In silico analysis of mismatches in RT-qPCR assays of 177 SARS-CoV-2 sequences from Brazil

Santos, Renan da Silva; Bret, Raissa Souza Caminha; Moreira, Ana Cristina de Oliveira Monteiro; Campos, Adriana Rolim; Silva, Angelo Roncalli Alves e; Lima, Danielle Malta; Tavares, Kaio César Simiano

doi:10.1590/0037-8682-0657-2020

Cited by 4 publications

(3 citation statements)

References 18 publications

(28 reference statements)

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“…Although SARS-CoV-2 has a slower mutation rate than, for example, influenza (Manzanares-Meza and Medina-Contreras, 2020), it nevertheless has accrued mutations in most of the primer and probe binding sites of globally used PCR assays within a few months of the start of the pandemic. Aligning 177 genomes collected up to July 2020 from across Brazil with primer sequences of 15 of the WHO-listed SARS-CoV-2 assays revealed that only 3 assays (NIID_2019-nCoV-N, nCoV-IP4 and CN-CDC-E) had a perfect match to all 177 genomes (Santos et al, 2020). A similar study using sequence data from 375 genomes collected around the world up to April 2020 showed that only 2 out of 12 WHO-listed assays had a perfect match to all analyzed genomes (CDC-N1 and Charité RdRP) (Toms et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

Zimmermann

Urban

Krüger

et al. 2022

Journal of Virological Methods

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Section: Introductionmentioning

confidence: 99%

In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

Zimmermann

Urban

Krüger

et al. 2022

Journal of Virological Methods

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“…Therefore, for both odd and even number nucleotides in the probe, central location i.e. -1, 0, + 1 nucleotides must align with the genome sequence [ 21 , 25 ]. Notwithstanding any of the above, primers/probes having degenerate nucleotides were considered acceptable if even a single combination of nucleotide meets all the above criteria.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, for both odd and even number nucleotides in the probe, central location i.e. -1, 0, + 1 nucleotides must align with the genome sequence [ 21 , 25 ].…”

Section: Methodsmentioning

confidence: 99%

A two-step process for in silico screening to assess the performance of qRTPCR kits against variant strains of SARS-CoV-2

Gupta

Kumar²,

Gupta³

et al. 2022

BMC Genomics

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Background Since inception of the COVID-19 pandemic, early detection and isolation of positive cases is one of the key strategies to restrict disease transmission. Real time reverse transcription polymerase chain reaction (qRTPCR) has been the mainstay of diagnosis. Most of the qRTPCR kits were designed against the target genes of original strain of SARS-CoV-2. However, with the emergence of variant strains of SARS-CoV-2, sensitivity of the qRTPCR assays has reportedly reduced. In view of this, it is critical to continuously monitor the performance of the qRTPCR kits in the backdrop of variant strains of SARS-CoV-2. Real world monitoring of assay performance is challenging. Therefore, we developed a two-step in-silico screening process for evaluating the performance of various qRTPCR kits used in India. Results We analysed 73 qRT-PCR kits marketed in India, against the two SARS-CoV-2 VoCs. Sequences of both Delta (B.1.617.2) and Omicron (B.1.1.529) VoCs submitted to GISAID within a specific timeframe were downloaded, clustered to identify unique sequences and aligned with primer and probe sequences. Results were analysed following a two-step screening process. Out of 73 kits analysed, seven were unsatisfactory for detection of both Delta and Omicron VoCs, 10 were unsatisfactory for Delta VoC whereas 2 were unsatisfactory for only Omicron VoC. Conclusion Overall, we have developed a useful screening process for evaluating the performance of qRTPCR assays against Delta and Omicron VoCs of SARS-CoV-2 which can be used for detecting SARS-CoV-2 VoCs that may emerge in future and can also be redeployed for other evolving pathogens of public health importance.

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True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?

Lima

Mesquita

Oliveira

et al. 2022

Expert Review of Molecular Diagnostics

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Introduction The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease has had a catastrophic impact on the world resulting in several deaths. Since World Health Organization declared the pandemic status of the disease, several molecular diagnostic kits have been developed to help the tracking of viruses spread. Areas Covered This review aims to describe and evaluate the currently reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) diagnosis kit. Several processes used in COVID-19 diagnostic procedures are detailed in further depth to demonstrate optimal practices. Therefore, we debate the main factors that influence the viral detection of SARS-COV-2 and how they can affect the diagnosis of patients. Expert Opinion Here is highlighted and discussed several factors that can interfere in the RT-PCR analysis, such as the viral load of the sample, collection site, collection methodology, sample storage, transport, primer, and probe mismatch/dimerization in different brand kits. This is a pioneer study to discuss the factor that could lead to the wrong interpretation of RT-qPCR diagnosis of SARS-CoV-2. This study aimed to help the readers to understand what very likely is behind a bad result of SARS-CoV-2 detection by RT-PCR and what could be done to reach a reliable diagnosis.

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In silico analysis of mismatches in RT-qPCR assays of 177 SARS-CoV-2 sequences from Brazil

Cited by 4 publications

References 18 publications

In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

A two-step process for in silico screening to assess the performance of qRTPCR kits against variant strains of SARS-CoV-2

True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?

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