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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.