Detection kit and simple method for detecting target nucleic acids

1. A nucleic acid detection kit comprising:

(i) a single-stranded circular DNA template containing:

a sequence of 10 to 30 bases complementary to a first portion of a target nucleic acid; and

a primer-binding sequence of 7 to 8 bases adjacent to 5′-side thereof; and

(ii) an oligonucleotide primer containing:

a sequence of 8 to 15 bases complementary to a second portion adjacent to the 3′-side of the first portion of the target nucleic acid; and

a sequence of 7 to 8 bases adjacent to the 3′-side thereof and complementary to the primer-binding sequence of the single-stranded circular DNA template.

2. A nucleic acid detection kit comprising:

(i) a first single-stranded circular DNA template containing:

a sequence of 10 to 30 bases complementary to a first portion of a target nucleic acid;

a first primer-binding sequence of 7 to 8 bases adjacent to the 5′-side thereof; and

a second single-stranded circular DNA-binding sequence;

(ii) a first oligonucleotide primer containing:

a sequence of 8 to 15 bases complementary to a second portion adjacent to the 3′-side of the first portion of the target nucleic acid; and

a sequence of 7 to 8 bases adjacent to the 3′-side thereof and complementary to the first primer-binding portion of the first single-stranded circular DNA template;

(iii) a second single-stranded circular DNA containing:

the same sequence as the second single-stranded circular DNA-binding sequence of the first single-stranded circular DNA; and

a second primer-binding sequence adjacent to the 5′-side thereof; and

(iv) a second oligonucleotide primer containing:

the same sequence as the portion adjacent to the 5′-side of the second single-stranded circular DNA-binding sequence of the first single-stranded circular DNA; and

a sequence adjacent to 3′-side thereof and complementary to the second primer-binding sequence of the second single-stranded circular DNA.

3. A method for detecting a target nucleic acid using the kit according to claim 1 , said method comprising the steps of:

hybridizing said single-stranded circular DNA template and said primer with the target nucleic acid;

performing nucleic acid amplification reaction based on the target nucleic acid by rolling circle amplification; and

detecting a product of the nucleic acid amplification reaction.

4. The nucleic acid detection kit according to claim 1 , wherein said kit further comprises (iii) a detection reagent and wherein said single-stranded circular DNA template further contains a sequence complementary to a detection reagent-binding sequence.

5. The nucleic acid detection kit according to claim 4 , wherein said detection reagent-binding sequence is a guanine quadruplex-forming sequence, and said detection reagent is a guanine quadruplex-binding reagent.

6. The nucleic acid detection kit according to claim 5 , wherein the sequence complementary to the guanine quadruplex-forming sequence comprises a C 3 N 1-10 C 3 N 1-10 C 3 N 1-10 C 3 sequence.

7. The nucleic acid detection kit according to claim 5 , wherein said guanine quadruplex-binding reagent comprises a compound represented by the following General Formula (I):

wherein R 1 represents hydrogen, or a hydrocarbon group which optionally contains one or more selected from the group consisting of O, S, and N;

R 2 , R 3 , and R 4 each independently represent a C 1 -C 5 hydrocarbon group;

n represents an integer of 0 to 5; and

X represents O, S, or NH.

8. The nucleic acid detection kit according to claim 7 , wherein the compound represented by General Formula (I) is represented by the following Formula (II) or (Ill):

9. The method according to claim 3 , wherein said target nucleic acid is RNA.

10. The nucleic acid detection kit according to claim 1 , wherein said target nucleic acid is RNA.

11. The nucleic acid detection kit according to claim 2 , wherein said target nucleic acid is RNA.

12. The nucleic acid detection kit according to claim 2 , wherein said kit further comprises (v) a detection reagent and wherein said second single-stranded circular DNA further contains a sequence complementary to a detection reagent-binding sequence.

13. The nucleic acid detection kit according to claim 12 , wherein said detection reagent-binding sequence is a guanine quadruplex-forming sequence, and said detection reagent is a guanine quadruplex-binding reagent.

14. The nucleic acid detection kit according to claim 13 , wherein the sequence complementary to the guanine quadruplex-forming sequence comprises a C 3 N 1-10 C 3 N 1-10 C 3 N 1-10 C 3 sequence.

15. The nucleic acid detection kit according to claim 13 , wherein said guanine quadruplex-binding reagent comprises a compound represented by the following General Formula (I):

wherein R 1 represents hydrogen, or a hydrocarbon group which optionally contains one or more

selected from the group consisting of O, S, and N;

R 2 , R 3 , and R 4 each independently represent a C 1 -C 5 hydrocarbon group;

n represents an integer of 0 to 5; and

X represents O, S, or NH.

16. The nucleic acid detection kit according to claim 15 , wherein the compound represented by General Formula (I) is represented by the following Formula (II) or (III):

17. A method for detecting a target nucleic acid using the kit according to claim 2 , said method comprising the steps of:

hybridizing said single-stranded circular DNA template and said primer with the target nucleic acid;

performing nucleic acid amplification reaction based on the target nucleic acid by rolling circle amplification; and

detecting a product of the nucleic acid amplification reaction.

18. The method according to claim 17 , wherein said target nucleic acid is RNA.