Optimization of reverse hybridization in microplates coated with rRNA targeted oligonucleotide probes

Among the modern molecular techniques for the identification of microorgani sms the most straightforward way is through direct hybridization with rRNA/ rDNA targeted probes. In this study, the optimization of the experimental p rocedures for the reverse hybridization technique in 96-well microplates is described using both synthetic model oligonucleotides (18 b) and amplified DNA (app. 4500 bp). Three different types of plates were compared (Maxi So rp, NucleoLink, CovaLink). Plates made from non-chemically modified polysty rene which are conventionally used in immunoassays (MaxiSorp) proved to be an economic alternative for plates offering chemically modified tailor-made surfaces. Phosphorylation of the oligonucleotide probe was not necessary f or successful immobilization whereas with 5'-terminal hexa-deoxyadenosine t ailed capture oligonucleotides an enhanced sensitivity of the assay was obs erved. Variation of the stringency by adjusting different concentrations of formamide during the washing step ensures high probe specificity and there fore allows reliable identification of the microorganisms. The assay can be performed in less than 4 hours using pre-coated plates which can be stored for several weeks. After dissociation of the target DNA/capture probe dupl ex with an alkaline denaturing solution rehybridization is possible.