Quantification ofCoxiella burnetii by polymerase chain reaction (PCR) and a colorimetric microtiter plate hybridization assay (CMHA)

A colorimetric microtiter plate hybridization assay (CMHA) for the quantitative determination ofCoxiella burnetii DNA after amplification by externally controlled polymerase chain reaction (PCR) is described. The quantification assay is based on an enzyme linked immunosorbent assay (ELISA) format. Cloned DNA, representing a sequence complementary to an internal part of the diagnostic amplicon, was noncovalently attached to the wells of a microtiter plate. Biotinylated PCR product was hybridized to the immobilized capture probe. Bound product was detected via streptavidin horseradish peroxidase. The devised nonisotopic technique allows specific, rapid, and convenient quantification ofC. burnetii DNA. Additionally, it is compatible with standard laboratory ELISA equipment, making this assay amenable to automation and permitting processing of large sample numbers.