Real-time detection of nucleic acid targets, comprises providing a primer containing a sequence-target binding sequence to amplify nucleic acid, introducing a fluorophore and providing a probe for detection, which carries a fluorophore

摘要

Real-time detection of nucleic acid targets as products of a nucleic acid-amplification reaction by a primer/probe system, comprises providing at least a primer, which contains at least one sequence-target binding sequence (TBS) to specifically amplify nucleic acid and carries modification at the 3'-end, which allows an enzymatic extension complementary to the target; introducing a fluorophore 1; and providing at least a probe for the detection, which carries a fluorophore 2 at least at the 3'-end, where one of both fluorophores is stimulated by light of suitable wavelength. Real-time detection of nucleic acid targets as products of a nucleic acid-amplification reaction by a primer/probe system, comprises providing at least a primer, which contains at least one sequence-target binding sequence (TBS) specifically to amplify nucleic acid and carries modification at the 3'-end, which allows an enzymatic extension complementary to the target; introducing a fluorophore 1; and providing at least a probe for detection, which carries a fluorophore 2 at least at the 3'-end, where at least one probe with the complementary strand is extended to the target sequence at the 3'-end primer or use of more primers hybridized with the target strand, and one of both fluorophores is stimulated by light of suitable wavelength in such a way a fluorescence resonance energy transfer takes place between fluorophore 1 and 2, which are detected by optical fluorescence method. Independent claims are included for: (1) the primer/probe system for the detection of nucleic acids, comprising: (a) at least a primer containing at least one sequence specific for the assigned nucleic acid target and exhibiting modification at the 3'-end, which allows an enzymatic extension, where optionally one of the primer is marked with a fluorophore 1; and (b) at least a probe, which at least carries a fluorophore 2 and optionally exhibits a further fluorophore 1; (2) a reagent kit for the detection of nucleic acids, comprising at least a primer/probe system and a suitable buffer and optionally further auxiliary materials; (3) a device system for the performance of real-time polymerase chain reaction (PCR) reactions, which is fully automated computer-assisted technique, comprising: thermo-cycler, which exhibits a reaction range with several temperaturable recordings for the reaction containers; optionally an immobilization system for micro-particles; a reaction range assigned lighting mechanisms (light emitting diode and/or laser) with which the stimulated light is radiationable; detector installation, which produces measured values as a function of a measured light intensity with optical installation; optionally a reference arrangement, which produces a reference value; and an evaluator, where the reaction containers contain optically transparent container bottom and/or container cap with low characteristic fluorescence, in which the fluorescence optical analysis facilitates during the thermocyclic or isothermic performance of the detection reaction; (4) a reaction container for PCR with transparent container bottom; and (5) an immuno-PCR, which conducts multiplex-immune reactions under the use of non-fluorescent beads, porous or non-porous two-dimensional or small rod-shaped carrier materials and the analyte specific amplification product of fluorescence coded microparticle.