Method for lowering both sequence variations and increase of base lines effects in diagnostic hybridisation assay, assay for performing such a method and probe for use in the assay

Use in a diagnostic hybridization assay of a probe for lowering the effect of sequence variations in a nucleic acid analyte, which assay comprises the steps of contacting a set of primers and a sample containing the nucleic acid analyte to amplify the analyte and detecting the amplified analyte or its complement by means of the probe, characterized in that the probe comprises:

• one or more nucleotides and/or nucleotide analogues, selected from 2′-O-methyl nucleotides or LNA nucleotides, that have an affinity increasing modification and the diagnostic assay is for assessing the amount of analyte present in the sample, and
• one or more unmodified nucleotides.

Use in a diagnostic hybridization assay of a probe for lowering the effect of sequence variations in a nucleic acid analyte, which assay comprises the steps of contacting a set of primers and a sample containing the nucleic acid analyte to amplify the analyte and detecting the amplified analyte or its complement by means of the probe, characterized in that the probe comprises:

• one or more nucleotides and/or nucleotide analogues, selected from 2′-O-methyl nucleotides or LNA nucleotides, that have an affinity increasing modification, i.e. at a constant temperature of hybridization, the melting temperature of the probe with any possible analyte's polymorphism is increased compared to the melting temperature of an unmodified probe with any analyte's polymorphism and the diagnostic assay is for assessing the presence of the analyte in the sample
• one or more unmodified nucleotides.

Use in a diagnostic hybridization assay of a molecular beacon probe for lowering the possible opening of the stem of the molecular beacons by way of at least one contaminant present in the amplification enzymes mixture, which assay comprises the steps of contacting a set of primers and a sample containing the nucleic acid analyte to amplify the analyte and detecting the amplified analyte or its complement by means of the probe, characterized in that the probe's stem comprises:

• one or more nucleotides and/or nucleotide analogues that have an affinity increasing modification, especially 2′-O-methyl nucleotides, and
• one or more unmodified nucleotides.

Molecular beacon probe for use in a diagnostic hybridization assay, said probe comprising one or more nucleotides and/or nucleotide analogues, selected from 2′-O-methyl nucleotides, that have an affinity increasing modification, i.e. at a constant temperature of hybridization, the melting temperature of the probe with any possible analyte's polymorphism is increased compared to the melting temperature of an unmodified probe with the same target.

Molecular beacon probe for use in a diagnostic hybridization assay, said probe allowing the lowering of the possible opening of the stem-loop structure of the molecular beacons by way of at least one contaminant present in the amplification enzymes mixture, which assay comprises the steps of contacting a set of primers and a sample containing the nucleic acid analyte to amplify the analyte and detecting the amplified analyte or its complement by means of the probe, characterized in that the probe's stem comprises:

• one or more 2′-methyl nucleotides, and
• one or more unmodified nucleotides.