Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-06-06
1998-10-06
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 72, 435 732, 435 735, 435 29, 4352872, 5303885, G01N 3353, G01N 33569
Patent
active
058174729
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to the detection of motile organisms in a sample, and to a device for use in this method. The method is particularly, but not exclusively, directed to the detection of zoospores of the pathogenic fungus Phytophthora cinnamomi. More generally, the method extends to the detection of other organisms that produce motile cells, including bacteria, fungi, algae and protozoa.
BACKGROUND OF THE INVENTION
Phytophthora cinnamomi Rands is one of the most important plant pathogens found throughout the tropical and temperate zones (34,36). It affects an increasingly diverse range of species from a wide variety of plant families (35). Hosts include economically important horticultural crops such as avocado, pineapple and macadamia, ornamental species and several valuable timber species. Effects of this fungus on the endemic flora of southeastern Australia and the southwestern part of Western Australia, where some plant species are on the brink of extinction are devastating examples of the impact of an introduced pathogen on a flora composed of many susceptible species (31,34,35). Control of this pathogen and improved understanding of its biology must be based in part on information on the location and density of inoculum in soil. To date, and depending on the method used, this has required relatively difficult and time-consuming procedures to first isolate and then identify P. cinnamomi from soil. It is possible to isolate and identify P. cinnamomi within 2-3 days (13) but many procedures have used baiting of soil samples for up to 7-10 days followed by plating of the infected bait onto one or more selective media. After several days growth on the selective medium it is necessary for identification to be carried out by someone familiar with Phytophthora taxonomy (14,33).
Currently, the most versatile and useful diagnostic assays are those based on antibodies which specifically recognise the target organism. These assays have been used with great success with plant viral and bacterial diseases (16) and also plant diseases caused by a range of fungi (9). Polyclonal antibodies have been used for the detection of P. cinnamomi. Antibodies were produced that permitted detection of chlamydospores of P. cinnamomi in soil, but the assay suffered from high background binding of the antibodies to soil particles and lacked specifies-specificity (24). Similar procedures were used to produce antibodies that labelled Phytophthora zoospore cysts and germ tubes, but again these were not species-specific (24). Several immunoassays were developed for a number of important plant pathogens including Phytophthora (27). For example, monoclonal antibodies were used to detect cysts of Phytophthora and Phythium collected from irrigation water on filter pads (1). These assays all have been based on the use of fungal mycelium or mycelial fractions as the immunogen but have also shown considerable lack of specificity. A collection of monoclonal antibodies raised to aldehyde-fixed zoospores of P. cinnamomi which, in preliminary studies, showed genus-, species- and isolate-specificities (18), have great potential for the development of a species-specific immunodiagnostic test for P. cinnamomi.
It is one object of the present invention to provide a simple, rapid "dipstick" diagnostic immunoassay which in one embodiment, enables the detection and quantitation of P. cinnamomi in soil within 1-2 days. A diagnostic immunoassay of this type is based on an antigen absorbed by, or a capture antibody adsorbed to, a dipstick. These assays involve movement of the dipstick from the test solution through solutions containing a labelled antibody and then into a final solution that allows visualisation of bound antibody. This form of immunoassay has been used successfully to detect several plant pathogens (5,10,11,26) and forms the basis of many medical diagnostic assays (21,32). The great advantages of the dipstick-type assays over other antibody-based assays are that they can be carried out quickly, cheaply and wi
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Cahill David Miles
Hardham Adrienne Ruth
Housel James C.
Portner Ginny Allen
The Australian National University
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