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

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 € 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by (i) Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 Programa Operacional Competitividade e Internacionalizacao (POCI), (ii) Fundacao para a Ciencia e a Tecnologia (FCT) through the project DETECT Ref 433_613549914 (20/7/153), under the scope of the 2nd edition of the programme RESEARCH4COVID19, (iii) the project STOP-COVID Strategies to prevent COVID-19 by early detection of asymptomatic carriers at increased risk: epidemiological studies and validation of a rapid in-house diagnostic test, Ref 072559, funded by FEDER Fundo Europeu de Desenvolvimento Regional from Programa Operacional Regional Lisboa and (iv) the Municipality of Oeiras ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was given by the Institutional Review Board of Hospital das Forcas Armadas (HFA), Portugal, (samples collected at HFA) or by the Instituto de Higiene e Medicina Tropical (IHMT) - Instituto de Tecnologia Quimica e Biologica (ITQB NOVA) Ethics Committee (samples collected at Fundicao de Oeiras), Portugal. The study was conducted in accordance with the European Statements for Good Clinical Practice and the Declaration of Helsinki of the World Health Medical Association. Written informed consent was obtained from all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript