Multiplex nested PCR for Brazilian Alphavirus diagnosis

Abstract
A reverse-transcriptase PCR (RT–PCR) and a multiplex nested PCR were developed for the rapid detection and identification of 14 Brazilian alphaviruses. Using Alphavirus genus-specific primers in a RT–PCR, we obtained amplified products of 434 bp. Species-specific primers were selected and simultaneously tested in a multiplex nested PCR. The nested PCR increased the test sensitivity 1000-fold and was capable of identifying Brazilian Alphavirus showing the expected bands with diagnostic sizes for Venezuelan (400 bp), Eastern (124 bp), and Western (208 bp) equine encephalitis, Aura (86 bp), and Mayaro (270 bp) viruses. This strategy for diagnosis is fast, sensitive, specific and it can be used as a reliable alternative for routine Brazilian Alphavirus diagnosis.