title: International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.
abstract: Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 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 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.
authors.affiliation: Korea Research Institute of Standards and Science , Daejeon 34113, Republic of Korea.|University of Science and Technology , Daejeon 34141, Republic of Korea.
authors.forename: Hee-Bong
authors.initials: HB
authors.lastname: Yoo
authors.affiliation: Korea Research Institute of Standards and Science , Daejeon 34113, Republic of Korea.|University of Science and Technology , Daejeon 34141, Republic of Korea.
authors.forename: Sang-Ryoul
authors.identifier: 0000-0003-4143-7906
authors.initials: SR
authors.lastname: Park
authors.affiliation: National Institute of Metrology , Beijing 100013, P.R. China.
authors.forename: Lianhua
authors.initials: L
authors.lastname: Dong
authors.affiliation: National Institute of Metrology , Beijing 100013, P.R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: National Institute of Metrology , Beijing 100013, P.R. China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Sui
authors.affiliation: National Institute of Biology , 1000 Ljubljana, Slovenia.
authors.forename: Jernej
authors.initials: J
authors.lastname: Pavšič
authors.affiliation: National Institute of Biology , 1000 Ljubljana, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Milavec
authors.affiliation: TUBITAK UME National Metrology Institute , Kocaeli 41470, Turkey.
authors.forename: Muslum
authors.initials: M
authors.lastname: Akgoz
authors.affiliation: TUBITAK UME National Metrology Institute , Kocaeli 41470, Turkey.
authors.forename: Erkan
authors.initials: E
authors.lastname: Mozioğlu
authors.affiliation: Institute for Reference Materials and Measurements, Joint Research Centre, European Commission , Geel 2440, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Corbisier
authors.affiliation: Institute for Reference Materials and Measurements, Joint Research Centre, European Commission , Geel 2440, Belgium.
authors.forename: Mátrai
authors.initials: M
authors.lastname: Janka
authors.affiliation: National Institute of Metrology, Quality and Technology , Xerém 25250-020, Brazil.
authors.forename: Bruno
authors.initials: B
authors.lastname: Cosme
authors.affiliation: National Institute of Metrology, Quality and Technology , Xerém 25250-020, Brazil.
authors.forename: Janaina J
authors.initials: JJ
authors.lastname: de V Cavalcante
authors.affiliation: National Institute of Metrology, Quality and Technology , Xerém 25250-020, Brazil.
authors.forename: Roberto Becht
authors.initials: RB
authors.lastname: Flatshart
authors.affiliation: National Measurement Institute Australia , Lindfield 2070, Australia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Burke
authors.affiliation: National Measurement Institute Australia , Lindfield 2070, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Forbes-Smith
authors.affiliation: National Measurement Institute Australia , Lindfield 2070, Australia.
authors.forename: Jacob
authors.identifier: 0000-0001-9995-1508
authors.initials: J
authors.lastname: McLaughlin
authors.affiliation: National Measurement Institute Australia , Lindfield 2070, Australia.
authors.forename: Kerry
authors.initials: K
authors.lastname: Emslie
authors.affiliation: LGC , Teddington TW11, United Kingdom.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Whale
authors.affiliation: LGC , Teddington TW11, United Kingdom.
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
authors.affiliation: LGC , Teddington TW11, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Parkes
authors.affiliation: National Institute of Standards and Technology , Gaithersburg, Maryland 20899-1070, United States.
authors.forename: Margaret C
authors.initials: MC
authors.lastname: Kline
authors.affiliation: National Institute of Standards and Technology , Gaithersburg, Maryland 20899-1070, United States.
authors.forename: Jo Lynne
authors.initials: JL
authors.lastname: Harenza
authors.affiliation: National Institute of Standards and Technology , Gaithersburg, Maryland 20899-1070, United States.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Vallone
mesh_terms: D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary; D005434:Flow Cytometry / Q000379:methods*; D013058:Mass Spectrometry; D009711:Nucleotides / Q000032:analysis; D010957:Plasmids / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D009711:Nucleotides; D004247:DNA