Saliva effect on RT-PCR performance

ABSTRACT

The use of saliva directly in RT-PCR reactions may be interesting, particularly in screenings for the detection of DNA/RNA of pathogens, due to the advantages of time and costs. However, its use faces some difficulties that essentially result from the saliva being a matrix with a variable composition and which contains inhibitors of the polymerization reaction. Saliva samples obtained from healthy donors were spiked with SARS-CoV-2 RNA and 3 viral targets were detected by RT-PCR. This work was considered exempt from review by an institutional ethical review board, because it comprises use of completely anonymized specimens obtained voluntarily and informed. The efficiency of RT-PCR reactions was calculated in the presence of variable amounts of saliva and the effect of saliva pre-treatment on the performance of each reaction was also analysed. It was found that saliva has no significant influence on the mean efficiency of each individual reaction, affecting mainly the beginning of the exponential phase of amplification, resulting in a significant increase in Ct values. This effect which is target dependent, presents a highly between-person variability (DELTACt = 0.93 to 11.36) and affects RNA/DNA detection and the limit-of-detection, but can be partly reversed by a treatment of saliva prior to the reaction. The work presented contributes to a better understanding of the advantages and limitations of using saliva directly in RT-PCR. This work was supported by ProjectNORTE-01-0145- FEDER-072545- SAICTCOVID/72545/2020 and was the result of the projects UIDB/05937/2020 and UIDP/05937/2020- Centre for Research and Development in Agrifood Systems and Sustainability-funded by national funds, through FCT-Fundacao para a Ciencia e a Tecnologia.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.