Detection of Cytomegalovirus in Whole Blood Using Three Different Real-Time PCR Chemistries

Vincent, Erica; Gu, Zheng; Morgenstern, Markus; Gibson, C.; Pan, Jianmin; Hayden, Randall T.

doi:10.2353/jmoldx.2009.080073

Cited by 13 publications

(7 citation statements)

References 30 publications

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“…Regarding routine monitoring of any immunosuppressed patient or patient's response to therapy, CMV DNA blood levels are usually not considered clinically signiWcant. However, it appears more relevant to consider the viral load change (between successive measures) instead of the absolute value given [26,28].…”

Section: Discussionmentioning

confidence: 97%

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR

Raggam¹,

Bozic

Salzer

et al. 2010

Med Microbiol Immunol

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Quantitation of cytomegalovirus (CMV) DNA in whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical performance of the artus™ CMV RG PCR kit in conjunction with automated sample preparation on the new QIAsymphony™ SP instrument was evaluated. Clinically referred samples were tested and results compared to those obtained with the routinely used molecular test system. Accuracy testing showed results to be within ±0.2 log(10) unit of the expected panel results. The assay was linear (R = 0.9972) from a lower quantification limit of 148 copies/ml to 1.3 × 10(7) copies/ml. The between-day imprecision CV was 8-63%, and the within-run imprecision CV was 16-61%. When 100 clinically referred samples were tested, results obtained with the new test system showed an acceptable concordance with those obtained with the routinely used easyMAG™ sample preparation and CMV HHV6,7,8 R-gene™ test system. Discrepant results were found with low-titer samples containing CMV DNA concentrations under the lower limit of quantification or within half a log unit above. The time to results was comparable for both test systems. The QIAsymphony™ sample preparation and artus™ CMV RG PCR test system allows for a rapid, sensitive, precise, and accurate high-throughput quantitation of CMV DNA in whole blood in the routine diagnostic laboratory.

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Section: Discussionmentioning

confidence: 97%

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR

Raggam¹,

Bozic

Salzer

et al. 2010

Med Microbiol Immunol

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“…of CMV DNA loads in plasma (5,11,12,18,24). Likewise, there are scarce data published on the performance of automated extraction systems for CMV plasma DNA quantitation (5,11,(14)(15)(16)20).…”

Section: Vol 49 2011 Qrt-pcr Automated Extraction Method and CMV mentioning

confidence: 99%

“…Nevertheless, a few studies directly compared the performance of these assays for the quantitation of CMV plasma DNAemia (5,11,12,18,24). In addition, to the best of our knowledge, data on the performance of the QRT-PCR from Nanogen have not been published.…”

Section: Vol 49 2011 Qrt-pcr Automated Extraction Method and CMV mentioning

confidence: 99%

“…The analytical performance and clinical usefulness of these assays have been assessed, mostly in comparison with the pp65 antigenemia test or quantitative endpoint PCR assays (1, 2, 6, 9, 10-13, 15, 17, 19, 21, 22, 24-26). Limited data are available on how these QRT-PCR tests compare to each other for the quantitation of CMV plasma DNAemia (5,11,12,18,24). This information may allow, at least to some extent, direct comparisons of CMV DNA loads measured at different centers.…”

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confidence: 99%

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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

et al. 2011

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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.Quantitative real-time PCR (QRT-PCR) assays are being increasingly used for the surveillance of active cytomegalovirus (CMV) infection in allogeneic stem cell transplant (Allo-SCT) recipients (23). Several CMV QRT-PCR assays targeting different CMV genes and using different chemistries are commercially available (23). The analytical performance and clinical usefulness of these assays have been assessed, mostly in comparison with the pp65 antigenemia test or quantitative endpoint PCR assays (1, 2, 6, 9, 10-13, 15, 17, 19, 21, 22, 24-26). Limited data are available on how these QRT-PCR tests compare to each other for the quantitation of CMV plasma DNAemia (5,11,12,18,24). This information may allow, at least to some extent, direct comparisons of CMV DNA loads measured at different centers.Nucleic acid extraction is a critical step in QRT-PCR testing, and it has been shown to be a major source of assay variability in viral DNA quantitation (7). Automated nucleic acid extraction systems are less time-consuming, less prone to analytical errors, and, overall, more efficient than manual methods. A few studies have directly compared the extraction efficiency of different automat...

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“…4 Nevertheless, studies comparing their relative analytical performance are scarce. 6,7 Since no universally accepted standards for CMV viral load quantitation are available, this issue is critical if clinical studies involving various participant centers that may use different assays are planned.…”

mentioning

confidence: 99%

Differences in cytomegalovirus plasma viral loads measured in allogeneic hematopoietic stem cell transplant recipients using two commercial real-time PCR assays

Gracia-Ahufinger

Tormo

Espigado

et al. 2010

Journal of Clinical Virology

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Detection of Cytomegalovirus in Whole Blood Using Three Different Real-Time PCR Chemistries

Cited by 13 publications

References 30 publications

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR

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

Differences in cytomegalovirus plasma viral loads measured in allogeneic hematopoietic stem cell transplant recipients using two commercial real-time PCR assays

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