IMPROVED MICROPLATE IMMUNOENZYMATIC ASSAY OF PCR PRODUCTS FOR RAPID DETECTION OF MYCOPLASMA-PNEUMONIAE

We developed a microtitre hybridization assay for the detection of pol ymerase chain reaction (PCR) amplified sequences. For this, cloned Myc oplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalenty bound to microtitre wells. These coated mi croplates can be stored for several months. Then, an aliquot of the PC R product, labelled with digoxigenin-dUTP during its synthesis is hybr idized 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 te chniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detec tion of M. pneumoniae in a series of clinical specimens.