A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva

Abstract

The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.

Keywords: COVID-19; RT-PCR SYBR Green; diagnostics; saliva test.

Figure 1.

Sensitivity of the SYBR Green RT-PCR assay for SARS CoV-2 detection. Amplification and melting temperature profiles obtained with the SYBR Green RT-PCR assay after (A) 40 or (B) 33 PCR cycles. (C) LoD of the assay after 33 PCR cycles. (D) Comparison of the CT values obtained with the SYBR Green test and the gold standard test with fluorogenic probes; 56 RNA samples were extracted from NP swab samples of SARS-CoV-2 infected patients and amplified using both strategies. The blue dots indicate RNA samples from infected individuals that did not amplify with the SYBR Green test. NTC, no template control.

Figure 2.

Performance of the SYBR Green RT-PCR assay in detecting SARS CoV-2 VOCα. (A) Specific primers designed to detect the ORF1a Δ3675-3677 mutation. Amplification plots for the SYBR Green RT-PCR assay using (B) known amounts of the vector carrying the mutation (pVOCα) or the original sequence (pWT) and (C) RNA extracted from NP swabs of SARS CoV-2 infected patients. NTC, no template control.

Figure 3.

Sensitivity of the saliva SYBR Green RT-PCR test. (A) Amplification and melting curves obtained after the direct amplification of saliva samples spiked with a known copy number of the IVT N-gene. (B) Analytical LoD of the assay. (C) Scheme of the experimental layout used in (D) created in Biorender.com. (D) Comparison of the CT values obtained after amplification of paired samples (i.e. RNA extracted from NP swabs and saliva samples from the same individual) using the SYBR Green (saliva samples) and the RT-PCR gold standard (NP swabs) tests. The blue circle pinpoints the sample, for which the results of both tests disagree. NTC—no template control.

Figure 4.

Phylogenetic tree based on the alignment of common SNPs of the Omicron clade highlighting the isolates analysed in this study. Isolates 35, 54, 62, and 72, clustered in clade 21K (BA.1 Pango lineage). Isolates 71 and 73 clustered in clade 21 L (BA.2 Pango lineage). SYBR Green-based one-step RT-PCR saliva assay was in agreement with pharmacy antigen test (RAT) for isolates 71, 72, and 73 (highlighted in orange); SYBR Green-based one-step RT-PCR saliva assay was in disagreement with RAT for isolates 35, 54, and 62 (highlighted in grey). The number of mutations relative to the reference strain used (SARS-CoV-2 Wuhan-Hu-1/2019 virus, GenBank accession number MN908947) is shown below in the xx scale. Tree was generated in Nextclade software (accessed on 04 August 2022).