Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients

Abstract

Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

Fig. 1.

Correlation and linear regression analysis of cytomegalovirus (CMV) DNA load values (copies/ml) obtained for all positive specimens by the Abbott CMV PCR kit following DNA extraction using the Abbott mSample preparation system DNA kit on the m24 SP instrument (M24), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (AMPLIPREP), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (EZ).

Fig. 2.

Bland-Altman representation of CMV DNA loads (copies/ml) measured using the Abbott CMV PCR kit in the m2000RT (ABBOTT), the LightCycler CMV Quant kit in the Light Cycler 2.0 instrument (ROCHE), and the Q-CMV complete kit in the LightCycler 2.0 instrument (NANOGEN), following DNA extraction by the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform.

Fig. 3.

Bland-Altman representation of CMV DNA loads (copies/ml) measured using the Abbott CMV PCR kit in the m2000RT (ABBOTT) after extraction by the Abbott mSample preparation system DNA kit on the m24 SP instrument and those measured by the LightCycler CMV Quant kit in the Light Cycler 2.0 instrument (ROCHE), following extraction by the High Pure viral nucleic acid kit on the COBAS AmpliPrep system.