Polymorphism (SNP) using lamp and blocking primers

A method for detecting a first allele of a single nucleotide polymorphism (SNP) in a nucleic acid sequence, wherein the method comprises:

• (a) amplifying a nucleic acid sequence from a sample using one of the group consisting of:
• Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), and Nucleic Acid Sequences Based Amplification (NASBA), in a reaction mixture comprising:
• (i) the nucleic acid sequence,
• (ii) a nucleic acid polymerase,
• (iii) a nucleoside triphosphate mixture,
• (iv) a forward inner primer targeting the first allele (FIPa1), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the first allele and a F2 region which anneals to a F2c region of the nucleic acid sequence,
• (v) a backward inner primer targeting the first allele (BIPa1), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the first allele and a B2 region which anneals to a B2c region of the nucleic acid sequence,
• (vi) a forward blocking primer targeting a second allele of the SNP (FBa2), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the second allele, and
• (vii) a backward blocking primer targeting the second allele (BBa2), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the second allele,
• wherein the F1 region of the nucleic acid sequence and the B1 region of the nucleic acid sequence both contain the SNP,
• wherein BBa2 primes extension from its 3′ end and/or FBa2 primes extension from its 3′ end;
• and
• (b) detecting an amplified nucleic acid sequence, wherein the detection of the amplified nucleic acid sequence indicates the presence of the first allele of the SNP.