Use of complementary single stranded fluorescent reporter complexes in pcr methods

An in vitro PCR method to detect and/or quantify, in a biological sample comprising nucleic acid molecules, the presence of at least one nucleic acid target sequence (TSi), said method comprising:

• A) contacting the sample with a set containing, for each TSi:
• i) at least one mediator probe containing:
• a 3′ terminal probe region which is complementary to said target sequence (TSi),
• a 5′ terminal mediator region containing one tag sequence, and
• a biological cleavage site located between the mediator region and the probe region, whereby an enzyme with nuclease activity can mediate the cleavage of the two regions during the amplification process of the nucleic acid target sequence (TSi)
• and
• ii) at least one Complementary Single Stranded Fluorescent Reporter (CSSFR) complex, said reporter complex containing:
• a main reporter oligonucleotide molecule (m-reporter) containing at least one sequence that is complementary to the tag sequence (“tag complementary sequence”, TCS) carried by a mediator probe of the set associated to said TSi, and, at its 5′ end, a reporter molecule which may be either a fluorophore or a quencher group;
• a complement reporter oligonucleotide molecule (c-reporter) containing a sequence that is complementary to the 5′ end sequence of the m-reporter and:
• a fluorophore group in case the m-reporter has a quencher group or at least one fluorophore group, or
• a quencher group in case the m-reporter has a fluorophore group,
• wherein the fluorescence of the fluorophore group of said c-reporter or m-reporter molecule is modified as a result of the hybridization of, or of the elongation of, the associated tag sequence of the 5′ terminal mediator region once cleaved from the mediator probe, on the tag complementary sequence (TCS) on the m-reporter,
• B) amplifying said at least one nucleic acid target in the presence of an enzyme with nuclease activity,
• C) detecting or measuring the intensity of fluorescence for each fluorophore group,
• D) optionally, processing the data collected in step C) in order to quantify the concentration of the at least one TSi in said biological sample.