Rapid subgroup identification of the flaviviruses using degenerate primer E-gene RT-PCR and site specific restriction enzyme analysis

A simplified and rapid method for the diagnosis of all flaviviruses could provide an important tool for understanding their epidemiology. A protocol based on the use of degenerate nested oligonucleotide primers and RT-PCR was developed for the identification of flaviviruses. The primers were selected to flank the three E-gene markers that identify the viruses, giving DNA products of 971-986 (outer primers) and 859-884 bp (inner primers). Eighty five percent of E genes from flaviviruses representing most of the genus were specifically amplified, representing viruses from each of the 14 virus groups defined by the seventh International Committee for the Taxonomy of Viruses. Categorisation of the flavivirus cDNA products into the corresponding virus groups was undertaken through restriction enzyme analysis by defining conserved restriction sites common to related viruses in appropriate virus groups. Ninety percent of the known vector-borne flaviviruses with published full length E-gene sequences could be identified within 10 h.