TABLE 1.
Summary of techniques employed to quantify plasma SIV RNA
| Assay | Quantification systema | Target region | RNA extraction (plasma vol [μl]) | Calibration/standards | Upper and lower assay detection limits (RNA copies/ml) | Real-time PCR platform | Reference(s) |
|---|---|---|---|---|---|---|---|
| 1 | Real-time qRT-PCR | p27-gag | QIAamp, Qiagen (140) | External SIV251 RNA plasma series | 1 × 109 - 1.69 × 102 | Mx3000P (Stratagene) | 1 |
| 2 | Conventional qRT-PCRc | p27-gag | Trizol, propan-2-ol (200) | Coamplified synthetic RNA transcript | 1 × 107 - 1.6 × 102 | n/ab | 27 |
| 3 | Real-time qRT-PCR | p27-gag | QIAamp, Qiagen (200) | SIV251 RNA transcript | 1.6 × 106 - 1.69 × 102 | ABI Prism (Applied Biosystems) | 5, 18 |
| 4 | qRT assay | n/a | Viral binding gel (1,000) | BrdU/poly(A) system/HIV standard curve | 1.2 ×106 - 2.3 × 102 | n/a | 3 |
| 5 | Real-time qRT-PCRv | p15-gag | Nuclisens, bioMérieux (200) | SIV251 and internal HIV-1 control | 1 × 106 - 2.39 × 102 | ABI Prism (Applied Biosystems) | 20, 24, 25 |
| 6 | Real-time qRT-PCR | p27-gag | Trizol (200) | Coamplified synthetic RNA transcript | 1 × 1010 - 2.3 × 102 | iQ5 multicolor system (Bio-Rad) | 10 |
a
qRT-PCR, quantitative reverse transcriptase PCR; qRT-PCRc, conventional qRT-PCR system based on endpoint estimation of preamplified product; qRT-PCRv, quantitative RT-PCR validated by a virion estimation technique. qRT assay (assay 4), ExaVir Load v. 2 (Cavidi AB, Uppsala, Sweden). Assay 6 is a previously established real-time PCR method (10), with a modification of the SIV probe sequence to the Black Hole Quencher 2 dye in place of TAMRA.
b
n/a, not applicable.