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

Autor: Bel Hadj Ali I; Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia. insaf.bha@gmail.com., Souguir H; Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia., Melliti M; Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia., Mohamed MVT; Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia., Ardhaoui M; Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia., Ayouni K; Laboratory of Clinical Virology, WHO Regional Reference Laboratory for Poliomyelitis and Measles for the EMR, Institut Pasteur de Tunis, Tunis, Tunisia.; Laboratory of Virus, Host and Vectors, LR20 IPT02, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, Tunisia., Haddad-Boubaker S; Laboratory of Clinical Virology, WHO Regional Reference Laboratory for Poliomyelitis and Measles for the EMR, Institut Pasteur de Tunis, Tunis, Tunisia.; Laboratory of Virus, Host and Vectors, LR20 IPT02, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, Tunisia., Saadi Ben Aoun Y; Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia., Triki H; Laboratory of Clinical Virology, WHO Regional Reference Laboratory for Poliomyelitis and Measles for the EMR, Institut Pasteur de Tunis, Tunis, Tunisia.; Laboratory of Virus, Host and Vectors, LR20 IPT02, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, Tunisia., Guizani I; Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia.
Jazyk: angličtina
Zdroj: BMC infectious diseases [BMC Infect Dis] 2024 Dec 18; Vol. 24 (1), pp. 1417. Date of Electronic Publication: 2024 Dec 18.
DOI: 10.1186/s12879-024-10296-1
Abstrakt: 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 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. 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.
Competing Interests: Declarations. Ethics approval and consent to participate: The study is approved by the Biomedical Ethics Committee of the Institut Pasteur de Tunis under the reference Ref: 2020/21/I/LR16IPT in accordance with the Declaration of Helsinki. The need for consent to participate was waived by the Biomedical Ethics Committee of Institut Pasteur de Tunis. We used RNA retrospectively collected in the frame of the routine diagnosis of the COVID-19. Anonymous extracted viral RNA samples were transferred to our laboratory for assays development. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
(© 2024. The Author(s).)
Databáze: MEDLINE
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