Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

Abstract

The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.

Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers

“…Moreover, Soo-PCR is user-friendly and can be used as an endpoint diagnosis without requiring a qPCR device, thanks to its on–off system. Therefore, it can be combined with various nano-based diagnostic platforms, leading to potential synergistic effects [ 44 , 45 ].…”

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“…The genome starts at 3' with an extragenic area of 44 nucleotides before the NS1 gene and ends with another extragenic region of 155 nucleotides after the L gene. The first nine genes are separated by a dynamics of other respiratory viruses such as influenza and, more recently, SARS-CoV-2, which involves the continuous emergence of new variants, the introduction of the same and new populations, and the extinction of the previous ones [41].…”

“…RSV's mutation rate, like that of other RNA viruses, is significant, resulting in constant genetic and antigenic drift with repercussions for humans. RSV, like influenza viruses, is very variable, and the lack of a proof-reading exonuclease (which exists in SARS-CoV-2) results in a mutation rate of roughly 10 -3 /10 -4 nucleotide substitutions/site/year [41][42][43][44], depending on the gene and strain. RSV-A viruses appear to have a lower mutation rate (1.48x10 -3 nucleotide substitutions/site/year) than RSV-B viruses (1.92x10 -3 nucleotide substitutions/site/ year) [41].…”

A dual-labeled fluorescence quenching lateral flow assay based on one-pot enzyme-free isothermal cascade amplification for the rapid and sensitive detection of pathogens

“…Nucleic acids have been classified as biomarkers for diseases, 1 food safety, 2 and environment monitoring. 3 Compared with proteins or living cells, nucleic acids are more stable in the environment and, more importantly, a series of amplification methods are available to enhance detection signals.…”