1 INTRODUCTION

The recent outbreak of the new coronavirus (severe acute respiratory syndrome coronavirus-2 [SARS-CoV-2]), firstly reported from Wuhan, China, in December 2019, has spread across the globe in the last few months, causing more than 40 million infections and over one million deaths. SARS-CoV-2 belongs to the subgenus sarbecovirus, orthocoronavirinae subfamily. Like other betacoronaviruses, the genome of SARS-CoV-2 has five typical open reading frame (ORFs) on the same coding strand.1

On 30 January 2020, the World Health Organisation (WHO) declared the Covid-19 outbreak as a global public health emergency. The virus spreads so rapidly that it has attracted global attention, and rapid and simple diagnosis is one of the most effective ways to control and prevent the disease.

Diagnosis of Covid-19 based on clinical symptoms, but diagnosis at early stages of infection, is difficult as patients can remain asymptomatic and there are no specific initial manifestations. Symptoms may appear in few days or up to 2 weeks after exposure.2 Chest computed tomography and reverse transcription polymerase chain reaction (RT-PCR) have been used for the coronavirus pneumonia clinical diagnosis.3 RT-PCR protocols have been rapidly developed for the quantitative and qualitative detection of SARS-CoV-2 in respiratory fluid, sputum, nasopharyngeal swabs and blood samples.4 However, the application of RT-PCR requires well-equipped laboratories, experienced personnel and optimal conditions, the minimum total run time of an RT-PCR test is 3–5 h from collecting the sample to result reporting.5

To address these challenges, a rapid, specific, sensitive, robust and quantitative diagnostic tool would be highly desirable. Loop-mediated isothermal amplification (LAMP) could be used for high-throughput screening applications in both referral and local laboratories. Recently, different studies have presented the applicability of the LAMP as an effective tool for simple and rapid detection of pathogens.5-8

Since the advent of the Covid-19 pandemic, various studies have developed several LAMP assay prototypes for SARS-CoV-2 diagnosis. This work presents a review of the actual status, diagnosis robustness and perspectives of the LAMP test for this purpose.

1.1 Developments and features of the LAMP technique

During the year 2000, Notomi et al.9 developed a new molecular technique, LAMP, as a simple field and cost-effective diagnostic tool.9 The LAMP technique uses a DNA polymerase from Bacillus stearothermophilus (Bst) that has polymerase and reverse transcriptase activity. Technically, LAMP uses two inner primers (FIP and BIP) and two outer primers (F3 and B3) that can recognize a total of six distinct regions in the target DNA. Two extra loop primers are also employed (LF and LB) to accelerate amplification and improve detection performance (Figure 1).10 The DNA synthesis initiation by multiple primers makes the technique highly specific.11 The Bst polymerase properties enable amplification using a normal water bath or heating block maintained at a fixed temperature avoiding the use of thermal cycler machines. Additionally, LAMP amplification product can be visualized using agarose gel electrophoresis and/or colorimetric naked-eye detection systems12 and real-time fluorimetry.13,14 Therefore, one of the major advantages of LAMP is its possible deployment in the field and in resource-limited settings.

Schematic principle of loop-mediated isothermal amplification

1.2 Search strategies and data sources

We went through articles in bioRxiv, Web of Science, PubMed, Embase, Google Scholar, Scopus and medRxiv, with different keywords: Covid-19 LAMP assay, SARS-CoV-2, molecular diagnosis, Rapid diagnosis, RT-PCR and LAMP, Covid-19 diagnosis, Coronavirus RNA, Coronavirus Pneumonia and related words. The selection criteria covered the studies interested Covid-19 LAMP development with or without clinical test studies.

1.3 Application of LAMP technique to SARS-CoV-2

Many scientific groups are interested in the development of the LAMP assay as a molecular tool for the development of a point-of-care (POC) test amplifying coronavirus RNA. Until July 2020, a total of 42 LAMP assays were developed and evaluated using clinical or simulated respiratory samples (Table 1).

