Table 1.
Specification of primers used in this study.
Fig 1.
Quantification of pBR322 plasmid copy number by digital droplet PCR.
E. coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A (Table 1A). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs, respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs. Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.
Fig 2.
Quantification of pBR322 plasmid copy number with a single colour, multiplex, digital droplet PCR.
(A) Column represents a single well of ~ 20,000 droplets containing E. coli DH5α total DNA template with multiplexed bla and dxs. (B) Representative droplet digital plot for PCR amplification of bla (blue colour), dxs (green colour) and double positive droplets (orange) in a multiplexed reaction for both targets. Grey colour represents droplets with no DNA. (C) E. coli DH5α total DNA served as a template for PCR amplification of bla (69 bp), dxs (160 bp), and a multiplexed reaction for both targets. PCR products were run in a 1.7% agarose gel. DNA marker- pUC19 DNA/MspI (HpaII) (Thermo Scientific). (D) Estimated pBR322 copy number by multiplex (tube no. 1) and single (tubes no. 2 and 3) droplet digital PCR. The plasmid copy number of pBR322 (PCN) was calculated by dividing the copy number of bla by the copy number of dxs.
Fig 3.
Construction of the standard curves for bla and dxs.
(A) The standard curves were calculated with serial 10-fold dilutions of pGEM-dxs, ranging from 1 × 105 to 1 × 109 copies μl-1. Each standard dilution was amplified by qPCR using bla and dxs primer sets (n = 2). For each gene, the determined CT values were plotted against the logarithm of their known initial copy number. A standard curve was generated by linear regression through these points. (B) Validation of the ΔΔCT calculation. The ΔCT deviation of bla vs. dxs was calculated for each dilution and plotted (n = 2). Average ΔCT = average ± SD (n = 10).
Table 2.
Estimated plasmid copy number by (A) absolute and (B) relative quantification after DNA isolation by QIAamp DNA Mini kit.
Fig 4.
Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns.
Genomic and plasmid DNA were isolated from E. coli DH5α and E. coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A&A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P<0.02; Student t test).
Table 3.
Estimated plasmid copy number by absolute quantification after DNA isolation by the bead-beating method.