Long-tail probe-mediated cycled strand displacement amplification: Label-free, isothermal and sensitive detection of nucleic acids

We design a long-tail shaped DNA probe for the label-free, isothermal and sensitive detection of nucleic acids based on cycled strand displacement amplification (Ltail-CSDA). The long-tail probe, a stem-loop structure with a long poly(T) tail at 5′-termini, integrates target-binding and amplification and signaling within one multifunctional design. The specific binding between the long-tail probe and the target triggers a polymerization reaction, during which a long dsDNA product is synthesized and the hybridized target is displaced by the strand displacement activity of polymerase. The displaced target forms another specific probe-target binding and prompts cycled polymerization reactions. The proposed Ltail-CSDA has the distinct advantages of its isothermal nature, free-label, simplicity and attomolar sensitivity compared with other existing technologies. More significantly, the dynamic range of the method is extremely large, covering nine orders of magnitude. Using total RNA samples extracted from hepatitis C virus (HCV) as targets, we further demonstrate the detection capability of the method for complex nucleic acid samples, indicating its potential applicability for clinic molecular diagnostic assays.