ACVP-06: Cell-Associated SIV/SHIV RNA and DNA Analysis on Cell/Tissue Specimens 

This testing is available only after mandatory prior consultation with Dr. Jeff Lifson (lifsonj@mail.nih.gov) of the Quantitative Molecular Diagnostics Core (QMDC).  

This assay is performed by the QMDC for measuring levels of cell- or tissue-associated SIV/SHIV DNA and RNA in cell pellets or tissue samples from nonhuman primates. Specimens for analysis must be prepared according to specified recommendations for processing, preparation, labelling, storage, and shipment (contact Dr. Jeff Lifson, lifsonj@mail.nih.gov).

Total DNA and RNA are extracted from the specimen, then tested 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. While the assay provides nominal single copy per reaction sensitivity, the clinical sensitivity will vary as a function of the specimen provided and the number of cell equivalents of RNA and DNA analyzed. Assay details can be found in Hansen et al, Nature. 2017 Jul 6;547(7661):123-124. doi: 10.1038/nature22984. Epub 2017 Jun 21. PMID:28636599. Sufficient basic consultation with a subject matter expert is included with the service.

AVCP-08: Cell-Associated SIV/SHIV RNA and DNA Analysis on Cell/Tissue Specimens

This testing is available only after mandatory prior consultation with Dr. Jeff Lifson (lifsonj@mail.nih.gov) of the Quantitative Molecular Diagnostics Core (QMDC).  

This assay is performed by the QMDC for measuring levels of cell- or tissue-associated SIV/SHIV RNA in cell pellets or tissue samples from nonhuman primates. Specimens for analysis must be prepared according to specified recommendations for processing, preparation, labelling, storage, and shipment (contact Dr. Jeff Lifson, lifsonj@mail.nih.gov). Total DNA and RNA are extracted from the specimen, then tested separately, 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 5 replicate reverse transcription polymerase chain reaction (RT-PCR) reactions. Testing is combined with a DNA PCR assay for a single copy genomic sequence (rhesus macaque CCR5) to permit normalization to cell numbers based on diploid genome equivalents. If all wells give positive RT PCR reactions for SIV/SHIV RNA, the average of the standard curve interpolated values is used to calculate the diploid genome cell equivalent normalized SIV RNA copy number value for the specimen. If not all wells give positive reactions, Poisson methods are used to calculate copy numbers, which are then normalized per diploid genome cell equivalents based on CCR5 DNA measurements from the same samples. Values for SIV/SHIV RNA are normalized to cell equivalents based on CCR5 DNA copy numbers in light of near quantitative recovery of RNA and DNA with the processing and extraction methods employed. While the assay provides nominal single copy per reaction sensitivity, the clinical sensitivitywill vary as a function of the specimen provided and the number of cell equivalents of RNA and DNA analyzed. 

Compared to ACVP-06, the ACVP-08 assay may be useful when specimens are expected to not contain very low levels of SIV/SHIV RNA requiring maximum sensitivity, and viral DNA measurements are not required. Prior consultation with Dr. Lifson will help determine which assay is best suited to a particular project or specimenset. Sufficient basic consultation with a subject matter expert is included with the service.