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Updated: Sep 7, 2025
Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots
Published on: February 11, 2022
Validation of a molecular assay to detect SARS-CoV-2 in saliva
Janet L Pitman1, Arthur J Morris2, Stephen Grice3
1Associate Professor, School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington.
Aim:
To validate a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay to detect SARS-CoV-2 in saliva in two independent Aotearoa New Zealand laboratories.
Methods:
An RT-qPCR assay developed at University of Illinois Urbana-Champaign, USA, was validated in two New Zealand laboratories. Analytical measures, such as limit of detection (LOD) and cross-reactivity, were performed. One hundred and forty-seven saliva samples, each paired with a contemporaneously collected nasal swab, mainly of nasopharyngeal origin, were received. Positive (N=33) and negative (N=114) samples were tested blindly in each laboratory. Diagnostic sensitivity and specificity were then calculated.
Results:
The LOD was <0.75 copy per µL and no cross-reactivity with MERS-CoV was detected. There was complete concordance between laboratories for all saliva samples with the quantification cycle values for all three genes in close agreement. Saliva had 98.7% concordance with paired nasal samples: and a sensitivity, specificity and accuracy of 97.0%, 99.1% and 99.1%, respectively.
Conclusion:
This saliva RT-qPCR assay produces reproducible results with a low LOD. High sensitivity and specificity make it a reliable option for SARS-CoV-2 testing, including for asymptomatic people requiring regular screening.
