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

Raggam, Reinhard B.; Bozic, Michael; Salzer, Helmut J. F.; Hammerschmidt, Sandra; Homberg, Cordula; Ruzicka, Katharina; Kessler, Harald H.

doi:10.1007/s00430-010-0164-z

Cited by 12 publications

(9 citation statements)

References 27 publications

(31 reference statements)

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“…Data with a version of this system employing manual rather than automated reaction setup have been reported previously (7). The measurable range was similar to that in our study (2.0 to 7.0 log 10 copies/ml), but no LOD was reported.…”

supporting

confidence: 88%

Cytomegalovirus DNA Quantification Using an Automated Platform for Nucleic Acid Extraction and Real-Time PCR Assay Setup

2011

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Analytical performance characteristics of the QIAsymphony RGQ system with artus cytomegalovirus (CMV) reagents were determined. Measurable range spanned 2.0 to >7.0 log 10 copies/ml. The detection limit was 23 copies/ml. Intrarun and interrun coefficients of variation were <2.1% at 3.0 and 5.0 log 10 copies/ml. In clinical specimens, RGQ values were ϳ0.2 log 10 copies/ml higher than those in an assay using a BioRobot M48 extraction/manual reaction setup/7500 Real-Time PCR instrument. No cross-contamination was observed.Cytomegalovirus (CMV) DNA quantification in peripheral blood has become the standard of care for disease diagnosis in symptomatic individuals; detection of CMV replication in asymptomatic patients, allowing for preemptive treatment; monitoring of antiviral treatment response; and detection of antiviral drug resistance (4, 5). Instrumentation systems that incorporate automated nucleic acid extraction with volumetric pipetting stations for reaction setup are highly desirable solutions to meet mounting testing needs. The aim of this study was to investigate the performance of the QIAsymphony RGQ system for CMV DNA quantification in plasma using artus CMV reagents (Qiagen). The QIAsymphony RGQ system is comprised of an automated extraction instrument (QIAsymphony SP), an automated volumetric pipetting system for reaction setup (AS module), and a real-time PCR instrument (Rotor-Gene Q). CMV reagents are available as analyte-specific reagents (ASR). They are currently designated research use only (RUO) when adapted for use on RGQ. Analytical performance characteristics of the RUO assay were determined.Extraction was performed on the QIAsymphony SP (QIAsymphony Virus/Bacteria Midi Test Kit reagents and Cell-free 1000 instrument protocol). Plasma input/elution volumes were 1.2 ml (1,000 l processed)/95 l, respectively. Internal control and carrier RNA volumes were calculated by instrument software. These were added manually to buffer AVE and then placed in the designated QIAsymphony SP slot. After extraction, eluates in 96-microwell plates were transferred by the instrument to the AS module. Instrument software calculated worktable requirements for assay setup. The AS module assembled the master mix and then aliquoted reaction components (30 l master mix plus 20 l assay standards, assay controls, or extracted samples) into individual strip tubes. Strip tubes were then manually removed, capped, placed in the 72-position rotor disc, and positioned onto the Rotor-Gene Q. Amplification parameters were as follows: 95°C for 10 min and then 45 cycles of 95°C for 15 s, 65°C for 30 s, and 72°C for 20 s.CMV Towne strain (VR-977; American Type Culture Collection, Manassas, VA) was used for analytical studies. Genome equivalents were determined with a laboratory-developed US17-based real-time PCR test (151-bp amplicon [6], using purified spectrophotometrically quantified US17 subclone plasmid as calibrator). The stock concentration was defined as the mean geometric (log 10

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supporting

confidence: 88%

Cytomegalovirus DNA Quantification Using an Automated Platform for Nucleic Acid Extraction and Real-Time PCR Assay Setup

2011

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“…The artus CMV PCR has been evaluated in a number of previous studies (4,6,7,12,22). Our results agree with the findings of the only previous report to compare artus CMV PCR reagents to the COBAS Amplicor CMV Monitor assay (4).…”

Section: Discussionsupporting

confidence: 88%

Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

Waggoner

Libiran

et al. 2012

J Clin Microbiol

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The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log 10 copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated 100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding 6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test. The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml to >200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low level.

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“…This threshold was validated by testing QCMD panel samples and clinical samples diluted in WB to maintain extraction conditions, thus confirming the high sensitivity of this assay for CMV quantification in WB. This new commercial assay, validated and released for WB samples, reaches a higher sensitivity than the tests already commercially available, which provide a higher LLQ (23,24).…”

Section: Discussionmentioning

confidence: 99%

Fully Automated Quantification of Cytomegalovirus (CMV) in Whole Blood with the New Sensitive Abbott RealTi m e CMV Assay in the Era of the CMV International Standard

et al. 2013

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f Fully standardized reproducible and sensitive quantification assays for cytomegalovirus (CMV) are needed to better define thresholds for antiviral therapy initiation and interruption. We evaluated the newly released Abbott RealTime CMV assay for CMV quantification in whole blood (WB) that includes automated extraction and amplification (m2000 RealTime system). Sensitivity, accuracy, linearity, and intra-and interassay variability were validated in a WB matrix using Quality Control for Molecular Diagnostics (QCMD) panels and the WHO international standard (IS). The intra-and interassay coefficients of variation were 1.37% and 2.09% at 5 log 10 copies/ml and 2.41% and 3.80% at 3 log 10 copies/ml, respectively. According to expected values for the QCMD and Abbott RealTime CMV methods, the lower limits of quantification were 104 and <50 copies/ml, respectively. The conversion factor between international units and copies (2.18), determined from serial dilutions of the WHO IS in WB, was significantly different from the factor provided by the manufacturer (1.56) (P ‫؍‬ 0.001). Results from 302 clinical samples were compared with those from the Qiagen artus CMV assay on the same m2000 RealTime system. The two assays provided highly concordant results (concordance correlation coefficient, 0.92), but the Abbott RealTime CMV assay detected and quantified, respectively, 20.6% and 47.8% more samples than the Qiagen/artus CMV assay. The sensitivity and reproducibility of the results, along with the automation, fulfilled the quality requirements for implementation of the Abbott RealTime CMV assay in clinical settings. Our results highlight the need for careful validation of conversion factors provided by the manufacturers for the WHO IS in WB to allow future comparison of results obtained with different assays.

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

Cited by 12 publications

References 27 publications

Cytomegalovirus DNA Quantification Using an Automated Platform for Nucleic Acid Extraction and Real-Time PCR Assay Setup

Cytomegalovirus DNA Quantification Using an Automated Platform for Nucleic Acid Extraction and Real-Time PCR Assay Setup

Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

Fully Automated Quantification of Cytomegalovirus (CMV) in Whole Blood with the New Sensitive Abbott RealTi m e CMV Assay in the Era of the CMV International Standard

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