Loop-Mediated Isothermal Amplification (LAMP): A Rapid, Sensitive, Specific, and Cost-Effective Point-of-Care Test for Coronaviruses in the Context of COVID-19 Pandemic

Abstract

The rampant spread of COVID-19 and the worldwide prevalence of infected cases demand a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. The most common molecular tests approved by regulatory bodies across the world for COVID-19 diagnosis are based on Polymerase Chain Reaction (PCR). While PCR-based tests are highly sensitive, specific, and remarkably reliable, they have many limitations ranging from the requirement of sophisticated laboratories, need of skilled personnel, use of complex protocol, long wait times for results, and an overall high cost per test. These limitations have inspired researchers to search for alternative diagnostic methods that are fast, economical, and executable in low-resource laboratory settings. The discovery of Loop-mediated isothermal Amplification (LAMP) has provided a reliable substitute platform for the accurate detection of low copy number nucleic acids in the diagnosis of several viral diseases, including epidemics like Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). At present, a cocktail of LAMP assay reagents along with reverse transcriptase enzyme (Reverse Transcription LAMP, RT-LAMP) can be a robust solution for the rapid and cost-effective diagnosis for COVID-19, particularly in developing, and low-income countries. In summary, the development of RT-LAMP based diagnostic tools in a paper/strip format or the integration of this method into a microfluidic platform such as a Lab-on-a-chip may revolutionize the concept of PoCT for COVID-19 diagnosis. This review discusses the principle, technology and past research underpinning the success for using this method for diagnosing MERS and SARS, in addition to ongoing research, and the prominent prospect of RT-LAMP in the context of COVID-19 diagnosis.

Keywords: COVID-19; LAMP; RT-LAMP; SARS-CoV-2; coronavirus; point-of-care tests; reverse transcription loop-mediated isothermal amplification.

Figure 1

Scheme showing the comparison of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with RT-PCR. Data is adapted from Dhama et al. [19].

Figure 2

Schematic representation displaying the process of the amplification for the RT-LAMP assay. Initially, the LAMP primers bind to the complementary target cDNA sequences (in the case of RNA viral detection), and dumbbell-shaped DNAs are produced. Then, during the cycling amplification step, several copies of such dumbbell DNAs are continuously produced. The products formed during the cycling amplification step are used in the elongation phase to generate amplified DNA with various sizes.

Figure 3

Steps in sample processing for the LAMP reaction, the LAMP reaction in a simple water bath, and the detection of amplicons.

Figure 4

Application of reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of SARS-CoV-2. (A) Location of RdRp primer sequence in the SARS-CoV-2 genome. (B) Cross-reactivity test of the novel SARS-CoV-2 RT-LAMP assay to other common respiratory viruses. Viral RNA isolated from a COVID-19 positive patient was used as a positive control (PC). NTC, non-template control. (C) RT-LAMP based colorimetric visual detection of SARS-CoV-2 RNA standards. (D) Clinical samples from positive (+) or negative (−) COVID-19 cases. NTC: non-template control. (E) Schematic representation of the SARS-CoV-2 DETECTR method workflow such as RNA extraction. The DETECTR method that involves the LAMP preamplification, and Cas12-based gene detection, and visualization by a fluorescent reader or lateral flow strip (TwistDx). (F) LoD for DETECTR assay showing the fluorescence values using SARS-CoV-2 DETECTR assay (n = 6) using SARS-CoV-2 N2 gene in vitro-transcribed (IVT) RNA. Representative lateral flow results for the assay of samples with 0 copies per μL and 10 copies per μL viral RNA. (G) Lateral flow strips showing SARS-CoV-2 DETECTR assay results after 3 min of flow. A–D are reproduced with permission from Lu et al. [70]. E–G are reproduced with permission from Broughton et al. [83].

Figure 5

Schematic representation of the future possible advances in RT-LAMP based point-of-care test (PoCT) for novel coronavirus disease (COVID-19). LAMP reagents can be loaded in a paper-based system for easy use, which can be heated after sample loading to get quick results. Another advancement will be the coating of the LAMP reagent on the walls of a reaction tube where a simple mixing of the sample with the buffer and heating will give the results. Similarly, an RT-LAMP test-strip, like a conventional pregnancy detection kit, can also be developed. The researchers will also explore the possibility of developing an RT-LAMP master mix that can react at room temperature, which will fully transform the current concept of RT-LAMP based diagnosis. Most advanced development will be a lab on a chip (or LAMP on a chip) with advanced microfluidics, sensors, and computer integration, which can also be connected with an electronic device like computer or mobile phone to analyze the results.