COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2

Abstract

The current coronavirus disease 2019 (COVID-19) pandemic is largely driven by community transmission, after 2019 novel Coronavirus (2019-nCoV or SARS-CoV-2) crosses the borders. To stop the spread, rapid testing is required at community clinics and hospitals. These rapid tests should be comparable with the standard PCR technology. Isothermal amplification technology provides an excellent alternative that is highly amenable to resource limited settings, where expertise and infrastructure to support PCR are not available. In this review, we provide a brief description of isothermal amplification technology, its potential and the gaps that need to be considered for SARS-CoV-2 detection. Among this emerging technology, loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA) and Nicking enzyme-assisted reaction (NEAR) technologies have been identified as potential platforms that could be implemented at community level, without samples referral to a centralized laboratory and prolonged turnaround time associated with the standard COVID-19 RT-PCR test. LAMP, for example, has recently been shown to be comparable with PCR and could be performed in less than 30 min by non-laboratory staff, without RNA extractions commonly associated with PCR. Interestingly, NEAR (ID NOW™ COVID-19 (Abbott, IL, USA) was able to detect the virus in 5 min. More so, isothermal platforms are cost effective and could easily be scaled up to resource limited settings. Diagnostics developers, scientific community and commercial companies could consider this alternative method to help stop the spread of COVID-19.

Keywords: COVID-19; LAMP, RPA, NEAR; novel coronavirus; point of care testing; rapid testing.

Figure 1

Importance of rapid testing of infectious diseases in controlling an outbreak. The y-axis represents the number of cases, and x-axis indicates the period of detection and response. The red color represents the number of cases without an intervention like testing, and yellow color is the drop in cases due to intervention, drastically reducing the response rate, unlike the graph on the left hand side [22].

Figure 2

Schematic representation of loop-mediated amplification reaction and its principle. Unlike PCR primer design, LAMP is characterized with four different primers, specifically designed to recognize six distinct regions of the target DNA. Forward inner primer (FIP) consists of a F2 region at the 3’-end and an F1c region at the 5’-end. While the F3 primer (forward outer primer) consists of a F3 region which is complementary to the F3c region of the template sequence. The Backward Inner primer (BIP) is made up of a B2 region at the 3’-end and a B1c region at the 5’-end. B3 primer (backward outer primer) consists of a B3 region which is complementary to the B3c region of the template sequence. In regards to LAMP reaction, amplification begins when F2 region of FIP anneals to F2c region of the target DNA and initiates complementary strand synthesis, and F3 primer anneals to the F3c region of the target and extends, displacing the FIP linked complementary strand. This displaced strand forms a loop at the 5’-end, which provides the template for BIP, and B2 anneals to B2c region of the template. DNA synthesis is initiated, which results in the formation of a complementary strand and opening of the 5’-end loop. Subsequently, B3 anneals to B3c region of the target DNA and extends, displacing the BIP linked complementary strand, which forms a dumbbell-shaped DNA. The nucleotides are added to the 3’-end of F1 by Bst DNA polymerase, which extends and opens up the loop at the 5’-end. The dumbbell-shaped DNA is converted to a stem–loop structure (a and b), which initiates LAMP cycling (second stage of LAMP reaction). The amplicons formed are a mixture of stem–loop and cauliflower-like structures with multiple loops [49].

Figure 3

Schematic naked-eye visualization strategy of LAMP reaction. (A) Detection of LAMP reaction by turbidity. Left tube, without turbidity (negative); right tube, with turbidity (positive); (B) detection of LAMP reaction by fluorescence using calcein. Left tube, without green color (negative); right tube, with green color (positive); (C) real-time detection of LAMP reaction using a portable equipment like Genie^® II. The equipment is portable and robust for point of care testing and it uses 24-h rechargeable battery. This equipment is available from OptiGene Limited, Horsham, UK.

Figure 4

Recombinase polymerase amplification schematic representation. It begins with the binding of recombinase (T4 uvsY and uvsX; green diamonds and orange circles, respectively) to forward and reverse primers, which forms a complex that search for homologous sequences in double stranded DNA. Strand exchange reaction occurs once the homology is found. The single strand binding proteins (SSB, T4 gp32 protein; brown circles) aligns to unwound DNA strand, allowing DNA polymerase (Bacillus subtilis Pol I, Bsu; green circles) to initiate template amplification using the two primers, forming two double stranded DNA. The repetition of the cycle leads to exponential amplification.

Figure 5

RPA detection mechanism. (A) The TwistAmp nfo is for lateral flow detection strategy, while (B) exo probe is for real-time detection. The probe annealed to double stranded DNA has a 3′ block (dark blue) that prevents extension. The Escherichia coli endonuclease IV (nfo) or exonuclease III (exo) recognizes and cleaves the tetrahydrofuran (THF) residue (as indicated with the arrow) within the probe, detaching the 3′-end block. This process helps the integration into the amplified products through Bsu polymerase elongation from the 3′-end hydroxide; (A) Regarding nfo amplification, fluorophore labeled amplicons (for example, with fluorescein amidites and biotin dyes) can be detected visually using lateral flow strips. This sandwich format allows the fluorophore (bright orange) to be captured through anti-fluorophore conjugated gold nanoparticles. It also can detect a second label like biotin (purple) by binding to a streptavidin detection line; (B) Regarding exo amplification, fluorescent signals are generated when exonuclease III (exo, pink) cuts the THF site like the nfo, separating the fluorophore (bright orange) from the quencher (red); (C) The lateral flow coupled with RPA nfo reaction can be performed within a broad range of temperatures (top) and a positive test is observed visibly after 10 min (bottom) [54]; (D) The exo fluorescent signals are detected by a real-time device, such as the T16-ISO equipment from TwistDx, Cambridge, UK.

Figure 6

Schematic representation of nicking enzyme-assisted reaction. Unlike PCR primers, NEAR primers are uniquely designed for a successful reaction. Each primer has three regions: restriction site (5′-GAGTCNNNN-3′, for example, Nt. BstNBI), stabilizing region and a target-binding region that is complementary to the target nucleic acid strand. First, the forward primer (primer 1) anneals with template 1 at the target-binding region and extended from its 3′ end by DNA polymerase. This results in the formation of an intermediate strand, complementary to template 1 (step I). At the same time, the nicking endonuclease recognizes the asymmetric restriction site (5′-GAGTC-3′) cleaves the strand with four base pairs after the recognized sequence introduces a new nick site (step II). At the nick site, DNA polymerase initiates another extension and displace the intermediate strand generated in step I (step III and IV). This displaced strand (template 2) carries the target-binding sequence complementary to reverse primer (primer 2), and DNA polymerase extends from the 3′ end of this primer (step VI). Again, nicking endonuclease recognizes and cleaves the restriction site, and the polymerase enzyme extends and displaces the initial template strand (template 1) from the nick site, which is recovered (step VII and IX) for another cycle, starting from step I. The target template is exponentially amplified by repeating this cycle of events [53].