A MICROTITER PLATE ASSAY USING CASCADE AMPLIFICATION FOR DETECTION OFNONISOTOPICALLY LABELED DNA

We describe a microtiter-plate-based, colorimetric assay for DNA, the enzyme-linked DNA-enzyme-linked coagulation assay (EDNA-ELCA). The EDN A-ELCA uses amplification of the common pathway of coagulation for the ultrasensitive detection of DNA which is tagged by incorporation of f unctional groups such as biotin and fluorescein. The EDNA-ELCA enables detection of attomole amounts of DNA (<1 pg per microtiter well), wit h a sensitivity 200-1000 times higher than other colorimetric techniqu es. The assay has been applied as an adjunct to PCR for quantitative d etermination of methicillin-resistant Staphylococcus aureus DNA at lev els corresponding to 1-10(5) organisms. The EDNA-ELCA can also be used to assay DNA by hybridization; <50 amol of an unlabeled DNA template is detected by hybridization to biotin- and fluorescein-labeled probes . (C) 1995 Academic Press, Inc.