Automated extraction of viral-pathogen RNA and DNA for high-throughput quantitative real-time 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 x 10(5) HCV IU/ml and from 100 to 1.0 x 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 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.
Beuselinck
Kurt
K
Laboratory of Molecular Diagnostics, University Hospital Gasthuisberg, Herestraat 49, BE-3000 Leuven, Belgium. kurt.beuselinck@uz.kuleuven.ac.be
van Ranst
Marc
M
van Eldere
J
J
D001331:Automation; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification; D004266:DNA Virus Infections / Q000175:diagnosis / Q000821:virology; D004279:DNA, Viral / Q000097:blood / Q000302:isolation & purification*; D004492:Edetic Acid; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods*; D012327:RNA Virus Infections / Q000175:diagnosis / Q000821:virology; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D007202:Indicators and Reagents; D012367:RNA, Viral; D004492:Edetic Acid