US 11,884,969 B2
Isothermal amplification components and processes
Andrew P. Miller, San Diego, CA (US); and Honghua Zhang, San Diego, CA (US)
Assigned to NAT DIAGNOSTICS, INC., San Diego, CA (US)
Filed by NAT DIAGNOSTICS, INC., San Diego, CA (US)
Filed on Aug. 13, 2021, as Appl. No. 17/402,358.
Application 17/402,358 is a continuation of application No. 15/930,958, filed on May 13, 2020, granted, now 11,118,219.
Application 15/930,958 is a continuation of application No. 16/089,063, granted, now 11,299,777, previously published as PCT/US2017/020921, filed on Mar. 6, 2017.
Application 16/089,063 is a continuation in part of application No. 15/090,405, filed on Apr. 4, 2016, granted, now 9,617,587, issued on Apr. 11, 2017.
Prior Publication US 2022/0064703 A1, Mar. 3, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6844 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/686 (2018.01); G01N 21/64 (2006.01); C12Q 1/6818 (2018.01); C12Q 1/689 (2018.01)
CPC C12Q 1/6844 (2013.01) [C12Q 1/686 (2013.01); C12Q 1/689 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6818 (2013.01); G01N 21/6428 (2013.01); C12Q 2521/101 (2013.01); C12Q 2521/107 (2013.01); C12Q 2527/101 (2013.01); C12Q 2527/113 (2013.01); C12Q 2531/113 (2013.01); C12Q 2561/113 (2013.01); C12Q 2600/16 (2013.01); G01N 2021/6432 (2013.01)] 30 Claims
 
1. A method for detecting a target nucleic acid sequence in a sample, the method comprising:
(a) amplifying a target nucleic acid sequence in a sample under an isothermal amplification condition, wherein the target nucleic acid sequence comprises a first strand and a second strand complementary to each other, and wherein the amplifying comprises contacting a double-stranded nucleic acid comprising the target nucleic acid sequence with:
(i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the second primer is capable to hybridizing to a sequence of the second strand of the target nucleic acid sequence; and
(ii) an enzyme having a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, wherein the nucleic acid amplification product comprises:
(1) the sequence of the first primer or a portion thereof, or the reverse complement thereof,
(2) the sequence of the second primer or a portion thereof, or the reverse complement thereof, and
(3) a spacer sequence flanked by (1) and (2), wherein the spacer sequence is 1 to 10 bases long; and
(b) detecting the nucleic acid amplification product, wherein the detecting is performed in 20 minutes or less from the time the double-stranded nucleic acid is contacted with (a)(i) the first and second primers and (a)(ii) the enzyme having a hyperthermophile polymerase activity,
wherein the method does not comprise using any enzymes that are not a polymerase.