Chapter Twenty - Demonstration of Lyssavirus Nucleic Acids by Pyrosequencing

Abstract

Although molecular methods cannot be considered as a replacement for standard laboratory techniques in rabies, over the last decade they have been used as an alternative means to diagnose viral diseases. The One-Step reverse transcription polymerase chain reaction (RT-PCR) followed by pyrosequencing as herein described is a mutation-resistant method because it is based on primers designed on conserved lyssavirus gene regions, while the most variable part of the nucleoprotein (N) gene is further pyrosequenced. Pyrosequencing is a novel DNA sequencing technology based on the sequencing-by-synthesis principle. It represents an alternative to the Sanger method. Although the applications of this innovative technology do not completely overlap with those of conventional methods, they are characterized by several advantages, such as the rapidity in obtaining a final diagnosis and the relatively lower cost for identifying and typing lyssavirus species involved in the infection.