Comparative Evaluation of a Commercially Available Automated System for Extraction of Viral DNA from Whole Blood: Application to Monitoring of Epstein-Barr Virus and Cytomegalovirus Load

Abstract

The NucliSENS easyMAG automated system was compared to the column-based Qiagen method for EpsteinBarr virus (EBV) or cytomegalovirus (CMV) DNA extraction from whole blood before viral load determination using the corresponding R-gene amplification kits. Both extraction techniques exhibited a total agreement of 81.3% for EBV and 87.2% for CMV.Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections represent a significant clinical threat for immunocompromised patients. The frequent determination of EBV and CMV viral load permits the early diagnosis of infection, start of preemptive or curative therapy, and monitoring of treatment efficiency (5,17,20). By comparison to serum, plasma, or white-blood-cell fractions, whole-blood samples are now recognized as the most suitable sample for the determination of viral loads for EBV and CMV (3,4,7,9,10,12,13,18,19).Although the methods relying on silica columns are timeconsuming and need trained experimenters, these methods are considered the gold standard for the extraction of nucleic acids from whole-blood samples. Due to the large amount of genetic material in such samples, new extraction methods must be carefully evaluated, including those relying on automated devices (1, 7, 10, 14, 15). The fully automated NucliSENS easyMAG instrument (bioMérieux) using magnetic silica particles (2) allows the simultaneous process of up to 24 extractions. The use of magnetic particles eliminates the several centrifugation steps that could be a source of cross-contamination, and manual steps are limited to the loading of samples, reagents, and disposables. The performance of this method in the extraction of DNA from whole-blood samples prior to viral quantification has not been yet evaluated. The present study was undertaken to answer this question in the clinical context of EBV or CMV infection.The whole-blood specimens selected for this study included 80 samples for EBV analysis and 94 samples for CMV analysis, taken from patients hospitalized at the University Hospital of Saint-Etienne, Saint-Etienne, France, from December 2007 to September 2008. The samples were kept frozen at Ϫ20°C. After the samples were thawed, whole-blood aliquots were tested and kept at 4°C for up to 24 h for potential retest. Two hundred microliters of each selected sample was extracted by two different technicians either by the reference manual method, i.e., QIAamp column DNA blood extraction kit according to the manufacturer's recommendations (Qiagen), or by the new specific B protocol on the NucliSENS easyMAG instrument. The latter protocol consists of the treatment of 200 l of whole blood in 2 ml of lysis buffer and the capture of nucleic acids by 140 l of magnetic silica. After incubation and washing procedures, nucleic acids were recovered in 50 l of elution buffer. EBV and CMV loads were quantified by using the respective R-gene amplification kit (Argene Biosoft) according to the manufacturer's recommendations. Both amplification kits have been previously validated for quantification of EBV and CM...

Optimization of Toxoplasma gondii DNA extraction from amniotic fluid using NucliSENS easyMAG and comparison with QIAamp DNA minikit

“…samples were randomly allocated to undergo manual or automated extraction, and the analyses were performed on the same days by the same operator who routinely used both methods. Our results are supported by earlier reports which showed that manual extraction methods were comparable [9][10][11] or less sensitive [12][13][14][15] than automated methods for other types of samples and micro-organisms. Besides, automated methods are associated with additional benefits, e.g.…”

Cytomegalovirus Load in Inflamed Intestinal Tissue Is Predictive of Resistance to Immunosuppressive Therapy in Ulcerative Colitis

“…Routine virological studies included the detection of IgM and IgG antibodies to CMV (Siemens, Saint Denis, France) and the measure of CMV DNA load in whole blood using the CMV Rgene kit (Argene, Verniolle, France ) as previously described ( 25 ).…”

Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

“…Although various extraction systems have been used, 6 -8 comparisons between automated and manual systems are limited in number. 9,10 The QIAamp spin column method (Qiagen, Valencia, CA) is a widely used manual extraction system based on the adsorption of DNA to a silica gel membrane. With the processing of large batches of specimens, this system becomes extremely labor-intensive, however, and the high number of manual micropipetting steps increases the risk for repetitive motion injury and the potential for technical error.…”