Precision of ddPCR assays
As shown in Fig. 3, the %RSD values of the uniplex, duplex and multiplex ddPCR assays were 2–36%, 0–35% and 1–41%, respectively, for detection of the pfmdr1 GCN, and 3–28%, 2–39% and 2–21%, respectively, for detection of the pfplasmepsin2 GCN. Meanwhile, the %RSD values of the uniplex and duplex ddPCR assays were 2–36% and 1–17% for detection of the pfgch1 GCN.
Limitation of GCN quantification
As shown in Table 1, the accepted range of λ values of the uniplex, duplex and multiplex ddPCR assays were 0.011–0.987, 0.005–2.178 and 0.003–0.842, respectively, for the pfmdr1 GCN, and 0.010–1.870, 0.002–1.890 and 0.003–0.941, respectively, for the pfplasmepsin2 GCN. The accepted range of λ values of the uniplex and duplex ddPCR assays were 0.006–1.915 and 0.003–1.877, respectively, for the pfgch1 GCN. Based on the limitation of GCN quantification, the average accuracy and %RSD value of the multiplex ddPCR assay were 95% and 5%, respectively, for measurement of the pfmdr1 GCN, and 91% and 5%, respectively, for measurement of the pfplasmepsin2 GCN. The accuracy and %RSD value of the duplex ddPCR were 95% and 4, respectively.
Table 1
Descriptive statistics of P. falciparum mdr1, plasmepsin2, and gch1 CNVs based on the accepted lamda(λ) value of ddPCR assay of pf-β-tubulin gene.
Statistic
|
pfmdr1 CNVs
|
pfplasmepsin2 CNVs
|
pfgch1 CNVs
|
Singleplex
|
Duplex
|
Multiplex
|
Singleplex
|
Duplex
|
Multiplex
|
Singleplex
|
Duplex
|
Range
|
0.253
|
0.177
|
0.225
|
0.315
|
0.276
|
0.214
|
0.324
|
0.236
|
Minimum
|
0.812
|
0.817
|
0.861
|
0.814
|
0.738
|
0.813
|
0.868
|
0.926
|
Maximum
|
1.065
|
0.994
|
1.086
|
1.129
|
1.014
|
1.027
|
1.192
|
1.162
|
Mean
|
0.922
|
0.919
|
0.964
|
0.943
|
0.895
|
0.906
|
1.016
|
1.039
|
SD
|
0.061
|
0.040
|
0.061
|
0.085
|
0.057
|
0.057
|
0.078
|
0.065
|
Variance
|
0.004
|
0.002
|
0.004
|
0.007
|
0.003
|
0.003
|
0.006
|
0.004
|
%RSD
|
6.954
|
3.273
|
4.755
|
8.675
|
5.947
|
5.385
|
5.282
|
3.705
|
%Accuracy
|
92.240
|
92.135
|
95.047
|
93.304
|
89.533
|
90.638
|
95.118
|
94.876
|
Accepted lamda(λ) range
|
0.011–0.976
|
0.005–2.173
|
0.003–0.839
|
0.010–1.860
|
0.002–1.890
|
0.003–0.938
|
0.006–1.909
|
0.003–1.874
|
Comparison between the uniplex, duplex and multiplex ddPCR assays
Two-fold serial dilutions of DNA from P. falciparum strain 3D7 (4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625 and 0.001953125 ng/µL) were used to compare the CNVs of the pfmdr1 and pfplasmepsin2 genes obtained from the uniplex, duplex and multiplex ddPCR assays. In addition, two-fold serial dilutions of DNA from P. falciparum strain D6 (8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125 and 0.00390625 ng/µL) were used for comparison of the CNVs of the pfgch1 gene obtained with the uniplex and duplex ddPCR assays. As shown in Fig. 4, there were no significant differences in the pfmdr1 (p = 0.363) and pfplasmepsin2 (p = 0.330) GCNs, as determined with the uniplex, duplex and multiplex ddPCR assays. In addition, there was no significant difference for detection of the pfgch1 GCN between the uniplex and duplex ddPCR assays (p = 0.276).
Standardised analytical workflow of ddPCR analysis for quantification of GCN
A flowchart, including validation criteria, for a standardised analytical workflow of ddPCR analysis was designed based on the observed limit of quantification of the optimal accuracy and precision (Fig. 5). Control samples, as well as positive and negative controls, were included for each ddPCR assay. The accepted criteria used for the ddPCR assay are that the results of the negative control are negative, those of the positive single copy control are positive (ratio = 0.80–1.20) and those of the positive multiple copy control are positive (ratio > 1.20). Each ddPCR reaction contained at least 12,000 droplets. The GCN results were considered acceptable at a λ value of 0.003–0.800 for quantification of the pfmdr1 and pfplasmepsin2 genes with the multiplex ddPCR assay, and 0.003–1.900 for quantification of the pfgch1 gene with the duplex ddPCR assay. GCNs were calculated as the ratio of the concentrations (copies/µL) of the target and references genes (single GCN ratio of 0.8–1.2 and multiple GCN ratio of > 1.20).
Agreement of GCN results between the ddPCR and qPCR assays
DNA samples from the P. falciparum reference strain (n = 7) and P. falciparum strains 3D7, 7G8, D10, DD2, HB3, W2 and D6 were collected to compare the pfmdr1, pfplasmepsin2 and pfgch1 GCNs determined with the ddPCR and qPCR assays. The results showed 100% agreement between the ddPCR and qPCR assays (κ = 1) (Additional file 2).
CNVs of the pfmdr1, pfplasmespsin2 and pfgch1 genes of P. falciparum isolates from Thailand
The extracted DNA samples were determined the CNVs in the pfmdr1, pfplasmespsin2 and pfgch1 genes following the a standardised analytical workflow obtained from this study. The results showed that the pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Northeastern Thailand, whereas the pfgch1 GCN was amplified in 50% of samples from Yala, Southern Thailand (Fig. 6, Additional file 3). Comparisons of the results of the ddPCR and qPCR assays were 100% in agreement for CNV assessments of the pfmdr1, pfplasmepsin2 and pfgch1 genes (Fig. 7)
Cost and turn-around time of ddPCR assays
The costs of the uniplex, duplex and multiplex ddPCR assays to determine the CNVs of the pfmdr1, pfplasmepsin2 and pfgch1 genes were 10.40, 5.50 and 5.70 USD per sample, respectively. The turn-around times for the uniplex, duplex and multiplex ddPCR assays of 96 samples were12, 6 and 6 h, respectively.