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

Abstract

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

Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma

“…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).…”

Outcomes in Transplant Recipients Treated With Foscarnet for Ganciclovir-Resistant or Refractory Cytomegalovirus Infection

“…CMV quantitative PCR assays were performed using a laboratory-developed assay based on Qiagen-artus analyte specific reagents until 4/2/2013 (copies/ml with lower limit of quantification 100 copies/ml) 24 . From 4/2/2013 onward, an FDA-approved real-time PCR assay was performed (Cobas AmpliPrep/Cobas TaqMan CMV, results in IU/ml with lower limit of quantification 137 IU/ml).…”

Comparison of BKV quantification using a single automated nucleic acid extraction platform and 3 real-time PCR assays

“…LOD was determined by probit regression analysis using R (Forman et al, 2011). Descriptive statistics for precision experiments were calculated using Microsoft Excel 2007.…”