We developed a low-cost molecular test for the diagnosis of COVID-19, in a centrifugal microdevice, based on RT-LAMP assay run at 72 °C for 10 min.

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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.

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...

Dengue is one of the most prevalent infectious tropical diseases in the world, with high incidence in over 100 countries. Rapid and reliable diagnosis of dengue is of great importance for public health. To simplify molecular diagnostics, isothermal amplification techniques have recently emerged as an alternative to conventional methods of deoxyribonucleic acid (DNA) amplification. Here, we developed a one-step method for dengue virus detection from real sample based on RT-LAMP (reverse transcription-loop mediated isothermal amplification) in a disposable microdevice. The reaction was thermally controlled with a thermoblock for 15 min at 72 °C. At the end of the incubation time, we either removed the solution for detection of fragments by gel electrophoresis or added DNA intercalator for visual detection on-chip. Our results demonstrated that it is possible to detect dengue virus through RT-LAMP directly from a serum sample, without previous ribonucleic acid (RNA) extraction. The success of RT-LAMP was confirmed in reactions initiated with 0.8 fg μL −1 of RNA, which represents 200 copies of RNA per μL. RT-LAMP in a polyester-toner (PeT) microdevice is a simple and inexpensive method that allows for rapid detection of dengue virus with high reliability and great potential for point-of-care applications.

The coronavirus pandemic has been causing damage to many nations, as public and private health systems deteriorate by the increasing demand. Some infected patients have culturable severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) even though not presenting any symptoms, and therefore, are probably able to transmit it. Correctly diagnosing and isolating infected patients is an important step towards preventing new infections. Current diagnostic methods rely mainly on reverse transcription quantitative polymerase chain reaction (RT-qPCR). Methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have risen as viable alternatives, as they are cheaper and require less infrastructure, they have the potential to be applied in low-resource scenarios and even at point-of-care. Here we report a colorimetric RT‑LAMP assay capable of detecting SARS-CoV-2 in ribonucleic acid (RNA) from saliva. In some cases, the test was able to detect viral RNA before symptom onset and even in a self-reported asymptomatic carrier. It had a limit of detection of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. The possibility of detecting positive cases even before the clinical manifestation shows great potential and can contribute to controlling the pandemic.

Introdução As Variantes de preocupação (VOC - Variants of Concern) são variantes do vírus SARS-CoV-2 que possuem uma maior capacidade de infecção, algum nível de escape de anticorpos, diminuição na eficácia de tratamentos e/ou possível impacto nos métodos de diagnóstico. Estas variantes podem ser responsáveis por novos surtos e sua prevalência deve ser monitorada na população. O método padrão ouro para monitoramento de variantes é o sequenciamento genético, que apesar de ser extremamente robusto, necessita de reagentes caros e de pessoal extremamente capacitado. A técnica de Amplificação Isotérmica Mediada por Loop com transcriptase reversa (RT-LAMP) é uma técnica barata, de fácil aplicação e com potencial de distinguir amostras que contenham pequenas regiões com mutações e/ou SNPs. Objetivos O objetivo do estudo é diferenciar e identificar amostras que contenham dois conjuntos de mutações definidoras de linhagem: a deleção de nove nucleotídeos localizada na posição 11288 e as mutações L18F, T20N e P26S no gene que codifica a proteína Spike. Com a identificação das amostras que possuem esses dois conjuntos de mutações, é possível inferir a que variante pertencem. Métodos Desenhamos dois conjuntos de primers capazes de detectar duas regiões com mutações no genoma do SARS-CoV-2. Para a reação de RT-LAMP utilizamos o kit colorimétrico WarmStart desenvolvido pela empresa New England Biolabs (NEB) e obtivemos o resultado em 70 minutos. Para visualização das reações, utilizamos o aplicativo PhotoMetrix Pro em um celular Xiaomi (Redmi Note 8). A diferenciação de amostras negativas e positivas se dá pela mudança de cor no tubo da reação, de rosa (negativa) para amarela (positiva). Todas as amostras analisadas haviam sido previamente sequenciadas. Resultados A técnica é capaz de amplificar amostras que possuem as mutações alvo com um limite de detecção de ct 23. Em amostras com até essa quantidade de cópias, a sensibilidade do teste é de 81,82%, a especificidade de 100,00% e a acurácia de 88,89%. O teste apresenta potencial para ser usado para triagem de amostras em locais onde o sequenciamento não está disponível. Conclusão A técnica de RT-LAMP pode ser utilizada para detecção de mutações em amostras de SARS-CoV-2, e a presença destas mutações podem indicar que as amostras pertencem à uma variante específica. Com estes dados, é possível inferir a prevalência da variante ao longo do tempo na população, mesmo sem o aparato de sequenciamento.