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Journal of Clinical Microbiology, November 2005, p. 5541-5546, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5541-5546.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

Kurt Beuselinck,* Marc van Ranst, and J. van Eldere

Laboratory of Molecular Diagnostics, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium

Received 28 April 2005/ Returned for modification 5 July 2005/ Accepted 7 September 2005

The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ± 0.06 for HCV and 0.97 ± 0.03 for HBV, indicating a linear extraction from 100 to 1.0 x 105 HCV IU/ml and from 100 to 1.0 x 106 HBV IU/ml. Intra- and interrun variability was below 0.23 log10 IU/ml for 2.98 to 5.28 log10 HCV IU/ml and 2.70 to 5.20 log10 HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log10 HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log10 HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log10 copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log10 copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.

* Corresponding author. Mailing address: Laboratory of Molecular Diagnostics, University Hospital Gasthuisberg, Herestraat 49, BE-3000 Leuven, Belgium. Phone: 32-16-347950. Fax: 32-16-347949. E-mail: kurt.beuselinck{at}uz.kuleuven.ac.be.

Journal of Clinical Microbiology, November 2005, p. 5541-5546, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5541-5546.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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