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Keeping the Lights on for SARS-CoV-2 Research

By 07.02.2022

LAMP Assays and surveillance of infectious disease: Keeping the Lights on for SARS-CoV-2 Research

Quantitative real-time PCR (qRT-PCR) is widely considered the “gold standard” for infectious disease research.1

The technique enables small quantities of RNA or DNA to be sensitively and specifically detected through exponential amplification. With the re-emergence of SARS-CoV-2, the adoption of qRT-PCR-based research was relatively rapid, given the existing infrastructure and foundational place in pathogen detection.2

As infectious disease research continues into present day, several challenges still exist. A surge in cases, driven by the highly-infectious omicron variant, has exposed major gaps in a testing capacity. Getting test results took days, a significant issue for starting appropriate treatments early and preventing further spread of the virus.3 These delays were driven by the need to transport samples to centralized testing facilities, the time required to process samples and run qRT-PCR (i.e., four to eight hours), and data collection and analysis.4

Given these limitations and additional financial constraints, other amplification methods have arisen as more attractive alternatives to qRT-PCR. While there are several options, we’ll discuss one promising replacement method: loop-mediated isothermal amplification (LAMP).

What is the LAMP Assay?

Initially developed in 2000 by Notomi et al., the LAMP assay can amplify single-digit copies of a target DNA or RNA (in the case of reverse transcriptase-LAMP, or RT-LAMP) into 109 copies in under an hour.5 One huge benefit of applying the LAMP assay in infectious disease research, is that diagnostics developers can utilize colorimetric results and an isothermal reaction, which requires minimal equipment. Such is the case with a wide range of LAMP-based research on pathogen detection, including many viruses, such as influenza, dengue, Chikungunya, and Zika.6-9

How Does the LAMP Assay Work?

LAMP and RT-LAMP amplify target DNA or RNA, respectively. Four to six total primers that bind to specific regions within the target DNA or RNA sequence drive amplification in the LAMP and RT-LAMP assay. If RT-LAMP is used, reverse transcriptase is included in the reaction mix to synthesize cDNA.

Amplification is initiated via strand invasion by the forward inner primer (FIP), which is extended by a strand displacing DNA polymerase, separating the original target DNA duplex. This first product is displaced by the extension of another forward outer primer (that binds to a target sequence upstream of the FIP), allowing it to form a loop structure, facilitated by the inclusion of a reverse complementary sequence, engineered into a the 5′ end of the FIP.

This same annealing and displacement cycle repeats on the opposite side of the target sequence using a reverse inner primer (RIP) and a reverse outer primer (that binds to a target sequence downstream of the RIP). The resulting product forms a dumbbell structure, which contains several initiation sites for exponential amplification: From the 3’ end of the open dumbbell, FIP or RIP binding and extension, or another set of forward and reverse primers that bind within the loop. Amplification at these sites results in long concatemeric, double-stranded DNA structures. This rapid and exponential accumulation of double-stranded DNA can then be detected using fluorescent or colorimetric readouts, measured using a plate reader or by eye, respectively.

Advantages and Disadvantages of the LAMP Assay vs. PCR

qRT-PCR has been the go-to method for pathogen detection as it provides highly selective and specific amplification of small amounts of template. Additional PCR-based techniques that improve the specificity of qRT-PCR have been developed more recently. Yet, as mentioned above, the accuracy of qRT-PCR comes at a cost: Equipment that combines temperature cycling (for the iterative cycling of denaturing, annealing, and extension) and an optical unit that can measure amplification is required. Technical prowess and time are also needed to run the assay.

In the areas where qRT-PCR is deficient, LAMP and RT-LAMP excel. First, the LAMP assay can be done quickly and at a constant temperature (RT-LAMP too!) without specialized instrumentation. Second, its amplification efficiency is high, even with low inputs of DNA or RNA. And lastly, its simplicity makes it cost-effective: There is no need for expensive equipment, trained personnel, or even nucleic acid extraction. The reaction is insensitive to many common PCR inhibitors, and crude samples can be readily assayed. DNA amplification can be easily detected using a pH indicator or intercalating fluorescent dye.

LAMP and RT-LAMP have a few challenges to overcome. For one, the design for LAMP method development is complicated and can require significant effort to optimize a workflow. In addition, because the amplified double-stranded DNA is complex, the resulting product can’t be used for any additional downstream analysis (i.e., cloning or sequencing). The most significant drawback is that LAMP is not as well vetted as PCR-based assays, making it challenging to design and implement LAMP workflows.

