Method for multiplex nucleic acid detection based on clustered regularly interspaced short palindromic repeat
AssigneeShandong Shunfeng Biotechnology Co., Ltd.
InventorsYafeng Liang, Jie Sun, Ruiheng Liu
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Abstract
A method for multiplex nucleic acid detection based on clustered regularly interspaced short palindromic repeat (CRISPR), a system, and a kit for detecting a target nucleic acid based on CRISPR are provided. The detection method includes: adding any one, any two, any three, or four from the group consisting of a first nucleic acid detection composition, a second nucleic acid detection composition, a third nucleic acid detection composition, and a fourth nucleic acid detection composition to a reaction system with a target nucleic acid to achieve the multiplex detection of the target nucleic acid.
Claims20
A method for detecting a target nucleic acid in a sample, comprising contacting the sample with at least three nucleic acid detection compositions;
- and detecting a detectable signal generated due to a cleavage of the Cas protein on the single-stranded nucleic acid reporter to detect the target nucleic acid;
- wherein the at least three nucleic acid detection compositions comprises at least three compositions selected from the group consisting of:
- a first nucleic acid detection composition comprising Cas12i, a gRNA, and a first single-stranded nucleic acid reporter;
- a second nucleic acid detection composition comprising Cas12b, a gRNA, and a second single-stranded nucleic acid reporter;
- a third nucleic acid detection composition comprising Cas12a, a gRNA, and a third single-stranded nucleic acid reporter;
- and
- a fourth nucleic acid detection composition comprising Cas12j, a gRNA, and a fourth single-stranded nucleic acid reporter;
- wherein,
- the first single-stranded nucleic acid reporter comprises at least two consecutive nucleotides selected from the group consisting of ribonucleotides, deoxyribonucleotides, nucleic acid analogues and combinations thereof;
- the second single-stranded nucleic acid reporter comprises at least one nucleotide and at least one abasic spacer or a locked nucleic acid (LNA);
- the third single-stranded nucleic acid reporter comprises at least one nucleotide and at least one abasic spacer;
- and
- the fourth single-stranded nucleic acid reporter comprises at least one nucleotide and at least one abasic spacer or 2′-O-methyl RNA.
A system or composition or kit for detecting a target nucleic acid in a sample, comprising at least three nucleic acid detection compositions selected from the group consisting of:
- i) a first nucleic acid detection composition comprising Cas12i, a gRNA, and a first single-stranded nucleic acid reporter;
- ii) a second nucleic acid detection composition comprising Cas12b, a gRNA, and a second single-stranded nucleic acid reporter;
- iii) a third nucleic acid detection composition comprising Cas12a, a gRNA, and a third single-stranded nucleic acid reporter;
- and
- iv) a fourth nucleic acid detection composition comprising Cas12j, a gRNA, and a fourth single-stranded nucleic acid reporter;
- wherein,
- the first single-stranded nucleic acid reporter comprises at least two consecutive nucleotides selected from the group consisting of ribonucleotides, deoxyribonucleotides, nucleic acid analogues and combinations thereof;
- the second single-stranded nucleic acid reporter comprises i) at least one nucleotide and at least one abasic spacer;
- or, ii) a locked nucleic acid (LNA);
- the third single-stranded nucleic acid reporter comprises at least one nucleotide and at least one abasic spacer;
- and
- the fourth single-stranded nucleic acid reporter comprises i) at least one nucleotide and at least one abasic spacer;
- or, ii) 2′-O-methyl RNA.
Dependencies are inferred from the claim text.