A new highly automated extraction system for quantitative real-time PCRs from whole blood samples: routine monitoring of opportunistic infections in immunosuppressed patients

J Clin Virol. 2012 Apr;53(4):314-9. doi: 10.1016/j.jcv.2012.01.002. Epub 2012 Jan 30.

Abstract

Background: Rapid, high throughput extraction systems are needed to monitor viral infections in immunosuppressed patients.

Objectives: Evaluate the performance of the MagNA Pure 96™ extraction system, and compare it to the COBAS Ampliprep™ for quantitative real-time PCR from whole blood samples.

Study design: Compare the MagNA Pure LC™, COBAS Ampliprep™ and MagNA Pure 96™ using ten-fold dilutions of blood samples containing cytomegalovirus. Evaluate analytical performances of the MagNA Pure 96™ from test samples containing cytomegalovirus. Evaluate clinical performances from 209 blood samples collected prospectively, extracted with the COBAS Ampliprep™ and the MagNA Pure 96™ systems and tested for cytomegalovirus, Epstein-Barr, BK and JC viruses.

Results: All three extraction systems gave similar results with dilutions of a cytomegalovirus-positive sample. Analytical tests showed that the limit of detection was 500 copies/ml, specificity was 100%, with no cross-contamination. Quantification was linear from 3.0 to 6.0 log(10)copies/ml. Intra-assay variation was 8.3-0.9% and inter-assay variation 8.8-5.2%. Clinical specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments agreed well for cytomegalovirus (r=0.54; p=0.07), Epstein-Barr virus (0.69; p=0.0005) and BK virus (0.85; p=0.01). All 55 samples were negative for JC virus. Mean loads were similar for cytomegalovirus (0.17 log(10)copies/ml) and BK virus (-0.24 log(10)copies/ml) while that of Epstein-Barr virus was slightly lower (1.02 log(10)copies/ml).

Conclusions: The MagNA Pure 96™ instrument is an easy-to-use, reliable high throughput platform for extracting nucleic acid from clinical whole blood specimens.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Automation / methods*
  • Cytomegalovirus / genetics
  • Cytomegalovirus / isolation & purification
  • Cytomegalovirus Infections / blood
  • Cytomegalovirus Infections / virology
  • DNA Virus Infections / blood
  • DNA Virus Infections / virology
  • DNA Viruses / genetics
  • DNA Viruses / isolation & purification*
  • DNA, Viral / blood
  • DNA, Viral / isolation & purification*
  • Humans
  • Immunocompromised Host*
  • Opportunistic Infections / blood*
  • Opportunistic Infections / virology
  • Reagent Kits, Diagnostic
  • Real-Time Polymerase Chain Reaction / instrumentation*
  • Real-Time Polymerase Chain Reaction / methods
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Viral Load / methods

Substances

  • DNA, Viral
  • Reagent Kits, Diagnostic