A Simple Method for Amplifying RNA Targets (SMART)

作者：McCalla Stephanie E; Ong Carmichael; Sarma Aartik; Opal Steven M; Artenstein Andrew W; Tripathi Anubhav^*

来源：Journal of Molecular Diagnostics, 2012, 14(4): 328-335.

DOI：10.1016/j.jmoldx.2012.02.001

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation micro-channel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl2 are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (<= 100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem. (J Mol Diagn 2012, 14:328-335; http://dx.doi.org/10.1016/j.jmoldx.2012.02.001)

• 出版日期2012-7