ACVP-02: Plasma SIV/SHIV RNA Viral Load Measurements through the AIDS and Cancer Virus Program Quantitative Molecular Diagnostics Core

The SIV plasma viral load assay performed by the Quantitative Molecular Diagnostics Core (QMDC) utilizes reagents specifically designed to detect and accurately quantify the full range of SIV/SHIV viral variants and clones in common usage in the research community. This is achieved by performing a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay, targeting a highly conserved sequence in the SIV genome. At positions of documented sequence heterogeneity within this targeted region, unconventional neutral bases are incorporated into primer and probe sequences used in the assay to avoid biasing quantification of different target virus sequences. 

Ethylenediamine-tetraacetic acid (EDTA) or acid citrate dextrose (ACD) anti-coagulated plasma specimens are acceptable; heparin anticoagulated specimens are not compatible with the assay procedures and are not acceptable. Specimens of 0.7-1.2 mL plasma should be prepared and provided according to mandatory specimen preparation, labelling, and shipment instructions (contact Dr. Jeff Lifson, lifsonj@mail.nih.gov). This test provides a sensitivity of 15 SIV RNA copy Equivalents/mL plasma. A detailed assay description can be found in Li, H, et al, Proc Natl Acad Sci USA. 2016 Jun 14;113(24):E3413-22. doi: 10.1073/pnas.1606636113. Epub 2016 May 31. Sufficient basic consultation with a subject matter expert is included with the service.

ACVP-13: High Sensitivity Plasma SIV/SHIV RNA Measurements through the AIDS and Cancer Virus Program Quantitative Molecular Diagnostics Core Assay

This high sensitivity assay performed by the Quantitative Molecular Diagnostics Core (QMDC) is for measuring low levels of virion associated SIV/SHIV RNA in plasma samples from nonhuman primates. The QMDC performsa quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that employs a hybrid assay format that combines a qRT-PCR procedure based standard curve interpolation with Poisson-based methods. For high sensitivity analysis, 1.6 mL of plasma is processed and the extracted RNA is resuspended and divided among 12 replicate RT qPCR reactions. If all 12 wells give positive reactions, the average of the standard curve interpolated values is used to calculate the volume normalized SIV RNA copy number value for the specimen. If not all wells give positive reactions, Poisson methods are used to calculate copy numbers. If the starting source plasma specimen volume is 1.6 mL, Poisson calculations for 1/12 wells giving a positive reaction would correspond to 1.57 copies/1.6 mL, or ~ 1 copy/mL. If there are no positive reactions in the 12 reactions tested, then the specimen is judged to contain < 1 copy/mL. 

Ethylenediaminetetraacetic acid (EDTA) or acid citrate dextrose (ACD) anti-coagulated plasma specimens are acceptable; heparin anticoagulated specimens are not compatible with the assay procedures and are not acceptable. Specimens of 1.7 mL plasma should be prepared and provided according to mandatory specimen preparation, labelling, and shipment instructions (contact Dr. Jeff Lifson, lifsonj@mail.nih.gov). Sufficient basic consultation with a subject matter expert is included with the service.