20.500.12556/DiRROS-19687 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. molecular diagnostics direct quantification viruses virus reference materials human cytomegalovirus digital PCR true false true Angleški jezik Ni določen Neznano 2024-07-24 03:44:49 2024-07-25 17:02:35 2024-07-26 04:28:04 0000-00-00 00:00:00 2016 0 0 str. 67-75 iss. 1 Vol. 408 2016 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2016-01-01 577.2 1618-2642 10.1007/s00216-015-9109-0 3637327 RAZ_Pavsic_Jernej_2016.pdf RAZ_Pavsic_Jernej_2016.pdf 1 7867B648B32BAB129FCA25007A4BA9F1 25f405f01dc4daf539bd5aed3778aea0c0ce95de9c4541aaa5a14abc8252b590 8e92f29d-4a86-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27530 https://doi.org/10.1007/s00216-015-9109-0 0 86bf8abc-4a86-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27529 Nacionalni inštitut za biologijo 0 0 0