Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction

Israeli, Ofir; Beth-Din, Adi; Paran, Nir; Stein, Dana; Lazar, Shirley; Weiss, Shay; Milrot, Elad; Atiya-Nasagi, Yafit; Yitzhaki, Shmuel; Laskar, Orly; Schuster, Ofir

doi:10.1016/j.ijid.2020.08.015

Cited by 21 publications

(26 citation statements)

References 6 publications

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“…The sensitivity of RT-qPCR assay is estimated to be 1–10 PFU/mL at Ct ≤ 40, 14 , 15 which is significantly greater than that shown in our assay (∼10 4 PFU/mL). To enable effective COVID-19 diagnosis, the assay’s sensitivity needs to be improved; therefore, several virus enrichment processes were evaluated.…”

Section: Results and Discussioncontrasting

confidence: 59%

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

et al. 2021

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SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 10 4 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

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Section: Results and Discussioncontrasting

confidence: 59%

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

et al. 2021

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“…The sensitivity of RT-qPCR assay is estimated to 1-10 PFU/ml at Ct ≤40 14,15 , which is significantly greater than that shown in our assay (~10 4 PFU/ml). To enable effective COVID-19 diagnosis, the assay's sensitivity of the needs to be improved, therefore several virus enrichment processes were evaluated.…”

Section: Sample Processing For Sensitivity Improvementcontrasting

confidence: 62%

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

Schuster

Zvi²,

Rosen³

et al. 2020

Preprint

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This study describes the development of a novel assay for SARS-CoV-2 identification using LC-MS/MS analysis. A multi-step procedure for the rational down-selection of a set of markers has leaded to the discovery of six SARS-CoV-2 specific and sensitive markers, enabling the reliable identification of the virus. A rapid and simple assay was developed, successfully applied to clinical nasopharyngeal samples. The assay may potentially serve as a complementary approach for SARS-CoV-2 identification.

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“…There have been recent advances in extraction-free protocols for COVID-19 amplification [154][155][156][157][158][159] and sequencing-based tests [160]. However, such protocols involve trade-offs between lower sensitivity and simplicity [22,161,162]. Automated extraction platforms utilizing liquid handling robots are useful for high-throughput, centralized COVID-19 diagnostic testing.…”

Section: Sample Preparationmentioning

confidence: 99%

SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations

et al. 2020

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The unprecedented global pandemic known as SARS-CoV-2 has exercised to its limits nearly all aspects of modern viral diagnostics. In doing so, it has illuminated both the advantages and limitations of current technologies. Tremendous effort has been put forth to expand our capacity to diagnose this deadly virus. In this work, we put forth key observations in the functionality of current methods for SARS-CoV-2 diagnostic testing. These methods include nucleic acid amplification-, CRISPR-, sequencing-, antigen-, and antibody-based detection methods. Additionally, we include analysis of equally critical aspects of COVID-19 diagnostics, including sample collection and preparation, testing models, and commercial response. We emphasize the integrated nature of assays, wherein issues in sample collection and preparation could impact the overall performance in a clinical setting.

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Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction

Cited by 21 publications

References 6 publications

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations

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