Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR

Garson, Jeremy A.; Badru, Samuel; Parker, Eleanor; Tedder, Richard S.; McClure, Myra O.

doi:10.1016/j.jcv.2021.105049

Cited by 16 publications

(19 citation statements)

References 15 publications

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“…The first sequenced Omicron case was reported in Botswana on 11 November 2021, and a few days later, another sequenced case was reported in Hong Kong in a subject traveling from South Africa [ 18 ]. Now, SARS-CoV-2 variants are screened by NGS methodology in positive samples and several other valid genotyping methods, such as the double-mismatch, allele-specific RT-PCR [ 19 ], Sanger-based approach [ 20 ], as well as high-resolution melting analysis [ 21 , 22 ]. Double-mismatch, allele-specific RT-PCR methodology requires two parallel RT-PCR reactions to detect any SARS-CoV-2 sequence other than the target variant of concern.…”

Section: Discussionmentioning

confidence: 99%

“…The primers and probes are custom-synthesized. Garson et al claim that these double-mismatch, allele-specific RT-PCR tests show a strong and reliable correlation with results obtained through genome sequencing [ 19 ]. Bezerra et al used a Sanger sequencing methodology and reported that they were able to identify and discriminate all known SARS-CoV-2 variants by sequencing a single PCR fragment [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

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A Rapid and Consistent Method to Identify Four SARS-CoV-2 Variants during the First Half of 2021 by RT-PCR

Fabiani¹,

Margiotti²,

Sabatino

et al. 2022

Vaccines

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Since 2020, the COVID-19 pandemic has spread worldwide, causing health, economic, and social distress. Containment strategies rely on rapid and consistent methodology for molecular detection and characterization. Emerging variants of concern (VOCs) are currently associated with increased infectivity and immune escape (natural defence mechanisms and vaccine). Several VOCs have been detected, including Alpha variant (B.1.1.7), Beta variant (B.1.351), Gamma variant (P.1/B.1.1.28.1) and Delta variant (B.1.617.2), first identified in the UK, South Africa, Brazil and India, respectively. Here, a rapid and low-cost technique was validated to distinguish the Alpha, Beta, Gamma, and Delta SARS-CoV-2 variants by detecting spike gene mutations using a real-time reverse transcription polymerase chain reaction methodology (RT-PCR). A total of 132 positive patients affected by coronavirus disease-19 (COVID-19) were analysed by employing RT-PCR to target single-nucleotide polymorphisms (SNPs) to screen spike protein mutations. All data were validated by the next-generation sequencing (NGS) methodology and using sequences from a public database. Among 132 COVID-19-positive samples, we were able to discriminate all of the investigated SARS-CoV-2 variants with 100% concordance when compared with the NGS method. RT-PCR -based assays for identifying circulating VOCs of SARS-CoV-2 resulted in a rapid method used to identify specific SARS-CoV-2 variants, allowing for a better survey of the spread of the virus and its transmissibility in the pandemic phase.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Rapid and Consistent Method to Identify Four SARS-CoV-2 Variants during the First Half of 2021 by RT-PCR

Fabiani¹,

Margiotti²,

Sabatino

et al. 2022

Vaccines

View full text Add to dashboard Cite

show abstract

“…They were then followed by a surge of reports on the detection of the Delta variant. Garson et al utilized double-mismatch allele-specific RT-PCR at L452R and T478K to differentiate the Delta variant from other VOCs among 42 U.K. patient samples ( 40 ). Aoki et al described an approach that combined nested PCR along with high-resolution melting analysis at the same mutations, which was validated in a small Japanese patient cohort ( 41 ).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification

et al. 2022

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The ability to distinguish between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, responses to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories.

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“…This was then followed by a surge of reports on the detection of the Delta variant. Garson et al utilized double-mismatch allele-specific RT-PCR at L452R and T478K to differentiate Delta variant from other VOCs in 42 UK patient samples (40). Aoki et al described an approach that combines nested PCR along with high-resolution melting analysis at those same mutations, which was validated in a small Japanese patient cohort (41).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Evaluation of a Rapid and Accessible RT-qPCR Approach for SARS-CoV-2 Variant of Concern Identification

Yeung

Wang

Sibai

et al. 2022

Preprint

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The ability to distinguish between SARS-CoV-2 variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, response to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories.We designed and analytically validated a two-reaction multiplex reverse transcription quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in Reaction 1, and del69-70, K417N, and T478K in Reaction 2. This assay had 95-100% agreement with WGS in 502 upper respiratory swabs collected between April 26 and August 1, 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement.This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

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Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR

Cited by 16 publications

References 15 publications

A Rapid and Consistent Method to Identify Four SARS-CoV-2 Variants during the First Half of 2021 by RT-PCR

A Rapid and Consistent Method to Identify Four SARS-CoV-2 Variants during the First Half of 2021 by RT-PCR

Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification

Evaluation of a Rapid and Accessible RT-qPCR Approach for SARS-CoV-2 Variant of Concern Identification

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