Rapid detection of enteric adenovirus and rotavirus: a simple method using polyacrylamide gel electrophoresis.

Abstract

SUMMARY A simple and rapid procedure for identifying adenovirus and rotavirus in stool extracts has been developed. The technique is based on polyacrylamide gel electrophoresis of the virus nucleic acid, but sample preparation is straightforward and does not entail phenol extraction or the use of a radioactive label. Furthermore, processing is not influenced by the amount of specimen obtained and is thus suitable for application as a batch testing method. This approach removes the need for specific antisera, which are not readily available since most of these viruses cannot be grown using routine tissue culture procedures. Trials in this laboratory have indicated that the technique is of comparable sensitivity to electron microscopy.

Restriction enzyme analysis of faecal adenoviruses in Newcastle upon Tyne

“…Polyacrylamide gel analysis was performed using the Laemmli (1970) discontinuous polyacrylamide gel system as described by Moosai, Carter & Madeley (1984), except that PAG Gel Bond (Miles Laboratories Ltd, Slough, England) was used to provide a rigid support for the gel to facilitate handling. The gel was subsequently silver-stained by the method of Herring et al (1982) using the modifications described by Moosai, Carter & Madeley (1984).…”

“…The gel was subsequently silver-stained by the method of Herring et al (1982) using the modifications described by Moosai, Carter & Madeley (1984). This method stains nucleic acids black in a light brown gel matrix.…”

“…In the absence of readily available type-specific antisera, and difficulties in virus culture, direct serotyping by neutralization or by other methods such as ELISA are inapplicable. In order to look at the prevalence of these 'fastidious' adenoviruses in the Newcastle area as well as the more readily propagated lower numbered serotypes, we decided to extend the polyacrylamide gel electrophoresis (PAGE) method, developed in Newcastle to identify rotavirus RNA in stools, and which can also detect adenovirus DNA (Moosai, Carter & Madeley, 1984). Adenovirus DNA was obtained from stool samples without prior growth in cell culture, and digested with the enzyme Sma I.…”

“…Faecal adenovirus restriction analysis 447 Moosai, Carter & Madeley (1984) found that treatment with 5 % sodium dodecyl sulphate (SDS) was sufficient to release adenovirus DNA from this material. This DNA could be clearly identified as a single band on a 75 % discontinuous polyacrylamide gel.…”

“…A 5 jd sample was removed, mixed with an equal volume of 2 x sample buffer (Laemmli, 1970) and applied to a 7-5 % polyacrylamide discontinuous gel to detect and roughly quantify any adenovirus DNA present in the sample (Moosai, Carter & Madeley, 1984).…”