Exonuclease cycling assay

A method for detecting a target polynucleotide in a test sample, wherein the target polynucleotide has a target sequence and is protected against digestion by a 5′ exonuclease, wherein the 5′ exonuclease is lambda exonuclease, comprising

• (a) incubating the test sample with a probe having a sequence complementary with the target sequence and lacking a phosphorylated 5′ end, wherein the probe has a quencher at its 5′ end and a fluor, whereby a hybrid of the probe and the target polynucleotide is formed, wherein the hybrid comprises a hybridization region formed between the target sequence and the probe, wherein the fluor of the probe is quenched by the quencher before forming the hybrid, and wherein the fluor of the probe is quenched by the quencher in the hybrid;
• (b) exposing the hybrid to the 5′ exonuclease to digest the probe in the hybrid and forming a reaction mixture, whereby a digested probe comprising the fluor is generated, wherein the fluorescence of the fluor of the digested probe is not quenched;
• (c) dissociating the target polynucleotide from the digested probe in the reaction mixture;
• (d) repeating steps (a)-(c) by adding the probe and the 5′ exonuclease to the reaction mixture after step (c), wherein step (b) of step (d) is performed for a first time period until no further increase in the fluorescence in the reaction mixture;
• (e) determining a first fluorescence intensity of the fluor in the reaction mixture after step (d);
• (f) repeating steps (a)-(d) with a control sample in place of the test sample, wherein step (b) in step (f) is performed for a second time period, wherein the second time period equals to the first time period, wherein the control sample is identical to the test sample except without the target polynucleotide;
• and
• (g) determining a second fluorescence intensity of the fluor in the control sample after step (f), wherein the first fluorescence intensity greater than the second fluorescence intensity indicates the presence of the target polynucleotide in the test sample.