J. Morris et al., Development of a highly sensitive nested RT-PCR method for Beet necrotic yellow vein virus detection, J VIROL MET, 95(1-2), 2001, pp. 163-169
A diagnostic test incorporating reverse-transcription polymerase chain reac
tion (RT-PCR) and nested polymerase chain reaction (nPCR) was developed for
the detection of Beet necrotic yellow vein virus (BNYVV). The RT-PCR used
the primers designed by (Henry et al.. J. Virol. Methods 54 (1995)15) but r
efinements were made to the protocol including simplification of the extrac
tion method, the use of standard reagents and adoption of a one-step proced
ure. None of these changes impaired sensitivity or specificity. The RT-PCR
could also be used to amplify immunocaptured virus but this was slightly le
ss sensitive than amplification from purified RNA. In nPCR, a second round
of amplification was performed using primers. which produce a specific 326
base-pair product. Both RT-PCR and nPCR detected a range of 21 isolates col
lected From Europe. America and Asia (including A. B and P pathotypes) isol
ated from either sugar beet or Chenopodium quinoa. Neither assay produced P
CR products using total RNA extracted from the roots of healthy sugar beet
or beet infected with Beet soil-borne virus. However. the sensitivity of th
e nPCR was 1000 times greater than the standard RT-PCR. The reliability of
the standard RT-PCR and nPCR was demonstrated using a range of cultivars co
llected from an infected field site. The use of the nPCR assay is recommend
ed for applications where its improved sensitivity over standard RT-PCR is
necessary. for trample in the early detection of infection from bait-test s
oils and for quarantine and breeding purposes. (C) 2001 Elsevier Science B,
V. All rights reserved.