A generic nested-RT-PCR followed by sequencing for detection and identification of members of the alphavirus genus

Abstract

A specific and sensitive nested RT-PCR method was developed for the detection of members of the alphavirus genus. Based on available sequences, degenerated primers were selected in the nsP4 gene. Reaction components and thermal cycling parameters were investigated and standardised, and optimal ones were selected. As few as 25 pfu/tube could be detected. The identities of the amplified fragments were confirmed by sequencing, and phylogenetic analysis was carried out. The resulting phylogenetic tree could be applied to classify every alphavirus according to its serogroup. This technique is suitable for rapid, sensitive and reliable detection of these viruses and may be very valuable for diagnostic applications and surveillance.