The Effectiveness of ddPCR for Detection of Point Mutations in Poor-Quality Saliva Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Statistical Analysis of Isolated Samples
2.3. Statistical Sample Analysis
2.4. Laboratory Sample Analysis
2.5. Statistical Sample Analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Jarrah, R.; Myers, W.K.A. Comparison of Modern Techniques for Saliva Screening. J. Forensic Sci. 2008, 53, 862–867. [Google Scholar]
- 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. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jingyi Liu, Y.D. Saliva: A Potential Media for Disease Diagnostics and Monitoring. Oral Oncol. 2012, 48, 569–577. [Google Scholar]
- 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. [Google Scholar] [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. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Biorad. Droplet Digital TM PCR Droplet Digital TM PCR Applications Guide; Biorad: Hercules, CA, USA, 2018; p. 145. [Google Scholar]
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef]
- 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. [Google Scholar] [CrossRef] [Green Version]
- 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. [Google Scholar] [CrossRef] [Green Version]
- 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. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- 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. [Google Scholar] [CrossRef]
- 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. [Google Scholar] [CrossRef] [Green Version]
- 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. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- 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. [Google Scholar] [PubMed]
Blood—Extract Purity (A260/280) | Blood—DNA—Concentration (ng/µL) | Saliva—Extract Purity (A260/280) | Saliva—DNA—Concentration (ng/µL) | |
---|---|---|---|---|
Number of samples | 47 | 47 | 47 | 47 |
Median (IQR) | 1.80 (1.7–1.84) | 29.20 (17.6–43.7) | 1.50 (1.26–2.13) | 5.13 (3.06–10.99) |
Arithmetic mean ± SD | 1.78 ± 0.11 | 32.00 ± 17.80 | 1.67 ± 0.59 | N/A |
Minimum | 1.52 | 8.50 | 0.76 | 0.79 |
Maximum | 2.02 | 86.00 | 3.09 | 54.27 |
Skewness/Kurtosis tests for Normality | 0.7805 | 0.0629 | 0.1996 | <0.001 |
Evaluation Using the Number of Copies | Evaluation Using Fractional Abundance | |||
---|---|---|---|---|
Positive (Mutation Present) | Negative (without Mutation) | Positive (Mutation Present) | Negative (without Mutation) | |
Positive (mutation present) | a = 5 | b = 0 | a = 6 | b = 0 |
Negative (without mutation) | c = 1 | d = 41 | c = 0 | d = 41 |
Sensitivity | 83.3% (35.9–99.6) | 100% (54.1–100) | ||
Specificity | 100% (92.0–100) | 100% (91.4–100) | ||
Positive predictive value | 100% (47.8–100) | 100% (54.1–100) | ||
Negative predictive value | 97.6% (87.7–99.9) | 100% (91.4–100) | ||
False positive rate | 0% | 0% | ||
False neagtive rate | 21% | 21% | ||
Accuracy | 97.9% (88.7–99.9) | 100% (92.4–100) |
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Riedlova, P.; Kramna, D.; Ostrizkova, S.; Tomaskova, H.; Jirik, V. The Effectiveness of ddPCR for Detection of Point Mutations in Poor-Quality Saliva Samples. Healthcare 2022, 10, 947. https://doi.org/10.3390/healthcare10050947
Riedlova P, Kramna D, Ostrizkova S, Tomaskova H, Jirik V. The Effectiveness of ddPCR for Detection of Point Mutations in Poor-Quality Saliva Samples. Healthcare. 2022; 10(5):947. https://doi.org/10.3390/healthcare10050947
Chicago/Turabian StyleRiedlova, Petra, Dagmar Kramna, Silvie Ostrizkova, Hana Tomaskova, and Vitezslav Jirik. 2022. "The Effectiveness of ddPCR for Detection of Point Mutations in Poor-Quality Saliva Samples" Healthcare 10, no. 5: 947. https://doi.org/10.3390/healthcare10050947