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    Moleculaire detectie van small round structured viruses (SRSV) in (riool) waterconcentraten

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    Authors
    Lodder WJ
    Mulders MN
    Vinje J
    Medema GJ
    Koopmans MPG
    Type
    Report
    Language
    nl
    
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    Title
    Moleculaire detectie van small round structured viruses (SRSV) in (riool) waterconcentraten
    Translated Title
    Molecular detection of small round structured viruses (SRSV) in (sewage) watersamples
    Publiekssamenvatting
    Doel van dit onderzoek was om een methode te ontwikkelen voor de detectie van SRSV, met behulp van de reverse transcriptase-polymerase chain reaction (RT-PCR), in riool- en oppervlakte water (concentraten). In eerste instantie werd de RT-PCR geoptimaliseerd met behulp van SRSV positieve faeces, en werden twee RNA opwerkmethoden vergeleken (polyethyleen glycol (PEG)/cetyltri-methylammonium bromide (CTAB) en guanidinium isothiocyanaat (GuSCN)/silica). Rioolwater en 3% beef extract werden kunstmatig besmet met poliovirus serotype 3 voor gebruik als model systeem, omdat poliovirus in tegenstelling tot SRSV wel gekweekt kan worden, en met behulp van de plaque overlay methode gekwantificeerd kan worden. Twee opwerkmethoden voor monsters water zijn met elkaar vergeleken: een methode gebaseerd op twee-fasen scheiding en een precipitatiemethode. De virus opbrengst (poliovirus) werd zowel met celkweek als met RT-PCR bepaald. Met de twee-fasen scheidingsmethode varieerde de opbrengst tussen 4,8-17,4% wanneer er kunstmatig besmet werd met 10 exp.3 TCID50 ('50% tissue culture infective dose') en tussen 0,7-9,1% wanneer er kunstmatig besmet werd met 10 exp. 5 TCID50. Met de precipitatie methode was de opbrengst ongeveer 0,5%. Gebaseerd op semi-kwantitatieve RT-PCR werden bij SRSV besmette monsters overeenkomstige opbrengsten gevonden. Gebaseerd op deze vergelijking werd de twee-fasen scheidingsmethode geselecteerd voor verdere evaluatie en voor gebruik in veld onderzoek.<br>
    This study was done to develop and evaluate a method for the detection of Small Round Structured Viruses (SRSV) in sewage and water samples by reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR was first optimized with SRSV positive stool specimens comparing two RNA isolation methods (polyethylene glycol (PEG)/ cetyltrimethylammonium bromide (CTAB) and guanidinium isothiocyanate (GuSCN)/silica). Sewage and 3% beef extract seeded with poliovirus serotype 3 were used as a model system because poliovirus can easily be quantified by plaque assay, unlike SRSV which cannot be cultured. Two methods of water sample concentration and purification were compared, one based on two-phase separation and one based on precipitation. Virus (polio virus) was recovered by cell culture isolation and by RT-PCR. With the two-phase separation method the recovery varied between 4.8-17.4% when samples were seeded with 10 exp. 3 TCID50 ('50% tissue culture infective dose') polio serotype 3 and between 0.7-9.1% when samples were seeded with 10 exp. 5 TCID50 polio serotype 3. With the precipitation method the recovery was approximately 0.5%. Based on semiquantitative RT-PCR, similar recoveries were found for SRSV specimens. Based on this comparison, the two-phase separation method was selected for further evaluation and for use in field studies.<br>
    Publisher
    Rijksinstituut voor Volksgezondheid en Milieu RIVM
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