A low-cost molecular test for SARS-CoV-2 detection suitable for variant discrimination and community testing using saliva (preprint)

The gold standard for COVID-19 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through RT-PCR with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection make 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 SARS-CoV-2 directly from saliva samples or RNA isolated from nasopharyngeal 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 of less than {euro} 0.8 per child, and in some cases, the saliva test outperformed nasopharyngeal 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. To further reduce testing costs, we produced all the necessary enzymes and established a new RT-PCR protocol based on the EvaGreen dye. 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.

A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples (preprint)

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 minutes. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, qRT-PCR, using 177 nasopharyngeal RNA samples. For Ct ≤ 32, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.