Effect of preparation method and storage period on the stability of saliva DNA

Saliva is an attractive source for oral microbial detection and quantification since sampling is non-invasive and rapid.To determine whether different saliva preparation methods or preservation time periods affect DNA stability.Saliva samples from 4 healthy adult volunteers were processed to obtain 3 different preparations: whole saliva, and after centrifugation pellet and supernatant. Purified DNA (MasterPure™) from each sample was divided into 4 aliquots, one for immediate analysis and 3 (stored at -80°C) for later analyses after 1 week and 2 and 6 months. DNA concentrations and qPCR based quantities of Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Filifactor alocis and Streptococcus mutans were determined.DNA concentration did not decrease (P0.05) during the 6-month period in any sample. Mean (SE) DNA concentrations (ng/μl) in whole saliva were 152.2 (51.2) and 147.8 (50) at day 0 and 6 months, respectively. Similarly, the values for pellet were 134.9 (42.5) and 133.6 (42.9), and for supernatant, 11 (1.9) and 8.9 (2.3), the difference being significant (P0.001) between supernatant and whole saliva or pellet. The quantities of most bacterial species found at day 0 remained stable over the 6-month period in all saliva preparations. In supernatant, species quantities were lower (P0.05) than in whole saliva or pellet.DNA concentrations were comparable between whole saliva and pellet, suggesting that either of them can be used for DNA-based analyses. Our results also demonstrated that DNA extracted from saliva can be preserved at -80°C for at least 6 months without decrease in DNA concentration.