Allele specific pcr assay for detection of nucleotide variants

A method of detecting a mutant first allele of a target nucleic acid molecule in a biological sample, comprising:

• (A) amplifying the target nucleic acid molecule from the biological sample by real-time polymerase chain reaction (qPCR) using a test primer pair in a test amplification, comprising
• a first plus primer comprising a locked nucleic acid at the 3′ end that is complementary to the mutant first allele of the target nucleic acid molecule, a mismatch nucleotide at the −1 position from the 3′ end that is not complementary to the target nucleic acid molecule, and remaining nucleotides that are complementary to the target nucleic acid molecule;
• and
• a reverse primer of the test primer pair;
• (B) amplifying the target nucleic acid molecule from the biological sample by qPCR using a control set of primers in a control amplification, comprising
• the first primer pair;
• and
• a first control primer comprising a locked nucleic acid at the 3′ end that is complementary to a wildtype first allele corresponding to the mutant first allele, and remaining nucleotides that are the same as the first plus primer;
• (C) determining a threshold cycle (Ct) value of the test amplification and a Ct value of the control amplification;
• and
• (D) comparing a difference between the Ct values of the test and control amplifications with a standard control generated using a mixture of target nucleic acid molecules comprising a pre-selected proportion of the mutant and wildtype first alleles to detect the mutant first allele of the target nucleic acid molecule in the biological sample.