A method for detection of the presence of a target polynucleotide in a sample, said method comprising a) providing a mixture of said sample, nucleic acid amplification reagents and a target binding agent, said target binding agent being capable of assuming a first position where said target binding agent is not bound to said target polynucleotide and a second position where said target binding agent is bound to said target polynucleotide, and a signal is measurable from said mixture, the intensity of said signal depending on the proportions of target binding agent being in said first and second positions; b) exposing said mixture to conditions that allow nucleic acid amplification to occur in said mixture and allowing said amplification to complete; c) exposing said mixture to a first condition by heating said mixture, at which first condition said target binding agent and all nucleic acids are in denaturated state and the target binding agent is not hybridized, resulting in that said target binding agent is in said first position, the temperature of the first condition is higher than that of the second condition in step d); d) exposing said mixture to a second condition by lowering the temperature of said mixture, at which second condition said target binding agent is hybridized with the target polynucleotide, resulting in that said target binding agent is in said second position, e) measuring at least once, after completion of said nucleic acid amplification and after said mixture has been exposed to said first condition, said signal of the mixture at a first time when less than in step f) of said target binding agent present in said mixture has assumed the second position; f) measuring at least once, after completion of said nucleic acid amplification and after said mixture has been exposed to said second condition, said signal of the mixture at a second time when, if the sample contains said target polynucleotide, more than in step e) of said target binding agent has assumed the second position; and g) determining the ratio of the measurement results obtained in steps e) and f) and comparing it to a pre-determined cut-off value that is characteristic for said mixture of sample and target binding agent to determine whether said target polynucleotide is present in the sample or not. a) providing a mixture of said sample, nucleic acid amplification reagents and a target binding agent, said target binding agent being capable of assuming a first position where said target binding agent is not bound to said target polynucleotide and a second position where said target binding agent is bound to said target polynucleotide, and a signal is measurable from said mixture, the intensity of said signal depending on the proportions of target binding agent being in said first and second positions; b) exposing said mixture to conditions that allow nucleic acid amplification to occur in said mixture and allowing said amplification to complete; c) exposing said mixture to a first condition by heating said mixture, at which first condition said target binding agent and all nucleic acids are in denaturated state and the target binding agent is not hybridized, resulting in that said target binding agent is in said first position, the temperature of the first condition is higher than that of the second condition in step d); d) exposing said mixture to a second condition by lowering the temperature of said mixture, at which second condition said target binding agent is hybridized with the target polynucleotide, resulting in that said target binding agent is in said second position, e) measuring at least once, after completion of said nucleic acid amplification and after said mixture has been exposed to said first condition, said signal of the mixture at a first time when less than in step f) of said target binding agent present in said mixture has assumed the second position; f) measuring at least once, after completion of said nucleic acid amplification and after said mixture has been exposed to said second condition, said signal of the mixture at a second time when, if the sample contains said target polynucleotide, more than in step e) of said target binding agent has assumed the second position; and g) determining the ratio of the measurement results obtained in steps e) and f) and comparing it to a pre-determined cut-off value that is characteristic for said mixture of sample and target binding agent to determine whether said target polynucleotide is present in the sample or not.