Digital PCR for direct quantification of viruses without DNA extraction.
DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.
Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia. jernej.pavsic@nib.si.|Jožef Stefan International Postgraduate School, Jamova cesta 39, 1000, Ljubljana, Slovenia. jernej.pavsic@nib.si.
Jernej
J
Pavšič
Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
Jana
J
Žel
Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
Mojca
M
Milavec
D003587:Cytomegalovirus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral
Digital PCR; Direct quantification; Human cytomegalovirus; Molecular diagnostics; Virus reference materials; Viruses