The COBAS AmpliPrep/COBAS TaqMan CMV Test is an FDA-approved in vitro nucleic acid amplification test for the quantification of cytomegalovirus (CMV) DNA in human plasma using the COBAS AmpliPrep instrument for automated viral nucleic acid extraction (generic silica-based capture technique) and the COBAS TaqMan analyzer for automated amplification and detection of the viral nucleic acid target. This assay targets the highly-conserved, nondrug target region of the CMV DNA polymerase (UL54) gene. Generic silica-based specimen preparation is used to capture the CMV DNA and an internal control CMV quantitation standard (QS) DNA, and defined oligonucleotides are used as primers in amplification of the CMV DNA and CMV QS DNA. A target specific and a QS-specific dual-labeled oligonucleotide probe permit independent identification of the CMV amplicon and of the CMV QS amplicon. The COBAS AmpliPrep/COBAS TaqMan CMV Test uses 2 amplification primers for PCR. A fluorescent, signal-generating probe modified with a 5' fluorochrome (FAM) and a 3' quencher hybridizes to 1 of the 2 strands and is cleaved by Z05 DNA polymerase during extension of the primers. Cleavage of the probe physically separates the quencher from the reporter, enabling light emitted by the latter to be detected by a photomultiplier tube. Because amplification and detection are performed simultaneously, amplification products are measured during the exponential phase of DNA amplification regardless of the initial target concentration.
The COBAS AmpliPrep/COBAS TaqMan CMV Test quantitates CMV viral DNA by analyzing the difference between the CMV target and QS Ct values. The CMV QS is a noninfectious DNA construct containing fragments of CMV sequences with primer binding regions identical to those of the CMV target sequence. The CMV QS contains CMV primer binding regions and generates an amplification product of the same length and base composition as the CMV target DNA. The detection probe binding region of the CMV QS has been modified to differentiate CMV QS amplicon from CMV target amplicon. During the extension phase of the PCR in the COBAS TaqMan Analyzer, the specimens are illuminated and excited by filtered light and filtered emission fluorescence data are collected for each specimen. The readings from each specimen are then corrected for instrumental fluctuations. These fluorescence readings are sent by the instrument to the AMPLILINK software and stored in a database. Pre-Checks are used to determine if the CMV DNA target and CMV QS DNA data represent sets that are valid, and flags are generated when the data lie outside the preset limits. After all Pre-Checks are completed and passed, the fluorescence readings are processed to generate Ct values for the CMV DNA target and the CMV QS DNA. The lot-specific calibration constants provided with the COBAS AmpliPrep/COBAS TaqMan CMV Test are used to calculate the titer value for the specimens and controls based on both the CMV DNA target and CMV QS DNA Ct values. Titer results are reported in International Units/mL (IU/mL).(Package insert: COBAS AmpliPrep/COBAS TaqMan CMV Test; Roche Molecular Systems, Inc., Branchburg, NJ, 3/2015)