Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR.
BACKGROUND
The accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest.


RESULTS
In this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed.


CONCLUSIONS
Based on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 10(10) copies/μL, (1.53 ± 0.10) × 10(10) copies/μL and (1.70 ± 0.11) × 10(10) copies/μL respectively.Graphical abstractWe developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively, and the quantification of three different methods (UV, dPCR, ICP) was studied.
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Wen
W
Liang
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Li
L
Xu
Division of Medical and Biological Measurement, National Institute of Metrology, No.18, Bei San Huan Dong Lu, Chaoyang District, Beijing, 100013 People's Republic of China.
Zhiwei
Z
Sui
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Yan
Y
Li
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Lanying
L
Li
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Yanli
Y
Wen
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Chunhua
C
Li
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Shuzhen
S
Ren
Laboratory of Biometrology, Shanghai Institute of Measurement and Testing Technology, 1500 Zhang Heng Road, Shanghai, 201203 People's Republic of China.
Gang
G
Liu
Digital PCR; Escherichia coli O157:H7; Plasmid DNA; Reference material