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The present invention relates to a novel multiplex detection of nucleic acid targets in a sample. This is accomplished by using novel padlock probes which contain a unique cleavage site for linearization and a first member of a binding pair for isolation of the padlock probe from the sample. Three differ ent designs of the padlock probes are described and examples are shown for ligation, amplification and qualitative and quantitative detection. The combination of these features gives a fast, accurate and specific multiplex detection assay