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

Gonzalez‐González, Everardo; Lara-Mayorga, Itzel Montserrat; Rodríguez‐Sánchez, Iram P.; Leon, Felipe Yee-de; García-Rubio, Andrés; Garciamendez‐Mijares, Carlos Ezio; Guerra-Alvarez, Gilberto Emilio; García-Martínez, Germán; Aguayo-Hernandez, Juan Andres; Márquez-García, Eduardo; Zhang, Yu Shrike; Martínez-Chapa, Sergio O.; Zúñiga, Joaquı́n; Santiago, Grissel Trujillo‐de; Álvarez, Mario Moisés

doi:10.1101/2020.04.09.20058651

Cited by 23 publications

(19 citation statements)

References 50 publications

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“…Most isothermal amplification steps require incubation at elevated temperatures around 60°C. To facilitate isothermal amplification at remote testing facilities, González-González et al developed a 3D-printed water circulator that can act as a heat block for LAMP amplification and have demonstrated the ability to detect as few as 62 viral RNA molecules after 1 hour of incubation (González-González et al 2020). To make RT-PCR more accessible for remote testing, Wee et al have demonstrated a rapid, extraction-free PCR protocol that can detect 6 SARS-CoV-2 RNA copies using a portable thermocycler (Wee et al).…”

Section: Other Nats ("Thinking Outside the Box")mentioning

confidence: 99%

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Esbin

Whitney

Chong

et al. 2020

RNA

483

474

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The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleicacid based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the United States Center for Disease Control & Prevention, takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own labs. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

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Section: Other Nats ("Thinking Outside the Box")mentioning

confidence: 99%

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Esbin

Whitney

Chong

et al. 2020

RNA

483

474

View full text Add to dashboard Cite

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“…Proteinase K treatment both protects free RNA by inactivating nucleases, and releases particle-bound RNA. Incorporating quantitative colorimetric decomposition and analysis will enhance sensitivity and interpretability of borderline samples in future experiments 22 .…”

Section: Validation On Clinical Samplesmentioning

confidence: 99%

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

Lalli¹,

Langmade²,

Chen³

et al. 2020

Preprint

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Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard diagnostic assays are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pre-treatment protocols that enabled rapid and sensitive detection of < 10 2 viral genomes per reaction in contrived saliva controls. We also observed high performance of this assay on a limited number of clinical saliva samples. While thorough validation on additional clinical samples will be needed before such an assay can be widely used, these preliminary results demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

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“…These results contrast sharply with previous studies showing that incubation times of forty to fifty minutes are required for reliable color development and end-point detection of SARS-CoV-2 colorimetric LAMP assays. 12,29,32 This indicates that our simple instrument is able to detect subtle changes in the optical characteristics of the reaction that occur before easily observable color changes. The second luminance derivative peaks (L’ MAX2 ) in our instrument are much larger than L’ MAX1 and are typically observed between 25-35 minutes into the reaction which is more consistent with times required for endpoint assays, suggesting that this shift is primarily due to the spectral shift in the pH indicator.…”

Section: Resultsmentioning

confidence: 92%

“…Colorimetric assays were run ten minutes longer than fluorescent assays for a total time of 40 minutes to compensate for the relatively slow color change of the phenol red indicator that has been demonstrated to take forty to fifty minutes to detect single copy numbers of SARS-CoV-2 genomes in previous studies. 29,30 All subsequent colorimetric assays were performed under optimal conditions: 40 mM GuHCl and incubated at 65°C for 40 minutes.…”

Section: Resultsmentioning

confidence: 99%

Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared to endpoint assessment

Diaz

Johnson

Jenkins

2021

Preprint

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Controlling the course of the COVID-19 pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel coronavirus SARS-CoV-2. Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current FDA-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than two hours to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS- CoV-2 at either 1X (50 genomes/reaction) or 2X (100 genomes/reaction) of the LOD. Importantly the quantitative method was based on dynamic optical changes during the reaction so was able to correctly classify samples that were misclassified by endpoint observation of color.

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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

Abstract: 2 3 be limited. Moreover, the portability, ease of use, and reproducibility of this strategy make 4 1 it a reliable alternative for deployment of point-of-care SARS-CoV-2 detection efforts 4 2during the pandemics. 4 3 4 4

Cited by 23 publications

References 50 publications

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared to endpoint assessment

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