| Autor: |
Yoo HB; Korea Research Institute of Standards and Science , Daejeon 34113, Republic of Korea.; University of Science and Technology , Daejeon 34141, Republic of Korea., Park SR; Korea Research Institute of Standards and Science , Daejeon 34113, Republic of Korea.; University of Science and Technology , Daejeon 34141, Republic of Korea., Dong L; National Institute of Metrology , Beijing 100013, P.R. China., Wang J; National Institute of Metrology , Beijing 100013, P.R. China., Sui Z; National Institute of Metrology , Beijing 100013, P.R. China., Pavšič J; National Institute of Biology , 1000 Ljubljana, Slovenia., Milavec M; National Institute of Biology , 1000 Ljubljana, Slovenia., Akgoz M; TUBITAK UME National Metrology Institute , Kocaeli 41470, Turkey., Mozioğlu E; TUBITAK UME National Metrology Institute , Kocaeli 41470, Turkey., Corbisier P; Institute for Reference Materials and Measurements, Joint Research Centre, European Commission , Geel 2440, Belgium., Janka M; Institute for Reference Materials and Measurements, Joint Research Centre, European Commission , Geel 2440, Belgium., Cosme B; National Institute of Metrology, Quality and Technology , Xerém 25250-020, Brazil., de V Cavalcante JJ; National Institute of Metrology, Quality and Technology , Xerém 25250-020, Brazil., Flatshart RB; National Institute of Metrology, Quality and Technology , Xerém 25250-020, Brazil., Burke D; National Measurement Institute Australia , Lindfield 2070, Australia., Forbes-Smith M; National Measurement Institute Australia , Lindfield 2070, Australia., McLaughlin J; National Measurement Institute Australia , Lindfield 2070, Australia., Emslie K; National Measurement Institute Australia , Lindfield 2070, Australia., Whale AS; LGC , Teddington TW11, United Kingdom., Huggett JF; LGC , Teddington TW11, United Kingdom., Parkes H; LGC , Teddington TW11, United Kingdom., Kline MC; National Institute of Standards and Technology , Gaithersburg, Maryland 20899-1070, United States., Harenza JL; National Institute of Standards and Technology , Gaithersburg, Maryland 20899-1070, United States., Vallone PM; National Institute of Standards and Technology , Gaithersburg, Maryland 20899-1070, United States. |
| Abstrakt: |
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. |