TABLE 1. Overview of LAMP assays for diagnosis of Covid-19 reported in previous studies Author Location Publishing date Gene target Type of samples Sampling Visual detection Sensitivity/specificity Lamb et al.15 USA 14/02/2020 ORF1ab Synthetized N/A SYBR Green I *** El-Tholoth et al.16 USA 19/02/2020 ORF1ab Synthetized N/A LCV dye 100%–100% Yu et al.17 China 24/02/2020 ORF1ab Respiratory samples 43 SYBR Green/Finder 97.6%–100% Zhang et al.18 UK 29/02/2020 ORF1a, N, A Nasopharyngeal swab 7 pH indicator dyes 100%–100% Yang et al.19 China 02/03/2020 ORF1ab, E and N Nasopharyngeal swab 208 Fluorescent calcein 99%–99% Broughton et al.20 USA 06/03/2020 N, E Nasopharyngeal swab 78 Lateral flow strip 95%–100% Nguyen et al.21 Denmark 14/03/2020 N/A N/A N/A N/A *** Jiang et al.22 China 15/03/2020 N Nasopharyngeal swab 260 RT-monitoring 91.4%–99.5% Zhu et al.23 China 17/03/2020 ORF1ab, N Nasopharyngeal swab 129 Dye streptavidin coated polymer nanoparticles 100%–100% Lu et al.54 China 22/03/2020 RdRp Synthetized N/A WarmStart® Colorimetric LAMP 100%–100% Park et al.24 Korea 27/03/2020 Nsp3, S, ORF8 Synthetized N/A Hydroxy-naphtol-blue *** Österdahl et al.50 UK 01/04/2020 ORF1a Nasopharyngeal swab 24 RT-monitoring 80%–73% Yan et al.25 China 02/04/2020 ORF1ab, S Respiratory samples 130 Fluorescent calcein 100%–100% Butt et al.26 Pakistan 08/04/2020 ORF1a, N Nasopharyngeal swab 70 WarmStart colorimetric LAMP 95%–100% Schmid-Burgk et al.27 USA 08/04/2020 ORF1a, N, A N/A N/A Agarose gel *** González et al.51 Mexico 09/04/2020 N Synthetized N/A WarmStart colorimetric LAMP *** Bhadra et al.28 USA 13/04/2020 ORF1a, N, A N/A N/A Fluorogenic oligonucleotide strand exchange *** Huang et al.29 UK 14/04/2020 ORF1ab, N, S Nasopharyngeal swab 16 WarmStart colorimetric LAMP 100%–100% Baek et al.30 Korea 20/04/2020 N Nasopharyngeal swab 154 WarmStart colorimetric LAMP 100%–98.7% Wang D31 China 21/04/2020 N Synthetized N/A EvaGreen *** Sun et al.32 USA 21/04/2020 Equine model N/A EvaGreen *** Kashir and Yaqinuddin10 Saudi Arabia 23/04/2020 N/A N/A N/A N/A *** Rabe et al.33 USA 23/04/2020 ORF1a, N, A Nasopharyngeal swab, saliva N/A WarmStart colorimetric LAMP *** Lee et al.34 Australia 28/04/2020 N Nasopharyngeal swab 157 RT-monitoring 87%–100% Mohon et al.35 Canada 07/05/2020 RdRp, S Nasopharyngeal swab 120 Colorimetric fluorescence indicator 97.6%–98.7 Ben-Assa et al.36 Israel 07/05/2020 ORF1a, N, A Nasopharyngeal swab/saliva 180 WarmStart colorimetric LAMP 25.9%–100% Dao Thi et al.37 Germany 09/05/2020 ORF1a, N, A Nasopharyngeal swab 95 WarmStart colorimetric LAMP 92%–99.7% Lalli et al.38 USA 11/05/2020 ORF1a, N, A Saliva 6 WarmStart colorimetric LAMP *** Anahtar et al.39 USA 18/05/2020 ORF1a, N Nasopharyngeal swab 62 WarmStart colorimetric LAMP 87.5%–100% Ganguli et al.40 USA 21/05/2020 ORF1ab, N, S Synthetized RT-monitoring *** Hu et al.41 China 23/05/2020 S Nasopharyngeal swab 205 Hydroxy-naphtol-blue 88.89%–99.00% Tran et al.42 Vietnam 25/05/2020 ORF1ab, N Synthetized N/A WarmStart colorimetric LAMP 100%–100% Haq et al.43 Pakistan 29/05/2020 ORF1ab, N, S Nasopharyngeal swab 72 WarmStart colorimetric LAMP 100%–100% Li et al.56 China 03/06/2020 ORF1ab, N Synthetized N/A RT-monitoring *** Lau et al.44 Malaysia 03/06/2020 N Nasopharyngeal swab 49 Hydroxy-naphtol-blue 100%–100% Zhang et al.18 UK 03/06/2020 N, E Synthetized N/A WarmStart colorimetric LAMP 87.5%–100 Kellner et al.45 Austria 23/06/2020 ORF1ab, N, E Nasopharyngeal swab N/A Hydroxy-naphtol-blue 100%–100% Matsumura et al.46 Japan 24/06/2020 N/A Nasopharyngeal swab, oropharyngeal, sputum 155 RT-monitoring 80.9%–100% Eckel et al.47 Germany 03/07/2020 N/A Nasopharyngeal swab 109 RT-monitoring 17%–88.7% Ooi et al.48 Singapore 03/07/2020 N, E Synthetized N/A Lateral flow strip *** Nagura-Ikeda et al.49 Japan 07/07/2020 N/A Saliva 103 RT-monitoring 70.9%–100% Note: *** = Not Applicable. Abbreviations: LAMP, loop-mediated isothermal amplification; ORF, open reading frame; RT, reverse transcription.

