Can a field molecular diagnosis be accurate? A performance evaluation of colorimetric RT-LAMP for the detection of SARS-CoV-2 in a hospital setting

Silva, Lívia do Carmo; Santos, Carlos Abelardo Dos; Mendes, Geovana de Melo; Oliveira, Kézia Gomes de; Júnior, Marcio Neres de Souza; Estrela, Paulo Felipe Neves; Costa, Sérgio Henrique Nascente; Silveira-Lacerda, Elisângela de Paula; Duarte, Gabriela Rodrigues Mendes

doi:10.1039/d1ay00481f

Cited by 8 publications

(7 citation statements)

References 45 publications

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“…The detection limit of 4.8 × 10 4 copies (Ct ~ 22.93) found in the present study is higher than those reported in the literature 24 – 27 , 34 . Nonetheless, it has excellent potential for the confirmatory diagnosis of the VOC Gamma.…”

Section: Discussioncontrasting

confidence: 81%

“…The reaction was incubated at 65 °C for 70 min in a ProFlex 3 × 32-well PCR System (Thermofisher, Massachusetts, USA). The WarmStart Colorimetric Assay uses phenol red as an indicator of target amplification, and the color of the reaction changes from pink to yellow in the presence of amplicons (Silva et al 34 ). After amplification, we analyzed the color of the tube visually and confirmed it by agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

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Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers

Santos

Silva

Júnior

et al. 2022

Sci Rep

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Despite the advance of vaccination worldwide, epidemic waves caused by more transmissible and immune evasive genetic variants of SARS-CoV-2 have sustained the ongoing pandemic of COVID-19. Monitoring such variants is expensive, as it usually relies on whole-genome sequencing methods. Therefore, it is necessary to develop alternatives that could help identify samples from specific variants. Reverse transcription loop-mediated isothermal amplification is a method that has been increasingly used for nucleic acid amplification, as it is cheaper and easier to perform when compared to other molecular techniques. As a proof of concept that can help distinguish variants, we present an RT-LAMP assay capable of detecting samples carrying a group of mutations that can be related to specific SARS-CoV-2 lineages, here demonstrated for the Variant of Concern Gamma. We tested 60 SARS-CoV-2 RNA samples extracted from swab samples and the reaction showed a sensitivity of 93.33%, a specificity of 88.89% and a kappa value of 0.822 for samples with a Ct ≤ 22.93. The RT-LAMP assay demonstrated to be useful to distinguish VOC Gamma and may be of particular interest as a screening approach for variants in countries with poor sequencing coverage.

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Section: Discussioncontrasting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers

Santos

Silva

Júnior

et al. 2022

Sci Rep

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“…POCTs rely on low budget solutions to proportionate wide access to essential diagnosis needs, turning these type of tests very useful in the pandemic scenery ( Silva et al., 2021 ). However, despite countless works devoted to demonstrate the ability of NAAT-based approaches to be used as POCTs, the wide number of solutions wasn´t enough to dislodge RT-qPCRs as the prime choice for performing SARS-CoV-2 detection ( Bustin et al., 2021 ).…”

Section: Conclusion and Near Future Perspectivesmentioning

confidence: 99%

“…RT-qPCR joins very high sensivity, good specificity and was successfully adapted to the screening of large numbers of samples, what contributed for the implementation of the technique as the gold-standard for SARS-CoV-2 detection and COVID-19 management ( Nörz et al., 2020 ). Isothermal nucleic acid amplification has been intensively explored for point-of-care tests (POCTs) ( Bektaş et al., 2021 ), although being also used in clinical settings, but continues to be less appealing than RT-qPCR ( Silva et al., 2021 ).…”

Section: Introductionmentioning

confidence: 99%

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

Vindeirinho

Pinho

Azevedo

et al. 2022

Front. Cell. Infect. Microbiol.

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COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.

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“…From the perspective of point-of-care tests (POCT), loopmediated isothermal amplification (LAMP) received great attention, especially after the COVID-19 pandemic, when it was widely used as an alternative technique to PCR to obtain diagnoses faster and cheaper. [28][29][30][31] Initially, methodologies RT-LAMP used primers designed to recognise well-conserved regions of the virus and thus could indiscriminately detect any variant, which is very important for a common diagnostic test. Recently, RT-LAMP methodologies have emerged with the opposite strategy, using primers designed to identify specific lineage-defining mutations.…”

Section: Introductionmentioning

confidence: 99%

Fast, low-cost and highly specific colorimetric RT-LAMP assays for inference of SARS-CoV-2 Omicron BA.1 and BA.2 lineages

Estrela

Santos

Resende

et al. 2022

Analyst

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Can a field molecular diagnosis be accurate? A performance evaluation of colorimetric RT-LAMP for the detection of SARS-CoV-2 in a hospital setting

Abstract: SARS-CoV-2 currently represents a serious global public health problem. Non-pharmaceutical intervention measures (NPIs) have been widely adopted, and the testing strategy since the beginning of the infection is the most...

Cited by 8 publications

References 45 publications

Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers

Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

Fast, low-cost and highly specific colorimetric RT-LAMP assays for inference of SARS-CoV-2 Omicron BA.1 and BA.2 lineages

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