Generic detection and differentiation of tobamoviruses by a spot nested RT-PCR-RFLP using dI-containing primers along with homologous dG-containing primers

A spot nested RT-PCR-RFLP method to detect and identify all members of the Tobamovirus genus is described. It involves a one-step RT-PCR, in which the combination of degenerate deoxyinosine (dI)-substituted primers amplified part of the polymerase region of tobamoviruses, followed by a nested PCR amplification that increased specificity and sensitivity of detection. Virus species differentiation was achieved by subsequent restriction enzyme analysis. The sensitivity of the method was increased further when along with one primer containing many dIs, another homologous primer in which dIs were substituted by dGs was used. The homologous primer was shorter than the dI-containing primer and with lower degeneracy, resulting in higher overall amplification efficiency due to the increased stability of the primer-target duplex. With this strategy, highly degenerate primers containing many dIs can be used effectively to improve detection sensitivity, alleviating problems of primer-target duplex destabilisation that can occur due to many dI substitutions. This method can be useful on the diagnosis, epidemiological investigation, and characterisation of known and unidentified Tobamovirus species. (C) 2004 Elsevier B.V. All rights reserved.