Concentration Techniques

2.2.2.1 Concentration of Viruses by Silicon Dioxide (SiO2) Method

Silicon dioxide particles (Appendix A) were prepared according to the protocol described by Boom et al. (1990). The method as outlined by Baggi and Peduzzi (2000) and Baggi et al. (2001) was followed to concentrate the viruses from water. The spiked water sample was firstly analysed for the presence of rotavirus and adenovirus by means of a Combi-Strip (Coris Bioconcept, Gembloux, Belgium). Briefly, 3 x 100 ml of spiked water (Section 2.2.1) was then acidified to pH 3.5 with acetic acid. Sterile SiO2 (20 µl) and 100 µl aluminium

chloride solution (0.5 M) was added and mixed on a magnetic stirrer for 30 minutes at room temperature. The samples were placed at 4ºC for 24 hours to allow the SiO2 to settle

undisturbed. Approximately 90 ml of the supernatant was discarded and the remaining 10 ml was centrifuged at 7500 x g for 10 minutes to pellet the virus using an Avanti JE centrifuge (Beckman Coulter, USA). The supernatant was discarded by decanting and the viruses were recovered from the pellet by homogenising it with beef extract glycine buffer (0.25 N glycine and 3% beef extract at pH 9.5). The samples were incubated in a 64ºC oven for 10 minutes. The suspension was transferred to a sterile labelled micro-centrifuge tube and centrifuged at 1600 x g for 2 minutes. The supernatant containing virus was removed and centrifuged at 12500 x g for one hour at 4ºC. The supernatant was again discarded and the pellet was resuspended in 150 µl nuclease–free water. Fifty microliters of each sample was used for transmission electron microscopy (TEM). RNA extraction was performed using the TRIzol extraction method (Section 2.2.3.1), DNA extraction was performed using the High Pure PCR Template Preparation kit (Section 2.2.3.2), while the QIAamp Ultrasens Virus kit (Section 2.2.3.3) was utilised for the simultaneous extraction of DNA and RNA.

2.2.2.2 Concentration of viruses using Positively Charged Filters

For the concentration of virus particles using positively charged filters triplicate samples of the 100 ml of spiked water (Section 2.2.1) were centrifuged at 3000 rpm for 30 minutes to

54 pellet the debris. The supernatant was analysed for the presence of rotavirus and adenovirus by means of a Combi-Strip (Coris Bioconcept, Gembloux, Belgium) according to the manufacturer’s instructions. The supernatant (about 90 ml) was then passed through a 47 mm, 0.45 µm pore size Zetapore positively charged filter (Cuno Inc., Meriden, Conn. USA) to which viral particles adhere. The Zetapore membrane was placed in a 50 ml centrifuge tube containing 4 ml of 50 mM glycine (pH 9.5) and 1% beef extract. The virus particles were eluted from the filter by shaking at 500 rpm for 20 minutes at room temperature. The virus-containing buffer was adjusted to pH 8 with HCl, and micro- concentrated with the Amicon Ultra centrifugal filter device (Millipore). The volume was adjusted to 200 µl with phosphate buffered saline (PBS) and a 50 µl aliquot was removed for TEM. The concentrate was again checked for the presence of rotavirus and adenovirus using the Combi-Strip. RNA extraction was performed using the TRIzol extraction method (Section 2.2.3.1), DNA extraction was performed using the High Pure PCR Template Preparation kit (Section 2.2.3.2), while the QIAamp Ultrasens Virus kit (Section 2.2.3.3) was utilised for the simultaneous extraction of DNA and RNA.

2.2.2.3 Concentration of viruses using Negatively Charged Filters

For the concentration of virus particles using negatively charged filters triplicate samples of the 100 ml of spiked water (Section 2.2.1) were also centrifuged at 3000 rpm for 30 minutes to pellet the debris. The supernatant was analysed for the presence of rotavirus and adenovirus antigens by means of a Combi-Strip. To prepare the filters, 2 ml of 250 m M AlCl3

was passed through the GF/F negatively charged filter (Whatman International Ltd, England). The supernatant (about 90 ml) was removed and then passed through the AlCl3 treated

47 mm, 0.45 µm pore size GF/F negatively charged filter. The filter was then rinsed with 200 ml of 0.5 mM H2SO4 (pH 3.0) to remove aluminium ions. The filter was rinsed with 4 ml

of a 1 mM NaOH (pH 10.8) solution to elute the viral particles and the filtrate was placed in a 25 ml sterile conical tube containing 25 µl of 100 mM H2SO4 (pH 1.0) and 50 µl 100 X Tris-

EDTA (TE) buffer (pH 8.0). The virus-containing buffer was micro-concentrated with the Amicon Ultra centrifugal filter device (Millipore). The volume was adjusted to 150 µl with 1x PBS and a 50 µl aliquot was removed for TEM. This filtrate was once again tested for the presence of rotavirus and adenovirus antigens using the Combi-Strip test. RNA extraction was performed using the TRIzol extraction method (Section 2.2.3.1), DNA extraction was performed using the High Pure PCR Template Preparation kit (Section 2.2.3.2), while the QIAamp Ultrasens Virus kit (Section 2.2.3.3) was utilised for the simultaneous extraction of DNA and RNA.

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2.2.2.4 Concentration using Non-charged Mixed-ester Filters

Triplicate samples of the 100 ml of spiked water (Section 2.2.1) were centrifuged at 3000 rpm for 30 minutes to pellet the debris, for the concentration of virus particles using non-charged mixed-ester filters. The supernatant was analysed for the presence of rotavirus and adenovirus antigens by means of a Combi-Strip. The supernatant (about 90 ml) was transferred to a sterile 100 ml Schott bottle and the sediment was discarded. Subsequently 1 ml of 1 M CaCl2 and 1 ml of 1 M Na2HPO4 was added. The mixture was stirred for 5

minutes to allow flocculation and filtered through a 47 mm, 0.45 µm pore size non-charged mixed-ester filter membrane (Whatman GmbH, Germany). The membrane was transferred to a 9 cm diameter petri dish and soaked in 4 ml of a 0.3 M citrate buffer (pH 3.5) for 3 minutes after which the membrane was discarded. The virus-containing buffer was micro- concentrated with the Amicon Ultra centrifugal filter device (Millipore). The volume was adjusted to 200 µl with phosphate buffered saline and a 50 µl aliquot was removed for TEM. This solution was again checked for rotavirus and adenovirus antigens using the Combi-Strip test. RNA extraction was performed using the TRIzol extraction method (Section 2.2.3.1), DNA extraction was performed using the High Pure PCR Template Preparation kit (Section 2.2.3.2), while the QIAamp Ultrasens Virus kit (Section 2.2.3.3) was utilised for the simultaneous extraction of DNA and RNA.