Cobas ampliprep system v2.0 taqman 48 system

The levels of HIV-1 viral RNA were measured and analyzed at designated time points using the automated COBAS Ampliprep system v2.0/Taqman-48 system (Roche Molecular Diagnostics, Basel, Switzerland) as described previously [72 (link)], and plotted using Prism V9.4.1 software. The assay’s detection limit is 200 viral RNA copies/mL, reflective of the known dilution factors [71 (link)].

Mice from a single donor were used for all dual treatment mice. Humanization of the animals was affirmed by flow cytometry (25 (link), 26 (link)) for the presence of human CD45- and CD3-positive blood immune cells. At 18 to 20 wk of age, 48 NSG-hu mice were infected intraperitoneally with HIV-1_ADA (28 (link)) at 1.5 × 10⁴ TCID₅₀/mL. Three control-uninfected animals were included in all test evaluations. Levels of viral RNA copies/mL were analyzed with the automated COBAS Ampliprep System V2.0/Taqman-48 system (Roche Molecular Diagnostics) (26 (link), 27 (link), 29 ). For this assay, 50 to 100 μL mouse plasma was diluted to 1 mL with sterile filtered normal human serum. The detection limit of the assay after dilution is 200 viral RNA copies/mL. Although the eclipse phase for viral infection in humans remains variable (30 (link)), the viral loads and CD4 + T cell depletion levels observed in our infected humanized mice are in point of fact reflective of the disease course in an infected human host. Indeed, only after weeks of infection we do observe significant cell loss (18 (link), 23 (link), 27 (link), 31 (link)). These findings can be viewed as an affirmation of the model including CD4 + T cell-timed restorations seen after ART as is seen in humans. For the validation study, nine humanized mice were infected with HIV-1_ADA at 2.0 × 10⁴ TCID₅₀/mL.