Solid-phase PCR with hybridization and time-resolved fluorometry for detection of HLA-B27

Background: Preactivated solid surfaces provide new possibilities for multi ple consecutive reactions in a microtiter plate format. In this study, a co mbination of PCR and subsequent hybridization in the same microtiter well w as applied for the detection of HLA-B27 alleles. Methods: A multiplex solid-phase PCR to amplify the HLA-B27 alleles togethe r with beta -actin as an amplification control gene was performed on the Nu cleoLink(TM) (Nunc) surface. PCR was followed by hybridization and detectio n with time-resolved fluorescence. For the covalent capture of the PCR prim ers onto the solid support via a 1-(3-dimethylamino-propyl)-3-ethylcarbodii mide hydrochloride-mediated reaction, different 5'-end modifications of oli gonucleotides were tested [amination, phosphorylation, and a poly(dT)(10) l inker]. Results: For covalent immobilization of the primers, amination of the 5' en d combined with use of the poly(dT)(10) linker was superior. At least 19.5% of the primer added per well was attached via a stable bond. When the stan dard time-resolved, fluorescence-based HLA-B27 detection system was compare d with the newly developed method in a sample series of 82 genomic DNAs and the corresponding dried-blood spots, all results were in full agreement. Conclusions: The new solid-phase PCR approach can be applied for multiple-t arget DNA detection. PCR followed by hybridization can be accomplished in a few hours using precoated strips and dried-blood spot PCR templates. (C) 2 001 American Association for Clinical Chemistry.