Method for lowering the dependency towards sequence variation of a nucleic acid target in a diagnostic hybridization assay

A method of amplifying in a sample a nucleic acid target sequence containing at least one variation, defined as either one non-conserved nucleotide, called genotype variation, or one nucleotide variation within one and the same genotype, comprising performing on the sample an amplification reaction that amplifies the presence of this target sequence utilizing

• 1) a first primer capable of specifically hybridizing in a region of the target sequence and capable of directing, under extension conditions, extension of the first primer toward the hybridizing region of a second primer, and
• 2) the second primer capable of specifically hybridizing to a complementary sequence of another region of the target sequence and capable of directing, under extension conditions, extension of the second primer toward the complementary sequence of the hybridizing region of said first primer, and
• wherein said at least one variation is located in the hybridizing region of at least one of the primers, and
• wherein at least one of the primers comprises a nucleic acid sequence that is complementary to said target sequence except for the at least complementary base of the variation(s), which is not present.

A method of detecting in a sample a nucleic acid target sequence containing at least one variation, defined as either one non-conserved nucleotide, called genotype variation, or one nucleotide variation within one and the same genotype, comprising

• (a) performing on the sample an amplification reaction that detects the presence of this target sequence utilizing
• 1) a first primer capable of specifically hybridizing in a region of the target sequence or to a complementary region of the target sequence and capable of directing, under extension conditions, extension of the first primer toward the hybridizing region of a second primer, and
• 2) the second primer capable of specifically hybridizing in another region of the target sequence or to another complementary region of the target sequence and capable of directing, under extension conditions, extension of the second primer toward the complementary sequence of the hybridizing region of said first primer, and
• (b) performing on the sample a detection reaction that detects the presence of said target sequence, with at least one probe capable of specifically hybridizing to a region of the target sequence between the region with which the first primer is capable of hybridizing and the region with which the second primer is capable of hybridizing,
• wherein said at least one variation is located in the hybridizing region of at least one of the primers, and
• wherein at least one of the primers comprises a nucleic acid sequence that is complementary to said target sequence except for the at least complementary base of the variation(s), which is not present.

A method of detecting in a sample a nucleic acid target sequence containing at least one variation, defined as either one non-conserved nucleotide, called genotype variation, or one nucleotide variation within one and the same genotype, comprising

• (a) at least one probe capable of specifically hybridizing to a region of the target sequence, and
• (b) detects the presence of said target sequence,
• wherein said at least one variation is located in the hybridizing region of at least one of the probe(s), and
• wherein at least one of the probes comprises a nucleic acid sequence that is complementary to said target sequence except for the at least complementary base of the variation(s), which is not present.