How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

Anantharajah, Ahalieyah; Helaers, Raphaël; Defour, Jean‐Philippe; Olive, Nathalie; Kabera, Florence; Croonen, Luc; Deldime, F.; Vaerman, Jp.; Barbée, Cindy; Bodéus, Monique; Scohy, Anaïs; Verroken, Alexia; Rodríguez-Villalobos, Hector; Kabamba-Mukadi, Benoît

doi:10.1016/j.jviromet.2021.114197

Cited by 20 publications

(15 citation statements)

References 23 publications

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“…As SARS-CoV-2 continues to spread throughout the globe and within communities, random nucleotide variations in both coding and non-coding areas of the viral genome are under continuous observation 31 . The assays are detecting genetic changes within the spike region which harbors higher variation compared to the relatively conserved N, E, RdRp and ORF1ab genes which most of the conventional RT-qPCR assays detects 32 . Normally, when designing RT-qPCR assays for the detection of SARS-CoV-2, primers should be placed in those regions with the least sequence variation and best conditions for PCR amplification 33 .…”

Section: Discussionmentioning

confidence: 99%

Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants

Juul¹,

Spiegelhauer

Petersen

et al. 2022

Sci Rep

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Reverse transcription quantitative PCR (RT-qPCR) assays are gold standard in diagnosing SARS-CoV-2 infection and play a major role in viral subtyping for rapid detection and monitoring of important mutations, containing the spread of new virus variants. We wanted to compare RT-qPCR melting curve analysis assays to Sanger Sequencing for detection of variants within the SARS-CoV-2 spike glycoprotein and examined their sensitivity and specificity. Samples positive for SARS-CoV-2 (n = 663 + 82) were subtyped using both Sanger sequencing and five RT-qPCR melting curve analysis assays specific for the mutations N501Y, P681H, E484K, K417N/T, and N439K. The results of the two methods were compared. The training cohort and the clinical validation cohort showed equally, or significantly better sensitivity of the assays compared to the Sanger sequencing. The agreement of the Sanger sequencing and the assays ranged from 92.6 to 100% for the training cohort and 99.4–100% for the clinical validation. The sensitivity, specificity, and turn-around time of the RT-qPCR melting curve analysis assays are well-suited for clinical monitoring of VOCs, making the assays an important tool in contact tracing and risk stratification. Furthermore, the assays were able to indicate the presence of new mutations in the complementary sequence to the mutation-specific probes.

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Section: Discussionmentioning

confidence: 99%

Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants

Juul¹,

Spiegelhauer

Petersen

et al. 2022

Sci Rep

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“…The characterization of sequence conservation between SARS-CoV-2 and MERS-CoV as well as other seasonal human coronaviruses such as HKU1, OC43, NL63 and 229E is important for the establishment of RT-PCR primers and probes. Anantharajah et al, reported that no significant homologies were found between these organisms, suggesting a low risk of false positives during the RT-qPCR diagnosis [ 51 ]. Our study confirms such findings as the number of shared kmers between SARS-CoV-2 and SARS coronavirus HSR 1, MERS-CoV and the human coronavirus HKU1, OC43, NL63 and 229E is 1628, 1, 18, 8, 0 and 18, respectively out of 29,884, i.e., 5% of the total kmers in the case of SARS coronavirus HSR 1, the most similar virus to SARS-CoV-2.…”

Section: Discussionmentioning

confidence: 99%

“…Low viral load could hinder proper pathogen identification. Moreover, a study showed that 40–60% of positive SARS-CoV-2 clinical samples are not detected when they exhibit Ct values lower than 30 [ 51 ]. Additionally, a systematic study quantitatively investigated the effect of different mismatches at the 3′-end region of real-time PCR primers.…”

Section: Discussionmentioning

confidence: 99%

CovDif, a Tool to Visualize the Conservation between SARS-CoV-2 Genomes and Variants

