Nucleic acid amplification method, primer set, probe, and kit for nucleic acid amplification method

A method for amplifying nucleic acids, comprising:

• step A including annealing a Bw adapter primer comprising the following structure (a):
• 5′-B1c-BL-B1-N3c-3′ (a)
• wherein N3c represents an annealing region composed of a base sequence complementary to a base sequence on a 3′-end side of a target region of a target nucleic acid, BL represents a loop region comprising base sequence B2, and B1c and B1 represent stem regions comprising mutually complementary base sequences and capable of forming a double strand,
• and the target region, synthesizing a base sequence complementary to a base sequence on a 5′-end side of the target region starting from a 3′-end of the Bw adapter primer, and obtaining a first template nucleic acid comprising the following structure (b):
• 5′-B1c-BL-B1-N3c-N5c-3′ (b)
• wherein N5c represents the base sequence complementary to the base sequence on the 5′-end side of the target region, and a base sequence comprising N3c and N5c represents base sequence Nc complementary to the target region,
• in which a stem-loop structure is added to a 5′-end of a complementary strand of the target region;
• step B including annealing an Fw adapter nucleotide comprising the following structure (c):
• 5′-F1c-FL-F1-N5′-3′ (c)
• wherein N5′ represents an annealing region composed of a base sequence complementary to base sequence N5c of the first template nucleic acid, FL represents a loop region comprising base sequence F2, and F1c and F1 represent stem regions comprising mutually complementary base sequences and capable of forming a double strand,
• and having an extension-inhibiting modification at a 3′-end
• and the first template nucleic acid, synthesizing a complementary strand of the Fw adapter nucleotide starting from a 3′-end of the first template nucleic acid, and obtaining a second template nucleic acid comprising the following structure (d):
• 5′-B1c-BL-B1-N3c-N5c-F1c-FLc-F1-3′ (d)
• wherein FLc represents a base sequence complementary to FL of the Fw adapter nucleotide,
• in which a stem-loop structure is added to a 3′-end of the first template nucleic acid;
• and
• step C including amplifying base sequence Nc of the second template nucleic acid by a LAMP method by using an Fw inner primer comprising the following structure (e):
• 5′-F1c-F2-3′ (e)
• and a Bw inner primer comprising the following structure (f):
• 5′-B1c-B2-3′ (f)
• with the second template nucleic acid as a template.