BACKGROUND: The noninvasive collection of saliva samples for DNA analyses is simple, and its potential for research and diagnostic purposes is great. However, DNA isolates from such samples are often of inferior quality to those from blood. AIM: The aim of this study was to investigate the robustness and sensitivity of the ddPCR instrument for genetic analyses from saliva samples of poor quality by comparing their results to those obtained using an established method from blood samples. METHODS: Blood and saliva were collected from 47 university students, which was followed by manual isolation of DNA and analysis on droplet digital PCR (ddPCR). Results of analyses were supplemented with values of fractional abundances. RESULTS: ddPCR proved to be highly suitable for analysis of even low-quality saliva samples (concentrations as low as 0.79 ng/µL), especially when augmented by fractional abundance data. This combination yielded 100% agreement with results obtained from blood samples. CONCLUSION: This study verified the applicability of ddPCR as a sensitive and robust method of genetic diagnostic testing even from low-quality saliva isolates. This makes it potentially suitable for a wide range of applications and facilitates the performance of large epidemiological studies, even if sampling or sample processing is suboptimal.

Centre for Epidemiological Research Faculty of Medicine University of Ostrava 70103 Ostrava Czech Republic

Department of Epidemiology and Public Health Faculty of Medicine University of Ostrava 70103 Ostrava Czech Republic

See more in PubMed

Jarrah R., Myers W.K.A. Comparison of Modern Techniques for Saliva Screening. J. Forensic Sci. 2008;53:862–867. PubMed

Hansen T.V.O., Simonsen M.K., Nielsen F.C., Hundrup Y.A. Collection of Blood, Saliva, and Buccal Cell Samples in a Pilot Study on the Danish Nurse Cohort: Comparison of the Response Rate and Quality of Genomic DNA. Cancer Epidemiol. Biomark. Prev. 2007;16:2072–2076. doi: 10.1158/1055-9965.EPI-07-0611. PubMed DOI

Ooi D.S.Q., Tan V.M.H., Ong S.G., Chan Y.H., Heng C.K., Lee Y.S. Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall. PLoS ONE. 2017;12:e0170767. doi: 10.1371/journal.pone.0170767. PubMed DOI PMC

Rogers N.L., Cole S.A., Lan H.-C., Crossa A., Demerath E.W. New saliva DNA collection method compared to buccal cell collection techniques for epidemiological studies. Am. J. Hum. Biol. 2007;19:319–326. doi: 10.1002/ajhb.20586. PubMed DOI PMC

Williamson S., Munro C., Pickler R., Grap M.J., Elswick R.K., Jr. Comparison of Biomarkers in Blood and Saliva in Healthy Adults. Nurs. Res. Pract. 2012;2012:246178. doi: 10.1155/2012/246178. PubMed DOI PMC

Durdiaková J., Kamodyová N., Ostatníková D., Vlková B., Celec P. Comparison of different collection procedures and two methods for DNA isolation from saliva. Clin. Chem. Lab. Med. (CCLM) 2012;50:643–647. doi: 10.1515/cclm.2011.814. PubMed DOI

Philibert R.A., Zadorozhnyaya O., Beach S.R., Brody G.H. A Comparison of the Genotyping Results Using DNA Obtained from Blood and Saliva. Psychiatr. Genet. 2008;18:275–281. doi: 10.1097/YPG.0b013e3283060f81. PubMed DOI PMC

Jingyi Liu Y.D. Saliva: A Potential Media for Disease Diagnostics and Monitoring. Oral Oncol. 2012;48:569–577. PubMed

Looi M.-L., Zakaria H., Osman J., Jamal R. Quantity and quality assessment of DNA extracted from saliva and blood. Clin. Lab. 2012;58:307–312. PubMed

Hayden R.T., Gu Z., Ingersoll J., Abdul-Ali D., Shi L., Pounds S., Caliendo A.M. Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus. J. Clin. Microbiol. 2013;51:540–546. doi: 10.1128/JCM.02620-12. PubMed DOI PMC

Biorad . Droplet Digital TM PCR Droplet Digital TM PCR Applications Guide. Biorad; Hercules, CA, USA: 2018. p. 145.

Riedlova P., Kramna D., Ostrizkova S., Tomaskova H. Examination of in Factor V Leiden and Prothrombin II Thrombo-philic Mutations in Czech Young Women Using DdPCR—Prevalence and Cost—Benefit Analysis. Healthcare. 2021;9:1656. doi: 10.3390/healthcare9121656. PubMed DOI PMC

Ying G.-S., Maguire M.G., Glynn R.J., Rosner B. Calculating Sensitivity, Specificity, and Predictive Values for Correlated Eye Data. Investig. Opthalmol. Vis. Sci. 2020;61:29. doi: 10.1167/iovs.61.11.29. PubMed DOI PMC

Uchiyama Y., Nakashima M., Watanabe S., Miyajima M., Taguri M., Miyatake S., Miyake N., Saitsu H., Mishima H., Kinoshita A., et al. Ultra–sensitive droplet digital PCR for detecting a low–prevalence somatic GNAQ mutation in Sturge–Weber syndrome. Sci. Rep. 2016;6:22985. doi: 10.1038/srep22985. PubMed DOI PMC

Abraham J.E., Maranian M.J., Spiteri I., Russell R., Ingle S., Luccarini C., Earl H.M., Pharoah P.P., Dunning A.M., Caldas C. Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping. BMC Med. Genom. 2012;5:19. doi: 10.1186/1755-8794-5-19. PubMed DOI PMC

Dillon M.C., Opris D.C., Kopanczyk R., Lickliter J., Cornwell H.N., Bridges E.G., Nazar A.M., Bridges K.G. Detection of Homocysteine and C-Reactive Protein in the Saliva of Healthy Adults: Comparison with Blood Levels. Biomark. Insights. 2010;5:57–61. doi: 10.4137/BMI.S5305. PubMed DOI PMC

Ye P., Cai P., Xie J., Wei Y. The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: A systematic review and meta-analysis. PLoS ONE. 2021;16:e0248775. doi: 10.1371/journal.pone.0248775. PubMed DOI PMC

Link-Lenczowska D., Pallisgaard N., Cordua S., Zawada M., Czekalska S., Krochmalczyk D., Kanduła Z., Sacha T. A comparison of qPCR and ddPCR used for quantification of the JAK2 V617F allele burden in Ph negative MPNs: A system-atic review and meta-analysis. Ann. Hematol. 2018;97:2299–2308. doi: 10.1007/s00277-018-3451-1. PubMed DOI PMC

Rylander-Rudqvist T., Håkansson N., Tybring G., Wolk A. Quality and Quantity of Saliva DNA Obtained from the Self-administrated Oragene Method—A Pilot Study on the Cohort of Swedish Men. Cancer Epidemiol. Biomark. Prev. 2006;15:1742–1745. doi: 10.1158/1055-9965.EPI-05-0706. PubMed DOI

Sun F., Reichenberger E.J. Saliva as a Source of Genomic DNA for Genetic Studies: Review of Current Methods and Appli-cations. Oral Health Dent. Manag. 2014;13:217–222. PubMed