A. Rosner et al., THE USE OF SHORT AND LONG PCR PRODUCTS FOR IMPROVED DETECTION OF PRUNUS NECROTIC RINGSPOT VIRUS IN WOODY-PLANTS, Journal of virological methods, 67(2), 1997, pp. 135-141
Citations number
17
Categorie Soggetti
Virology,"Biochemical Research Methods","Biothechnology & Applied Migrobiology
The reverse transcriptase-polymerase chain reaction (RT-PCR) was used
for detection of prunus necrotic ringspot virus (PNRSV) in dormant pea
ch and almond trees by the application of two different pairs of prime
rs yielding a short and a long product, respectively. The relative amo
unt of the short (200 base pair, bp) product was higher than the longe
r (785 bp) product. PNRSV was detected better in plant tissues with a
low virus concentration (e.g. dormant trees) by amplification of the s
hort PCR product, whereas the long product was produced at higher viru
s titers. Simultaneous amplification of both short and long products w
as demonstrated using a three-primer mixture in a single reaction tube
. In this assay, amplification of either PCR product indicated the pre
sence of PNRSV-specific sequences in the plant tissue examined, thus c
overing a wide range of virus concentrations in a single test. Dilutio
n of the RNA extracted from infected plant material resulted in a stee
p decline in the amplification of both short and long PCR products. In
contrast, serial dilutions of the intermediate cDNA template differen
tially affected the amplification patterns: the relative amount of the
short product increased whereas that of the long product decreased. T
hese results may explain the preferential amplification of the short P
CR product observed in samples containing low virus concentrations. (C
) 1997 Elsevier Science B.V.