Digital PCR for direct quantification of viruses without DNA extraction

Significance Statement

DNA extraction before amplification is considered an essential step for the measurement of any DNA targets, as it releases the DNA from cells, viruses and bacteria and eliminates any PCR-inhibitory substances that are initially present in the matrix. However, different extraction procedures have different DNA yields and efficiency of inhibitory substances removal, consequently reducing quantification accuracy and hampering inter-laboratory comparability of data. In the past some viruses and bacteria have already been directly quantified using qPCR without DNA extraction, however drawbacks related to the qPCR formats such as susceptibility to inhibitory substances and dependence on the calibration material probably hampered the precise and reliable measurements of the actual concentrations therefore direct quantification using dPCR has a potential to overcome these.

To investigate the potential of digital PCR for direct quantification of viruses, human cytomegalovirus (HCMV) has been quantified without prior extraction using two digital PCR platforms and two standard materials. Up to 50% higher concentrations of DNA targets were measured using direct quantification compared to concentrations measured after the extraction step. Results of direct quantification were equally or even more repeatable, than that of the extracted DNA.

Direct quantification can be used to determine absolute DNA extraction efficiencies and for the evaluation of the influence of matrix on the extraction yield and therefore supports laboratories in decisions on fit for purpose extraction methods as well as in comparability of results especially in diagnostics.

Direct quantification is also a valuable approach for the characterization of reference materials and materials for external quality assurance schemes that underpin quality of results in diagnostics of human pathogens. This approach is of particular importance in the field of plant pathogen diagnostics, where no reference materials are commercially available as it enables preparation of well characterized in-house reference materials.

About the author

Jernej Pavšič is a PhD student at the National Institute of Biology in Ljubljana, Slovenia.

His research is focused on evaluation of novel nucleic-acid based techniques to standardize and improve diagnostics of human pathogens, enhance characterisation of reference materials and facilitate cancer diagnostics. He is especially interested is exploring capabilities of digital PCR and high-throughput qPCR.

About the author

Jana Žel is head of the genetically modified organisms group at the National Institute of Biology, Slovenia. She has been working for several years on cutting-edge approaches and methods for GMO testing and also the establishment of an internationally harmonized and metrologically sound system for molecular analyses.

About the author

Mojca Milavec is deputy head of GMO detection at the National Institute of Biology in Ljubljana, Slovenia, where she is responsible for introduction of new methods, in house validations and staff training for the National Reference Laboratory for GMO detection. Her research is focused on challenges associated with established and novel technologies for nucleic acid measurements as well as standardization and quality control of these technologies.

Journal Reference

Anal Bioanal Chem. 2016;408(1):67-75.

Pavšič J^1,2, Žel J³, Milavec M³.

[expand title=”Show Affiliations”]

Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia. [email protected].
Jožef Stefan International Postgraduate School, Jamova cesta 39, 1000, Ljubljana, Slovenia. [email protected].
Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia. [/expand]

Abstract

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 digital PCR and two different human cytomegalo virus whole-virus materials. Two digital PCR 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 digital PCR 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.

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