Method for detecting nucleic acid
View Patent ↗A method for detecting the presence and/or amount of a target nucleic acid molecule in a biological sample and a related kit. The method comprises: (a) contacting the biological sample with: i) Cas12b protein, ii) a gRNA targeting a target sequence of the target nucleic acid molecule, and iii) a single-stranded DNA reporter molecule generating a detectable signal after being cleaved to form a reaction mixture; and (b) detecting the presence and/or level of the detectable signal generated in the reaction mixture.
1 . A method of detecting the presence and/or amount of a target nucleic acid molecule in a biological sample, the method comprising the following steps:
(a) contacting the biological sample with: i) a Cas12b protein, ii) a gRNA directed against a target sequence in the target nucleic acid molecule, and iii) a single-strand DNA reporter molecule which produces a detectable signal after being cleaved, thereby forming a reaction mixture;
(b) detecting the presence and/or level of the detectable signal produced in the reaction mixture,
wherein the presence and/or level of the detectable signal indicates the presence and/or amount of the target nucleic acid molecule,
wherein the Cas12b protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1.
2 . The method of claim 1 , wherein the target nucleic acid molecule is a double-strand DNA molecule or a single-strand DNA molecule.
3 . The method of claim 1 , wherein the Cas12b protein is from Alicyclobacillus acidiphilus.
4 . The method of claim 1 , wherein the Cas12b protein comprises
an amino acid sequence set forth in SEQ ID NO: 1.
5 . The method of claim 1 , wherein the guide RNA is sgRNA.
6 . The method of claim 5 , the sgRNA comprises a scaffold sequence encoded by a nucleic acid sequence selected from one of SEQ ID NOs: 11-66.
7 . The method of claim 5 , wherein the sgRNA comprises a spacer sequence at the 3′-end of the scaffold sequence that specifically hybridizes to the target sequence or the complement of the target sequence.
8 . The method of claim 7 , wherein the spacer sequence has at least one nucleotide mismatch with the target sequence.
9 . The method of claim 1 , wherein the single-strand DNA reporter molecule comprises a fluorophore at one end of the single-stranded DNA reporter molecule, and a quenching group at the other end of the single-strand DNA reporter molecule.
10 . The method of claim 9 , wherein the fluorophore is selected from FAM, TEX, HEX, Cy3 and Cy5, and the quenching group is selected from BHQ1, BHQ2, BHQ3 and TAMRA.
11 . The method of claim 1 , wherein the single-strand DNA reporter molecule is about 2 to about 100 nucleotides in length.
12 . The method of claim 1 , wherein the single-strand DNA reporter molecule is polyA, polyC, or polyT.
13 . The method of claim 1 , further comprising a step of amplifying the target nucleic acid molecule in the biological sample prior to step (a).
14 . The method of claim 13 , the amplification comprises PCR amplification or Recombinase Polymerase Amplification (RPA).
15 . The method of claim 14 , the Recombinase Polymerase Amplification is performed for about 10 minutes to about 60 minutes.
16 . The method of claim 1 , the Cas12b protein has been pre-combined with the gRNA to form a Cas12b-gRNA complex prior to the contact with the biological sample.
17 . The method of claim 1 , the reaction of step (a) is carried out for from about 20 minutes to about 180 minutes.
18 . The method of claim 1 , wherein step (a) is carried out in a buffer comprising
(i) a final concentration of 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.9;
(ii) a final concentration of 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 100 μg/ml BSA, pH 7.9; or
(iii) a final concentration of 50 mM potassium acetate, 20 mM Tris-acetic acid, 10 mM magnesium acetate, 100 μg/ml BSA, pH 7.9.
19 . The method of claim 1 , wherein the biological sample is selected from whole blood, plasma, serum, cerebrospinal fluid, urine, feces, cell and tissue extracts.