Identification of a Polymorphism in the N Gene of SARS-CoV-2 That Adversely Impacts Detection by Reverse Transcription-PCR

Vanaerschot, Manu; Mann, Sabrina A; Webber, James T.; Kamm, Jack; Bell, Sidney M; Bell, John; Hong, Si Noon; Nguyễn, Minh Phương; Chan, Lienna Y.; Bhatt, Karan; Tan, Michelle; Detweiler, Angela M.; Espinosa, Alex; Wu, Wesley; Batson, Joshua; Dynerman, David; Wadford, Debra A.; Puschnik, Andreas S.; Neff, Norma; Ahyong, Vida; Miller, Steve; Ayscue, Patrick; Tato, Cristina M.; Paul, Simon; Kistler, Amy; DeRisi, Joseph L.; Crawford, Emily

doi:10.1128/jcm.02369-20

Cited by 70 publications

(77 citation statements)

References 6 publications

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“…The sensitive PCR tests are likely to miss mutant antigen and likely to cause a missed diagnosis [ 48 ]. A single nucleotide change can alter the primer/probe binding site on the target gene, as reported by Vanaerschot et al on reduced sensitivity of the RT-PCR diagnostic assay owing to a single mutation (Q289H) in the forward N gene primer [ 49 ].…”

Section: Discussionmentioning

confidence: 99%

Structure-Function Analyses of New SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications

Singh

Samal

Kumar

et al. 2021

Viruses

118

109

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SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) has accumulated multiple mutations during its global circulation. Recently, three SARS-CoV-2 lineages, B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1), have emerged in the United Kingdom, South Africa and Brazil, respectively. Here, we have presented global viewpoint on implications of emerging SARS-CoV-2 variants based on structural–function impact of crucial mutations occurring in its spike (S), ORF8 and nucleocapsid (N) proteins. While the N501Y mutation was observed in all three lineages, the 501Y.V1 and P.1 accumulated a different set of mutations in the S protein. The missense mutational effects were predicted through a COVID-19 dedicated resource followed by atomistic molecular dynamics simulations. Current findings indicate that some mutations in the S protein might lead to higher affinity with host receptors and resistance against antibodies, but not all are due to different antibody binding (epitope) regions. Mutations may, however, result in diagnostic tests failures and possible interference with binding of newly identified anti-viral candidates against SARS-CoV-2, likely necessitating roll out of recurring “flu-like shots” annually for tackling COVID-19. The functional relevance of these mutations has been described in terms of modulation of host tropism, antibody resistance, diagnostic sensitivity and therapeutic candidates. Besides global economic losses, post-vaccine reinfections with emerging variants can have significant clinical, therapeutic and public health impacts.

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Section: Discussionmentioning

confidence: 99%

Structure-Function Analyses of New SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications

Singh

Samal

Kumar

et al. 2021

Viruses

118

109

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“…RT-PCR detection of SARS-CoV-2 was performed at the CLIA-certified lab operated by UCSF and the Chan Zuckerberg Biohub as described [10,11]. Briefly, anterior nasal swabs collected in DNA/RNA Shield (Zymo Research) were subjected to RT-PCR using probes specific to the viral N and E genes, and to an internal human positive control (RNAse P).…”

Section: Methodsmentioning

confidence: 99%

Performance characteristics of a rapid SARS-CoV-2 antigen detection assay at a public plaza testing site in San Francisco

Pilarowski

Lebel

Sunshine

et al. 2020

Preprint

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We evaluated the performance of the Abbott BinaxNOWTM Covid-19 rapid antigen test to detect virus among persons, regardless of symptoms, at a public plaza site of ongoing community transmission. Titration with cultured clinical SARS-CoV-2 yielded a human observable threshold between 1.6x104-4.3x104 viral RNA copies (cycle threshold (Ct) of 30.3-28.8 in this assay). Among 878 subjects tested, 3% (26/878) were positive by RT-PCR, of which 15/26 had a Ct<30, indicating high viral load. 40% (6/15) of Ct<30 were asymptomatic. Using this Ct<30 threshold for Binax-CoV2 evaluation, the sensitivity of the Binax-CoV2 was 93.3% (14/15), 95% CI: 68.1-99.8%, and the specificity was 99.9% (862/863), 95% CI: 99.4-99.9%.

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“…Using a single target to detect the presence of SARS-CoV-2 in a specimen may increase the risk for false-negative results 13 . One way to improve this is by integrating a second viral target into the assay, such as the N2 nucleocapsid locus 17 ; however, published data show that N2 is less sensitive than N1 14 .…”

Section: Resultsmentioning

confidence: 99%

“…Taken together, these data establish that duplexed reactions to detect N1 and RNase P can be adapted to 384-plate format and alternate instrumentation with minimal loss of sensitivity, while allowing for four-times greater sample throughput. Using a single target to detect the presence of SARS-CoV-2 in a specimen may increase the risk for false-negative results 13 . One way to improve this is by integrating a second viral target into the assay, such as the N2 nucleocapsid locus 17 ; however, published data show that N2 is less sensitive than N1 14 .…”

Section: Concentration B Citationmentioning

confidence: 99%

“…One commercially available test entered the scene early, allowing clinical laboratories to rapidly detect multiple viral genes and control RNA in a single reaction; however, this test has reported risks of yielding inaccurate results 11 and suffers from supply shortages and high cost. Another less-expensive test detects only a single viral locus, which can lead to false-negative results 12,13 and several tests require 2-3 separate PCR reactions to yield clinical results 9,14,15 . To address these shortcomings, the IGI Clinical Laboratory 16 developed a new multiplexed assay with optimized viral and control RNA detection using inexpensive, widely available reagents.…”

Section: Introductionmentioning

confidence: 99%

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IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

Stahl

Hamilton

et al. 2020

Preprint

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Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

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Identification of a Polymorphism in the N Gene of SARS-CoV-2 That Adversely Impacts Detection by Reverse Transcription-PCR

Abstract: Since April 7, 2020, our COVID-19 diagnostic laboratory (CLIAHUB) has received samples from multiple counties in California — our RT-PCR protocol (1) employs N-gene (NIID_2019-nCov_N_F2/R2ver3/P2 (Japan) (2)) and E-gene (E_Sarbeco_F/R/P1 (Germany) (3)) simplex assays.…

Cited by 70 publications

References 6 publications

Structure-Function Analyses of New SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications

Structure-Function Analyses of New SARS-CoV-2 Variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, Diagnostic, Therapeutic and Public Health Implications

Performance characteristics of a rapid SARS-CoV-2 antigen detection assay at a public plaza testing site in San Francisco

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

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