The first studies were reported by Lamb et al.15 and El-Tholoth et al.16 The objective of the first study15 was to develop a fast screening diagnostic test. Simulated samples were generated by spiking biological samples with a fraction of the SARS-CoV-2 nucleic sequence. Primers were designed based on the publicly available SARS-CoV-2 data and also compared to other coronavirus sequences. To select the optimal conditions for reverse transcription-LAMP (RT-LAMP), different set-up modifications were evaluated, diverse primer sets, several ranges of temperatures (55–65°C) and incubation times (20–45 min) were also assessed. The best amplification conditions were reached at 63°C for 30 min. To determine the LAMP detection limit, titred virus was serially diluted. The specificity of the LAMP assays was tested by testing samples extracted from different pathogens, including viruses, fungi and bacteria.

Similarly, El-Tholoth et al.16 reported the design of a two-step LAMP (Covid-19 Penn-RAMP) achieved in closed tubes with colorimetric or fluorescence detection. When testing purified targets, the LAMP assay performance was different to conventional RT-PCR assays, showing 10-fold higher sensitivity.16

Since these first two publications, the number of studies has grown, and so many countries are now focusing on this technique (Figure 2).

Evolution of the number of Covid-19 loop-mediated isothermal amplification studies

2 THE SARS-CoV-2 LAMP ASSAYS STUDIES 2.1 Geographic distribution of developed LAMP

Like the distribution of the pandemic, the highest number of studies related to LAMP was registered in China and the United States (10) (Figure 3). Then, the United Kingdom with four publications. Germany, Japan, Pakistan and Korea published two studies each; one of the Korean studies developed and evaluated RT-LAMP assay using samples collected from Covid-19 patients. The results revealed high agreement with the RT-PCR tested on 154 clinical samples.30

Country origin of loop-mediated isothermal amplification assays studies

Only seven studies were reported from Europe (UK, Germany and Denmark) despite the large number of infections and deaths (WHO). Two assays were tested on biological samples,37,50 the UK researchers piloted an RT-LAMP assay on nasopharyngeal samples from 21 residents in a dependency care home, with two index Covid-19 cases, and compared it to RT-PCR. The German researchers used purified RNA and crude pharyngeal samples and revealed that RT-LAMP assays have excellent specificity, despite a lower sensitivity compared to RT-PCR.

