# US 12,110,538 B2 · Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof

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- Number: US 12,110,538 B2
- Document ID: US12110538B2
- Status: Granted
- Kind: B2
- Application number: 17/045,393
- Filed: 2018-10-12
- Published: 2021-06-03
- Granted: 2024-10-08
- Earliest claimed: 2018-04-03
- Assignees: Jiaohong Biotechnology (Shanghai) Co., Ltd.
- Applicants: Jiaohong Biotechnology (Shanghai) Co., Ltd.
- Inventors: Yan Feng; Guanhua Xun; Qian Liu; Yuesheng Chong
- Web page: https://patentgrep.com/patent/US12110538B2

## CPC

- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/708: {for papilloma}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Provided are a method for detecting a nucleic acid based on prokaryotic Argonaute protein and an application thereof. In particular, provided is a system for detecting a target nucleic acid molecule. The system comprises guide ssDNA, a gene-editing enzyme Pyrococcus furiosus Argonaute (PfAgo), and a fluorescent reporter nucleic acid.

## Claims

### Claim 1

A detection system for detecting a target nucleic acid molecule, wherein the system comprises:
(a) a guide ssDNA pair;
(b) a gene editing enzyme Pyrococcus furiosus (Pf Ago); and
(c) a fluorescent reporter nucleic acid, which has a fluorescent group and a quenching group;
wherein:
the target nucleic acid molecule is target DNA and is cleaved by the PfAgo enzyme to produce a secondary guide ssDNA,
the sequences of the secondary guide ssDNA and the fluorescent reporter nucleic acid are complementary; and
after complementary binding between the secondary guide ssDNA and the fluorescent reporter nucleic acid, the PfAgo enzyme is guided to cleave the fluorescent reporter nucleic acid, thereby generating a detectable signal.

### Claim 2 (depends on 1)

The detection system of claim 1, wherein the length of the guide ssDNA is 14-60 nt, preferably 16-40 nt.

### Claim 3 (depends on 1)

The detection system of claim 1, wherein the PfAgo enzyme is derived from the archaea Pyrococcus furiosus.

### Claim 4 (depends on 1)

The detection system of claim 1, wherein the mutation sites corresponding to different types of the target nucleic acid molecules are at positions 10 and 11 of the guide SSDNA.

### Claim 5 (depends on 1)

The detection system of claim 1, wherein the target nucleic acid molecules (or the amplification product thereof) are cleaved by the PfAgo enzyme to produce a secondary guide SSDNA.

### Claim 6 (depends on 1)

The detection system of claim 1, wherein the detection system further comprises the target nucleic acid molecules to be detected, and the concentration of the target nucleic acid molecules to be detected in the detection system is 1 fM-200 pM.

### Claim 7 (depends on 1)

The detection system of claim 1, the target DNA is selected from the group consisting of: single-stranded DNA (including cDNA), double-stranded DNA, and a combination thereof.

### Claim 8 (depends on 1)

A kit for detecting target nucleic acid molecules, wherein the kit comprises:
(i) the detection system according to claim 1 or reagents used for the preparation of the detection system; and
(ii) instructions for use, which describe the method for detecting target nucleic acid molecules with the detection system.

### Claim 9 (depends on 1)

A method for detecting the presence or absence of target nucleic acid molecules in a sample, comprising the steps of:
(a) providing the detection system for detecting target nucleic acid molecules according to claim 1; and
(b) reacting the detection system with the sample to be tested at a certain temperature, to form a first reaction solution;
(c) performing a fluorescence detection on the first reaction solution, to obtain a fluorescence signal value;
wherein, if the fluorescence signal value is detected in the first reaction solution, it indicates that there is target nucleic acid molecules in the sample; and if the fluorescence signal value is not detected in the first reaction solution, it indicates that there is no target nucleic acid molecule in the sample.

### Claim 10 (depends on 1)

The detection system of claim 1, wherein the guide ssDNA is a 5′-phosphorylated single-stranded DNA molecule.

### Claim 11 (depends on 1)

The detection system of claim 1, wherein the detection system further comprises primers for amplifying the target nucleic acid molecules.

### Claim 12 (depends on 6)

The detection system of claim 6, wherein the molar ratio of the fluorescent reporter nucleic acid to the target nucleic acid molecules is 103:1 to 108:1, preferably 104:1 to 107:1.

### Claim 13 (depends on 9)

The method of claim 9, wherein the sample to be detected comprises an unamplified sample and an amplified (or nucleic acid amplified) sample.

### Claim 14 (depends on 9)

The method of claim 9, wherein the nucleic acid amplification method is selected from the group consisting of: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification and rolling circle amplification.