Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications

Abstract

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

FIGURE 1

“We have a LAMP for that”: major design choices when developing RT-LAMP tests. At each stage in the design process, a series of decisions affect the final configuration of the test, be that for an individual patient or for surveillance testing with pooled screening. The inherent flexibility and comparative simplicity of LAMP means that, for almost all settings and uses, there is 1 configuration of the LAMP toolbox that is fit for the purpose. The LAMP tests for use in any 2 settings or geographies can be dramatically different and can use NPS, ANS, OPS, TCEP, EDTA, DIY, HNB, LCV, DARQ, QuasR, OSD, or MolBeac. Abbreviations: ANS, anterior nares swab; DIY, do-it yourself; DARQ, dark quenching technique; EDTA, ethylenediamine tetraacetic acid; HNB, hydroxynaphthol blue; LAMP, loop-mediated isothermal amplification; LCV, leuco crystal violet; MolBeac, Molecular Beacons. NPS, nasopharyngeal swabs; OPS, oropharyngeal swab; OSD, oligonucleotide strand displacement; QuasR, quenching of unincorporated amplification signal reporters; RT-LAMP, reverse transcription–LAMP; TCEP, tris(2-carboxyethyl) phosphine.

FIGURE 2

LAMP mechanism. The LAMPs employ 2 sets of primers, forward/backward internal primers (FIP and BIP) and outer primers (F3 and B3) to target 6 distinct regions (F1c, F2c, F3c sites on 1 end, and B1, B2, B3 sites on the other). The reaction is initiated by the binding of FIP to the F2c region on the double-stranded DNA. As the polymerase elongates the DNA from the FIP, the outer primer F3, which is shorter in length and lower in concentration than the FIP, binds onto its complementary region on the DNA and starts to displace the newly synthesized DNA. The replaced strand then forms a loop structure at one end because of the complementarity of F1 and F1c. This results in a single-stranded, double-stem-loop DNA structure (the so-called “dumb-bell” structure) with similar performance for BIP and B3. This dumbbell-structured DNA enters the amplification cycle because it is already self-primed. Elongation by the polymerase can occur from the free 3′-end of the single-stranded DNA (ssDNA) and from binding of the FIP/BIP primers to the single-stranded loop or from the optional accelerating loop primers (see Supplemental Fig. S1, with permission from Alhassan et al. 2015).

FIGURE 3

A) Biospecimens taken from the patient are inactivated, and the virus is lysed by heating or enzymatic treatment, with or without the addition of chemical agents. B) RNA can be extracted and purified from contaminating proteins and inhibitory contaminants, or C) the step can be omitted (direct methods). F) After transfer of the processed RNA sample into the reverse transcription–loop-mediated isothermal amplification (RT-LAMP) reaction mixture. D) Detection of positive reactions can be achieved through a variety of methods (“Amplicon Detection”), often using real-time fluorescent or visual endpoint readouts E).

FIGURE 4

A flowchart for SARS-CoV-2 reverse transcription–loop-mediated isothermal amplification (RT-LAMP) primer design and selection. A) Having chosen a preferred viral target genomic sequence based on, for example, abundance or mutagenesis consideration, B) primer sets are designed and selected in silico, considering potential primer-dimers or other undesired interactions, inclusivity across SARS-CoV-2 variants, and exclusivity from other coronaviruses (i.e., Middle East respiratory syndrome [MERS]) or species. C) Having selected promising primer sets in silico, empirical testing D) (time-to-threshold, limit-of-detection, etc.), and reaction optimization in the laboratory identifies the set(s) with the desired empirical properties.

FIGURE 5

Representation of the physical and genomic RNA structure of SARS-CoV-2. The genome of the virus is shown at the bottom, and a rendering of the viral structure is shown on the top.

FIGURE 6

Uracil-DNA-glycosylase (UDG)-supplemented reverse transcription–loop-mediated isothermal amplification (RT-LAMP): the system (Kim et al. 2016) removes carryover DNA contamination from one experiment (N) to subsequent ones (N + 1). In the first experiment, uracil is incorporated into contaminants through the use of approximately one-third of the deoxyuridine triphosphate (dUTP): approximately two-thirds of the deoxythymidine triphosphate (dTTP) is used in the amplification reaction—amplicons so derived contain a mixture of T and U bases. In the subsequent (N + 1) experiment, UDG is added to the input sample before amplification. The UDG specifically cleaves uracil-containing contaminants that were inadvertently carried over from the first (N) experiment at room temperature. Upon elevation of the reaction to approximately 65°C, the UDG is heat inactivated, ensuring that only the target RNA (or 100% thymine-containing DNA) target is amplified.

FIGURE 7

LAMP detection methods overview. A) Visual endpoint readouts use dyes that exhibit simple color or turbidimetric changes upon amplification. B) Similar to qPCR, real-time detection methods use fluorescent dyes to monitor the increase in viral load as the amplification progresses. The fluorescent signal can be sequence-independent (e.g., DNA intercalating; see “Sequence-Independent Detection of RT-LAMP Amplification Products”) or sequence-dependent (hybridization-based; see “Sequence-dependent detection of RT-LAMP amplification products”). C) The loop-mediated isothermal amplification (LAMP) products can, in principle, be verified by agarose gel electrophoresis followed by DNA staining, although that requires postamplification manipulation and the corresponding very real risk of between-experiment cross-contamination.

FIGURE 8

Categorization of detection methods. Reactions can be monitored using simpler, but less-specific, sequence-independent methods (e.g., pH changes) or the somewhat more complex but equally more-specific sequence-dependent methods. These, in turn, can either be monitored in real time (e.g., detection of amplification by releasing of quenching [DARQ], intercalating dyes) allowing amplicon formation to be monitored kinetically or as an end-point, stopping/recording the result at a defined time (e.g., quantify and annotate short reads in R [QuasR]). DNA sequencing is the ultimate sequence-dependent endpoint method. When sequence-independent methods are used, false-positive results can be an issue. Most sequence-dependent methods also allow for multiplexing multiple targets in the same reaction. Sequencing of amplicons can allow detection of different variants. Variations on these themes have been described—the location of the icon in the 4-box is purely indicative.

FIGURE 9

Schematic illustrations of some sequence-dependent fluorescent detection methods. The A) detection of amplification by releasing of quenching (DARQ) and quantify and annotate short reads in R (QuasR), B) one-step strand displacement (OSD), and C) molecular beacon methods can improve specificity of detection and processing for viral and other genome targets.

FIGURE 10

Schematic of the diagnostics with coronavirus enzymatic reporting (DISCoVER) loop-mediated isothermal amplification (LAMP)-CRISPR-Cas (Agrawal et al. 2020). Viral RNA is reverse transcribed and amplified via LAMP then converted back to RNA using T7 polymerase. Cas13 enzymes are programmed with a guide RNA to specifically recognize the desired RNA molecules over non-specifically amplified products. Subsequent activation of Cas13 ribonuclease activity results in cleavage of quenched fluorescence reporter molecules. The CRISPR-Cas provides additional layers of specificity and sensitivity, albeit at increased cost and complexity.