Skip to main content
. 2021 Feb 18;4:227. doi: 10.1038/s42003-021-01751-9

Fig. 4. PANDAA adaptation of the probe-binding site rescues qPCR performance.

Fig. 4

Conventional qPCR using PANDAA primers lacking the 3′ ADR was compared to PANDAA for the four most common K103 probe-binding sites (001 to 004).Template 001 (blue circles), which does not contain probe-binding site sequence variation, was included as a reference in each experiment against which the target containing a single-nucleotide variation was compared when using PANDAA (blue squares) versus conventional qPCR (yellow triangles). a PANDAA restored detection of template 002 to the same level as a target with no probe-binding site sequence variation. b Detection of template 003 was increased almost 40-fold when using PANDAA compared to conventional qPCR. c Conventional qPCR did not detect template 004, whereas PANDAA restored detection close to that observed with template 001. d Single-clone sequencing of the PANDAA amplicon demonstrated adaptation of the probe-binding site when using template 004 as the target. Adaptation was seen in 42/44 clones (95.5%). e Sequential adaptation overcomes multiple ADR mismatches. Template 011 contains two mismatches in the 5′ ADR, which must be adapted by the forward primer. By including a low concentration of 2830 F primers that contain only one of the two mismatches, adaptation can be performed in a stepwise manner such that the 2830 F PANDAA primer must only adapt one nucleotide variant rather than two. f Two primers, 2830 F [−3A] and 2830 F [−6G], each of which contains only a single mismatch, were added to separate PANDAA reactions at 10% of the 2830 F forward PANDAA primer concentration, and adaptation performance was evaluated with 103 copies per reaction template 001 or 011 DNA. 2830 F [−3A] resulted in a 0.9-cycle decrease compared to the 011 template with only the standard PANDAA primer. 2830 F [−6G] reduced the Cq by 4.8 cycles, whereas both sequential adaptation primers together led to a 4.1-cycle decrease. g A dose response was evident with 2830 F [−6G] sequential adaptation from 2.5 to 20% of the 2830 F forward PANDAA primer concentration. Results are the representative median of six replicates.