Single primer pair designs that facilitate simultaneous detection and differentiation of peach mosaic virus and cherry mottle leaf virus

Peach mosaic virus (PMV) and cherry mottle leaf virus (CMLV) are viruses wh ich are related serologically and share common Prunus hosts, but cause dist inct diseases. An RT-PCR procedure using a single oligonucleotide primer pa ir that allows simultaneous detection and differentiation of the two viruse s was developed. A sense primer with 100% complementarity to PMV and 83% co mplementarity to the corresponding site of the CMLV genome was combined wit h either of two antisense primers (one of PMV origin and the other of CMLV origin) with 3' end complementarity at variable sites. This allowed the dif ferential amplification of PMV and CMLV specific fragments, 419 and 705 bp, respectively. When oligo (dT) was used to generate the cDNA template, diff erential amplification was not observed, only amplification of the homologo us virus associated with the antisense primer. This indicates polyadenylati on of both viruses. Incorporation of the antisense primer into cDNA at the reverse transcription step was shown to be essential for this approach. The PMV primer pair reliably detected all isolates of PMV tested by RT-PCR ana lysis, both in peach leaf and budwood tissue. (C) 1999 Elsevier Science B.V . All rights reserved.