Modification of DENV1/3 BIP prevents self-extension in QUASR. (A)
Original DENV1/3 BIP-Cy5 was incubated with or without BIPc-10 quencher (no
other primers) in RT-LAMP assay with enzyme (solid trace) or without enzyme
(dashed trace), indicating that the primer self-extends in the presence of
enzyme, inhibiting quenching upon cooling down. For comparison, a reaction with
no quencher is shown (short dashes) illustrating the full (unquenched)
fluorescence of the primer. (B) DENV3 QUASR with modified DENV1/3 BIP-Cy5
primer, positive control (104 copies DENV3 RNA), or no-template
control, displaying the expected behavior (quenching of no-template control).
(C) Endpoint fluorescence images of original and modified
(“bumped”) DENV3 QUASR reactions in PCR tubes. Positive controls
have 5000 copies DENV3 RNA as a template.