Reduced PCR sensitivity due to impaired DNA recovery with the MagNA Pure LC total nucleic acid isolation kit.
The increasing demand for molecular diagnostics in clinical microbiology laboratories necessitates automated sample processing. In the present study, we evaluated the performance of the MagNA Pure LC total nucleic acid isolation kit (M extraction) in comparison with the manual method (Si extraction) according to Boom et al. (R. Boom, C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990) for the detection of viral DNA by competitive quantitative PCR. Reconstruction experiments with HindIII-digested phage lambda DNA and HaeIII-digested phiX174 DNA showed that the recovery of DNA from phosphate-buffered saline, cerebrospinal fluid, EDTA-anticoagulated plasma, and EDTA-anticoagulated whole blood by M extraction is, on average, 6.6-fold lower compared to Si extraction. PCR signals of spiked PCR control DNAs for Epstein-Barr virus and varicella-zoster virus were also between 1.9- and 14.2-fold lower after M extraction compared to Si extraction, also suggesting impaired DNA recovery. M extraction of spiked cytomegalovirus strain AD 169 in whole blood showed a 5- to 10-fold reduction in PCR sensitivity compared to Si extraction. This reduction of PCR sensitivity was also observed when clinical whole blood samples were processed by M extraction. Before implementing M extraction, the clinical consequences of the reduced recovery should first be considered, especially when maximal sensitivity is required.
Schuurman
Tim
T
Department of Research and Development, Laboratory for Infectious Diseases, Groningen, The Netherlands. t.schuurman@infectielab.nl
van Breda
Alex
A
de Boer
Richard
R
Kooistra-Smid
Mirjam
M
Beld
Marcel
M
Savelkoul
Paul
P
Boom
René
R
D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000821:virology; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification*; D020031:Epstein-Barr Virus Infections / Q000821:virology; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic