Development of a highly sensitive nested RT-PCR method for Beet necrotic yellow vein virus detection

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.