final program.qxd - Parallels Plesk Panel

PP 2.4
The Cobas Ampliprep/Cobas Taqman 48 HIV-1 test is compatible with the Primagen
dried fluid spot technology for viral load measurement
Michel P. de Baar, John T. Dekker, Esther de Rooij, Vincent Geutjes, Suzanne Jurriaans#,
Harm B. van Schijndel
Primagen, Amsterdam, the Netherlands, and #Department of Human Retrovirology,
Academic Medical Center, Amsterdam, the Netherlands
Objective
to develop and evaluate protocols to combine the Cobas Ampliprep/Cobas Taqman 48
HIV-1 test with Primagen's proprietary dried fluid spot (DFS) technology.
Methods
HIV-1 negative whole blood (WB) and plasma (P) was obtained via the blood bank. For
reconstruction experiments, the WB and P were spiked with various concentrations of a
viral stock, of which the viral load has been determined by repetitive testing using the
Cobas Amplicor HIV-1 v1.5. These samples were tested according to the manufacturer's
protocol as well as spotted on the Primagen filter papers for comparisons. The DFS were
dried on the air for at least 15 minutes and stored upon use. The spots were punched out
of the filter by a manually operated puncher or by laser cutter. Nucleic acids were
recovered from the spots by dissolving them in lysis buffer for at least 3 hrs at room
temperature before quantifying on the Cobas system. The clinical P samples were
obtained, with permission, from the clinic of the Academic Medical Center in Amsterdam.
Results
The lower limit of detection for DFS was 360 copies HIV-1 RNA per ml P, comparable with
the standard protocol when corrected for an input of only 200 µl P (4 spots of 50 µl each).
10 P samples with RNA levels varying from 3log10 to 8 log 10 were tested both directly and
via DFS. After the correction for 1 ml input in the direct method and 200 µl input via DFS,
the Pearson correlation coefficient was 0.996 (P