Cedeño-Pérez

Gómez-Romero

2022

Viruses

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The spread of the newly emerged severe acute respiratory syndrome (SARS-CoV-2) virus has led to more than 430 million confirmed cases, including more than 5.9 million deaths, reported worldwide as of 24 February 2022. Conservation of viral genomes is important for pathogen identification and diagnosis, therapeutics development and epidemiological surveillance to detect the emergence of new viral variants. An intense surveillance of virus variants has led to the identification of Variants of Interest and Variants of Concern. Although these classifications dynamically change as the pandemic evolves, they have been useful to guide public health efforts on containment and mitigation. In this work, we present CovDif, a tool to detect conserved regions between groups of viral genomes. CovDif creates a conservation landscape for each group of genomes of interest and a differential landscape able to highlight differences in the conservation level between groups. CovDif is able to identify loss in conservation due to point mutations, deletions, inversions and chromosomal rearrangements. In this work, we applied CovDif to SARS-CoV-2 clades (G, GH, GR, GV, L, O, S and G) and variants. We identified all regions for any defining SNPs. We also applied CovDif to a group of population genomes and evaluated the conservation of primer regions for current SARS-CoV-2 detection and diagnostic protocols. We found that some of these protocols should be applied with caution as few of the primer-template regions are no longer conserved in some SARS-CoV-2 variants. We conclude that CovDif is a tool that could be widely applied to study the conservation of any group of viral genomes as long as whole genomes exist.

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“…Two groups of primer-probe sets (Charité/Berlin and CDC designed Primer probe sets) which are recognized globally and used in many diagnostic assays was primarily chosen to be evaluated. Evaluation of these primer probes to find an optimum combination was conducted by thoroughly reviewing the literature [ 25 , 26 , 27 , 28 , 29 , 30 ]. WHO recommended the Charité/Berlin assay which detects two viral targets E and RdRp gene [ 16 ].…”

Section: Discussionmentioning

confidence: 99%

Performance Evaluation of Developed Bangasure™ Multiplex rRT-PCR Assay for SARS-CoV-2 Detection in Bangladesh: A Blinded Observational Study at Two Different Sites

Razu¹,

Ahmed²,

Hossain³

et al. 2022

Diagnostics

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In this study, we evaluated the performance of the in-house developed rRT-PCR assay for SARS-CoV-2 RNA targeting the envelope (E) and nucleocapsid (N) genes with internal control as human RNase P. A total of 50 positive samples and 50 negative samples of SARS-CoV-2 were tested by a reference kit at site 1 and a subset (30 positives and 16 negatives) of these samples are tested blindly at site 2. The limit of detection (LoD) was calculated by using a replication-deficient complete SARS-CoV-2 genome and known copy numbers, where Pseudo-virus samples were used to evaluate accuracy. On site 1, among the 50 SARS-CoV-2 positive samples 24, 18, and eight samples showed high (Ct < 26), moderate (26 < Ct ≤ 32), and low (32 < Ct ≤ 38) viral load, respectively, whereas in site 2, out of 30 SARS-CoV-2 positive samples, high, moderate, and low viral loads were found in each of the 10 samples. However, SARS-CoV-2 was not detected in the negative sample. So, in-house assays at both sites showed 100% sensitivity and specificity with no difference observed between RT PCR machines. The Ct values of the in-house kit had a very good correlation with the reference kits. LoD was determined as 100 copies/mL. It also displayed 100% accuracy in mutant and wild-type SARS-CoV-2 virus. This Bangasure™ RT-PCR kit shows excellent performance in detecting SARS-CoV-2 viral RNA compared to commercially imported CE-IVD marked FDA authorized kits.

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How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

Cited by 20 publications

References 23 publications

Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants

Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants

CovDif, a Tool to Visualize the Conservation between SARS-CoV-2 Genomes and Variants

Performance Evaluation of Developed Bangasure™ Multiplex rRT-PCR Assay for SARS-CoV-2 Detection in Bangladesh: A Blinded Observational Study at Two Different Sites

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