Biomedical subjects

D J Barbara

Publications and source records attributed to D J Barbara.

5 recordsLinked to original sources

PubMed1999

Taxonomic status of putative Verticillum alboatrum isolates.

Two fungal isolates, formerly classified as Verticillium alboatrum and proposed as forming the basis of a new sub-group ('Group 2') within the species, have been shown to be non-pathogenic to known hosts of V. alboatrum and, on the basis of molecular evidence, to be closely related to Verticillium psalliotae and Verticillium fungicola. We propose that the taxon V. alboatrum be confined to those closely related isolates, usually plant pathogenic and usually producing dark resting mycelium, referred to by other authors as Group 1. The only sub-specific groupings which appear valid (based on pathological and molecular evidence) comprise: (1) host-adapted isolates from lucerne; and (2) all other isolates.

Phylogeny↗

PubMed1997

Detection of a single base exchange in PCR-amplified DNA fragments using agarose gel electrophoresis containing bisbenzimide-PEG.

Using PCR fragments of known sequences derived from isolates of two related fungal species, simple submarine electrophoresis in agarose gels containing a bisbenzimide-PEG conjugate (H.A.-Yellow) has been shown to be capable of distinguishing DNA fragments 567 bp long which differ by as little as a single base change. However, only changes affecting bisbenzimide binding sites (which consist of at least four consecutive A/T bases) alter mobility; other changes are ineffective. A second ligand (H.A.-Red) with high G/C specificity is suggested which may be as effective in detecting other sequence changes.

Bisbenzimidazole↗

PubMed1995

Rapid differentiation of closely related isolates of two plant viruses by polymerase chain reaction and restriction fragment length polymorphism analysis.

Immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the product has been shown to be an effective procedure for discriminating serologically indistinguishable isolates of two plant viruses, raspberry bushy dwarf (RBDV) and zucchini yellow mosaic (ZYMV). For both viruses, only limited sequence information was available at the time of primer design, but most of the isolates which were tested could be amplified (the one exception being a serologically quite distinct isolate of ZYMV). Restriction endonucleases revealing diagnostic RFLPs were readily identified. Each of two isolates of ZYMV could be detected in the presence of the other and the relative proportions approximately quantified by visual estimation of the relative intensity of the appropriate bands. A range of isolates of different RBDV pathotypes were compared; isolates were grouped in ways that accorded with their known history. Computer analysis of the published sequence from which the primers had been derived showed the sequenced isolate to be identical with an isolate imported from the USSR. The PCR/RFLP procedure is rapid (it can be completed in less than 2 days), effective and will probably be generally applicable to distinguishing closely related virus isolates, even where little sequence information is available.

Base Sequence↗

PubMed1987

A method for the quantitative analysis of ELISA data.

A method is described for measuring relationships among virus serotypes. An indirect ELISA was used to obtain antibody-response curves for homologous and heterologous combinations of virus and dilutions of antibody. Data from the proportional response region of each curve were converted to logarithmic values and subjected to a form of regression analysis to derive relative titres for the various combinations of virus and antibody. SDI values were then obtained by calculating the mean difference in relative titres between homologous and reciprocal heterologous combinations. Virus models in two different groups were examined using this technique. Relative titres obtained were consistent and reproducible over several experiments indicating that the technique was capable of reliably discriminating among serotypes which differed by as little as 0.2 SDI.

Enzyme-Linked Immunosorbent Assay↗

PubMed1982

A simple indirect ELISA using F(ab')2 fragments of immunoglobulin.

An indirect ELISA is described in which (i) virus is trapped by F(ab')2 fragments of specific IgG immobilized on a solid phase support, (ii) trapped virus is detected by intact IgG (from the same or a different antiserum) and (iii) positive reactions are identified using an enzyme conjugate specific for the Fc portion of IgG. Pepsin digestion of the Fc portion of the trapping antibody permits the use of a general purpose enzyme conjugate to discriminate between trapping and detecting antibody. Consequently, the assay requires only a single virus-specific antiserum which is often all that is available to the plant virologist. The assay was at least as sensitive for detecting small amounts of antigen as the standard double-antibody sandwich procedure and, for some viruses, two- to fourfold more sensitive. The improvement in performance resulted largely from lower and more consistent background reactions. Both assays were equally effective in revealing heterologous reactions when optimized for detecting homologous antigen. However, increased cross-reactions were obtained in the F(ab')2 procedure by the use of higher concentrations of detecting antibody. The assay is considered particularly suited for comparing antisera from different sources or of different bleeds from the same source, and for investigations involving so few tests that the effort or expense of preparing individual virus-specific conjugates is not justified.

Antibodies, Viral↗