Plasma or CSF SIV/SHIV RNA measurements

• Detects and quantifies SIV/SHIV Gag RNA sequences in cell free samples. 
• Testing available at two levels of sensitivity, depending on study needs and available sample amount. 
  • Standard: 15 copies/mL  
  • High sensitivity: 1.0 copies/mL 
• Covers isolates used most commonly in SIV experimental studies. 

The process: 

• The Quantitative Molecular Diagnostics Core performs 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. 
• 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. 

Acceptance criteria: 

• 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 for standard analysis and 1.7 mL plasma for ultrasensitive analysis should be prepared and provided according to mandatory specimen preparation, labelling, and shipment instructions. 
  • Contact Jeff Lifson, M.D. for details.  

Includes sufficient basic consultation with a subject matter expert.