Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

Alves, Pedro Augusto; Oliveira, Ellen G. de; Franco-Luiz, Ana Paula Moreira; Almeida, Letícia Trindade; Gonçalves, Amanda Bonoto; Borges, Isabela Nascimento; Rocha, Flávia Souza; Rocha, Raíssa Prado; Bezerra, Matheus Filgueira; Miranda, Pâmella; Capanema, Flávio Diniz; Martins, Henrique; Weber, Gerald; Teixeira, Santuza Maria Ribeiro; Wallau, Gabriel Luz; Monte-Neto, Rubens L. do

doi:10.3389/fmicb.2021.713713

Cited by 32 publications

(25 citation statements)

References 83 publications

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“…Detailed conditions of RT-LAMP reaction, including primer sequences, are described in our previous work. (4) Briefly, reactions were performed at 65ºC, during 30 min, using WarmStart ® Colorimetric LAMP 2× Master Mix (New England Biolabs #M1804). To validate the visual colorimetric output, samples resolved in 2% agarose gel stained with DNA intercalator GelRed ® (Biotium #41003), confirming the specific amplification when compared to the positive control (RNA of inactivated parental lineage B of SARS-CoV-2 extracted from the supernatant infected Vero E6 cells).…”

Section: Methodsmentioning

confidence: 99%

“…(1,2,3) As an alternative to RT-qPCR, reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used with success to detect SARS-CoV-2 RNA from nasopharyngeal swabs and saliva samples. (4) During RT-LAMP reaction, the amplification of genetic material occurs at constant temperature (65ºC) without the need for sophisticated thermal cyclers as in RT-qPCR. (5) Furthermore, the colorimetric reaction allows the result to be interpreted faster than RT-qPCR, since the amplified products can be visually detected due to pH-dependent sensors that changes reaction color from pink to yellow (positive result).…”

mentioning

confidence: 99%

“…(6) Previous results obtained by our group, showed that RT-LAMP reaction targeting SARS-CoV-2 N and E genes, can be equivalent to the gold standard RT-qPCR using nasopharyngeal swabs samples. (4) Given the fact that genomic mutations in SARS-CoV-2 omicron VOC are also present in N and E coding sequences, the main goal of this work was to verify whether RT-LAMP reaction targeting N and E genes (in a multiplex or singleplex manner), would works to detect the virus reinforcing the use of it as an affordable and robust COVID-19 diagnostic tool.…”

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confidence: 99%

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Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

Almeida

Gonçalves

Franco-Luiz

et al. 2022

Mem. Inst. Oswaldo Cruz

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BACKGROUND Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis.OBJECTIVES To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages.METHODS SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E.FINDINGS SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL.MAIN CONCLUSIONS Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

Almeida

Gonçalves

Franco-Luiz

et al. 2022

Mem. Inst. Oswaldo Cruz

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“…A range of molecular biology methods have been developed to diagnose SARS-CoV-2 infections, such as RT-qPCRs, RT-LAMP, immunoassays, and Sanger sequencing [5][6][7][8]. However, only whole-genome sequencing can provide enough genetic information (genome-wide mutation patterns) for the reliable lineage discrimination that is…”

Section: Introductionmentioning

confidence: 99%

ViralFlow: A Versatile Automated Workflow for SARS-CoV-2 Genome Assembly, Lineage Assignment, Mutations and Intrahost Variant Detection

Dezordi

Neto

Campos

et al. 2022

Viruses

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The COVID-19 pandemic is driven by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) that emerged in 2019 and quickly spread worldwide. Genomic surveillance has become the gold standard methodology used to monitor and study this fast-spreading virus and its constantly emerging lineages. The current deluge of SARS-CoV-2 genomic data generated worldwide has put additional pressure on the urgent need for streamlined bioinformatics workflows. Here, we describe a workflow developed by our group to process and analyze large-scale SARS-CoV-2 Illumina amplicon sequencing data. This workflow automates all steps of SARS-CoV-2 reference-based genomic analysis: data processing, genome assembly, PANGO lineage assignment, mutation analysis and the screening of intrahost variants. The pipeline is capable of processing a batch of around 100 samples in less than half an hour on a personal laptop or in less than five minutes on a server with 50 threads. The workflow presented here is available through Docker or Singularity images, allowing for implementation on laptops for small-scale analyses or on high processing capacity servers or clusters. Moreover, the low requirements for memory and CPU cores and the standardized results provided by ViralFlow highlight it as a versatile tool for SARS-CoV-2 genomic analysis.

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“…The loop-mediated isothermal amplification (LAMP) method is cost-effective (1.50 USD/test), simple (the isothermal reaction requires a simple heating device), fast (results within 60 min) [10], and visually detected [11]. Because of this, LAMP has been used to detect parasites, [12][13][14][15], bacterias, [16,17], sexually transmitted diseases [18][19][20] , and viruses including SARS-CoV-2 [21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation

Fernandes

Rocha

Guerra-Duarte

et al. 2022

Preprint

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Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and swab samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.

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Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

Cited by 32 publications

References 83 publications

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

ViralFlow: A Versatile Automated Workflow for SARS-CoV-2 Genome Assembly, Lineage Assignment, Mutations and Intrahost Variant Detection

Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation

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