Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets

Vogels, Chantal B.F.; Brito, Anderson; Fauver, Joseph R.; Ott, Isabel M.; Kalinich, Chaney C.; Petrone, Mary E.; Casanovas‐Massana, Arnau; Muenker, M. Catherine; Moore, Adam J.; Klein, Jonathan; Lu, Peiwen; Lu-Culligan, Alice; Jiang, Xiaodong; Kim, Daniel J.; Kudo, Eriko; Mao, Tianyang; Moriyama, Miyu; Oh, Ji Eun; Park, Annsea; Silva, Julio; Song, Eric; Takahashi, Takehiro; Taura, Manabu; Tokuyama, Maria; Venkataraman, Arvind; Weizman, Orr-El; Wong, Patrick; Yang, Yexin; Cheemarla, Nagarjuna R.; White, Elizabeth B.; Lapidus, Sarah; Earnest, Rebecca; Geng, Bertie; Vijayakumar, Pavithra; Odio, Camila D.; Fournier, John; Bermejo, Santos; Farhadian, Shelli; Cruz, Charles S. Dela; Iwasaki, Akiko; Ko, Albert Icksang; Landry, Marie L.; Foxman, Ellen F.; Grubaugh, Nathan D.

doi:10.1038/s41564-020-0761-6

Cited by 734 publications

(851 citation statements)

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“…The study found high specificity among all primer sets and variable sensitivity, with the CDC N2 and the Germany E-gene sets being more sensitive than others. 11 Another study (in preprint) examined the sensitivity and efficiency of four 12 Other studies have also compared the US CDC N1 and N2 against commercial test kits, reporting similar specificity with variable sensitivity. 13,14 To accelerate testing capacity, the US Food and Drug Administration (FDA) released updated guidance on February 29, 2020, opening up the production of diagnostic kits to state laboratories, laboratories certified under Clinical Laboratory Improvement Amendments (CLIA), and commercial diagnostic developers.…”

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confidence: 99%

COVID‐19 Clinical Diagnostics and Testing Technology

2020

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Given the global nature of the coronavirus disease 2019 (COVID‐19) pandemic, the need for disease detection and expanding testing capacity remains critical priorities. This review discusses the technological advances in testing capability and methodology that are currently used or in development for detecting the novel coronavirus. We describe the current clinical diagnostics and technology, including molecular and serological testing approaches, for severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) testing as well as address their advantages and limitations. Nucleic acid amplification technology for molecular diagnostics remains the gold standard for virus detection. We highlight alternative molecular detection techniques used for developing novel COVID‐19 diagnostics on the horizon. Antibody response against SARS‐CoV‐2 remains poorly understood and proper validation of serology tests is necessary to demonstrate their accuracy and clinical utility. In order to bring the pandemic under control, we must speed up the development of rapid and widespread testing through improvements in clinical diagnostics and testing technology as well as access to these tools.

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confidence: 99%

COVID‐19 Clinical Diagnostics and Testing Technology

2020

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“…The SARS-CoV-2 reference genome NC 045512.2 was obtained from the National Center for Biotechnology Information GenBank database [29]. The sequence of the RNA transcripts used by Vogels et al [4] to determine the primer efficiency were input into the programs Iterative HFold [26] to predict their secondary structure. This output is the secondary structure of the transcript prior to the interaction with the reverse primer.…”

Section: System and Experiments Setupmentioning

confidence: 99%

“…The primer-probe set used to detect SARS-CoV-2 depends on which qRT-PCR assay is used. The China Center for Disease Control (China CDC), United States CDC (US CDC), Charité Institute of Virology, Universitätsmedizin Berlin (Charité), and Hong Kong University (HKU) have developed the most common assays to detect SARS-CoV-2 that all target different areas of the SARS-CoV-2 genome [4]. In a recent publication, Vogels et al compared the analytical efficiencies and sensitivities of the primer-probe sets used in these assays [4].…”

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confidence: 99%

“…We aimed to determine whether the RNA structure of the SARS-CoV-2 genome affects the binding of the reverse primers in the qRT-PCR assay and whether this correlated to the analytical efficiencies and sensitivities shown by Vogels et al [4]. To study the structure of such interactions, we developed DinoKnot, a program that given two nucleic acid strands predicts their interaction structure.…”

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confidence: 99%

“…We used DinoKnot to predict how the RNA structure of SARS-CoV-2 changes when the reverse primer binds to the RNA genome and whether the primer was binding in the location that was expected. We further predicted how mutations in the primer/probe binding region of the SARS-CoV-2 genome provided by Vogels et al could affect the primer/probe sensitivity for SARS-CoV-2 detection [4].…”

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In silico prediction of COVID-19 test efficiency with DinoKnot

