Dual-Gene-Controlled Rolling Circle Amplification Strategy for SARS-CoV-2 Analysis.

The development of sensitive, accurate, and conveniently operated methods for the simultaneous assay of two nucleic acids is promising while still challenging. In this work, by using two genes (the N gene and RdRp gene) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as examples, we have designed an ingenious dual-gene-controlled rolling circle amplification (RCA) strategy to propose an accurate and sensitive electrochemical method. Specifically, the coexistence of the two target genes can trigger the RCA reaction to generate a number of repeated G-quadruplex (G4)-forming sequences. These sequences then switch into G4/hemin complexes with redox activity after the incubation of hemin, which can catalyze the TMB/H2O2 substrates to produce significantly enhanced current responses. Experimental results reveal that the proposed method exhibits satisfying feasibility and analytical performance, enabling the sensitive detection of SARS-CoV-2 in the range of 0.1-5000 pM, with the detection limit of 57 fM. Meanwhile, because only the simultaneous existence of the two target genes can effectively trigger the downstream amplification reaction, this method can effectively avoid false-positives and ensure specificity as well as accuracy. Furthermore, our method can distinguish the COVID-19 samples from healthy people, and the outcomes show a satisfying agreement with the results of RT-PCR, manifesting that our label-free dual-gene-controlled RCA strategy exhibits great possibility in clinical application.

Electrochemical Gene Amplification Signal Detection of Disposable Biochips Using Electrodes.

Real-time Polymerase Chain Reaction (RT-PCR), a molecular diagnostic technology, is spotlighted as one of the quickest and fastest diagnostic methods for the actual coronavirus (SARS-CoV-2). However, the fluorescent label-based technology of the RT-PCR technique requires expensive equipment and a sample pretreatment process for analysis. Therefore, this paper proposes a biochip based on Electrochemical Impedance Spectroscopy (EIS). In this paper, it was possible to see the change according to the concentration by measuring the impedance with a chip made of two electrodes with different shapes of sample DNA.

Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription.

Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2 fM and 200 fM within 1 h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.

Isothermal gene amplification coupled MALDI-TOF MS for SARS-CoV-2 detection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide for more than a year and has undergone several mutations and evolutions. Due to the lack of effective therapeutics and long-active vaccines, accurate and large-scale screening and early diagnosis of infected individuals are crucial to control the pandemic. Nevertheless, the current widely used RT-qPCR-based methods suffer from complicated temperature control, long processing time and the risk of false-negative results. Herein, we present a three-way junction induced exponential rolling circle amplification (3WJ-eRCA) combined MALDI-TOF MS assay for SARS-CoV-2 detection. The assay can detect simultaneously the target nucleocapsid (N) and open reading frame 1 ab (orf1ab) genes of SARS-CoV-2 in a single test within 30 min, with an isothermal process (55 °C). High specificity to discriminate SARS-CoV-2 from other coronaviruses, like SARS-CoV, MERS-CoV and bat SARS-like coronavirus (bat-SL-CoVZC45), was observed. We have further used the method to detect pseudovirus of SARS-CoV-2 in various matrices, e.g. water, saliva and urine. The results demonstrated a great potential of the method for large scale screening of COVID-19, which is an important part of the pandemic control.

An update on ACE2 amplification and its therapeutic potential.

The renin angiotensin system (RAS) plays an important role in the pathogenesis of variety of diseases. Targeting the formation and action of angiotensin II (Ang II), the main RAS peptide, has been the key therapeutic target for last three decades. ACE-related carboxypeptidase (ACE2), a monocarboxypeptidase that had been discovered 20 years ago, is one of the catalytically most potent enzymes known to degrade Ang II to Ang-(1-7), a peptide that is increasingly accepted to have organ-protective properties that oppose and counterbalance those of Ang II. In addition to its role as a RAS enzyme ACE2 is the main receptor for SARS-CoV-2. In this review, we discuss various strategies that have been used to achieve amplification of ACE2 activity including the potential therapeutic potential of soluble recombinant ACE2 protein and novel shorter ACE2 variants.

Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: Getting the message across.

The recent approval of messenger RNA (mRNA)-based vaccines to combat the SARS-CoV-2 pandemic highlights the potential of both conventional mRNA and self-amplifying mRNA (saRNA) as a flexible immunotherapy platform to treat infectious diseases. Besides the antigen it encodes, mRNA itself has an immune-stimulating activity that can contribute to vaccine efficacy. This self-adjuvant effect, however, will interfere with mRNA translation and may influence the desired therapeutic outcome. To further exploit its potential as a versatile therapeutic platform, it will be crucial to control mRNA's innate immune-stimulating properties. In this regard, we describe the mechanisms behind the innate immune recognition of mRNA and provide an extensive overview of strategies to control its innate immune-stimulating activity. These strategies range from modifications to the mRNA backbone itself, optimization of production and purification processes to the combination with innate immune inhibitors. Furthermore, we discuss the delicate balance of the self-adjuvant effect in mRNA vaccination strategies, which can be both beneficial and detrimental to the therapeutic outcome.

A straightforward molecular strategy to retrospectively investigate the spread of SARS-CoV-2 VOC202012/01 B.1.1.7 variant.

The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of 4 RNA viral genomes demonstrated that those curves were associated with B.1.1.7 variant strains. Our results strongly suggest that B.1.1.7 variant spread has begun in this area at least since January and imply the potential of these analytical methods to track and characterize the spread of B.1.1.7 strains in those areas where Allplex SARS-CoV-2 datasets have been previously recorded.

Disentangling primer interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR.

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.