International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction

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• 作者：Hee-Bong Yoo ; Sang-Ryoul ParkLianhua Dong ; Jing Wang ; Zhiwei Sui ; Jernej Pavšič ; Mojca Milavec ; Muslum Akgoz ; Erkan Mozioğlu ; Philippe Corbisier ; Mátrai Janka ; Bruno Cosme ; Janaina J. de V. Cavalcante ; Roberto Becht Flatshart ; Daniel Burke ; Michael Forbes-Smith ; Jacob McLaughlin ; Kerry EmslieAlexandra S. Whale ; Jim F. Huggett ; Helen Parkes ; Margaret C. Kline ; Jo Lynne Harenza ; Peter M. Vallone
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：December 20, 2016
• 年：2016
• 卷：88
• 期：24
• 页码：12169-12176
• 全文大小：368K
• ISSN：1520-6882

文摘

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.