Eldere J. Automated extraction of viral-pathogen RNA and DNA for high-throughput quantitative realtime PCR

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 1.0 105 HCV IU/ml and from 100 to 1.0 106 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 th