JPWO2020245808A5

JPWO2020245808A5 -

Info

Publication number: JPWO2020245808A5
Application number: JP2021572506A
Authority: JP
Publication date: 2023-06-09

Claims

i) a first nucleic acid sequence capable of hybridizing to at least a portion of a target nucleic acid sequence or its reverse complement and further comprising an aptamer-encoding template sequence, wherein the aptamer-encoding template sequence is at the 3' end of the first nucleic acid sequence; and ii) a second nucleic acid sequence capable of hybridizing to at least a portion of the target nucleic acid sequence or its reverse complement, the 5′ end of the second nucleic acid sequence comprising: a nucleic acid molecule comprising a second nucleic acid sequence covalently linked to the 3' end of the first nucleic acid sequence, wherein the 3' end of the second nucleic acid sequence does not substantially hybridize to the first nucleic acid sequence, or Analogue.

2. The nucleic acid molecule of claim 1, wherein at least the terminal 3 nucleotides of the 3' end of the second nucleic acid sequence do not hybridize to the first nucleic acid sequence.

3. The nucleic acid molecule of claim 1 or 2, wherein the first nucleic acid sequence is about 20 to about 100 nucleotides in length.

The nucleic acid molecule comprises a terminal stem structure, wherein at least the terminal nucleotide at the 5' end of the second nucleic acid sequence is complementary to at least the terminal nucleotide at the 5' end of the first nucleic acid and forms at least a portion of the terminal stem structure. A nucleic acid molecule according to any one of claims 1 to 3 which forms.

The nucleic acid molecule of any one of claims 1-4 , wherein the second nucleic acid sequence comprises a degenerate sequence.

6. The nucleic acid molecule of any one of claims 1-5 , wherein the target nucleic acid sequence is of viral, microbial, fungal, animal or plant origin, or is a synthetic construct.

A composition comprising the first nucleic acid molecule of any one of claims 1-6 .

further comprising a second nucleic acid molecule hybridizable to at least a portion of the target nucleic acid sequence or its reverse complement and comprising a first RNA polymerase promoter sequence, wherein the first and second nucleic acid molecules are 8. The composition of claim 7 , forming a first primer pair capable of amplifying a first sequence.

further comprising a third nucleic acid molecule and a fourth nucleic acid molecule;
the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule;
either the third nucleic acid molecule or the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence;
9. The composition of claim 7 or 8 , wherein the second primer pair hybridizes to the target nucleic acid molecule at a location external to that of the first primer pair and is capable of amplifying the first and second sequences. thing.

further comprising a fifth nucleic acid molecule and a sixth nucleic acid molecule;
the fifth and sixth nucleic acid molecules form a third primer pair capable of amplifying a third sequence of the target nucleic acid molecule;
either the fifth nucleic acid molecule or the sixth nucleic acid molecule comprises a third RNA polymerase promoter sequence;
7- wherein the third primer pair is capable of hybridizing to the target nucleic acid molecule at a location external to that of the first and second primer pairs and amplifying the first, second and third sequences. 11. The composition of any one of 10 .

A kit comprising a nucleic acid molecule according to any one of claims 1 to 6 or a composition according to any one of claims 7 to 10 together with instructions for amplification of a target nucleic acid sequence.

A method of amplifying a target nucleic acid sequence, comprising:
i) providing a sample suspected of containing the target nucleic acid molecule;
ii) providing a first nucleic acid molecule according to any one of claims 1-6 ;
iii) providing a second nucleic acid molecule hybridizable to at least a portion of the target nucleic acid sequence or its complement and comprising a first RNA polymerase promoter sequence,
said step wherein the first and second nucleic acid molecules form a first primer pair capable of amplifying a first sequence of the target nucleic acid sequence; and iv) a first primer pair comprising the target nucleic acid molecule and the first primer pair; The method comprising the step of performing an amplification reaction to obtain a first amplification product, wherein the first amplification product comprises the first sequence of the target nucleic acid sequence.

v) providing a third nucleic acid molecule and a fourth nucleic acid molecule,
the third and fourth nucleic acid molecules form a second primer pair capable of amplifying a second sequence of the target nucleic acid molecule;
either the third nucleic acid molecule or the fourth nucleic acid molecule comprises a second RNA polymerase promoter sequence;
the second primer pair is capable of hybridizing to the target nucleic acid molecule at a location external to that of the first primer pair and amplifying the first and second sequences of the target nucleic acid molecule; and vi a) performing a second amplification reaction comprising the first amplification product and the second primer pair to obtain a second amplification product, the second amplification reaction prior to the first amplification reaction; 13. The method of claim 12 , further comprising the steps performed and wherein the second amplification product comprises the first sequence and the second sequence of the target nucleic acid molecule.

14. The method of claim 12 or 13 , further comprising detecting the target nucleic acid sequence.

A method according to any one of claims 12-14 , further comprising the step of quantifying the target nucleic acid sequence.

A method according to any one of claims 12 to 15 , wherein the amplification is isothermal amplification.

A method according to any one of claims 12 to 16 , wherein the sample is of viral, microbial, fungal, animal, plant or environmental origin.

18. The method of claim 17 , wherein the virus is SARS, MERS, or SARS-CoV-2.

A method according to any one of claims 12 to 18 , wherein the sample is obtained from water, soil, saliva, faeces, urine, blood, tracheal aspirate, or nasal aspirate.

18. The method of claim 17 , wherein the animal is human.