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.
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