Laboratory management for SARS-CoV-2 detection: a user-friendly combination of the heat treatment approach and rt-Real-time PCR testing

Mancini, Fabiola; Barbanti, Fabrizio; Scaturro, Maria; Iacobino, Angelo; Bella, Antonino; Riccardo, Flavia; Marsili, Giulia; Stefanelli, Paola; Pezzotti, Patrizio; Rezza, Giovanni; Ciervo, Alessandra

doi:10.1080/22221751.2020.1775500

Cited by 42 publications

(47 citation statements)

References 11 publications

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“…CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 3, 2020. ; https://doi.org/10.1101/2020.12.03.409714 doi: bioRxiv preprint sequencing [38]. Heating samples can form free radicals, such as 8-hydroxy-20-deoxyguanine (8-Oxo-dG), that could cause high levels of C->A and G ->U mutations and promote the hydrolytic deamination of C->U [30][31][32][33]35,37,39,40].…”

Section: Discussionmentioning

confidence: 99%

Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect

Rocheleau

Laroche

et al. 2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a genome comprised of a ~30K nucleotides non-segmented, positive single-stranded RNA. Although its RNA-dependent RNA polymerase exhibits exonuclease proofreading activity, viral sequence diversity can be induced by replication errors and host factors. These variations can be observed in the population of viral sequences isolated from infected host cells and are not necessarily reflected in the genome of transmitted founder viruses. We profiled intra-sample genetic diversity of SARS-CoV-2 variants using 15,289 high-throughput sequencing datasets from infected individuals and infected cell lines. Most of the genetic variations observed, including C->U and G->U, were consistent with errors due to heat-induced DNA damage during sample processing, and/or sequencing protocols. Despite high mutational background, we confidently identified intra-variable positions recurrent in the samples analyzed, including several positions at the end of the gene encoding the viral S protein. Notably, most of the samples possesses a C->A missense mutation resulting in the S protein lacking the last 20 amino acids (SΔ20). Here we demonstrate that SΔ20 exhibits increased cell-to-cell fusion and syncytia formations. Our findings are suggestive of the consistent emergence of high-frequency viral quasispecies that are not horizontally transmitted but involved in intra-host infection and spread.Author summaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease, COVID-19, has caused significant worldwide mortality and unprecedented economic burden. Here we studied the intra-host genetic diversity of SARS-CoV-2 genomes and identified a high-frequency and recurrent non-sense mutation yielding a truncated form of the viral spike protein, in both human COVID-19 samples and in cell culture experiments. Through the use of a functional assay, we observed that this truncated spike protein displays an elevated fusogenic potential and forms syncytia. Given the high frequency at which this mutation independently arises across various samples, it can be hypothesized that this deletion mutation provides a selective advantage to viral replication and may also have a role in pathogenesis in humans.

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Section: Discussionmentioning

confidence: 99%

Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect

Rocheleau

Laroche

et al. 2020

Preprint

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“…The copyright holder for this this version posted September 12, 2020. ; https://doi.org/10.1101/2020.09.10.20191189 doi: medRxiv preprint the direct qRT-PCR protocol of COVID-19 detection and also assess the impact of different swab storage media composition on PCR efficiency [7]- [10]. However, most of the studies are done with small sample size, and needs to be expanded for better understanding performance and caveats associated with direct sample testing,…”

Section: Orf1abmentioning

confidence: 99%

SARS-CoV-2 detection by extraction-free qRT-PCR for massive and rapid COVID-19 diagnosis during a pandemic

Avetyan¹,

Chavushyan²,

Ghazaryan³

et al. 2020

Preprint

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COVID-19 pandemic severely impacted the healthcare and economy on a global scale. It is widely recognized that mass testing is an efficient way to contain the infection spread as well as the development of informed policies for disease management. However, the current COVID-19 worldwide infection rates increased demand in the rapid and reliable screening of SARS-CoV-2 infection. We compared the performance of qRT-PCR in direct heat-inactivated, heat-inactivated/pelleted samples against RNA in a group of 74 subjects (44 positive and 30 negative). In addition, we compared the sensitivity of heat-inactivated/pelleted in another group of 196 COVID-19 positive samples. Our study suggests that swab sample heat-inactivation and pelleting show higher accuracy for SARS-CoV-2 detection PCR assay compared to heat-inactivation only (89% vs 83% of the detection in RNA). The accuracy of detection using direct samples varied depending on the sample transport and storage media as well as the viral titer. Our study suggests that purified RNA provides more accurate results, however, direct qRT-PCR may help to significantly increase testing capacity. Switching to the direct sample testing is justified if the number of tests is doubled at least.

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“…The droplets produced when COVID-19 patients coughed or sneezed could be transported over a long distance by airflow before settled on the surface, and the SARS-CoV-2 could stay viable for a long time after the droplets completely evaporated 8 , 9 . The RNA purification is the gold standard for the detection of SARS-CoV-2 in samples, and the existence of SARS-CoV-2 RNA in the specimens of respiratory tract and feces of confirmed and asymptomatic patients have been confirmed by several researches 10 , 11 . Therefore, the environmental contamination could be attributable to direct contact with infected respiratory fluids, feces and depositing of aerosol particles 12 .…”

Section: Introductionmentioning

confidence: 99%

Evaluation of disinfection procedures in a designated hospital for COVID-19

Yan

Zheng

et al. 2021

American Journal of Infection Control

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Laboratory management for SARS-CoV-2 detection: a user-friendly combination of the heat treatment approach and rt-Real-time PCR testing

Abstract: The RNA purification is the gold standard for the detection of SARS-CoV-2 in swab samples, but it is dependent on the availability of chemical reagents. In this study, we evaluated the heat treatment method without RNA extraction as a reliable option to nucleic acid purification.

Cited by 42 publications

References 11 publications

Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect

Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect

SARS-CoV-2 detection by extraction-free qRT-PCR for massive and rapid COVID-19 diagnosis during a pandemic

Evaluation of disinfection procedures in a designated hospital for COVID-19

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