Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

Abstract

Background: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis.

Objectives: To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages.

Methods: SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E.

Findings: SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL.

Main conclusions: Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

Fig. 1:. colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes allows the detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) variant omicron. Selected nasopharyngeal swab-derived samples, previously detected positive for SARS-CoV-2 by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and identified as the new omicron variant of concern (VOC) by next generation sequencing, were included as input for colorimetric RT-LAMP reaction targeting N and E genes. RT-LAMP reactions were performed combining the primer sets N2/E1 in the same reaction (multiplex) or independently.⁽⁴⁾ Both strategies resulted in positive reaction as evidenced by yellow colour. Negative or absence of specific amplification is represented by pink colour and no bands on gel electrophoresis. RT-LAMP reaction was performed at 65ºC during 30 min, using the WarmStart^® colorimetric LAMP 2X master mix (NEB #M1804). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed^® (Biotium #41003) to confirm DNA amplification. +C: positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC: non-template control. Upper panel cartoon is showing SARS-CoV-2 genome where N and E gene regions are zoomed in. Polymorphisms are indicated in their corresponding positions. The SNP C26270U, highlighted by the red arrow, is the only polymorphism that overlaps with F2 primer (magnifying glass). F3: forward; B3: backward; LF: loop forward; LB: loop backward; F2 and F1c: parts of FIP - forward internal primer; B1 and B2c: parts of BIP - backward inner primer. P: proline; L: leucine; R: arginine; K: lysine; G: glycine; S: serine; T: threonine; I: isoleucine; E: glutamic acid. BA.2, also known as 21L, is considered a sub-lineage of SARS-CoV-2 omicron. Genomic representation was created using SnapGene. Parts of this figure were created with BioRender.com and are licensed under the agreement number: QC23MCKTFE.

Fig. 2:. reverse transcription loop-mediated isothermal amplification (RT-LAMP) of Omicron variant sub-lineages. (A) The test was performed using multiplex N/E strategy. All samples were positive (yellow colour) after 30 min reaction. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed^® (Biotium #41003) to confirm DNA amplification. +C: positive control using severe acute respiratory syndrome coronavirus (SARS-CoV-2) RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC: non-template control. From the left to the right hand side, clinical samples corresponds to Supplementary data (Table samples 11 to 20. (B) Limit of detection (LoD) of Omicron samples. The test was performed using as input a serial dilution of the sample 3 (randomly chosen). The absolute quantification was performed based on a standard curve prepared using the template SARS-CoV-2 E gene-harboring plasmid 2 × 10⁵ copies/µL; Biogene Coronavirus disease 19 (COVID-19) polymerase chain reaction (PCR), Bioclin/Quibasa #K228-1; Lot: 0007. The equation of a straight line is y = -3,476x+38,063, where Y is the Ct for E gene of the sample 3 in reverse transcription quantitative polymerase chain reaction (RT-qPCR) and X is the value of quantification. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed^® (Biotium #41003) to confirm DNA amplification. +C: positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC: non-template control. Figure representative of independent assays and signs of cutted and overlapping images can be seen for this reason. Original raw material can be send upon request.