Basic Procedure: RNA is extracted from lysed mosquitoes and vertebrates, either by hand or with a robot workstation. Various types of RT-PCR (RT= reverse transcription) assays are used to detect specific sequences of nucleotides within the RNA. In each type (standard, real-time, multiplex), different primers where included in the mixture so that amplification reactions could take place. The procedures for PCR differ mainly by the time at which they detect specific sequences during the reaction and what kind/how many primers are used. These procedures were done both by hand and with a robot workstation as well. IFA was also performed on frozen avian tissues for comparison.

Why is this better? The main difference between detection with PCR and IFA is that the former detects the actual presence of the virus, as opposed to the bodies’ reaction to the virus. IFA takes time because cells must be cultured for growth so that enough of the virus is present and then the body must respond to its presence. PCR is quicker and very specific in identification of a nucleotide sequence. Also, in mammalian tissues, chemicals can bond nonspecifically so that confirmation of the disease is not clear. It was also shown that use of the robot workstation was very accurate and time-saving, especially in preparing the assay.