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Abstract

The present invention relates to a method for detecting the amount of a target polynucleotide in a sample. A combination is provided in a medium. The combination comprises (i) a sample suspected of containing the target polynucleotide, the target polynucleotide being in single stranded form, (ii) a reference polynucleotide comprising a sequence that is common with a sequence of the target polynucleotide, and (iii) a predetermined amount of an oligonucleotide probe that has a sequence that hybridizes with the sequence that is common. The combination is subjected to conditions for amplifying the target polynucleotide and the reference polynucleotide. The conditions permit formation of substantially non-dissociative complexes of the target polynucleotide and the reference polynucleotide, respectively, with the oligonucleotide probe. Furthermore, the predetermined amount of the oligonucleotide probe is less than the expected amount of the amplified target polynucleotide. The ratio of the amount of the complex of the target polynucleotide with the oligonucleotide probe to the amount of the complex of the reference polynucleotide with the oligonucleotide probe is determined. Determination of the ratio is facilitated by employing second and third oligonucleotide probes. The second oligonucleotide probe has a sequence that hybridizes only with the second sequence of the target polynucleotide. The third oligonucleotide probe has a sequence that hybridizes only with a respective second sequence of the reference polynucleotide. The ratio is related to the known amount of the reference polynucleotide to determine the amount of the target polynucleotide in the sample. One or more reference polynucleotides may be employed with a corresponding third oligonucleotide probe for each reference probe. Kits for carrying out the above methods are also disclosed. The method is particularly applicable to the amplification and detection of RNA.

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First Claim