Highly specific DNA detection from massive background nucleic acids based on rolling circle amplification of target dsDNA

摘要: An advanced rolling circle amplification (RCA) strategy based on the target-circularization of targeted double-stranded DNA (dsDNA) was established. Different from traditional padlock-RCA, in which single-stranded probe circularized and amplified as signal amplification, our new approach could amplify a target. This special circularization target realized by ligation with biotin-labelled duplex adaptor containing 9 nt sticky ends complementary base pairing. High specificity obtained using two primers targeting sequence but not itself padlock-RCA. With help streptavidin magnetic beads that immobilized ligated dsDNA amplicon, background nucleic acids contributing most to non-specific were eliminated. Under optimized conditions, less than 60 copies be detected presence massive (>1012 unrelated sequences). The sensitivity can rival canonical PCR. Without thermal cycles, reduced handling simpler equipment requirements render this assay simple rapid alternative conventional methods. Based these advantages, method is promising candidate practical applications such detecting contaminated food-borne pathogens comprehensive food samples.