Clinical Validation of a Colorimetric Loop-Mediated Isothermal Amplification Using a Portable Device for the Rapid Detection of SARS-CoV-2

Raddatz, Bruna Winkert; Rabello, Felipe J.; Benedetti, Rafael; Steil, Gisleine Jarenko; Imamura, Louise Matiê; Kim, Edson Y. S.; Santiago, Erika Bergamo; Hartmann, Luís F.; Predebon, João V.; Delfino, Bruna M.; Nogueira, Meri Bordignon; Santos, Jucélia Stadinicki dos; Silva, Breno G. da; Nicollete, Diego Rinaldi Pavesi; Almeida, Bernardo Montesanti Machado de; Rogal, Sérgio Renato; Figueredo, Marcus Vinícius Mazega

doi:10.3390/diagnostics13071355

Cited by 3 publications

(2 citation statements)

References 35 publications

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“…1 B). Due to the sensitivity of the test, its variability and standard deviation are high at target copy numbers close to the Limit of Detection (LoD) 21 , 22 To avoid these non-linear regions, the coefficient of determination (R 2 ) was calculated using the logarithm of reactions containing 1E2 to 1E6 copies.…”

Section: Methodsmentioning

confidence: 99%

Data treatment methods for real-time colorimetric loop-mediated isothermal amplification reactions

Kim,

Imamura,

Winkert Raddatz

et al. 2023

Sci Rep

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With the SARS-CoV-2 pandemic and the need for affordable and rapid mass testing, colorimetric isothermal amplification reactions such as Loop-Mediated Isothermal Amplification (LAMP) are quickly rising in importance. The technique generates data that is similar to quantitative Polymerase Chain Reaction (qPCR), but instead of an endpoint color visualization, it is possible to construct a signal over a time curve. As the number of works using time-course analysis of isothermal reactions increases, there is a need to analyze data and standardize their related treatments quantitatively. Here, we take a step forward toward this goal by evaluating different available data treatments (curve models) for amplification curves, which allows for a cycle threshold-like parameter extraction. In this study, we uncover evidence of a double sigmoid equation as the most adequate model to describe amplification data from our remote diagnostics system and discuss possibilities for similar setups. We also demonstrate the use of multimodal Gompertz regression models. Thus, this work provides advances toward standardized and unbiased data reporting of Reverse Transcription (RT) LAMP reactions, which may facilitate and quicken assay interpretation, potentially enabling the application of machine learning techniques for further optimization and classification.

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Section: Methodsmentioning

confidence: 99%

Data treatment methods for real-time colorimetric loop-mediated isothermal amplification reactions

Kim,

Imamura,

Winkert Raddatz

et al. 2023

Sci Rep

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show abstract

“…Most of the methods described for the RT-LAMP detection of SARS-CoV-2 are based on commercial colorimetric pH-sensitive detection kits where the pH change of the amplification reaction changes, in turn, the colour of the sample from pink to yellow [14,[18][19][20][21]. However, these pH-sensitive RT-LAMP methods are most of the time incompatible with the extraction-free process due to the pH of the sample, especially saliva, and the lack of washing steps, resulting in low sensitivity and false-positive results [22].…”

Section: Introductionmentioning

confidence: 99%

Extraction-Free Colorimetric RT-LAMP Detection of SARS-CoV-2 in Saliva

et al. 2023

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The pandemic situation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need of fast, simple, and cost-effective tests for the diagnosis of emerging pathogens. RT-qPCR has been established as the reference technique for the diagnosis of SARS-CoV-2 infections. This method requires a time-consuming protocol for the extraction of the nucleic acids present in the sample. A colorimetric reverse transcription loop-mediated isothermal amplification using the calcein molecule combined with a simple extraction-free method for saliva samples (calcein RT-LAMP) has been developed. Samples are heated 95 °C for 10 min before amplification at 63 °C for 40 min. The results can be observed by fluorescence or by the naked eye with a color change from orange to green. The method was compared with commercialized available colorimetric and fluorescent RT-LAMP kits. The developed method shows better sensitivity and specificity than the colorimetric commercial RT-LAMP and the same as the fluorescent RT-LAMP, without the need of a fluorescent reader. Moreover, the calcein RT-LAMP has, compared to RT-qPCR, a sensitivity of 90% and a specificity of 100% for saliva samples with a Ct ≤ 34, without the need for expensive RT-qPCR instruments, demonstrating the potential of this method for population screening.

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Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

de Souza,

Silva,

Celis-Silva

et al. 2023

Exp Biol Med (Maywood)

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The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised awareness in the scientific community about the importance of being prepared for sanitary emergencies. Many measures implemented during the COVID pandemic are now being expanded to other applications. In the field of molecular and immunological diagnostics, the need to massively test the population worldwide resulted in the application of a variety of methods to detect viral infection. Besides gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) arose as an alternative and sensitive method to amplify and detect viral genetic material. We have used openly available protocols and have improved the protein production of RT-LAMP enzymes Bst polymerase and HIV-reverse transcriptase. To optimize enzyme production, we tested different protein tags, and we shortened the protein purification protocol, resulting in reduced processing time and handling of the enzymes and, thus, preserved the protein activity with high purity. The enzymes showed significant stability at 4 °C and 25 °C, over 60 days, and were highly reliable when used as a one-step RT-LAMP reaction in a portable point-of-care device with clinical samples. The enzymes and the reaction setup can be further expanded to detect other infectious diseases agents.

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Clinical Validation of a Colorimetric Loop-Mediated Isothermal Amplification Using a Portable Device for the Rapid Detection of SARS-CoV-2

Cited by 3 publications

References 35 publications

Data treatment methods for real-time colorimetric loop-mediated isothermal amplification reactions

Data treatment methods for real-time colorimetric loop-mediated isothermal amplification reactions

Extraction-Free Colorimetric RT-LAMP Detection of SARS-CoV-2 in Saliva

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

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