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

1. A molecular beacon probe, comprising: a stem comprising one or more unmodified nucleotides, and in the 3′ strand of the stem, one or more nucleotides or nucleotide analogues having an affinity increasing modification, wherein said one or more nucleotides or nucleotide analogues are selected from the group consisting of a 2′-O-derivatized nucleotide, a locked nucleic acid, and a peptide nucleic acid, wherein each base pair of said stem comprises no more than one 2′-O-derivatized nucleotide, and further wherein said molecular beacon probe has better stability and does not open spontaneously in the presence of contaminants present in an amplification enzyme mixture as compared to a molecular beacon probe without said stem.

2. The molecular beacon probe as claimed in claim 1 , wherein the 2′-O-derivatized nucleotide is a 2′-O-methyl-nucleotide.

3. The molecular beacon probe as claimed in claim 1 , wherein at least one base pair of said stem contains no nucleotide or nucleotide analogue having an affinity increasing modification.

4. The molecular beacon probe as claimed in claim 1 , wherein only one base pair of said stem comprises no nucleotide or nucleotide analogue having an affinity increasing modification.

5. A kit for performing a diagnostic amplification assay, comprising the appropriate primers, polymerase(s) and reagents for performing amplification of an analyte to be diagnosed and the molecular beacon probe of claim 1 for detecting the amplified analyte.

6. A molecular beacon probe comprising a stem and a loop, wherein said loop comprises: one or more nucleotides and/or nucleotide analogues that have an affinity increasing modification, and one or more unmodified nucleotides; and said stem comprises one or more unmodified nucleotides, and in the 3′ strand of the stem, one or more 2′-O-methyl nucleotides, wherein each base pair of said stem comprises no more than one 2′-O-methyl nucleotide, wherein the sensitivity of said molecular beacon probe with said loop to polymorphisms in the target nucleic acid sequence is lowered as compared to a molecular beacon probe without said loop and wherein the spontaneous opening of the molecular beacon probe with said stem in the presence of contaminants present in an amplification enzyme mixture is lowered as compared to a molecular beacon probe without said stem.

7. The molecular beacon probe as claimed in claim 6 , wherein the nucleotides or nucleotide analogues having an affinity increasing modification are selected from the group consisting of 2′-O-derivatized nucleotides, locked nucleic acids, and peptide nucleic acids.

8. The molecular beacon probe as claimed in claim 6 , wherein at least one base pair of said stem contains no nucleotide or nucleotide analogue having an affinity increasing modification.

9. The molecular beacon probe as claimed in claim 6 , wherein only one base pair of said stem comprises no nucleotide or nucleotide analogue having an affinity increasing modification.

10. A kit for performing a diagnostic amplification assay, comprising the appropriate primers, polymerase(s) and reagents for performing amplification of an analyte to be diagnosed and the molecular beacon probe of claim 6 for detecting the amplified analyte.

11. A molecular beacon probe, comprising: a stem comprising one or more nucleotides or nucleotide analogues having an affinity increasing modification, wherein said one or more nucleotides or nucleotide analogues are selected from the group consisting of a 2′-O-derivatized nucleotide, a locked nucleic acid, and a peptide nucleic acid, and one or more unmodified nucleotides, wherein each base pair of said stem comprises no more than one 2′-O-derivatized nucleotide and only one base pair of said stem comprises no nucleotide or nucleotide analogue having an affinity increasing modification, and further wherein said molecular beacon probe with said stem has better stability and does not open spontaneously in the presence of contaminants present in an amplification enzyme mixture as compared to a molecular beacon probe without said stem.

12. The molecular beacon probe as claimed in claim 11 , wherein the 2′-O-derivatized nucleotide is a 2′-O-methyl-nucleotide.

13. A molecular beacon probe comprising a stem and a loop, wherein said loop comprises: one or more nucleotides and/or nucleotide analogues that have an affinity increasing modification, and one or more unmodified nucleotides; and said stem comprises: one or more 2′-O-methyl nucleotides, and one or more unmodified nucleotides, wherein each base pair of said stem comprises no more than one 2′-O-methyl nucleotide and only one base pair of said stem comprises no nucleotide or nucleotide analogue having an affinity increasing modification,

wherein the sensitivity of said molecular beacon probe with said loop to polymorphisms in a target nucleic acid sequence is lowered as compared to a molecular beacon probe without said loop and wherein the spontaneous opening of said molecular beacon probe with said stem in the presence of contaminants present in an amplification enzyme mixture is lowered as compared to a molecular beacon probe without said stem.

14. A molecular beacon probe, comprising: a stem comprising one or more unmodified nucleotides, and in each strand of said stem, at least one nucleotide or nucleotide analogue having an affinity increasing modification, wherein said at least one nucleotide or nucleotide analogue is selected from the group consisting of a 2′-O-derivatized nucleotide, a locked nucleic acid, and a peptide nucleic acid, wherein each base pair of said stem comprises no more than one 2′-O-derivatized nucleotide, and further wherein said molecular beacon probe with said stem has better stability and does not open spontaneously in the presence of contaminants present in an amplification enzyme mixture as compared to a molecular beacon probe without said stem.

15. The molecular beacon probe as claimed in claim 14 , wherein the 2′-O-derivatized nucleotide is a 2′-O-methyl-nucleotide.

16. The molecular beacon probe as claimed in claim 14 , wherein only one base pair of said stem comprises no nucleotide or nucleotide analogue having an affinity increasing modification.

17. A molecular beacon probe comprising a stem and a loop, wherein said loop comprises: one or more nucleotides and/or nucleotide analogues that have an affinity increasing modification, and one or more unmodified nucleotides; and said stem comprises: one or more unmodified nucleotides, and in each strand of said stem, at least one 2′-O-methyl nucleotide, wherein each base pair of said stem comprises no more than one 2′-O-methyl nucleotide, wherein the sensitivity of said molecular beacon probe with said loop to polymorphisms in a target nucleic acid sequence is lowered as compared to a molecular beacon probe without said loop and wherein the spontaneous opening of said molecular beacon probe with said stem in the presence of contaminants present in an amplification enzyme mixture is lowered as compared to a molecular beacon probe without said stem.

18. The molecular beacon probe as claimed in claim 17 , wherein only one base pair of said stem comprises no nucleotide or nucleotide analogue having an affinity increasing modification.

19. A method for assessing the presence of a nucleic acid analyte in a sample using a molecular beacon probe, the method comprising 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 using the molecular beacon probe of claim 1 , thereby assessing the presence of nucleic acid analyte in the sample.

20. The method of claim 19 wherein the method is a homogenous assay.

21. The method of claim 19 wherein the method is a heterogeneous assay.

22. A method for assessing the presence of a nucleic acid analyte in a sample using a molecular beacon probe, the method comprising 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 using the molecular beacon probe of claim 6 , thereby assessing the presence of nucleic acid analyte in the sample.

23. The method of claim 22 wherein the method is a homogenous assay.

24. The method of claim 22 wherein the method is a heterogeneous assay.