A Multiallelic Molecular Beacon-Based Real-Time RT-PCR Assay for the Detection of SARS-CoV-2

Chrysostomou, Andreas; Rodosthenous, Johana Hezka; Topcu, Cicek; Papa, Christina; Aristokleous, Antonia; Stathi, Georgia; Christodoulou, Christina; Eleftheriou, Christina; Stylianou, Dora C.; Kostrikis, Leondios G.

doi:10.3390/life11111146

Cited by 6 publications

(9 citation statements)

References 41 publications

(57 reference statements)

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“…Unlike the TaqMan probe, the molecular beacon probe does not require a polymerase with exonuclease activity [ 20 ]. The molecular beacon probe has recently been used in a real-time PCR assay for the detection of SARS-CoV-2 [ 21 ].…”

Section: Basic Principlesmentioning

confidence: 99%

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Artika

Dewi

Nainggolan

et al. 2022

Genes

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Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

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Section: Basic Principlesmentioning

confidence: 99%

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Artika

Dewi

Nainggolan

et al. 2022

Genes

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“…Specifically, these incorrect targets are based on Wuhan-Hu-1 and include the relative regions of the genome targeted by the molecular beacons; however, as this reference genome is classified as B lineage [ 12 ], it does not contain any of the deletions/insertion found in the five VOCs targeted by the molecular beacons. Moreover, we provide the option of an internal positive control (IPC) designed using sections of the S , E , M , and N genes of the SARS-CoV-2 reference genome Wuhan-Hu-1 (MN908947.3) (explained in detail in [ 36 ]).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, molecular beacons were selected as probes due to the high specificity they offer and the extensive expertise and experience of our laboratory in molecular beacon technology [ 23 , 24 , 25 , 26 , 36 , 37 ]. Molecular beacons comprise a loop section (the target recognition sequence), a stem composed of two short sequences complementary to each other, a fluorescent dye covalently attached at the 5′-end, and a quencher attached at the 3′-end [ 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay

Chrysostomou

Aristokleous

Rodosthenous

et al. 2023

Life

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Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which can be used for monitoring and screening of the population for these variants. Thus, we developed a molecular beacon-based real-time RT-PCR assay according to the principles of spectral genotyping. This assay employs five molecular beacons that target ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔΝ211, S:ins214EPE, and S:ΔL242/A243/L244, deletions and an insertion found in SARS-CoV-2 VOCs. This assay targets deletions/insertions because they inherently provide higher discrimination capacity. Here, the design process of the molecular beacon-based real-time RT-PCR assay for detection and discrimination of SARS-CoV-2 is presented, and experimental testing using SARS-CoV-2 VOC samples from reference strains (cultured virus) and clinical patient samples (nasopharyngeal samples), which have been previously classified using NGS, were evaluated. Based on the results, it was shown that all molecular beacons can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. Furthermore, this assay was able to confirm the genotype of each of the tested samples from various VOCs, thereby constituting an accurate and reliable method for VOC detection and discrimination. Overall, this assay is a valuable tool that can be used for screening and monitoring the population for VOCs or other emerging variants, contributing to limiting their spread and protecting public health.

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“…Impressively, the technology works adequately for individuals testing toward the start of their infection, detecting as little as 0.1 copies/μL in saliva samples with a minimal reaction volume of 10 μL (USD 0.03 per test). The molecular beacon is becoming more attractive over conventional intercalating dyes due to the concern over accidental amplification of nucleic acid targets not of interest [ 94 , 95 , 96 , 97 ].…”

Section: Readout Modalitiesmentioning

confidence: 99%

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

et al. 2022

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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.

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A Multiallelic Molecular Beacon-Based Real-Time RT-PCR Assay for the Detection of SARS-CoV-2

Cited by 6 publications

References 41 publications

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

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