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Unified-Amplifier Based Primer Exchange Reaction (Uniamper) Enabled Detection of Sars-Cov-2 From Clinical Samples Publisher



Tavakolikoopaei R1 ; Javadizarnaghi F1 ; Mirhendi H2, 3
Authors
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Authors Affiliations
  1. 1. Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
  2. 2. Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Core Facilities Research Laboratory, Mycology Reference Laboratory, Isfahan University of Medical Sciences, Isfahan, Iran

Source: Sensors and Actuators B: Chemical Published:2022


Abstract

Primer exchange reaction (PER) is an emergent method for non-templated synthesis of single stranded DNA molecules. PER has been shown to be effective in cell imaging systems and for detection of macromolecules. A particular application of PER is to detect a specific target nucleic acid. To this endeavor, two coupled DNA hairpins, a detector and an amplifier, play in accordance to extend a target nucleic acid with a concatemer DNA sequence. Here we introduced unified-amplifier based primer exchange reaction (UniAmPER) that beneficially extends the target by a unified-amplifier. The unified-amplifier operates as both detector and amplifier hairpins. The extension resulted in synthesis of concatemer G-rich sequences. The G-rich sequences were expected to form G-quadruplex (GQ) structures. Presence of the GQ structures were investigated by peroxidase activity of GQs in presence of hemin, H2°2 and 3,3′,5,5′-Tetramethylbenzidine (TMB) as well as by fluorescence signal generation upon intercalation of thioflavin T (ThT). The presented unified-amplifier in this study facilitates application of PER systems for development of colorimetric or fluorogenic biosensors. As a proof of principle, the method has been applied for detection of reversely transcribed cDNAs from clinical SARS-CoV-2 samples. © 2022