Haq et al.43 and Butt et al.26 implemented the RT-LAMP protocol for the qualitative detection of viral RNA. Extracted RNA from 70 nasopharyngeal swabs was analysed using RT-PCR and RT-LAMP. The second study from Pakistan developed and validated an RT-LAMP to propose a potential RT-PCR alternative for rapid testing of suspected Covid-19 individuals. Comparative RT-LAMP assay assessment showed good specificity and sensitivity.

A single study was reported from two other countries. Lee et al.34 from Australia evaluated their LAMP assay on 157 clinical specimens previously screened by E-gene RT-PCR and revealed a specificity and sensitivity of 100% and 87%. A Canadian study reported that the validation of LAMP targeting the S gene compared to RT-PCR reference, exhibited a negative percent agreement (NPA) and positive percent agreement (PPA) of 98.72% and 97.62%, respectively. RT-LAMP targeting S and RdRp gene showed an NPA 100% and PPA of 91.97% when discrepant samples were included.35

The first Latin American study51 demonstrated the mixed use of a colorimetric embodiment of LAMP. This strategy was used to amplify and detect SARS-CoV-2 RNA using a set of in house designed initiators that target the N protein.

2.2 Gene targets

The LAMP specificity and sensitivity depend generally on the primer sets used; hence, when designing the primers, care must be taken. Despite the fact it is difficult to choose a valid and specific target for amplification (species-specific target and/or highly conserved region), it is essential to confirm that the primers are specific and amplifies the selected target; for that, preliminary optimisation is required before final primer validation. Although the Eiken Primer Explorer software was the most used tool for LAMP primer design, proper primers can be manually designed. For the detection of SARS-CoV-2 RNA different target regions were selected. Most of the studies targeted the conserved sequence of nucleocapsid gene (N gene) and ORF1ab (Figure.4) because of their high homology and their divergence from the other coronaviruses.29 The N gene is at the 3'-end of the SARS-CoV-2 RNA,52 ORF1ab encodes the replicase polyprotein and it is about 21-kb long.53 Dao Thi et al.37 compared several primers and selected one primer set to detect N gene as the best. The same target was selected by Badhra et al.28 and Zhang et al.18 This corresponds to the results of Viehweger et al.27 who reported that the N gene has the highest read coverage of all coronavirus genes after they sequenced RNA from cell cultures infected with coronavirus HCoV-229E. Using RNA isolated in vitro, they confirmed that the RT-LAMP detection limit using N gene primers is 100 copies.54

Proportion of targeted genes used for loop-mediated isothermal amplification development studies

2.3 LAMP sensitivity, specificity and application

One of the major advantages of the LAMP technique is the high degree of sensitivity and specificity. Since the first tests, articles reported the successful application of RT-LAMP assays to detect SARS-CoV-2 RNA in patient samples, demonstrating that 1–10 copies of viral RNA in a sample can lead to a successful detection, 10–100 fold more sensitive than classical RT-PCR. The LAMP assays tested on clinical samples showed a high specificity (80%–100%) and sensitivity (73%–100%), applying repeated RT-PCR as reference.

One principal limitation of LAMP assays for the diagnosis of SARS-CoV-2 arises from their dependence on time intensive and laboratory-based procedures for viral isolation, lysis and removal of possible inhibiting materials. Recently, respiratory samples were directly tested by the variplex SARS-CoV-2 ready to use test based on LAMP method. However, the variplex test system failed to accurately detect SARS-CoV-2 without RNA extraction.47

To avoid the time-consuming and expensive sample preparation step and further increase the sensitivity, several studies focused on innovative approaches in sample preparation. Anahtar et al.39 reported that RT-LAMP can be applied directly from a nasopharyngeal sample; furthermore, LAMP sensitivity has risen by 30% with chemical RNase inactivation using TCEP/EDTA and heat-mediated lysis. Moreover, this inactivation step reduces the sample infectivity as well decreasing the risk for laboratory personnel. Rabe et al.33 developed a rapid process capable of inactivating virions and endogenous nucleases; this inactivation was coupled with a purification protocol. The purification and inactivation protocols, associated with RT-LAMP, increased the sensitivity to at least one viral SARS-CoV-2 RNA copy per microliter.