Assessment of template quality by the incorporation of an internal controlinto a RT-PCR for the detection of rabies and rabies-related viruses

A method is described to assess RNA template quality by the incorporation o f a ribosomal RNA (rRNA) internal (in tube) control into a standard rabies and rabies-related virus specific RT-PCR. Specific virus and rRNA templates were co-amplified in a duplex reaction from RNA extracts derived from 60 i solates representing all six of the established lyssavirus genotypes. To en sure a wide species applicability of this technique we demonstrated that th e rRNA assay was capable of functioning using the cells or tissues of 14 di fferent mammals. Parallel studies between the duplex and the unlinked lyssa virus assay demonstrated only a minor reduction in the sensitivity of the f ormer test. The ribosomal and viral targets (unlike p-actin RNA) were shown to have similar degradation kinetics making rRNA amplification a good cont rol for viral target integrity. As a consequence, the use of this system wo uld reduce the likelihood of obtaining false negative RT-PCR results from l yssavirus infected material. Crown copyright (C) 2000 Published by Elsevier Science B.V. All rights reserved.