Single Primer Based Multisite Strand Displacement Reaction Amplification Strategy for Rapid Detection of Terminal Deoxynucleotidyl Transferase Activity.

A fluorescence-based multisite strand displacement reaction (MSSDR) amplification strategy is developed for the rapid, sensitive, and selective detection the activity of terminal deoxynucleotidyl transferase (TdT). Oligo dT primer was used for the TdT extension reaction, then the left oligo dT primers were hybridized to the TdT extension reaction product by end to end tiled style and initiated the MSSDR by Klenow polymerase, subsequently, 3' terminals of these single-strand DNA produced by MSSDR are folded back to complement themselves with the adjacent sequences, and Klenow polymerase makes it into double-stranded DNA (dsDNA). The final dsDNA products were analyzed via dsDNA specific fluorescent dye. This method enables rapid (less than 100 min) and sensitive (limit of detection, LOD, 1.35 × 10-5 U) detection and has been demonstrated to work well using a real biosample. Our design would not only serve as a new prototype for high-throughput automated analysis and clinic diagnostic application but also has promising potential for improving the sensitivity of those TDT related biosensing system.