Rolling Circle Extension-Actuated Loop-Mediated Isothermal Amplification (RCA-LAMP) for Ultrasensitive Detection of MicroRNAs

Abstract

Rolling circle amplification (RCA) is an elegant and well-recognized isothermal nucleic acid amplification mechanism that has been widely used for the detection of various kinds of genetic biomarkers. However, traditional RCA is a linear signal amplifying mechanism so that the amplification efficiency is generally not satisfactory. Herein, we rationally combine RCA with efficient loop-mediated isothermal amplification (LAMP) to establish a rapid and ultrasensitive RCA-LAMP method for the detection of microRNAs (miRNAs). In the RCA-LAMP, the target let-7a miRNA can directly template the ligation of a padlock probe to trigger RCA reaction, producing long and tandem amplification products. Only such RCA-produced long DNA repeats can act as the template to generate a lot of double stem-loop DNAs with functional sequences, which are the essential starting materials to initiate subsequent LAMP. Finally, the products of LAMP reaction, the amount of which is dependent on the initial miRNA dosage, can be fluorescently monitored in a real-time manner. Through the combination of ligation-mediated RCA with LAMP, the amplification efficiency and the detection sensitivity has been significantly improved. As a result, even 10 aM miRNA target can be clearly and accurately detectable. Despite the excellent analytical performance for miRNA analysis, compared with conventional RCA-based miRNA assays, the combination of RCA with LAMP does not introduce any additional reaction steps or sample transfer operations. Both the RCA and LAMP are fulfilled in a single step. Therefore, this facile and ultrasensitive RCA-LAMP assay provides a new promising tool for miRNA analysis and can be extended to the detection of various kinds of genetic biomarkers.