New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants

Marchini, Antonio; Petrillo, Mauro; Parrish, Amy; Buttinger, Gerhard; Tavazzi, Simona; Querci, Maddalena; Betsou, Fay; Elsinga, Goffe; Medema, Gertjan; Abdelrahman, Tamir; Bernd, Gawlik; Corbisier, Philippe

doi:10.3390/v15010206

Cited by 18 publications

(13 citation statements)

References 54 publications

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“…Therefore, real-time RT-PCR genotyping is being considered as a practical solution for identifying SARS-CoV-2 variants. It has become pivotal to track SARS-CoV-2 variants to ensure a rapid public health response because the finding of this study is in concordance with other similar studies [16] , [17] , [18] , [19] , [20] .…”

Section: Discussionsupporting

confidence: 90%

SARS-CoV-2 variant typing using real-time reverse transcription-polymerase chain reaction–based assays in Addis Ababa, Ethiopia

G/meskel,

Desta,

Diriba

et al. 2024

IJID Regions

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Section: Discussionsupporting

confidence: 90%

SARS-CoV-2 variant typing using real-time reverse transcription-polymerase chain reaction–based assays in Addis Ababa, Ethiopia

G/meskel,

Desta,

Diriba

et al. 2024

IJID Regions

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“…Subsequently, the identi cation of a loss of sensitivity of the N1 assay led us to investigate the discrepancy between N1 and N2 concentrations following the emergence of Omicron sub-lineages. Basically, all COVID-19 diagnostic targets have undergone mutations and the N gene, targeted by commonly used primer/probe assays for diagnosing COVID-19, exhibits some of the highest number of mutations across the viral genome 29 . A study from Wisconsin, USA rst reported diminished uorescence intensity in SARS-CoV-2 positive-wastewater samples as proportion of Omicron infections rose when quantifying N1 gene with droplet digital PCR 9 .…”

Section: Discussionmentioning

confidence: 99%

A Dual Loci Quality Assurance and Control Framework for Real-Time Evaluation of Signal Accuracy in Wastewater Surveillance of Pathogens with High Rates of Mutation

Thakali,

Mercier,

Eid

et al. 2023

Preprint

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Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation.

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“…It is important to mention that real-time RT-PCR is a molecular technique that is used to amplify and simultaneously quantify a targeted DNA or RNA molecule. It is commonly used to detect, identify and measure the amount of a specific RNA molecule in a sample [ 42 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular Amplification and Cell Culturing Efficiency for Enteroviruses’ Detection in Cerebrospinal Fluids of Algerian Patients Suffering from Meningitis

Rai,

Ammi,

Anes-Boulahbal

et al. 2024

Viruses

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Enteroviruses (EVs) represent a major cause of viral meningitis, being responsible for nearly 1 billion infections each year worldwide. Several techniques were developed to obtain better diagnostic results of EV infections. Herein, we evaluated the efficiency of EV detection through isolation on both Rhabdomyosarcoma (RD) and Vero cell line cultures, conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. Thus, 50 cerebrospinal fluid (CSF) samples belonging to patients suspected to have viral meningitis in northern Algeria were collected, anonymously numbered from 1 to 50 and subjected to the above-mentioned techniques for EV detection. Using real-time RT-PCR, 34 CSF samples were revealed to be positive for viral origin of meningitis (68%). Thirteen of them were positive when the conventional RT-PCR was used (26%), and only three samples gave positive results when the cell culture technique was used (6%). Surprisingly, two cell culture-positive CSF samples, namely, 31 and 39, were negative using RT-PCR directly on the original samples. However, they turned to be positive when amplification was carried out on their corresponding cell culture supernatant. The cell-cultured viral isolates were then identified by sequencing their viral genome’s VP1 regions. All of them were revealed to belong to the echovirus 27 strain. This investigation demonstrates that RT-PCR techniques are often more sensitive, accurate and much faster, providing reliable results within a clinically acceptable timeframe. However, viral isolation on cell cultures remains crucial to obtain enough viral load for serological tests or even to avoid the rare, but existing, false negative PCR.

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New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants

Cited by 18 publications

References 54 publications

SARS-CoV-2 variant typing using real-time reverse transcription-polymerase chain reaction–based assays in Addis Ababa, Ethiopia

SARS-CoV-2 variant typing using real-time reverse transcription-polymerase chain reaction–based assays in Addis Ababa, Ethiopia

A Dual Loci Quality Assurance and Control Framework for Real-Time Evaluation of Signal Accuracy in Wastewater Surveillance of Pathogens with High Rates of Mutation

Molecular Amplification and Cell Culturing Efficiency for Enteroviruses’ Detection in Cerebrospinal Fluids of Algerian Patients Suffering from Meningitis

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