos) ou não estão disponíveis comercialmente (filtros de fibra de vidro). O desenvolvimento de processos de concentração de vírus tenha focado principalmente na optimização das técnicas de concentração primária (filtros), a fim de melhorar a recuperação de vírus a partir de água. No entanto, os procedimentos de concentração secundários, os quais reduzem o volume de eluente, tipicamente de 1 L para mililitro volumes, também pode ter um impacto significativo sobre a recuperação de vírus 8. Concentração Secundária de vírus entéricos normalmente se baseia em um agente de floculação, como alguns tipos de extrato de carne bovina (floccu orgânicamento) ou floculação leite desnatado 9-12 para remover partículas do vírus a partir de superfícies de filtro. Recentemente, um outro processo de concentração secundária utilizando extracto de carne juntamente com a adição de Celite (partícula fina) mostrou-se promissor para a recuperação de adenovírus, enterovírus, e norovírus 8,13,14. Obras de concentração de celite sob princípios semelhantes aos do método de floculação orgânico em que as partículas de vírus para anexar e são libertados de partículas (ou flocos de celite), alterando o pH da solução de suspensão. As comparações entre estas duas técnicas de concentração secundários foram avaliadas em recuperação de adenovírus cravado (Adv) tipos 40 e 41 8. Este estudo concluiu que as duas técnicas de concentração secundários foram estatisticamente semelhantes em recuperação de adenovírus. No entanto, o método de floculação biológica requer um 30 min. de incubação a pH 3,5, enquanto que a técnica de celite requer uma incubação mais curto (10 minutos) a pH 4,0. O flocculat orgânicaião também requer a utilização de equipamento de laboratório caro (centrífugas), para recolher as partículas de flocos durante a concentração terciária, a técnica de celite em contraste utiliza apenas equipamentos básicos de laboratório (filtração no vácuo) para separar as partículas de celite suspensão. Determinadas combinações de filtros e técnicas de eluição secundárias pode também afectar as recuperações de vírus. Um estudo concluiu que certas combinações de (filtros) electropositivos primárias e secundárias técnicas de concentração (Celite ou floculação orgânica) teve um impacto significativo de recuperação de adenovirus 13. Estes resultados sugerem que a optimização é necessária, a fim de recuperar o vírus alvo de forma óptima a partir de uma dada matriz de água quando se utiliza estas técnicas. Optimization é um demorado, árduo processo muitos pesquisadores evitam ativamente desde inúmeras variáveis ​​serão avaliadas (tipo de filtro / marca, solução de eluição pH, Celite / floculação orgânica). Para este study, foi desenvolvido um processo para identificar as condições óptimas para a concentração de vírus a partir da água utilizando adenovírus humano cravado estirpes 40 e 41. Presumivelmente, uma vez que cada tipo de vírus exibe uma morfologia única e capsídeo cápside carga específica, protocolos de concentração pode necessitar de ser optimizado para cada vírus alvo, a fim de alcançar a recuperação viral ideal. Este estudo fornece uma abordagem para AdV 40 e 41 concentração por: 1) avaliar as recuperações de vírus na água da torneira usando discos de filtro electropositivos seguidos de 2) avaliação de um método de floculação orgânica estabelecida versus a técnica de celite como uma concentração secundária, e 3) avaliação da tampões de eluição para a concentração terciária.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:295px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 40 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 41 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Thiosulfate</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">72051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Celites #577</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">22142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">NanoCeram 47mm</td>
			<td style="width:143px;">Argonide</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1MDS 47mm</td>
			<td style="width:143px;">3M</td>
			<td style="width:119px;">6408502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AP-20 Prefilter 47mm</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td style="width:119px;">AP2004700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glycine&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">50046-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Polyphosphate</td>
			<td style="width:143px;">Acros Organics</td>
			<td style="width:119px;">390930010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypsin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">PBS</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hydrochloric Acid</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">A481-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">BBL Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">212303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Difco Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">211520</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:231px;">ABI 7900 Real-time PCR system</td>
			<td style="width:143px;">ABI</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stainless Steel Filter Housing</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td>XX2004720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Blood DNA Extraction Kit</td>
			<td style="width:143px;">Qiagen&#160;</td>
			<td style="width:119px;">51104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EPA MPN Calculator</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.epa.gov/nerlcwww/online.html</td>
		</tr>
	</tbody>
</table>

a small volume procedure for viral concentration from water

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.

<html> Seleção Celite Três tipos diferentes de celite foram testados antes da selecção da variante com melhor desempenho. Celites com multa de partículas de tamanho médio produzido as maiores recuperações de adenovírus. Uso de celites maiores percentagens de recuperação mais baixas para ambos AdV40 e 41 (gama de 32% -100%) (Figura 1). A recuperação média de adenovirus 40 eram de 144% ± 52% (multa), 115% ± 28% (média) e 82% ± 53% celites (grande) de partícu...

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A Small Volume Procedure for Viral Concentration from Water

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.

Um Procedimento volume pequeno para Concentração Viral da Água

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 ...

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