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Updated: Oct 26, 2025
Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots
Published on: February 11, 2022
Validation and implementation of a direct RT-qPCR method for rapid screening of SARS-CoV-2 infection by using
Pedro Brotons1, Amaresh Perez-Argüello2, Cristian Launes3
1Institut de Recerca Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain; Department of Medicine, Universitat Internacional de Catalunya, Barcelona, Spain; Consorcio de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Madrid, Spain.
Objective:
To validate and implement an optimized screening method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining use of self-collected raw saliva samples, single-step heat-treated virus inactivation and RNA extraction, and direct RT-qPCR.
Methods:
This was a three-phase study conducted in Barcelona (Spain) during June to October, 2020. The three phases were (1) analytical validation against standard RT-qPCR in saliva samples; (2) diagnostic validation against standard RT-qPCR using paired saliva-nasopharyngeal samples obtained from asymptomatic teenagers and adults in a sports academy; and (3) pilot screening of asymptomatic health workers in a tertiary hospital.
Results:
In phase 1, the detection yield of the new method was comparable to that of standard RT-qPCR. In phase 2, the diagnostic sensitivity and specificity values in 303 self-collected saliva samples were 95.7% (95% confidence interval 79.0-99.2%) and 100.0% (95% confidence interval 98.6-100.0%), respectively. In phase 3, only 17 (0.6%) of the saliva samples self-collected by 2709 participants without supervision were invalid. The rapid analytical workflow with the new method (up to 384 batched samples could be processed in less than 2 hours) yielded 24 (0.9%) positive results in the remaining 2692 saliva samples. Paired nasopharyngeal specimens were all positive by standard RT-qPCR.
Conclusions:
Direct RT-qPCR on self-collected raw saliva is a simple, rapid, and accurate method with potential to be scaled up for enhanced SARS-CoV-2 community-wide screening.
