Method for the quantitative detection of nucleic acids

A method for the quantitative detection of a nucleic acid (target) from a sample, which comprises the following steps:

• a) extraction of the nucleic acid from the sample with another nucleic acid (calibrator) previously added to the sample itself, said calibrator having the same sequence of the target nucleic acid, said calibrator having i) the same sequence of the target nucleic acid, apart from the region hybridizing to the probe, which is randomized with respect to the corresponding region of the target nucleic acid, maintaining the same nucleotide composition, and ii) a Tm equal to the target nucleic acid Tm +/−4° C. with respect to the corresponding region of the target nucleic acid, having the same nucleotide composition, but with a random sequence, and a similar Tm,
• b) mixing the extracted target nucleic acid and calibrator with primers (forward and reverse) annealing to the corresponding regions on the calibrator and on the target nucleic acid, with probes bearing a reporter and a quencher and annealing to the target nucleic acid and to the corresponding randomized region on the calibrator, and with a nucleic acid polymerase with 5′-3′ nuclease activity, in suitable conditions to carry out a polymerization reaction, and
• c) determination of the signal associated with the reporters released due to the 5′ polymerase nuclease activity.

A method for the quantitative detection of a nucleic acid (target) from a sample, which comprises the following steps:

• a) extraction of the nucleic acid from the sample with another nucleic acid (calibrator) previously added to the sample itself, said calibrator having the same sequence of the target nucleic acid, said calibrator having i) the same sequence as the target nucleic acid, apart from the regions hybridizing to the probe or to the primers, which are randomized with respect to the corresponding regions of the target nucleic acid, maintaining the same nucleotide composition, and ii) a Tm equal to the target nucleic acid Tm +/−4° C.,
• b) mixing the extracted target nucleic acid and calibrator with primers (forward and reverse) annealing to the target nucleic acid and to the corresponding randomized regions on the calibrator, with probes bearing a reporter and a quencher and annealing to the target nucleic acid and to the corresponding randomized region on the calibrator, and with a nucleic acid polymerase with 5′-3′ nuclease activity, in suitable conditions to carry out a polymerization reaction, and
• c) determination of the signal associated with the reporters released due to the 5′ polymerase nuclease activity.

Kit for the quantitation of a nucleic acid from a sample, comprising one or more calibrators, a probe specific for each target nucleic acid and a probe specific for the calibrator, two or more primers and a thermostable nucleic acid polymerase with 5′-3′ nuclease activity.