Verfahren zum Nachweis von Nukleotidsequenzen, Verwendung des Verfahrens und Testbesteck

Method for detecting at least one target nucleotide sequence (I) that uses unlabeled, free oligonucleotides; labeled, free oligonucleotides and unlabeled, immobilized oligonucleotides able to hybridize to at least one (I). Detecting at least one target nucleotide sequence (I) comprises: (a) provision of unlabeled, free oligonucleotides (NMO); labeled, free oligonucleotides (MO) and unlabeled, immobilized oligonucleotides (CO) able to hybridize to at least one (I); (b) providing test material, i.e. (I), in the reaction chamber (b1) or preparation of (I) from the sample by reverse transcription (b2) in which RNA is used to generate labeled and/or unlabeled (I); (c) amplification (d1) of a mixture comprising b1, NMO, MO and CO to produce labeled amplicon, non-labeled amplicon and capture amplicon, or (d2) abandonment of amplification in so far as the target sequence of (b2) is recovered in sufficient amount by reverse transcription using MO, and in this case CO is used for preparation of MO; (d) hybridization of labeled amplicon (d1) with CO (e1), where the density of CO is high enough so that in the immediate vicinity of CO a reduction in concentration of free labeled and unlabeled amplicons occurs, i.e. saturation of CO is not the limiting factor for hybridization of labled amplicons, or (e2) hybridization of sequences (d2) with CO; and (e) detection of e1 or e2, and after step (b) all reagents needed for the process are present in a reaction chamber and none are added during the process. An independent claim is included for a test kit containing, in a single reaction chamber, NMO, MO and CO, also carrier for CO and reagents.