US 12,173,354 B2
Method of detecting nucleic acid and detection reagent
Masayuki Ogino, Taito-ku (JP); and Yoichi Makino, Taito-ku (JP)
Assigned to TOPPAN PRINTING CO., LTD., Taito-ku (JP)
Filed by TOPPAN PRINTING CO., LTD., Taito-ku (JP)
Filed on Jan. 31, 2020, as Appl. No. 16/778,618.
Application 16/778,618 is a continuation of application No. PCT/JP2018/028598, filed on Jul. 31, 2018.
Claims priority of application No. 2017-148402 (JP), filed on Jul. 31, 2017.
Prior Publication US 2020/0224253 A1, Jul. 16, 2020
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6818 (2018.01); C12Q 1/6823 (2018.01); C12Q 1/6837 (2018.01); C12Q 1/6876 (2018.01)
CPC C12Q 1/6818 (2013.01) [C12Q 1/6823 (2013.01); C12Q 1/6837 (2013.01); C12Q 1/6876 (2013.01); C12Q 2520/00 (2013.01)] 20 Claims
 
1. A method of detecting a nucleic acid, comprising:
preparing a detection reagent comprising a first enzyme and a second enzyme, wherein the first enzyme is a 5′-nuclease, and the second enzyme is FEN-1 such that a concentration of the first enzyme is in a range of 0.63 times to 2.46 times a concentration of the second enzyme;
mixing, with the detection reagent, a fluid comprising a target nucleic acid, a first nucleic acid having a first flap, a second nucleic acid, and a fourth nucleic acid having a second flap such that a cleaved product is produced, wherein the cleavage activity of the second enzyme for a first invasive structure is lower than the cleavage activity of the first enzyme for the first invasive structure, the cleavage activity of the first enzyme for a second invasive structure is lower than the cleavage activity of the second enzyme for the second invasive structure, the mixing comprises a first reaction in which the first nucleic acid forms a complex comprising the first invasive structure having the first flap with the target nucleic acid and the second nucleic acid, and the first enzyme cleaves the first flap of the first invasive structure and produces a third nucleic acid, and a second reaction in which the third nucleic acid forms a complex comprising the second invasive structure having the second flap with the fourth nucleic acid, and the second enzyme cleaves the second flap of the second invasive structure and produces the cleaved product; and
detecting the cleaved product such that the target nucleic acid is detected, wherein the fluid is mixed with the detection reagent such that the concentration of the first enzyme is 0.27 mg/mL or less and the concentration of the second enzyme is 0.12 mg/mL or less.