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Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing.

ABSTRACT: SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific variants. This method can easily distinguish Omicron from other variants.

SUBMITTER: Bloemen M

PROVIDER: S-EPMC8896866 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing.

Bloemen Mandy M Rector Annabel A Swinnen Jill J Ranst Marc Van MV Maes Piet P Vanmechelen Bert B Wollants Elke E

Journal of virological methods 20220304

SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific ...[more]

PMID: 35257682

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Project description:BackgroundThe 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).MethodsWe 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.ResultsThe 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.ConclusionOur 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.

Project description:BackgroundDengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.MethodsThe DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.ResultsThe RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 10(2) to 10(6) copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.ConclusionsOur results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.

Dataset Information

Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing.

Publications

Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing.

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