A nucleic acid detection method involving the direct generation of a measurable signal, by the action of an enzyme with 3′-5′ nuclease activity, and the applications for it, the signal generated can be detectable and quantifiable and can be carried out in real-time, in this method, the nucleic acid is placed in contact with at least one oligonucleotide that does not hybridize perfectly with it, so that the enzyme will split it at the unpaired bases generating the signal, the oligonucleotide can be labeled.