A deletion in the N gene of SARS-CoV-2 may reduce test sensitivity for detection of SARS-CoV-2

Wang, Huanyu; Jean, Sophonie; Wilson, Sandra Jo; Lucyshyn, Jocelyn M.; McGrath, Sean; Wilson, Richard K.; Magrini, Vincent; Leber, Amy

doi:10.1016/j.diagmicrobio.2021.115631

Cited by 15 publications

(18 citation statements)

References 15 publications

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“…4b ). We also considered a deletion of three nucleotides in the N gene (N-delQ9) identified in a Delta variant sublineage [31], which we were able to detect in a multiplexed fashion using test samples containing the E and N1/N1-delQ9 amplicons ( Fig. S9 ).…”

Section: Resultsmentioning

confidence: 99%

“…The DNA amplicon from SARS-CoV-2 was generated by PCR from the aforementioned plasmid with the CDC N1 primers, and the DNA amplicons from SARS-CoV-1 and MERS-CoV were chemically synthesized (IDT). Third, two DNA amplicons from the S gene, one being the wild-type version and another carrying the amino acid H69-V70 deletion[30], and a DNA amplicon from the N gene carrying the CAG deletion at position 28298 (deleting the ninth residue Q)[31] were chemically synthesized.Patient samples. Nasopharyngeal swabs corresponding to infected and non-infected patients with SARS-CoV-2 (RT-qPCR diagnostics) were obtained from the Clinic University Hospital of Valencia (Spain).…”

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confidence: 99%

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Multiplexable virus detection by CRISPR-Cas9-mediated strand displacement

Márquez-Costa

Montagud-Martínez

Marqués

et al. 2022

Preprint

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Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so better and handier virus detection methods would enable a faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a pre-amplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. Moreover, we show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Collectively, this CRISPR-Cas9 R-loop usage for molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic potential.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Multiplexable virus detection by CRISPR-Cas9-mediated strand displacement

Márquez-Costa

Montagud-Martínez

Marqués

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…27 Samples for this study were collected between July 2020 and May 2021, and while there were several variants of SARS-CoV-2 that circulated in Mexico and the United States between those dates, mutations affecting the target regions of the U.S. CDC's RT-qPCR assays for variants prior to October 2021 were extremely rare. 28 2019-CoV plasmids and customized gBlocks (gene fragments) containing the appropriate amplicons (Integrated DNA Technologies) were used as positive controls (standards) for SARS-CoV-2 RNA and PMMoV RNA, respectively. For quality control and avoidance of freeze/thaw cycling, which negatively affects RNA integrity, the 2019-CoV plasmids and gBlocks were aliquoted into 10 μL portions, and any unused thawed standards were disposed of.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…PMMoV RNA was quantified using a previously published assay . Samples for this study were collected between July 2020 and May 2021, and while there were several variants of SARS-CoV-2 that circulated in Mexico and the United States between those dates, mutations affecting the target regions of the U.S. CDC’s RT-qPCR assays for variants prior to October 2021 were extremely rare …”

Section: Materials and Methodsmentioning

confidence: 99%

Detection, Quantification, and Simplified Wastewater Surveillance Model of SARS-CoV-2 RNA in the Tijuana River

et al. 2022

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The COVID-19 pandemic and the detection of SARS-CoV-2 RNA in sewage has expanded global interest in wastewater surveillance. However, many underserved communities throughout the world lack improved sanitation and use informal combined sanitary and storm sewer systems. Sewage is transported via open channels, ditches, and rivers, where it mixes with surface water and/or stormwater. There is a need to develop better methods for the surveillance of pathogens such as SARS-CoV-2 RNA in this context. We developed a simplified surveillance system and monitored flow rates and concentrations of SARS-CoV-2 RNA in the Tijuana River at two locations downstream of the United States–Mexico border in California, United States. SARS-CoV-2 RNA was detected in the upstream location on six out of eight occasions, two of which were at concentrations as high as those reported in untreated wastewater from California sanitary sewer systems. The virus was not detected in any of the eight samples collected at the downstream (estuarine) sampling location, despite the consistent detection of PMMoV RNA. Synchrony was observed between the number of cases reported in Tijuana and the SARS-CoV-2 RNA concentrations measured with the CDC N1 assay when the latter were normalized by the reported flow rates in the river.

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“…False-negative results occur from time to time in molecular tests, and now that the virus genome has mutated, the probability of false-negative results will be much greater. It has been found that the deletion of the N gene may reduce the sensitivity of the detection of SARS-CoV-2 [ 59 ]. Most of the current antigen tests used to detect SARS-CoV-2 are based on antibodies against SARS-CoV-2.…”

Section: Virus Receptor-ace2mentioning

confidence: 99%