DETECTION OF ASPERGILLUS-FUMIGATUS PCR PRODUCTS BY A MICROTITRE PLATEBASED DNA HYBRIDIZATION ASSAY

Aims-To develop a DNA based plate hybridisation assay for the detectio n of polymerase chain reaction (PCR) products amplified from Aspergill us fumigatus DNA; and to determine the sensitivity of this technique a nd compare it with Southern blotting. Methods-A half-log dilution seri es of DNA extracted from A fumigatus was amplified with specific prime rs, one of which was 5' end labelled with biotin. PCR products were su bsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made. Results-A DNA concent ration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specif ic probe; 1.5 pg was detectable by streptavidin binding of the PCR pro ducts to a microtitre plate. The signal from the microtitre plate dete ction was proportional to the amount of DNA in the PCR reaction on a l og-log scale between 100 and 1 pg of DNA. Conclusions-A DNA based plat e hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in t hree hours rather than the three days required for agarose gel electro phoresis, blotting, hybridisation, and detection.