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      Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

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          Abstract

          The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the “gold standard” test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the “gold-standard” CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

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          Most cited references18

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          Virological assessment of hospitalized patients with COVID-2019

          Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
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            Loop-mediated isothermal amplification of DNA.

            T. Notomi (2000)
            We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
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              CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

              CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing based on its ability to generate targeted, double-stranded DNA (dsDNA) breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
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                Author and article information

                Journal
                RNA
                RNA
                RNA
                RNA
                Cold Spring Harbor Laboratory Press
                1355-8382
                1469-9001
                July 2020
                July 2020
                : 26
                : 7
                : 771-783
                Affiliations
                [1 ]Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94720, USA
                [2 ]The Howard Hughes Medical Institute, University of California Berkeley, Berkeley, California 94720, USA
                Author notes
                Corresponding author: jmlim@ 123456berkeley.edu
                Author information
                http://orcid.org/0000-0002-2157-3831
                Article
                9509184 RA
                10.1261/rna.076232.120
                7297120
                32358057
                7edd2823-a834-4102-b996-a9cc5b2434b3
                © 2020 Esbin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

                This article, published in RNA, is available undera Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

                History
                Page count
                Pages: 25
                Funding
                Funded by: Howard Hughes Medical Institute , open-funder-registry 10.13039/100000011;
                Award ID: CC34430
                Funded by: The National Institutes of Health , open-funder-registry 10.13039/100000002;
                Award ID: T32GM098218
                Award ID: T32GM007232
                Categories
                Review

                crispr,lamp,rt-pcr,covid-19,sars-cov-2
                crispr, lamp, rt-pcr, covid-19, sars-cov-2

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