Reverse transcription loop‑mediated isothermal amplification has a high performance in the detection of SARS‑CoV‑2 in saliva samples and nasal swabs from asymptomatic and symptomatic individuals

Reyes‑Morales, Rodolfo; Segundo‑Ibañez, Patricia; Flores‑de Los Ángeles, César; Vizcarra‑Ramos, David; Ibañez‑Galeana, Damián; Salas‑Cuevas, Gabriela; Olvera‑Serrano, Ángel; Pérez‑Silva, Nancy; Rocha‑Rocha, Valeria; El‑Kassi, Elie; Escobedo‑Straffon, Jorge; Contreras‑Mioni, Laura; Rosas‑Díaz, Marisol; Lopez‑Martinez, Karla; Arias‑Matus, Carlos; Bautista‑Rodriguez, Elizabeth; Nolasco‑Quiroga4, Manuel

doi:10.3892/etm.2023.12097

Cited by 3 publications

References 33 publications

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Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay

Bel Hadj Ali,

Souguir,

Melliti

et al. 2024

BMC Infect Dis

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Background The COVID-19 has put emphasis on pivotal needs for diagnosis and surveillance worldwide, with the subsequent shortage of diagnostic reagents and kits. Therefore, it has become strategic for the countries to access diagnostics, expand testing capacity, and develop their own diagnostic capabilities and alternative rapid accurate nucleic acid diagnostics that are at lower costs. Here, we propose a visual SARS-CoV-2 detection using a one-step fast multiplex reverse transcription-PCR (RT-PCR) amplification coupled to lateral flow immunoassay detection on a PCRD device (Abingdon Health, UK). Methods We developed various simplex fast-PCRs for screening sets of primer pairs newly designed or selected from literature or from validated WHO diagnostics, targeting S , N , E , RdRp or ORF1ab genes. We retained primers showing specific and stable amplification to assess for their suitability for detection on PCRD. Thus, fast RT-PCR amplifications were performed using the retained primers. They were doubly labeled with Fam and Biotin or Dig and Biotin to allow visual detection of the labeled amplicons on the lateral flow immunoassay PCR D etection (PCRD) device, looking at lack of interaction of the labeled primers (or primer dimers) with the test-lines in negative or no RNA controls. We set up all the assays using RNAs isolated from patients’ nasopharyngeal swabs. We used two simplex assays, targeting two different viral genomic regions ( N and E ) and showing specific detection on PCRD, to set up a one-step fast multiplex RT-PCR assay (where both differently labeled primer pairs were engaged) coupled to amplicons’ detection on a PCRD device. We evaluated this novel assay on 50 SARS-CoV-2 positive and 50 SARS-CoV-2 negative samples and compared its performance to the results of the quantitative RT-PCR (RT-qPCR) assays used for diagnosing the patients, here considered as the standard tests. Results The new assay achieved a sensitivity of 88% (44/50) and a specificity of 98% (49/50). All patients who presented Ct values lower than 33 were positive for our assay. Except for one patient, those with Ct values above 33 returned negative results. Conclusion Our results have brought proof of principle on the usefulness of the one-step fast multiplex RT- PCR assay coupled to PCRD as a new assay for specific, sensitive, and rapid detection of SARS-CoV-2 without requiring costly laboratory equipment, and thus, at reduced costs in a format prone to be deployed when resources are limited. This assay offers a viable alternative for COVID-19 diagnosis or screening at points of need. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-02...

