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. Utarbeidelse av Glass og filterhus <ol style="margin-left: 40px;"><li> Med mindre annet er angitt, sterilisere alle glass, filterhus og løsninger ved 121 ° C i 15 min. For å sikre sterilitet, dekke alle åpninger eller utsatte overflater med enten aluminiumsfolie eller tape-sikret papir før sterilisering. </li><li> Montere filtreringsapparat ved å feste filterhuset (47 mm diameter) til 1 L sidearm Erlenmeyerkolbe. Samle filtre som kreves: mm diameter 47 elektropositive / elektroskivefiltre. </li><li> Forbered ...

<ol>
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Elektropositive filtre er nyttige i å konsentrere virus fra vann; men disse filtre kan variere i deres struktur og sammensetning som i sin tur kunne forandre deres effektivitet. Compounding dette problemet, kapsidbindende strukturer og kostnadene varierer mellom virusstammer som krever konsentrasjon teknikker skreddersys for å sikre optimal utvinning 15. Gjennom enkle modifikasjoner av eksisterende konsentrasjonsteknikker (f.eks elektropositive filtre, kjøttekstrakt elusjonstester), mer effektiv k...

Menneskelige enteriske virus er viktige utløsende agenter for vannbårne sykdommer 1-3, men er generelt til stede i lave tall i forurenset miljø farvann, noe som gjør deteksjon deres vanskelig uten konsentrasjon. Fremgangsmåtene anvendt for å konsentrere virus typisk omfatte et filtreringstrinn, etterfulgt av filter eluering, og sekundære konsentrasjon av filter eluat. En vanlig filtrering prosedyre er avhengig av bruk av ladede membraner som elektropositive filtre (nylig anmeldt i 4,5). Disse filtrene er avhengige av å fange virus suspendert i vann ved hjelp av elektrostatiske interaksjoner mellom filterflaten (positivt ladet), og målrettede viruspartikler (negativt ladet). To elektropositive filtre som er kommersielt tilgjengelig stole på denne teknologien, de glass / cellulose og nano-alumina / glassfiberfiltre. De glass / cellulose filter kostnadene er opptil 10 ganger så stort som i nano-alumina / glassfiber, som begrenser bruken av glass / cellulose filtre for rutine virus overvåking. Nyere studier har konkludert forskjellene er nominell mellom disse to filtre i utvinningen av enterovirus fra ambient vann 6,7, forklarer bruken av et billigere filter alternativ. Andre filteret slik som elektronegative og glass-ull filtre har vært undersøkt, men de krever enten forbehandling av kilde vann (elektrofiltre) eller ikke er kommersielt tilgjengelige (glass-ull filter). Utviklingen av viruskonsentrasjons prosedyrer har hovedsakelig fokusert på å optimalisere primære konsentrasjonsteknikker (filtre) for å bedre virus inngang fra vann. Men sekundære konsentrasjons prosedyrer, som reduserer volumet av elueringsmiddel vanligvis fra en L til milliliter volumer, kan også ha en betydelig innvirkning på virus inngang 8. Sekundær konsentrasjon av enteriske virus avhenger typisk av et flokkulerende middel, så som noen typer kjøttekstrakt (organisk floccuning) eller skummet melk flokkulering 9-12 for å fjerne viruspartikler fra filterflater. Nylig har en annen sekundær-konsentrasjonen Fremgangsmåten ved bruk av biff-ekstrakt kombinert med tilsetning av celitt (fine partikler) vist seg lovende for gjenvinning av adenovirus, enterovirus, og norovirus 8,13,14. Celite konsentrasjons arbeider under lignende måte som den for den organiske flokkuleringsmetoden ved at viruspartiklene fester seg til, og blir frigjort fra partiklene (flokkulat eller celitt) ved å endre pH-verdien i suspensjonen løsning. Sammenligninger mellom disse to sekundære konsentrasjons teknikker har blitt evaluert i utvinningen av piggete adenovirus (AdV) typene 40 og 41 8. Denne studien konkluderte med at de to sekundære konsentrasjonsteknikker var statistisk lik i utvinningen av adenovirus. Imidlertid, den organiske flokkulering metoden krever en 30 min. inkubering ved pH 3,5, mens celitt teknikken krever en kortere inkubasjon (10 min) ved pH 4,0. Den organiske flocculation krever også anvendelse av kostbart laboratorieutstyr (sentrifuger) for å samle flokkulatsentre partikler under tertiært konsentrasjon på celitt teknikk i motsetning bare bruker enkel laboratorieutstyr (vakuumfiltrering) for å separere celit-partikler fra suspensjonen. Visse kombinasjoner av filtre og sekundære elueringsmetoder kan også påvirke virus inngang. En studie konkluderte med at visse kombinasjoner av primære (elektropositive filtre) og sekundære konsentrasjonsteknikker (Celite eller organisk flokkulering) hadde en betydelig innvirkning på utvinning av adenovirus 13. Disse funnene tyder på at en optimalisering er nødvendig for optimalt å gjenvinne target-virus fra en gitt vannmassen ved hjelp av disse teknikkene. Optimalisering er en tidkrevende, krevende prosess mange forskere aktivt unngå siden mange variabler vil bli evaluert (filtertype / merkevare, pH elueringsoppløsning, celitt / organisk flokkulering). For denne study, en prosedyre ble utviklet for å identifisere optimale forhold for virus konsentrasjon fra vann ved hjelp av piggete menneskelige adenovirus stammer som 40 og 41. Antagelig siden hver virus type viser en unik capsid morfologi og spesifikk capsid kostnad, kan konsentrasjonsprotokoller må være optimalisert for alle virus målrette for å oppnå optimal viral utvinning. Denne undersøkelsen gir en tilnærming for AdV 40 og 41 konsentrering ved: 1) å evaluere virusgjenvinninger i springvann ved hjelp av elektropositive filterskiver, etterfulgt av 2) evaluering av en etablert organisk flokkuleringsmetoden versus celitt teknikken som en sekundær konsentrasjon, og 3) evaluering av elueringsbuffere for høyere konsentrasjon.

<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>
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		<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>
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		<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> Celitt utvalg Tre forskjellige typer celitt ble testet før valg av beste resultater variant. Celites med fin mellomstore partikler produsert de høyeste adenovirus inngang. Bruk av større celites gitt lavere inngang for både AdV40 og 41 (område 32% -100%) (figur 1). Gjennomsnittlig gjenfunn av adenovirus 40 var 144% ± 52% (fin), 115% ± 28% (medium) og 82% ± 53% (stor) partikkel celites og for AdV41, 132% ± 39% (bot), 83% ± 25% (medium) og 50% ± 11% (stor). En...

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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.

Et lite volum Prosedyre for Viral Konsentrasjon fra 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 ...

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