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Biomedical subjects

A P Wyn-Jones

Publications and source records attributed to A P Wyn-Jones.

12 recordsLinked to original sources
PubMed2004

Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage.

AIMS: Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. METHODS AND RESULTS: Noroviruses in environmental water samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3'-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. CONCLUSIONS: Sewage was found to contain up to 1.8 x 10(6) norovirus cDNA copies per 100 ml and effluent contained up to 1.7 x 10(6) copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. SIGNIFICANCE AND IMPACT OF THE STUDY: The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods.

Benzothiazoles↗
PubMed2002

Antibodies in crevicular fluid: an epidemiological tool for investigation of waterborne disease.

A significant challenge in the epidemiological investigation of recreational waterborne disease is the establishment of a definite association between exposure to a contaminated water and infection. An increase in specific antibodies as a result of infection is a potent measure of disease exposure and its determination would enhance epidemiological studies of waterborne diseases. We report on the automated detection of HAV antibodies in crevicular fluid and its use in a field study. The method is easy to use, non-invasive, could be applied to volunteers of all ages and is comparable in sensitivity to serological procedures. Application to an epidemiological study of water recreationalists demonstrated that surfers were three times more likely to be immune to hepatitis A virus than either wind-surfers or a control group without recreational water contact.

Adolescent↗
PubMed2000

The detection of small round-structured viruses in water and environmental materials.

A procedure for concentrating small round-structured viruses (SRSVs) (Norwalk-like viruses) from water and other environmental materials is described. Primers based on the helicase region of the SRSV genome were confirmed as specific by reaction with typed specimens, and used to detect virus in concentrates of unseeded and seeded samples. Virus was detected in estuarine recreational water polluted by untreated sewage, although not in seawater samples taken some distance from outfall discharges. It was also detected in river water downstream of a sewage treatment plant. Virus could be detected in all matrices when they were seeded with a positive stool extract, including sewage seeded with as little as 2 microl stool extract, thus confirming the suitability of the method for environmental monitoring.

DNA Primers↗
PubMed1997

The detection of enteroviruses in large volume concentrates of recreational waters by the polymerase chain reaction.

A rapid and simple method was developed to detect enteroviruses in large-volume water samples. It relies on the adsorption of the virus capsids to silica particles under acidic conditions, allowing their recovery by relatively gentle centrifugation. Different reagents used in enterovirus concentration and detection were seeded with Coxsackievirus B5 and used to optimise the recovery method, which was then used to detect the enteroviruses from seeded and unseeded 101 seawater samples in one PCR tube rather than in up to 50 sub-sample volumes, demonstrating its use for routine environmental monitoring. Concentrates from 36 recreational water samples from three sites in N.E. England were analysed for enteroviruses by regular and the new method semi-nested PCR, and infectivity in cell culture. Some of the samples were also analysed for faecal indicator bacteria and F-specific bacteriophage. The results showed a marked increase in detection sensitivity when the whole sample concentrate was assayed as compared with a small volume aliquot.

Adsorption↗
PubMed1996

An evaluation of methods for the simultaneous detection of Cryptosporidium oocysts and Giardia cysts from water.

Methods for the simultaneous detection of Cryptosporidium parvum oocysts and Giardia cysts from water are described and their relative recovery efficiencies are assessed for seeded samples of both tap and river water. Cartridge filtration, membrane filtration, and calcium carbonate flocculation were evaluated, and steps to optimize the concentration procedures were undertaken. Increasing centrifugation to 5,000 x g, coupled with staining in suspension, was found to increase the overall efficiency of recovery of both cysts and oocysts. Cartridge filtration for both cysts and oocysts was examined by use of 100-liter volumes of both tap and river water. Improvements in recovery were observed for Cryptosporidium oocysts after extra washes of the filters. Calcium carbonate flocculation gave the maximum recovery for both Cryptosporidium oocysts and Giardia cysts and for both water types. A variety of 142-mm membranes was examined by use of 10-liter seeded samples of tap and river water. Cellulose acetate with a 1.2-micron pore size provided the best results for Cryptosporidium oocysts, and cellulose nitrate with a 3.0-micron pore size did so for Giardia cysts.

Animals↗
PubMed1985

Detection of rotavirus by a latex agglutination test, Rotalex: comparison with electron microscopy, immunofluorescence, polyacrylamide gel electrophoresis, and enzyme linked immunosorbent assay.

A commercially available latex agglutination test, Rotalex (Orion Diagnostics, Finland), for detecting rotaviruses was evaluated in comparison with four other tests (electron microscopy, immunofluorescence, polyacrylamide gel electrophoresis, and enzyme linked immunosorbent assay) routinely used in our laboratories. Although Rotalex was the least complex method, it showed lack of specificity and sensitivity when carried out according to the manufacturer's instructions. Four basic modifications of Rotalex are described. These include the use of Hank's balanced salt solution, increasing the incubation time to 20 min, reading the agglutination result by an experienced observer, and the use of 50 mm square glass plates. The modified procedure gave results which were comparable with those obtained by electron microscopy, immunofluorescence, polyacrylamide gel electrophoresis, and enzyme linked immunosorbent assay. The latter techniques, when used to detect rotavirus, all gave similar results.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Induction of virus-neutralizing antibody by glycoproteins isolated from chicken cells infected with a herpesvirus of turkeys.

The production of virus-induced proteins in chicken embryo fibroblast cells infected with a herpesvirus of turkeys was studied. It was found that glycoproteins isolated from membrane-rich fractions of infected cells by affinity chromatography using concanavalin A induced neutralizing and precipitating antibody in rabbits and chickens. After analytical electrophoresis, such isolates were found to contain three polypeptide bands of between 100 x 10(3) and 120 x 10(3) molecular weight not present in glycoprotein extracts of uninfected cells, and these polypeptides were further purified by preparative polyacrylamide gel electrophoresis. Inoculation of chickens with purified material also resulted in the production of precipitating and neutralizing antibody, showing that these high-molecular-weight polypeptides play a role in the humoral immunity to Marek's disease. Challenge of these chickens with virulent Marek's disease virus revealed that a partial protection was afforded by the inoculated glycoproteins.

Animals↗