Unified-amplifier based primer exchange reaction (UniAmPER) enabled detection of SARS-CoV-2 from clinical samples

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, H 2 O 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.

Malachite Green-Based Detection of SARS-CoV-2 by One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

“…These methods have been validated as a point of care (POC) test as well (Bodulev and Sakharov 2020 ). Methods such as loop-mediated isothermal amplification (LAMP) (Nzelu et al 2019 ), helicase-dependent amplification (HDA) (Ma et al 2017 ), rolling circle amplification (RCA) (Shen et al 2019 ), strand displacement amplification (SDA) (Becker et al 2019 ), recombinase polymerase amplification (RPA) (Rostron et al 2019 ), and nucleic acid sequence-based amplification (NASBA) (Zhai et al 2019 ), unified amplifier-based primer exchange reaction (UniAmPER) (Tavakoli-Koopaei et al 2022 ) are examples of isothermal amplification methods that have been developed in recent years for the detection of genetic materials of viruses. Among these methods, LAMP was able to achieve governmental approvals as IVD (Zhao et al 2016 ; Zhang et al 2015 ; Etchebarne et al 2017 ).…”

An Ultrasensitive Electrochemical Biosensor Integrated by Nicking Endonuclease-Assisted Primer Exchange Reaction Cascade Amplification and DNA Nanosphere-Mediated Electrochemical Signal-Enhanced System for MicroRNA Detection

“…19,20 More importantly, only when the right set of PER hairpin and the corresponding PER primer coexist is a new strand produced autonomously in situ, 21 which makes little signal leakage and thus effectively reduces the reaction background. Inspired by this specific reaction, the PER cascade has been extensively applied to highly sensitive detection of miRNAs by colorimetric, 22 fluorescent, 23 and electrochemical technologies. 24 Among them, the electrochemistry-based detection methods, especially the label-free strategies, have increasingly attracted attention due to the advantages of simplicity, speediness, high sensitivity, and repeatability.…”

“…The PER cascade uses a catalytic DNA hairpin (named as PER hairpin) as a template, and the short single-stranded DNA (ssDNA) primer can continually grow nascent ssDNA according to the prescribed reaction pathways with the help of the strand-displacing polymerase to achieve the stable amplification of target sequences. , More importantly, only when the right set of PER hairpin and the corresponding PER primer coexist is a new strand produced autonomously in situ, which makes little signal leakage and thus effectively reduces the reaction background. Inspired by this specific reaction, the PER cascade has been extensively applied to highly sensitive detection of miRNAs by colorimetric, fluorescent, and electrochemical technologies . Among them, the electrochemistry-based detection methods, especially the label-free strategies, have increasingly attracted attention due to the advantages of simplicity, speediness, high sensitivity, and repeatability. , Considering the unique characteristics of miRNAs, it is still a great challenge of combining diverse nuclease-assisted target amplification techniques in series for the multiple amplification and highly sensitive electrochemical detection of miRNAs.…”

A sensitive isothermal fluorescence biosensor for microRNA detection coupling primer exchange reaction with catalytic hairpin assembly

“…The PER has been used in a series of high-performance biosensors for proteins, DNA, and RNA in cells and tissues. 30–33…”