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Publication Number
US-12110542-B2
Publication Date
2024-10-08
Expiration Date
Abstract
Disclosed herein are “dual-priming” isothermal amplification method (including “self-priming” and “pairing-priming” strand extension, termed “DAMP”) for rapid nucleic acid detection.
Core Innovation
The invention relates to a method of amplifying a target nucleic acid by contacting a nucleic acid sample suspected of containing the target nucleic acid with six primers and a DNA polymerase with strand displacement activity. The target nucleic acid includes defined F3, F2, F1, R1, R2, and R3 sites, and the complement includes corresponding F3c, F2c, F1c, R1c, R2c, and R3c sites. The six primers include forward outer primer, reverse outer primer, forward inner primer, reverse inner primer, forward reverse pairing-competition primer, and reverse pairing-competition primer with specified single-site and double-site primer roles.
The method produces a first basic structure by synthesis events that include annealing and strand displacement and release. A first synthesis event produces DNA from the forward outer primer annealing to the F3c site and DNA from the F2 region of the forward inner primer annealing to the F2c site, with the strand elongated from the forward inner primer displaced and released by extension from the forward outer primer. A complementary synthesis event produces DNA from the reverse outer primer annealing to the R3 site and from the R2c region of the reverse inner primer annealing the R2 site of the released nucleic acid strand, with the strand elongated from the reverse inner primer displaced and released by extension from the reverse outer primer.
The method cyclically amplifies the first basic structure by contacting the first basic structure with the DNA polymerase, the forward inner primer, the reverse pairing-competition primer, the forward reverse pairing-competition primer, and the reverse inner primer to provide amplified target nucleic acid. The cyclic amplification operates by additional synthesizing events that include annealing of the pairing-competition primers to the FI site and extension and strand displacement and released basic structures.
Claims Coverage
Independent claim clm-00001 describes a target-site-defined six-primer DAMP method with a strand displacement DNA polymerase. The independent features include specific primer-site complementarity, primer type, sequential production of a first basic structure, and cyclic amplification using selected primers.
Six-primer strand-displacement amplification with defined F/R site architecture
A method amplifying a target nucleic acid by contacting a nucleic acid sample suspected of containing the target nucleic acid with six primers and a DNA polymerase with strand displacement activity, wherein the target nucleic acid comprises from 5′ to 3′ an F3, F2, F1, R1, R2, and R3 site and the complement comprises from 3′ to 5′ corresponding F3c, F2c, F1c, R1c, R2c, and R3c sites, and the six primers comprise FO, RO, FI, RI, FC, and RC with specified complementarity and primer types.
First basic structure production via FO/FI and RO/RI strand displacement and release
Amplifying comprises producing a first basic structure comprising from 5′ to 3′ the R1, R2c, R1c, F1c, F2c, and F1 sites, wherein producing comprises synthesizing DNA from FO annealing to F3c and from the F2 region of FI annealing to F2c with displacement and release by extension from FO, and synthesizing DNA from RO annealing to R3 and from the R2c region of RI annealing the R2 site of the released nucleic acid strand with displacement and release by extension from RO.
Cyclic amplification of the first basic structure using inner primers and pairing-competition primers
Cyclically amplifying the first basic structure by contacting the first basic structure with the DNA polymerase, the forward inner primer, the reverse pairing-competition primer, the forward reverse pairing-competition primer, and the reverse inner primer to provide amplified target nucleic acid.
Across the independent claim, the inventive coverage centers on a six-primer set with defined primer-to-site complementarity, formation of a first basic structure through strand-displacement-mediated synthesis events using FO/FI and RO/RI, and cyclic amplification of the produced basic structure using inner primers together with forward and reverse pairing-competition primers.
Stated Advantages
Ultralow nonspecific/background signals.
Faster amplification than conventional six-primer LAMP/PCR.
Versatile detection of HIV-1 DNA/RNA and E. coli targets.
Documented Applications
Detection of HIV-1 DNA/RNA using RT-DAMP.
Detection of HIV-1 p24 gene DNA.
Detection of E. coli targets, including the E. coli malB gene.
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