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

Abstract

ABSTRACT The performance of the m 1000 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 × 10 5 HCV IU/ml and from 100 to 1.0 × 10 6 HBV IU/ml. Intra- and interrun variability was below 0.23 log 10 IU/ml for 2.98 to 5.28 log 10 HCV IU/ml and 2.70 to 5.20 log 10 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 log 10 HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log 10 HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log 10 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 log 10 copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m 1000 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.