Detection of Strawberry crinkle virus in plants and aphids by RT-PCR using conserved L gene sequences

Posthuma, K.; Adams, A.; Hong, Y.; Kirby, M.

doi:10.1046/j.1365-3059.2002.00725.x

About 10% of the large (L) protein gene of Strawberry crinkle virus (SCV) was sequenced after amplification with degenerate primers designed to conserved regions of the rhabdovirus L protein. The virus sequence was extended to 1362 nucleotides through rapid amplification of cDNA ends. One pair of degenerate L gene primers amplified a 683-bp fragment from four different isolates of SCV cultured in the experimental host Physalis pubescens; the nucleotide sequences of these fragments differed by [<] 1% to 10% indicating the suitability of this region as a diagnostic target. This information enabled the development of a reverse transcription polymerase chain reaction (RT-PCR) detection method for SCV using primers designed to the L gene sequence. SCV was amplified from infected P. pubescens (573 bp fragment) and from infected Chaetosiphon fragaefolii aphids (770 bp fragment). SCV was also detected by RT-PCR in total RNA extracts from three strawberry plants showing symptoms typical of SCV infection but failed when the intensity of the disease symptoms decreased. However, both SCV positive-sense RNA, and negative-sense genomic RNA, were detected by nested PCR in chronically infected strawberry plants sampled in September.