Fig 1.
Graphical abstract.
Table 1.
The DNA sequence of oligonucleotides used in this study.
Table 2.
Sample and extraction kit used in this study.
Fig 2.
Absolute quantification of the serially diluted recombinant plasmid.
A representative 1D amplitude plot of ddPCR reactions (A) and amplification curve of qPCR (B). ddPCR threshold is shown by a pink line that divides the results as positive (above the threshold) and negative droplets (under the threshold). The vertical yellow line divides the ddPCR reactions with different diluted targets. A blue-dark line with the position shows qPCR threshold is in the middle of the logarithmic phase of the amplification curve.
Table 3.
Comparison of droplet digital PCR and qPCR systems using serially diluted pUC57 recombinant plasmid.
Fig 3.
Standard curves and quantification correlation were constructed by (A) ddPCR and (B) qPCR systems for linearity assessment.
A quantification correlation was obtained by measuring copy number per reaction against the assigned copy number per reaction (A). A quantification correlation was obtained by Cq value against log starting concentration for QPCR (B). The correlation coefficient (R2) assay for recombinant plasmid pUC57 in ddPCR and qPCR were 0,9998 and 0,9971, respectively.
Fig 4.
The linearity and quantification correlation of ddPCR between measured copy number per reaction and assigned copy number per reaction.
Fig 5.
Quantification of serially diluted recombinant plasmid using qPCR and ddPCR systems.
A representative LOD and LOQ amplification curve of qPCR (A) and 1D amplitude plot of ddPCR reactions (B).
Table 4.
Comparing the limit of detection and quantification of pUC57 recombinant plasmid using qPCR and ddPCR systems.
Quantification using ddPCR was shown in copy number due to its capability to perform absolute quantification.
Fig 6.
Results of inter-assay variability performed by qPCR systems (shown in CV%).
The assessment was carried out by twelve different laboratories using the pUC57 plasmid with a concentration of 104 copies/reaction using CFX Real-Time PCR Systems (Bio-Rad).
Fig 7.
Results of robustness test performed by qPCR systems (shown in CV%).
The assessment was carried out by various qPCR models using the pUC57 plasmid with a concentration of 104 copies/reaction.
Table 5.
Assay performance on various sample analyses using qPCR.