Automated extraction of viral-pathogen RNA and DNA for high-throughput quantitative real-time PCR

J Clin Microbiol. 2005 Nov;43(11):5541-6. doi: 10.1128/JCM.43.11.5541-5546.2005.

Abstract

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.

MeSH terms

  • Automation
  • Cytomegalovirus / genetics
  • Cytomegalovirus / isolation & purification
  • DNA Virus Infections / diagnosis
  • DNA Virus Infections / virology
  • DNA, Viral / blood
  • DNA, Viral / isolation & purification*
  • Edetic Acid
  • Hepacivirus / genetics
  • Hepacivirus / isolation & purification
  • Hepatitis B virus / genetics
  • Hepatitis B virus / isolation & purification
  • Herpesvirus 4, Human / genetics
  • Herpesvirus 4, Human / isolation & purification
  • Humans
  • Indicators and Reagents
  • Polymerase Chain Reaction / methods*
  • RNA Virus Infections / diagnosis
  • RNA Virus Infections / virology
  • RNA, Viral / blood
  • RNA, Viral / isolation & purification*
  • Sensitivity and Specificity

Substances

  • DNA, Viral
  • Indicators and Reagents
  • RNA, Viral
  • Edetic Acid