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Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay

Bel Hadj Ali,

Souguir,

Melliti

et al. 2024

BMC Infect Dis

View full text Add to dashboard Cite

show abstract

Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay

Ali,

Souguir,

Melliti

et al. 2024

Preprint

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Background The COVID-19 pandemics has put emphasis on pivotal needs for diagnosis and surveillance worldwide, with the subsequent shortage of diagnostic reagents and kits. Therefore, it has become strategic for the countries to be able to access diagnosis, expand it, and acquire its own capacity to deploy diagnostics and alternative rapid accurate nucleic acid tests that are at lower costs. Here, we propose a visual SARS-CoV-2 detection using a one-step fast multiplex reverse transcription-PCR (RT-PCR) amplification coupled to lateral flow immunoassay detection on a PCRD device (Abingdon Health, UK). Methods Various simplex fast-PCRs were developed for screening sets of primer pairs newly designed or selected from literature or from validated WHO tests, targeting S, N, E, RdRp or ORF1ab genes. Primers showing specific and stable amplification were retained to assess for their suitability for detection on PCRD. Thus, fast RT-PCR amplifications were performed using the retained primers. They were doubly labeled with Fam and Biotin or Dig and Biotin to allow visual detection of the labeled amplicons on the lateral flow immunoassay PCR Detection (PCRD) device, looking at lack of interaction of the labeled primers (or primer dimers) with the test lines in negative or no RNA controls. All the assays were set up using RNAs isolated from patients’ nasopharyngeal swabs. Two simplex assays, targeting two different viral genomic regions (N and E) and showing specific detection on PCRD, were used to set up a one-step fast multiplex RT-PCR assay (where both differently labeled primer pairs were engaged) coupled to amplicons’ detection on a PCRD device. This novel method was evaluated on 50 SARS-CoV-2 positive and 50 SARS-CoV-2 negative samples and its performance was compared to the results of the quantitative RT-PCR (RT-qPCR) tests used for diagnosing the patients, here considered as the standard methods. Results This way, the new method showed a sensitivity of 88% (44/50) and a specificity of 98% (49/50). All patients who presented Ct values lower than 33 were positive for our assay. Except for one patient, those with Ct values greater than 33 showed negative results. Conclusion Our results have brought proof of principle on the usefulness of the one-step fast multiplex RT- PCR assay coupled to PCRD as new method for specific, sensitive, and rapid detection of SARS-CoV-2 without requiring costly laboratory equipment, and thus at reduced costs, in a format prone to be deployed when resources are limited. This new method of SARS-CoV-2 detection appears to be a good alternative for COVID-19 diagnosis or screening at points of need.

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Mass Spectrometry-Based Metabolomics Reveals a Salivary Signature for Low-Severity COVID-19

Lopes de Lima,

Ap. Rosini Silva,

Brites

et al. 2024

IJMS

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Omics approaches were extensively applied during the coronavirus disease 2019 (COVID-19) pandemic to understand the disease, identify biomarkers with diagnostic and prognostic value, and discover new molecular targets for medications. COVID-19 continues to challenge the healthcare system as the virus mutates, becoming more transmissible or adept at evading the immune system, causing resurgent epidemic waves over the last few years. In this study, we used saliva from volunteers who were negative and positive for COVID-19 when Omicron and its variants became dominant. We applied a direct solid-phase extraction approach followed by non-target metabolomics analysis to identify potential salivary signatures of hospital-recruited volunteers to establish a model for COVID-19 screening. Our model, which aimed to differentiate COVID-19-positive individuals from controls in a hospital setting, was based on 39 compounds and achieved high sensitivity (85%/100%), specificity (82%/84%), and accuracy (84%/92%) in training and validation sets, respectively. The salivary diagnostic signatures were mainly composed of amino acids and lipids and were related to a heightened innate immune antiviral response and an attenuated inflammatory profile. The higher abundance of thyrotropin-releasing hormone in the COVID-19 positive group highlighted the endocrine imbalance in low-severity disease, as first reported here, underscoring the need for further studies in this area.

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Reverse transcription loop‑mediated isothermal amplification has a high performance in the detection of SARS‑CoV‑2 in saliva samples and nasal swabs from asymptomatic and symptomatic individuals

Cited by 3 publications

References 33 publications

Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay

Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay

Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay

Mass Spectrometry-Based Metabolomics Reveals a Salivary Signature for Low-Severity COVID-19

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