	US 12,104,204 B2
	Use of high-temperature-resistant Cas protein, and method and reagent kit for detecting target nucleic acid molecule
Shiyuan Li, Shanghai (CN); and Jin Wang, Shanghai (CN)
Assigned to SHANGHAI TOLO BIOTECHNOLOGY COMPANY LIMITED, Shanghai (CN)
Appl. No. 15/734,760
Filed by SHANGHAI TOLO BIOTECHNOLOGY COMPANY LIMITED, Shanghai (CN)
PCT Filed Jun. 3, 2019, PCT No. PCT/CN2019/089856 § 371(c)(1), (2) Date Dec. 3, 2020, PCT Pub. No. WO2019/233385, PCT Pub. Date Dec. 12, 2019.
Claims priority of application No. 201810560284.8 (CN), filed on Jun. 3, 2018.
Prior Publication US 2021/0238666 A1, Aug. 5, 2021
Int. Cl. C12Q 1/6858 (2018.01)

CPC C12Q 1/6858 (2013.01) [C12Q 2600/158 (2013.01)]

10 Claims

1. A method for detecting whether target nucleic acid molecules are present in a sample, which comprises the steps of:

(i) providing a detection system, comprising:

(a) a Cas protein, wherein the Cas protein is Cas12b;

(b) a guiding RNA, wherein the guiding RNA directs the Cas protein to specifically bind to the target nucleic acid molecules;

wherein, the target nucleic acid molecule is a target DNA;

(d) buffer;

(e1) a polymerase, which is used for amplifying target DNA;

(e2) an optional reverse transcriptase, which is used for reverse transcription;

(e3) dNTP, which is used for amplification reaction and/or reverse transcription reaction; and

(f) a sample to be tested;

(ii) performing a reverse transcription and/or amplification reaction in the detection system, thereby obtaining a detection system reverse transcripted and/or amplified; and

(iii) detecting whether the nucleic acid probe in the detection system obtained in the previous step is cleaved by the Cas protein, wherein the cleavage is a trans-cleavage of a bypass single-stranded DNA;

wherein, if the nucleic acid probe is cleaved by the Cas protein, it indicates that the target nucleic acid molecule is present in the sample; and if the nucleic acid probe is not cleaved by the Cas protein, it indicates that the target nucleic acid molecule is not present in the sample;

wherein in the detection, a nucleic acid amplification reaction and a collateral cleavage reaction are included, and the nucleic acid amplification reaction and the collateral cleavage reaction are performed simultaneously, and the detection system is a one-step reaction system and the temperature of the detection system is maintained at 50-70° C.