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

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developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories.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.

Keywords: CRISPR; LAMP; RT-PCR; covid-19; sars-cov-2.

FIGURE 1.

An overview of COVID-19 nucleic acid testing. Samples collected via nasopharyngeal swab are lysed and inactivated, and an amplification reaction is performed using either a crude swab sample or purified RNA. Amplification of specific viral sequences by RT-PCR, LAMP, or RPA is detected using fluorescent or colorimetric dyes, sequence-specific CRISPR-Cas nuclease cleavage of a reporter, or separation of reaction products on a lateral flow dipstick.

FIGURE 2.

An overview of sample processing. Patient nasopharyngeal swabs are collected and transported for testing. Viral particles are inactivated and lysed by heat and/or lysis buffer addition. Swab sample is then added directly to amplification reactions or RNA is purified from the sample and then amplified.

FIGURE 3.

An analysis of the total workflow time and calculated cost (in U.S. dollars) of published COVID-19 nucleic acid tests. Calculated costs are estimated from available online pricing for consumables and do not include labor or equipment. Protocols which required key reagents to be synthesized or created in a laboratory are not included but are likely to be even cheaper than commercially priced reagents. All raw data available in Supplemental Tables S1, S2.

FIGURE 4.

Examination of the total workflow for published COVID-19 testing methods. Each step of the workflow is shown with colored bars. Four example commercial RT-PCR kits are included for reference (blue) and were directly compared within a single publication. The CDC RT-PCR test is shown in red. (*) Sequencing typically takes 4–12 h but can vary significantly depending on library preparation and the platform used, and was not specifically stated in the cited protocols. Raw data available in Supplemental Table S1.

FIGURE 5.

Molecular overview of the RT-PCR reaction. Taqman probes are used to visualize increased fluorescence during each cycle of amplification. Amplification is quantified by C_q readout and a threshold is set for positive detection of the target amplicon.

FIGURE 6.

The limit of detection for published tests equivalent to the fewest number of molecules accurately assayed in a single reaction. For some spike-in controls, authors used viral DNA, plasmid DNA, or a pseudovirus instead of viral RNA (shown as open diamonds), which may have a different amplification efficiency than SARS-CoV-2 RNA and thus alter their calculated limit of detection. Raw data available in Supplemental Table S1.

FIGURE 7.

Molecular overview of isothermal amplification techniques. LAMP uses specially designed nested primers with complementary regions that form hairpins to permit priming of subsequent rounds of amplification. RPA uses recombinase-catalyzed strand invasion to prime amplification. Colorimetric pH indicators can be used to detect hydrogen ion release during dNTP incorporation.

FIGURE 8.

Molecular overview of CRISPR detection of amplified products. Binding to specific target sequences in amplified RNA or DNA activates Cas nucleases, which cleave reporter molecules. Reporter cleavage can then be assayed using a lateral dipstick.