Nucleic acid test kit, test method, and use
Disclosed is a nucleic acid test kit, comprising a Cpf1 test system, said Cpf1 test system comprising: a guide RNA, a Cpf1 protein, a nucleic acid probe, and a manganese ion-containing solution. Also provided is a mutated Cpf1 protein and a nucleic acid test kit containing the mutated Cpf1 protein. Also provided is a crRNA for testing coronavirus nucleic acid, and a nucleic acid test kit containing said crRNA. Further provided is a use of the nucleic acid test kit, and a test method for the nucleic acid. When the nucleic acid test kit, mutated Cpf1 protein, or crRNA of the present invention are used for nucleic acid testing (for example, testing the nucleic acid of a SARS-related coronavirus or MERS coronavirus), the signal strength during the test is substantially enhanced, and the test time is thus reduced and the test efficiency improved. In addition, the sensitivity, specificity, and accuracy are high, the test is visual, the cost is low, the operation is simple and convenient, and no large-scale complex t equipment is required.
1 . A nucleic acid detection kit, comprising a Cpf1 detection system comprising: a guide RNA, a mutated Cpf1 protein, a nucleic acid probe, and a manganese ion-containing solution;
wherein the mutated Cpf1 protein comprises the amino acid sequence of SEQ ID NO: 54 having one or more substitutions selected from the group consisting of T148R, T152R, G532R and K538R.
2 . The nucleic acid detection kit of claim 1 , wherein
the nucleic acid probe is a single-stranded DNA probe.
3 . The nucleic acid detection kit of claim 1 , wherein the guide RNA is capable of guiding the mutated Cpf1 protein to specifically bind to a target nucleic acid.
4 . The nucleic acid detection kit of claim 1 , wherein:
(i) the manganese ion-containing solution has a concentration of manganese ions of 5-600 mM;
(ii) the nucleic acid probe has a concentration of 1-100 μmol/μL;
(iii) the Cpf1 protein has a concentration of 20-1,000 ng/μL;
(iv) the guide RNA has a concentration of 0.1-50 μM; or,
(v) the manganese ion-containing solution is a manganese sulfate solution, a manganese chloride solution, or a manganese acetate solution;
optionally, the nucleic acid detection kit further comprises an RNase inhibitor and a buffer.
5 . A method for detecting a nucleic acid, comprising:
(a) contacting a sample to be tested containing the nucleic acid with the Cpf1 detection system in the nucleic acid detection kit of claim 1 ; and,
(b) detecting a signal indicative of the presence of the nucleic acid.
6 . The nucleic acid detection kit of claim 1 , wherein the mutated Cpf1 protein comprises the amino acid sequence of SEQ ID NO: 54 having one substitution selected from the group consisting of T148R, T152R, G532R and K538R.
7 . The nucleic acid detection kit of claim 1 , wherein the mutated Cpf1 protein comprises the amino acid sequence of SEQ ID NO: 54 having substitutions of T152R and G532R, or, the mutated Cpf1 protein comprises an amino acid sequence of SEQ ID NO: 54 having substitutions of T152R, G532R and K538R.
8 . The nucleic acid detection kit of claim 1 , wherein the mutated Cpf1 protein comprises the amino acid sequence of SEQ ID NO: 54 having substitutions of T148R, T152R, G532R and K538R.
9 . The nucleic acid detection kit of claim 1 , wherein the mutated Cpf1 protein is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39.
10 . The nucleic acid detection kit of claim 1 , wherein the mutated Cpf1 protein is encoded by a nucleotide sequence of SEQ ID NO: 40 or SEQ ID NO: 41.
11 . The nucleic acid detection kit of claim 2 , wherein the nucleic acid probe comprises a fluorescent label.
12 . The nucleic acid detection kit of claim 11 , wherein the nucleic acid probe comprises a fluorescent group and a fluorescence quenching group at its 5′ and 3′ terminals, respectively.
13 . The nucleic acid detection kit of claim 12 , wherein the fluorescent group is selected from the group consisting of 6-FAM, TET, CY3, CY5, and ROX; or, the fluorescence quenching group is selected from the group consisting of BHQ1, BHQ2, and BHQ3.
14 . The nucleic acid detection kit of claim 3 , wherein the guide RNA is a crRNA.
15 . The nucleic acid detection kit of claim 14 , wherein the crRNA is capable of detecting a viral nucleic acid.
16 . The nucleic acid detection kit of claim 15 , wherein the crRNA is capable of detecting a SARS-associated coronavirus or MERS-COV virus.
17 . The nucleic acid detection kit of claim 16 , wherein the SARS-associated coronavirus is a SARS-COV or SARS-COV-2 coronavirus.
18 . The nucleic acid detection kit of claim 17 , wherein the crRNA is capable of detecting a target selected from the group consisting of: an orf1ab gene of the SARS-CoV coronavirus, an orf1a gene of the MERS-COV virus, an upE gene of the MERS-COV virus, a E gene of the SARS-COV-2 coronavirus, an S gene of the SARS-COV-2 coronavirus, an M gene of the SARS-COV-2 coronavirus, and an N gene of the SARS-CoV-2 coronavirus;
wherein the nucleotide sequence of the orf1ab gene is set forth in SEQ ID NO: 11, the nucleotide sequence of the orf1a gene is set forth in SEQ ID NO: 12, the nucleotide sequence of the upE gene is set forth in SEQ ID NO: 13, and the nucleotide sequence of the E gene is set forth in SEQ ID NO: 46; the nucleotide sequence of the S gene of the SARS-COV-2 coronavirus is set forth in SEQ ID NO: 79; the nucleotide sequence of the M gene of the SARS-COV-2 coronavirus is set forth in SEQ ID NO: 80; and the nucleotide sequence of the N gene of the SARS-COV-2 coronavirus is set forth in SEQ ID NO: 81.
19 . The nucleic acid detection kit of claim 18 , wherein the nucleotide sequence of the crRNA is selected from the group consisting of SEQ ID NOs: 1-10, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 53, and SEQ ID NOs: 56-72.
20 . The method of claim 5 , wherein:
(i) the nucleic acid in the sample to be tested is released by using a nucleic acid rapid release reagent;
(ii) the nucleic acid is obtained by amplifying the nucleic acid in the sample to be tested;
(iii) a reaction temperature of the Cpf1 detection system is 37 ±5° C.; or,
(iv) a reaction time of the Cpf1 detection system is 25 ±5 min;
optionally, the method further comprising: (c) reading a result.