Improved microplate immunoenzymatic assay of PCR products for rapid detection of Mycoplasma pneumoniae.

We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalently bound to microtitre wells. These coated microplates can be stored for several months. Then, an aliquot of the PCR product, labelled with digoxigenin-dUTP during its synthesis is hybridized to the immobilized DNA. The use of a rapid hybridization buffer makes this step very short (5 min). Finally, the hybridization signal is detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase. Compared to Southern or other microplate hybridization techniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detection of M. pneumoniae in a series of clinical specimens.