Sensitive and specific colorimetric dot assay to detect eastern equine XXXX viral RNA in mosquitoes (Diptera: Culicidae) after polymerase chain reaction amplification.

@entity1472 @entity198 @entity2036 @entity1859 @entity377 @entity1651 @entity6498 @entity37600
A sensitive and specific colorimetric dot assay following polymerase chain reaction (PCR) method has been developed to detect 0.1 pg of eastern equine @entity1651 viral (EEEV) RNA. The assay is 250-fold more sensitive than analysis by electrophoresis and is based on converting a 291- @entity377 sequence of the viral coat protein @entity1472 terminus into a double-stranded DNA (dsDNA) and amplifying the DNA using a specific primer pair and PCR. The amplified complementary DNA (cDNA) is denatured adsorbed onto a nylon strip, baked, and detected with a @entity1859 -labeled probe. Dots with viral cDNA are stained dark red, whereas controls do not stain or stain lightly. The assay is very specific and sensitive and detects only EEEV. RNA of @entity37600 , St. Louis @entity198 , Keystone, Flanders, Tensaw, and western equine @entity198 viruses were not detected. EEEV (Ten Broeck) RNA was detected at the 10-ng level, indicating that the prototype we used may have different @entity2036 in the region where the primer pair binds. The PCR amplified EEEV cDNA that was 92% homologous to the consensus sequence of EEEV. The detection of EEEV in the liver of an infected @entity6498 bird and in field-collected mosquitoes from Florida and Massachusetts that were analyzed concurrently as blind samples by tissue culture plaque assay and by PCR dot analysis proved that the assay is sensitive and can be used to detect infected mosquitoes. The assay can detect at least 1 infected mosquito in a pool of 1,000 uninfected mosquitoes.
