Method for detecting single-stranded RNA virus
A method for detecting a single-stranded RNA virus in a sample includes: bringing a primer set into contact with a sample to perform a RT-LAMP reaction, wherein the primer set is designed based on a nucleotide sequence of a target RNA and a nucleotide sequence of a nucleic acid complementary to the target RNA, and includes following (i) to (v): (i) an FIP primer; (ii) a BIP primer; (iii) an F3 primer, which is an outer primer; (iv) a B3 primer, which is an outer primer; and (v) one or more additional outer primers, each additional outer primer having the same nucleotide sequence as an arbitrary region present on a 5′ terminal side from the region B3 or the region F3 in the nucleic acid complementary to the target RNA. According to the present invention, a single-stranded RNA virus can be detected with high sensitivity.
1 . A method for detecting a single-stranded RNA virus in a sample, the method comprising:
bringing a primer set into contact with a sample to perform a reverse transcription loop-mediated isothermal amplification reaction,
wherein the primer set is designed based on a nucleotide sequence of a target RNA of a single-stranded RNA virus and a nucleotide sequence of a nucleic acid complementary to the target RNA,
wherein the primer set comprises following (i) to (v):
(i) an FIP primer having the same nucleotide sequence as the region F1c at a 5′ terminal and having the same nucleotide sequence as the region F2 at a 3′ terminal;
(ii) a BIP primer having the same nucleotide sequence as the region B1c at a 5′ terminal and having the same nucleotide sequence as the region B2 at a 3′ terminal;
(iii) an F3 primer which is an outer primer having the same nucleotide sequence as the region F3;
(iv) a B3 primer which is an outer primer having the same nucleotide sequence as the region B3; and
(v) one additional outer primer, wherein either (a) or (b):
(a) the single-stranded RNA virus is a plus-strand single-stranded RNA virus, the target RNA comprises the arbitrary regions F3, F2, F1, B1c, B2c, B3c, and B4c in this order in the direction from the 5′ terminal to 3′ terminal, wherein the nucleic acid complementary to the target RNA comprises the arbitrary regions F3c, F2c, F1c, B1, B2, B3, and B4 in this order in the direction from 3′ terminal to 5′ terminal, and the one additional outer primer has a nucleotide sequence present on 5′ terminal side from the region B4 in the nucleic acid complementary to the target RNA; or
(b) the single-stranded RNA virus is a minus-strand single-stranded RNA virus, the target RNA comprises the arbitrary regions F4c, F3c, F2c, F1c, B1, B2, and B3 in this order in the direction from 3′ terminal to 5′ terminal, the nucleic acid complementary to the target RNA comprises the arbitrary regions F4, F3, F2, F1, B1c, B2c, and B3c in this order in the direction from the 5′ terminal to 3′ terminal, and the one additional outer primer has a nucleotide sequence present on 5′ terminal side from the region F4 in the nucleic acid complementary to the target RNA, and
wherein uracil that may be present in the nucleotide sequences of the FIP primer, the BIP primer, the F3 primer, the B3 primer, and the one additional outer primer may be substituted with thymine.
2 . The method according to claim 1 ,
wherein the primer set further comprises:
(vi) a loop primer F having the same nucleotide sequence as an arbitrary region between the region F1c and the region F2c; and
(vii) a loop primer B having the same nucleotide sequence as an arbitrary region between the region B1c and the region B2c, and
wherein uracil that may be present in the nucleotide sequences of the loop primer F and the loop primer B may be substituted with thymine.
3 . The method according to claim 1 ,
wherein a melting temperature of the one additional outer primer of (v) is 30° C. to 55° C.
4 . The method according to claim 1 , the method further comprising:
detecting an amplification product of the reverse transcription loop-mediated isothermal amplification reaction.