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 which are related serologically and share common Prunus hosts, but cause distinct diseases. An RT-PCR procedure using a single oligonucleotide primer pair that allows simultaneous detection and differentiation of the two viruses was developed. A sense primer with 100% complementarity to PMV and 83% complementarity to the corresponding site of the CMLV genome was combined with 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 differential amplification of PMV and CMLV specific fragments, 419 and 705 bp, respectively. When oligo (dT) was used to generate the cDNA template, differential amplification was not observed, only amplification of the homologous virus associated with the antisense primer. This indicates polyadenylation 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 analysis, both in peach leaf and budwood tissue.
James
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Centre for Plant Health, Canadian Food Inspection Agency, Sidney, BC. jamesd@em.agr.ca
Upton
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D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005069:Evaluation Studies as Topic; D005638:Fruit / Q000821:virology; D005784:Gene Amplification; D009029:Mosaic Viruses / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary