A PCR-ELISA procedure for the simultaneous detection and identification of prunus necrotic ringspot (PNRSV) and apple mosaic (ApMV) ilarviruses

Prunus necrotic ringspot virus (PNRSV) and apple mosaic virus (ApMV) are important and widely distributed ilarviruses infecting fruit trees. They induce a variety of symptoms in stone fruits and, in the case of ApMV, in pome fruits. Although they differ in host range, these two viruses are closely related and show serological cross-reactivity. We have determined the sequence of the coat protein (CP) gene of an isolate of each of these viruses. Multiple alignments of PNRSV and ApMV CP gene sequences allowed the selection of PCR primers in regions conserved between the two viruses and of virus-specific oligonucleotides to be used a capture probes in PCR-ELISA assays. An immunocapture-PCR protocol was developed for the sensitive detection of PNRSV and ApMV from fruit tree samples. Analysis of a range of isolates of PNRSV and ApMV showed that the selected primer pair allowed the detection of both viruses. Simultaneous use of the PNRSV-specific and ApMV-specific capture probes permitted the sensitive detection of both viruses in PCR-ELISA. Use of a single capture probe allowed the specific detection of one of the viruses only, even in the presence of amplified material derived from the other virus. The assay developed thus offers the possibility to detect both PNRSV and ApMV using a single amplification reaction and, if necessary, to identify the amplified virus. It also opens new opportunities for the analysis of the variability of these viruses.