Amplified fragments specific to each of the target pathogens
were obtained from tested field samples, whereas
no nonspecific band was detected in healthy citrons.
Fig.1 shows the amplification results of 5 citrus viroids
whole genome by uniplex RT-PCR. The size (bp) of
the amplified products for each viroid was 371 for
CEVd; 318 for CBLVd; 302 for HSVd; 294 for CVdIII,
and 284 for CVd-IV, respectively. Sequencing confirmed
that these amplicons corresponded to full-length
sequences of those viroids.
To differentiate cachexia variants and non-cachexia
variants of HSVd isolates, the same RT-PCR procedures
have been performed using two discriminating
primer pairs designed by Bernad and Duran-Vila (2006).
PCR yielded 220 bp amplicon (primer pair: HSVd-R2/
HSVd-F2´) only with samples infected by non-cachexia
variants of HSVd, whereas yielded 283 bp amplicon
(primer pair: HSVd-R2/HSVd-F2) only with samples
infected by cachexia isolates (Fig.2). To date, only one
cachexia variants of HSVd from Newhall navel orange
collected from Guizhou Province has been identified
by RT-PCR for strain characterization.
One-step multiplex RT-PCR assay for
simultaneous detection of citrus viroids
In the present study we have developed a one-step
multiplex RT-PCR for the simultaneous detection of
CEVd, CBLVd, HSVd, CVd-III, and CVd-IV, plus an
internal control, and meanwhile, amplified fragments
can easily be differentiated by agarose gel electrophoresis
because of redesigning partial primer sets.
Fig.3 shows the amplified fragments by one-step multiplex
RT-PCR from extracts of 7 infected samples and