- Detects and quantified SIV/SHIV RNA and DNA Gag sequences in cell pellet or tissue specimens.
- Testing available at two levels of sensitivity, depending on study needs and available sample amount.
- The Quantitative Molecular Diagnostics Core extracts total DNA and RNA from the specimen and tests them separately in each case using a hybrid assay format that combines quantitative, real-time, procedure-based standard curve interpolation and Poisson-based methods.
- Extracted RNA is resuspended and divided among 10 replicate quantitative polymerase chain reaction (qPCR) for DNA or quantitative reverse transcription polymerase chain reaction (qRT-PCR) for RNA reactions.
- Testing for SIV DNA is multiplexed with an assay for a single copy genomic sequence from rhesus macaque CCR5 to permit normalization to cell numbers based on diploid genome equivalents.
- If all wells give positive PCR reactions, the average of the quantitative real-time assay standard curve interpolated values is used to calculate the SIV DNA copy number value for the specimen, and then normalized for the number of cells tested, based on diploid genome cell equivalents.
- If not all wells give positive reactions, Poisson methods are used to calculate viral copy numbers, which are then normalized for the number of cells tested, based on diploid genome cell equivalents.
- Values for SIV/SHIV RNA are determined by qRT-PCR, and normalized to cell numbers based on diploid genome equivalents based on CCR5 DNA copy numbers, in light of near-quantitative recovery of RNA and DNA with the processing and extraction methods employed.
- Specimens for analysis must be prepared according to specified recommendations for processing, preparation, labelling, storage, and shipment.
Includes sufficient basic consultation with a subject matter expert.