Newman

Chang

Jabbari

2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus spreading across the world causing the disease COVID-19. The diagnosis of COVID-19 is done by quantitative reverse-transcription polymer chain reaction (qRT-PCR) testing which utilizes different primer-probe sets depending on the assay used. Using in silico analysis we aimed to determine how the secondary structure of the SARS-CoV-2 RNA genome affects the interaction between the reverse primer during qRT-PCR and how it relates to the experimental primer-probe test efficiencies. We introduce the program DinoKnot (Duplex Interaction of Nucleic acids with pseudoKnots) that follows the hierarchical folding hypothesis to predict the secondary structure of two interacting nucleic acid strands (DNA/RNA) of similar or different type. DinoKnot is the first program that utilizes stable stems in both strands as a guide to find the structure of their interaction. Using DinoKnot we predicted the interaction of the reverse primers used in four common COVID-19 qRT-PCR tests with the SARS-CoV-2 RNA genome. In addition, we predicted how 12 mutations in the primer/probe binding region may affect the primer/probe ability and subsequent SARS-CoV-2 detection. While we found all reverse primers are capable of interacting with their target area, we identified partial mismatching between the SARS-CoV-2 genome and some reverse primers. We predicted three mutations that may prevent primer binding, reducing the ability for SARS-CoV-2 detection. We believe our contributions can aid in the design of a more sensitive SARS-CoV-2 test.Author summaryThe current testing for the disease COVID-19 that is caused by the novel cornonavirus SARS-CoV-2 uses oligonucleotides called primers that bind to specific target regions on the SARS-CoV-2 genome to detect the virus. Our goal was to use computational tools to predict how the structure of the SARS-CoV-2 RNA genome affects the ability of the primers to bind to their target region. We introduce the program DinoKnot (Duplex interaction of nucleic acids with pseudoknots) that is able to predict the interactions between two DNA or RNA molecules. We used DinoKnot to predict the efficiency of four common COVID-19 tests, and the effect of mutations in the SARS-CoV-2 virus on ability of the COVID-19 tests in detecting those strains. We predict partial mismatching between some primers and the SARS-CoV-2 genome but that all primers are capable of interacting with their target areas. We also predict three mutations that prevent primer binding and thus SARS-CoV-2 detection. We discuss the limitations of the current COVID-19 testing and suggest the design of a more sensitive COVID-19 test that can be aided by our findings.

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Association Between COVID-19 Exposure and Self-reported Compliance With Public Health Guidelines Among Essential Employees at an Institution of Higher Education in the US

et al. 2021

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IMPORTANCE Detailed analysis of infection rates paired with behavioral and employee-reported risk factors is vital to understanding how transmission of SARS-CoV-2 infection may be exacerbated or mitigated in the workplace. Institutions of higher education are heterogeneous work units that supported continued in-person employment during the COVID-19 pandemic, providing a test site for occupational health evaluation. OBJECTIVE To evaluate the association between self-reported protective behaviors and prevalence of SARS-CoV-2 infection among essential in-person employees during the first 6 months of the COVID-19 pandemic in the US. DESIGN, SETTING, AND PARTICIPANTS This cross-sectional study was conducted from July 13 to September 2, 2020, at an institution of higher education in Fort Collins, Colorado. Employees 18 years or older without symptoms of COVID-19 who identified as essential in-person workers during the first 6 months of the pandemic were included. Participants completed a survey, and blood and nasal swab samples were collected to assess active SARS-CoV-2 infection via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and past infection by serologic testing. EXPOSURE Self-reported practice of protective behaviors against COVID-19 according to public health guidelines provided to employees. MAIN OUTCOMES AND MEASURES Prevalence of current SARS-CoV-2 infection detected by qRT-PCR or previous SARS-CoV-2 infection detected by an IgG SARS-CoV-2 testing platform. The frequency of protective behavior practices and essential workers' concerns regarding contracting COVID-19 and exposing others were measured based on survey responses. RESULTS Among 508 participants (305 [60.0%] women, 451 [88.8%] non-Hispanic White individuals; mean [SD] age, 41.1 [12.5] years), there were no qRT-PCR positive test results, and only 2 participants (0.4%) had seroreactive IgG antibodies. Handwashing and mask wearing were reported frequently both at work (480 [94.7%] and 496 [97.8%] participants, respectively) and outside work (465 [91.5%] and 481 [94.7%] participants, respectively). Social distancing was reported less frequently at work (403 [79.5%]) than outside work (465 [91.5%]) (P < .001). Participants were more highly motivated to avoid exposures because of concern about spreading the infection to others (419 [83.0%]) than for personal protection (319 [63.2%]) (P < .001). CONCLUSIONS AND RELEVANCEIn this cross-sectional study of essential workers at an institution of higher education, when employees reported compliance with public health practices both at and (continued)

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Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets

Cited by 734 publications

References 15 publications

COVID‐19 Clinical Diagnostics and Testing Technology

COVID‐19 Clinical Diagnostics and Testing Technology

In silico prediction of COVID-19 test efficiency with DinoKnot

Association Between COVID-19 Exposure and Self-reported Compliance With Public Health Guidelines Among Essential Employees at an Institution of Higher Education in the US

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