Sensitivity and specificity of a commercially available enzyme-linked immunoassay for the detection of polymerase chain reaction amplified DNA

The explosive growth of polymerase chain reaction (PCR) based diagnostics has led to the introduction of many different techniques that allow convenient detection of PCR products. We compared a commercial enzyme-linked immunosorbent assay (ELISA) to standard detection techniques and analyzed its sensitivity and specificity for use in a routine microbiological diagnostic laboratory. Optimal hybridization conditions were at high temperature with a short incubation time. The sensitivity found for the PCR ELISA is about 10-20 times higher compared to the detection by ethidium bromide staining after agarose gel electrophoresis. Conventional hybridization techniques have an additional increase in sensitivity but they are much more time consuming and not suited for large-scale use. To specifically discriminate two species (Bartonella henselae and Bartonella quintana) by PCR ELISA a three-nucleotide difference in the composition of the hybridization probes was sufficient.