PATHOGEN
DETECTION
LABORATORY

Road 98 & Hutchison
CNPRC – UC Davis
Davis, CA 95616

530-752-8249

PCR (Polymerase Chain Reaction)

Real-time PCR assays are used to detect viral DNA and RNA, and proviral DNA in whole blood of macaques. Probes and primers were designed to amplify specific gene sequences. In addition, probe and primers were designed to amplify the housekeeping gene oncostatin M (OSM) which encodes a cytokine. The OSM gene is diploid in all primate cells allowing for cell quantification for each reaction. Real-time PCR is the ability to measure the development of PCR product as it is produced “in real-time”. Taqman chemistry takes advantage of flourogenic-labeled probes that use the 5’ nuclease activity of Taq polymerase. As in Endpoint PCR, a primer set is designed around the gene-fragment of interest. A probe is also designed containing a reporter fluorescent dye on the 5’ end and a quencher dye on the 3’ end. The proximity of the quencher to the reporter greatly reduces the fluorescence. If the gene of interest is present the probe anneals downstream from the primer and then is cleaved by the aforementioned 5’ nuclease activity. Separation of the reporter from the quencher allows for an increase of reporter fluorescence. With each cycle, reporter dye is cleaved from it’s probe resulting in an increase in fluorescence proportional to the amount of amplicon that is produced. The fluorescence is measured throughout the PCR reaction and a cycle threshold (Ct) is reported.

The minimum sample requirement is 3 ml of heparinized whole blood sent at room temperature to arrive within 24 hours of collection.