The present invention provides methods of detecting a target nucleic acid in a sample using a duplex-specific nuclease (DSN), such as Kamchatka crab nuclease or RNaseH, and compositions for DSN reactions. For example, a composition of the invention may include a sample having a target nucleic acid, a nucleic acid probe, a DSN, and a buffer, and the composition may be maintained at about 90° C. to about 97° C. The target nucleic acid may be detected, for example, by hybridizing the target nucleic acid to a detection probe and digesting the resultant duplex using the duplex-specific nuclease, thus releasing a detectable component of the probe, which can be separated from unbound probe for detection or detected in situ. The invention also features methods of catalyzing hybridization or stabilizing hybridization between nucleic acid strands using DSNs. The methods and compositions described herein are therefore useful for rapid, efficient, sensitive, and accurate detection of target nucleic acids in a variety of applications, including, for example, diagnostic tests and laboratory assays.