Colorimetric ReadiLAMP Kit for SARS-CoV-2 Detection

LAMP Assays and SARS-CoV-2 research

Since the beginning of the SARS-CoV-2 outbreak, several publications declaring the successful development of RT-LAMP assays for SARS-CoV-2 detection research have been published.10-13 Dao Thi et al. developed an assay that targets the N gene and has excellent sensitivity (97.5%) and specificity (99.7%). Purified viral RNA serves as the template, but crude swab samples can also be used, though it reduces the sensitivity (86%).10 The assay included phenol red for an easy colorimetric readout of positive tests at 30 minutes. Yan et al. developed RT-LAMP assays for the orf1ab and S genes with similar sensitivity, specificity, and reaction time.11 More recently, Huang et al. developed a bioinformatics algorithm for accurate RT-LAMP primer design and developed a highly-sensitive assay, detecting SARS-CoV-2 titers between 10 to 100,000 copies/µl in crude saliva, comparable with commercially-available qRT-PCR assays.13

Thermo Fisher Scientific has developed and optimized a simple, intuitive, rapid kit for SARS-CoV-2 detection research, the Colorimetric ReadiLAMP™ Kit. Purified viral RNA or saliva/swab samples can be quickly assayed, visually or using a plate reader, in a low- or high-throughput workflow.14

 

Introducing the Invitrogen Colorimetric ReadiLAMP kit, Sars-CoV-2

Introducing the Invitrogen Colorimetric ReadiLAMP kit, Sars-CoV-2 available in 100 or 1,000 reactions. The kit is easy to use and compatible with multiple sample types including saliva, nasal swabs, preserved saliva.

Colorimetric kits have simple protocols for use and display clearly defined results. Find out more about how our sensitive LAMP assay can streamline and simplify your SARS-CoV-2 surveillance efforts by visiting the resource section for Colorimetric ReadiLAMP application notes.

For other related products see below:

This article is for Research Use Only. Not for use in diagnostic procedures.

References:

  1. Kralik P, Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front Microbiol. 2017;8:108. doi:10.3389/fmicb.2017.00108
  2. Esbin MN, Whitney ON, Chong S, Maurer A, Darzacq X, Tjian R. Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection. RNA. 2020;26(7):771-783. doi:10.1261/rna.076232.120
  3. Omicron leaves testing labs overwhelmed, causing frustrating delays to get results. LA Times website: https://www.latimes.com/california/story/2022-01-20/omicron-coronavirus-pcr-test-results-delays. Published January 20, 2022. Accessed April 20, 2022.
  4. Kashir J, Yaqinuddin A. Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19. Med Hypotheses. 2020;141:109786. doi:10.1016/j.mehy.2020.109786
  5. Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. doi:10.1093/nar/28.12.e63
  6. Ito M, Watanabe M, Nakagawa N, Ihara T, Okuno Y. Rapid detection and typing of influenza A and B by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation. J Virol Methods. 2006;135(2):272-275. doi:10.1016/j.jviromet.2006.03.003
  7. Lopez-Jimena B, Bekaert M, Bakheit M, et al. Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype. PLoS Negl Trop Dis. 2018;12(5):e0006381. doi:10.1371/journal.pntd.0006381
  8. Lopez-Jimena B, Wehner S, Harold G, et al. Development of a single-tube one-step RT-LAMP assay to detect the Chikungunya virus genome. PLoS Negl Trop Dis. 2018;12(5):e0006448. doi:10.1371/journal.pntd.0006448
  9. Kutsuna S, Saito S, Ohmagari N. Simultaneous diagnosis of dengue virus, Chikungunya virus, and Zika virus infection using a new point-of-care testing (POCT) system based on the loop-mediated isothermal amplification (LAMP) method. J Infect Chemother. 2020;26(12):1249-1253. doi:10.1016/j.jiac.2020.07.001
  10. Dao Thi VL, Herbst K, Boerner K, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med. 2020;12(556):eabc7075. doi:10.1126/scitranslmed.abc7075
  11. Yan C, Cui J, Huang L, et al. Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay. Clin Microbiol Infect. 2020;26(6):773-779. doi:10.1016/j.cmi.2020.04.001
  12. Huang WE, Lim B, Hsu CC, et al. RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2. Microb Biotechnol. 2020;13(4):950-961. doi:10.1111/1751-7915.13586
  13. Huang X, Tang G, Ismail N, Wang X. Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva. EBioMedicine. 2022;75:103736. doi:10.1016/j.ebiom.2021.103736
  14. A high-throughput workflow for SARS-CoV-2 detection using the Colorimetric ReadiLAMP Kit. Thermo Fisher Scientific website: https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/sars-cov-2-colorimetric-readilamp-kit-app-note.pdf. Published September 23, 2021. Accessed April 25, 2022.

 

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