The sensitivity of PDRA for the detection of two recombinant plasmids was 103 copies (Figure 2) and the reproducibility was shown in Table 1. No cross reaction between two recombinant plasmids (G and D) was observed within at least 3-minute interval by corresponding probes in the detection range (Figure 3). A total of 63 clinical samples were typed by PDRA and direct sequencing. The sequencing results of 63 clinical samples were compared with the results of PDRA (Figure 4), and the coincidence rate was 100% (Table 2). Among the 63 clinical samples, there were 22 S-D614 mutants, 31 S-G614 mutants, and 10 negative samples.Figure 2.
Sensitivity result of PDRA with recombinant plasmids. (A) Probe D tested S-D614 mutant recombinant plasmids; (B) Probe G tested S-D614 mutant recombinant plasmid; (C) Probe D tested S-G614 mutant recombinant plasmids; (D) Probe G tested S-G614 mutant recombinant plasmids.Note: When the S-D614 strain recombinant plasmid was detected, the sensitivity of Probe D and Probe G was 102 copies/reaction and 103 copies/reaction, respectively, and the time to positivity by probe D at each concentration was less (at least 3 min interval) than that by probe G. When the S-G614 mutant recombinant plasmid was detected, the sensitivity of both Probe D and Probe G was 102 copies/reaction. The time to positivity by probe G at each concentration was less (at least 3 min interval) than that by probe D.
Serial diluted DNA No. of replicates tested No. detected Detection rate (%) 107 20 20 100 106 20 20 100 105 20 20 100 104 20 20 100 103 20 20 100 102 20 2 10
Table 1. The reproducibility of probe-directed recombinase amplification (PDRA) assay.Figure 3.
Specificity result of PDRA with recombinant plasmids. (A) S-D614 strain recombinant plasmid; (B) S-G614 mutant recombinant plasmid.Note: When the 107 copies of S-D614 strain recombinant plasmid were detected, the time to positivity by Probe D was less than that of Probe G by 6 min. When the 103 copies of recombinant plasmid were detected, the time to positivity by Probe D was less than that of Probe G by 4 min. When the 107 copies and 103 copies of S-G614 mutant recombinant plasmids were detected, the time to positivity by Probe G was less than that of Probe D by 3 min.Figure 4.
Comparison of the PDRA assay and direct sequencing results. (A) The PDRA results of sample 18 and sample 34; (B) The direct sequencing results of sample 18 and sample 34.Note: Sample 18 was S-D614 mutant, and sample 34 was S-G614 mutant. Sample 18 was S-D614 mutant, and sample 34 was S-G614 mutant.
Type Sequencing PDRA Coincidence rate (%) S-D614 22 22 100 S-G614 31 31 100 Negative 10 10 100 Abbreviation: PDRA=probe-directed recombinase amplification.
Table 2. Clinical sample typing results.