International comparison of enumeration-based quantification of DNA copy- concentration using flow cytometric counting and digital polymerase chain reaction

Enumeration-based determination of DNA copy-concentration has been assessed through an international comparison among national metrology institutes (NMIs) and designated institute (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to ‘absolute quantification’, which offers the potential for reliable value assignment of DNA reference materials. In this study, two enumeration methods, flow cytometric counting (FCM counting) and digital polymerase chain reaction (d-PCR), were compared to quantify a solution of plasmid pBR322 at a concentration of several thousand copies per microliter. In addition, two orthogonal chemical analysis methods based on nucleotide quantification, isotope dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although nine d-PCR results from eight laboratories showed some dispersion (RSD=11.8%), their mean was closely aligned with the results of the FCM counting method as well as the results of orthogonal chemical analysis corrected with gravimetric dilution factors. If the mean of the d-PCR results is taken, RSD of all four methods is 1.8%, which strongly supports the validity of newer enumeration approaches. Despite the good agreement as a whole, scattering of individual d-PCR results was not sufficiently covered by the reported measurement uncertainties. This suggests that some laboratories may not have considered all factors contributing to the measurement uncertainty of d-PCR, and further investigation of this possibility is warranted.

YOO Hee-Bong;  PARK Sang-Ryoul;  DONG L.;  WANG J.;  SUI Zhiwei;  PAVŠIČ Jernej;  MILAVEC Mojca;  AKGOZ M;  MOZIOĞLU Erkan;  CORBISIER Philippe;  MATRAI Janka;  COSME Bruno;  CAVALCANTE Janaina J. De V.;  BECHT FLATSHART Roberto;  BURKE Daniel;  FORBES-SMITH Michael;  MCLAUGHLIN Jacob;  EMSLIE K.;  WHALE Alexandra S.;  HUGGETT Jim;  PARKES H.;  KLINE Margaret C;  HARENZA Jo Lynne;  VALLONE Peter M.; 

2017-01-19

AMER CHEMICAL SOC

JRC101717

0002-7863,   

https://publications.jrc.ec.europa.eu/repository/handle/JRC101717,   

10.1021/acs.analchem.6b03076,