Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts

Abstract

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.

Keywords: COVID-19; Coronavirus; Digital PCR; Droplet digital PCR; Quantitative assays; SARS-CoV-2; Subgenomic RNA; Viral transcription/replication.

Fig. 1

Schematic representation of SARS-CoV-2 genome organization, virion structure, assay design and sgRNA targets. SARS-CoV-2 employs discontinuous transcription to generate subgenomic RNAs. (A) The genome organization of SARS-CoV-2. The genome features two large genes, ORF1a (yellow) and ORF1b (blue), which encode 16 non-structural proteins (NSP1–NSP16). The structural genes encode the structural proteins, spike (S; green), envelope (E; blue), membrane (M; purple), and nucleocapsid (N; gold). Assay locations of each assay designed for this study are indicated. The SARS-CoV-2 virion structure is shown in the lower panel. (B) Assay design. Assays for a given subgenomic RNA and the corresponding “genomic” RNA region share the same probe and reverse primer (located in a body gene) but differ in the forward primers. The sgRNA-targeting forward primer is located in the 5’ UTR (upstream of the leader-body TRS junction), whereas the cognate gRNA-targeting forward primer is located upstream of the body TRS and coding region. (C) Schematic representation of negative strand RNA synthesis from the full-length positive strand gRNA. A template-switch occurs at the body TRS [TRS-B] to the 5’ leader TRS [TRS-L] to give rise to the negative-strand sgRNA. (D) The panel of validated canonical subgenomic RNA targets is shown, including S, 3a, E, M, 7a, 8 and N. Figure adapted from Telwatte et al., 2021 and Kim et al., 2020. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Comparing efficiency of subgenomic assays and total SARS-CoV-2 assays. The efficiency of selected subgenomic assays (sgE, sgM, and sgN_2) relative to previously validated SARS-CoV-2 assays (targeting total E, M or N coding region; grey symbols) were measured using the same plasmids. S indicates slope (efficiency).

Fig. 3

Efficiency and linearity of ddPCR assays for SARS-CoV-2 subgenomic transcripts determined using plasmid DNA. Specially designed subgenomic plasmids containing the 5′ UTR sequence upstream of the coding regions of individual body genes were quantified by UV spectroscopy and diluted (expected copies) to test the absolute number of copies detected by each primer/probe set using duplicate ddPCR reactions (measured copies). Coloured symbols denote the best-performing of alternate primer/probe sets for a given sgRNA.

Fig. 4

Efficiency and linearity of ddPCR assays for SARS-CoV-2 “genomic” and subgenomic transcripts determined using supernatant from in vitro infection. A SARS-CoV-2 ‘supernatant’ standard was prepared by extracting the RNA from the supernatant of an in vitro infection and quantified using the Abbott Real Time SARS-CoV-2 assay. Various inputs of the supernatant standard (which were used to calculate ‘Expected Copies’ per ddPCR well) were applied to a common reverse transcription reaction, from which aliquots of cDNA were used to measure the absolute number of copies detected by each ddPCR assay (measured copies) for ‘genomic’ or subgenomic transcripts. Each assay was tested with expected inputs of 10–10,000 copies/ddPCR well in duplicate. S (slope) and R² are indicated for each assay. Coloured symbols denote the best-performing of alternate primer/probe sets for a given gRNA or sgRNA.

Fig. 5

Expression of sgRNA and gRNA in supernatant from a SARS-CoV-2 in vitro infection. Aliquots of cDNA from a common RT reaction containing SARS-CoV-2 RNA were added to ddPCR reactions (predicted to contain 1000 copies of SARS-CoV-2/well) and the levels of each (A) sgRNA and (B) gRNA target were measured. (C) The copies of each sgRNA were divided by the copies of genomic S RNA to express the ratio of each subgenomic RNA to genomic RNA.. Each target region is depicted by a different colour (red: S; orange: ORF3a; yellow: E; green: M; blue: ORF7a; teal: ORF8; and grey: N). Symbols represent the following: squares: subgenomic RNA; circles: genomic RNA; and diamonds: ratio of sgRNA/gRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Expression of sgRNAs in pharynx of one acutely SARS-CoV-2- infected individual. Total cell-associated RNA was isolated from a nasopharyngeal swab. A common RT reaction was divided across ddPCR reactions to measure seven sgRNA targets and one genomic target (S). Independent measurements of two other genomic targets, RDRP and Main Proteinase, from the same sample are included as a reference. Levels of each target are expressed as copies per μL extract.

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