Detection of Chrysanthemum stunt viroid (CSVd) in cultivars of Argyranthemum frutescens by RT-PCR-ELISA

A reverse transcription-polymerase chain reaction-enzyme-linked immunosorbe nt assay (RT-PCR-ELISA) for the detection of Chrysanthemum stunt viroid (CS Vd) in Argyranthemum frutescens was developed. A sequence-specific, modifie d primer pair (5'-end labelled with digoxigenin or biotin) was used, defini ng a target sequence of 281 bp. The digoxigenin and biotin-labeled PCR prod ucts were directly bound to streptavidin-coated microtiter plates and detec ted using anti-dixoxigenin Fab fragments conjugated with alkaline phosphata se. The described technique does not require denaturation and hybridization of a detection probe to the amplified products and operates without the us e of hazardous organic solvents and toxic or mutagenic stains. Only 6.5 h a re required for the detection of CSVd from infected tissue. This ELISA tech nique could potentially replace common electrophoretic techniques for the d etection of PCR products.