An internal amplification control system based on primer-dimer formation for PCR product detection by DNA hybridization.

The detection of PCR products by DNA hybridization techniques can suffer from inhibition of the amplification process by sample matrix components. We have designed a simple internal control system for PCR based on the incorporation of a primer pair with complementary 3' ends, resulting in the generation of a unique "primer-dimer" detectable by hybridization with a specific capture probe immobilized on polyester cloth as part of an array of amplicon-specific probes. The inclusion of this primer pair did not adversely affect the amplification and subsequent detection of target gene sequences by hybridization with immobilized probes in either single gene amplification or multiplex PCR systems.

Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye.

A high-throughput, 96-well microplate fluorescence assay (MFA) was developed for DNA quantification using the double-stranded DNA-binding dye SYBR Green I. Samples mixed with SYBR Green I in the wells of a microtiter plate produced fluorescence in proportion with DNA concentration which was measured using a fluorescence plate reader. The performance characteristics of the assay were compared with spectrophotometric quantification based on ultraviolet absorption and the Hoefer DyNA Quant assay utilizing the fluorescent dye, Hoechst 33258.

Detection of Escherichia coli O157 in foods by a novel polymyxin-based enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) system was developed using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for Escherichia coli O157 lipopolysaccharide (LPS) antigens. Extracts from cell suspensions were reacted with polymyxin-coated microwells followed by immunoenzymatic detection of captured LPS antigens using a commercially available anti-E. coli O157 antibody-peroxidase conjugate and a 3,3',5,5'-tetramethylbenzidine substrate.