SENSITIVE AND SPECIFIC COLORIMETRIC DOT ASSAY TO DETECT EASTERN EQUINE ENCEPHALOMYELITIS VIRAL-RNA IN MOSQUITOS (DIPTERA, CULICIDAE) AFTER POLYMERASE CHAIN-REACTION AMPLIFICATION

A sensitive and specific colorimetric dot assay following polymerase c hain reaction (PCR) method has been developed to detect 0.1 pg of east ern equine encephalomyelitis viral (EEEV) RNA. The assay is 250-fold m ore sensitive than analysis by electrophoresis and is based on convert ing a 291-nucleotide sequence of the viral coat protein amino terminus into a double-stranded DNA (dsDNA) and amplifying die DNA using a spe cific primer pair and PCR. The amplified complementary DNA (cDNA) is d enatured, adsorbed onto a nylon strip, baked, and detected with a digo xigenin-labeled probe. Dots with viral cDNA are stained dark red, wher eas controls do not stain or stain lightly. The assay is very specific and sensitive and detects only EEEV. RNA of Venezuelan equine encepha litis, St. Louis encephalitis, Keystone, Flanders, Tensaw, and western equine encephalitis viruses were net detected. EEEV (Ten Broeck) RNA was detected at the 10-ng level, indicating that die prototype we used may have different nucleotides in the region where the primer pair bi nds. The PCR amplified EEEV cDNA that was 92% homologous to the consen sus sequence of EEEV. The detection of EEEV in the liver of an infecte d Emu bird and in field-collected mosquitoes from Florida and Masschus etts that were analyzed concurrently as blind samples by tissue cultur e plaque assay and by PCR clot analysis proved that the assay is sensi tive and can be used to detect infected mosquitoes. The assay can dete ct at least 1 infected mosquito in a pool of 1,000 uninfected mosquito es.