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

Abstract

A diagnostic test incorporating reverse-transcription polymerase chain reaction (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 refinements were made to the protocol including simplification of the extraction method, the use of standard reagents and adoption of a one-step procedure. None of these changes impaired sensitivity or specificity. The RT-PCR could also be used to amplify immunocaptured virus but this was slightly less 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 collected from Europe, America and Asia (including A, B and P pathotypes) isolated from either sugar beet or Chenopodium quinoa. Neither assay produced PCR products using total RNA extracted from the roots of healthy sugar beet or beet infected with Beet soil-borne virus. However, the sensitivity of the 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 collected from an infected field site. The use of the nPCR assay is recommended for applications where its improved sensitivity over standard RT-PCR is necessary, for example in the early detection of infection from bait-test soils and for quarantine and breeding purposes.