key: cord-0906389-w425dz89 authors: Chaouch, Melek title: Loop‐mediated isothermal amplification (LAMP): An effective molecular point‐of‐care technique for the rapid diagnosis of coronavirus SARS‐CoV‐2 date: 2021-01-21 journal: Rev Med Virol DOI: 10.1002/rmv.2215 sha: e1fdb79103ba759132816d255d7badfbad9dfff2 doc_id: 906389 cord_uid: w425dz89 The novel coronavirus disease‐2019 (Covid‐19) public health emergency has caused enormous loss around the world. This pandemic is a concrete example of the existing gap between availability of advanced diagnostics and current need for cost‐effective methodology. The advent of the loop‐mediated isothermal amplification (LAMP) assay provided an innovative tool for establishing a rapid diagnostic technique based on the molecular amplification of pathogen RNA or DNA. In this review, we explore the applications, diagnostic effectiveness of LAMP test for molecular diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2. Our results show that LAMP can be considered as an effective point‐of‐care test for the diagnosis of Covid‐19 in endemic areas, especially for low‐ and middle‐income countries. mediated isothermal amplification (LAMP) could be used for highthroughput 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] [6] [7] [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. 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 systems 12 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. We went through articles in bioRxiv, Web of Science, PubMed, Many scientific groups are interested in the development of the LAMP assay as a molecular tool for the development of a point-ofcare (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) . The first studies were reported by Lamb et al. 15 and El-Tholoth et al. 16 The objective of the first study 15 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 ). 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 2 of 9 -CHAOUCH 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 study 51 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. 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. 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 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 Various studies chose to combine LAMP with other techniques to improve its efficiency. Broughton et al. 20 Another innovative approach followed by Zhu et al. 23 11 Usually, amplicons are stable, which can lead to unintended carry-over contamination. Thereby, it is recommended to select a closed tube detection system to avoid post-amplification contamination. 11 To deploy more effectively LAMP in field settings and adapt it for processing a large numbers of samples, new protocols and kits may need to be developed as well as frontline personnel trainings. The development and application of SARS-CoV-2 RT-LAMP assays is matching the global objective of seeking simple, rapid and affordable tests. Focusing on the above-cited points will certainly improve the applicability of the LAMP assay, which is a good diagnostic tool suitable for this globally spread disease. Furthermore, it is in line with the WHO guidelines for diagnostics in developing countries, being ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end users. 57 In this review, we discussed the diagnostic performances and advances of the LAMP assay to detect SARS-CoV-2. The study outputs revealed that the RT-LAMP method has reliable application for SARS-CoV-2 diagnosis due to its simple application and low technical requirements, thus presenting a potentially effective test to help us to fight the ongoing Covid-19 pandemic. Novel coronavirus: From discovery to clinical diagnostics A review of coronavirus disease-2019 (COVID-19) Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing Combating the coronavirus pandemic: early detection, medical treatment, and a concerted effort by the global community. Research (Wash D C) Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of subtype H7N9 avian influenza virus Loop-mediated isothermal amplification of DNA Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19 Loop-mediated isothermal amplification (LAMP): an advanced molecular point-of-care technique for the detection of leishmania infection Development of a loopmediated isothermal amplification method for rapid mass-screening of sand flies for leishmania infection Evaluation of fluorimetry and direct visualization to interpret results of a loop-mediated isothermal amplification kit to detect leishmania DNA Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products Rapid detection of novel coronavirus (COVID-19) by reverse transcription-loop-mediated isothermal amplification A single and two-stage, closed-tube, molecular test for the 2019 novel coronavirus (COVID-19) at home, clinic, and points of entry Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv Rapid detection of SARS-CoV-2 using reverse transcription RT-LAMP method Rapid detection of 2019 novelcoronavirus SARS-CoV-2 using a CRISPR-based DETECTR lateral Flow assay 2019 novel coronavirus disease (COVID-19): paving the road for rapid detection and point-of-care diagnostics. Micromachines (Basel) Development and validation of a rapid single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19 Reverse transcription loop-mediated isothermal amplification combined with nanoparticles-based biosensor for diagnosis of COVID-19. medRxiv Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays targeting SARS-CoV-2 Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: a preliminary study Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2 Development of a reverse transcription-loop-mediated isothermal amplification as a rapid 8 of 9 -CHAOUCH early-detection method for novel SARS-CoV-2 One-pot Detection of COVID-19 with Real-time Reversetranscription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract SARS-CoV-2 detection using an isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification. medRxiv Validation of a single-step, singletube reverse transcription-loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2 SARS-CoV-2 On-the-Spot Virus Detection Directly From Patients Screening for SARS-CoV-2 infections with colorimetric RT-LAMP and LAMP sequencing Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reversetranscription loop-mediated isothermal amplification Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2 Development and clinical application of a rapid and sensitive loop-mediated isothermal amplification test for SARS-CoV-2 infection A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care Development optimization and validation of RT-LAMP based COVID-19 facility in Pakistan Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2 rapid and highly sensitive isothermal detection of SARS-CoV-2 for laboratory and home testing Comparison of 12 molecular detection assays for SARS-CoV-2. bioRxiv Variplex test system fails to reliably detect SARS-CoV-2 directly from respiratory samples without RNA extraction A CRISPR-based SARS-CoV-2 diagnostic assay that is robust against viral evolution and RNA editing Clinical evaluation of self-collected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19 Detecting SARS-CoV-2 at point of care: preliminary data comparing Loop-mediated isothermal amplification (LAMP) to PCR. medRxiv Scaling diagnostics in times of COVID-19: colorimetric loop-mediated isothermal amplification (LAMP) assisted by a 3D-printed incubator for cost-effective and scalable detection of SARS-CoV-2. medRxiv Origin and evolution of pathogenic coronaviruses Coronavirus genomics and bioinformatics analysis A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2 LAMP-seq: population-scale COVID-19 diagnostics using combinatorial barcoding Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow How to cite this article: Chaouch M. Loop-mediated isothermal amplification (LAMP): An effective molecular point-of-care technique for the rapid diagnosis of coronavirus SARS-CoV-2 The author declares that there is no conflict of interest. https://orcid.org/0000-0001-5868-4204