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Construction of a Quencher-Free Cascade Amplification System for Highly Specific and Sensitive Detection of Serum Circulating miRNAs
journal contribution
posted on 2020-05-25, 04:48 authored by Li-juan Wang, Ming Ren, Hou-xiu Wang, Jian-Ge Qiu, BingHua Jiang, Chun-yang ZhangCirculating
miRNAs are a newly emerging class of noninvasive biomarkers,
and the accurate quantification of their expression is essential to
the biological research and early clinic diagnosis. Herein, we demonstrate
the construction of a quencher-free cascade amplification system for
highly sensitive detection of serum circulating miRNAs. The target
miRNA can hybridize with the linear probe to induce the cyclic strand
displacement amplification (SDA) (cycle I) for the production of the
binding probes. The binding probe can subsequently react with the
2-aminopurine (2-AP)-hairpin probe to induce the recycling exonuclease
cleavage of 2-AP-hairpin probes (cycle II), releasing the triggers
and 2-AP molecules simultaneously. The released trigger can hybridize
with the free linear probe to start new cycles I and II amplifications.
Through multiple rounds of cascade amplifications, a large number
of 2-AP molecules are released, generating an enhanced fluorescence
signal. This method exhibits a large dynamic range of 8 orders of
magnitude and a detection limit of 0.16 aM. It can differentiate a
single-base mismatch in miR-486-5p, quantify miR-486-5p in lung cancer
cells at various stages, and even discriminate the expressions of
serum circulating miR-486-5p in healthy persons from that in nonsmall-cell
lung carcinoma (NSCLC) patients. Moreover, this assay can be rapidly
carried out in one step under isothermal condition in a label-free
manner, holding promising applications in point-of-care diagnosis
and prognosis of lung cancers.