Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples
(1) Introduction: Due to the non-invasive nature of saliva, many methods have been used to isolate and collect DNA from saliva samples for microbial screening. Many oral microbes also inhabit the oral biofilm, which may represent significantly different microbial constituents that may contribute to...
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| Format: | Article |
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MDPI AG
2020-07-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/3/3/48 |
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| author | Jason Emett Roxanne David Jaydene McDaniel Steven McDaniel Karl Kingsley |
| author_facet | Jason Emett Roxanne David Jaydene McDaniel Steven McDaniel Karl Kingsley |
| author_sort | Jason Emett |
| collection | DOAJ |
| description | (1) Introduction: Due to the non-invasive nature of saliva, many methods have been used to isolate and collect DNA from saliva samples for microbial screening. Many oral microbes also inhabit the oral biofilm, which may represent significantly different microbial constituents that may contribute to oral health and disease, including caries and periodontal disorders. Moreover, the biofilm may vary within the same patient at different sites. Few studies have evaluated the comparison between DNA isolated from saliva and DNA from site-specific biofilm, with virtually no studies addressing this analysis among pediatric patients. (2) Methods: An existing repository of paper point derived biofilm, gingival crevicular fluid (GCF), and unstimulated saliva samples previously collected from pediatric patients (<i>n</i> = 47) was identified. DNA was isolated from biofilm sites (tongue, upper buccal molar, mandibular lingual incisor), and GCF and saliva were used for quantitative DNA comparison using a phenol:chloroform extraction. A quantitative and qualitative analysis was performed using the NanoDrop 2000 spectrophotometer using absorbance readings at A230 nm, A260 nm and A280 nm. (3) Results: These data demonstrated the successful isolation of DNA from all of the patient samples, with the highest concentrations observed among unstimulated saliva (4264.1 ng/μL) and the lowest derived from GCF (1771.5 ng/μL). No differences were observed between males and females or minorities and non-minority patients. In addition, comparison of the overall concentrations of DNA obtained from adult samples was slightly higher than, but not significantly different from, the concentrations obtained from pediatric samples (<i>p</i> = 0.2827). A real-time quantitative qPCR screening revealed that all of the samples evaluated harbored bacterial and human DNA of sufficient quantity and quality for a molecular screening greater than the limit of detection (ΔRn = 0.01). (4) Conclusions: Many methods are currently available to provide the sampling and screening of saliva and specific sites within the oral cavity, but the validation and comparison of simple and low-cost methods, that include paper point sampling and unstimulated saliva collection, may suggest these methods and protocols provide sufficient DNA quality and quantity for molecular screening and other comparison applications. In addition, although heterogeneity will be a constant and consistent feature between patient samples, standardized methods that provide similar and consistent DNA from various oral sites may provide needed consistency for screening and molecular analysis. |
| first_indexed | 2024-03-10T18:34:47Z |
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| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-10T18:34:47Z |
| publishDate | 2020-07-01 |
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| spelling | doaj.art-aa4f6b60bff44280a6fa99bd2e5731992023-11-20T06:18:48ZengMDPI AGMethods and Protocols2409-92792020-07-01334810.3390/mps3030048Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm SamplesJason Emett0Roxanne David1Jaydene McDaniel2Steven McDaniel3Karl Kingsley4Department of Clinical Sciences, University of Nevada, Las Vegas—School of Dental Medicine, 1700 W. Charleston Blvd, Las Vegas, NV 89106, USADepartment of Clinical Sciences, University of Nevada, Las Vegas—School of Dental Medicine, 1700 W. Charleston Blvd, Las Vegas, NV 89106, USADMD—Department of Advanced Education in Pediatric Dentistry, University of Nevada, Las Vegas—School of Dental Medicine, 1700 W. Charleston Blvd, Las Vegas, NV 89106, USADMD—Department of Advanced Education in Pediatric Dentistry, University of Nevada, Las Vegas—School of Dental Medicine, 1700 W. Charleston Blvd, Las Vegas, NV 89106, USADepartment of Biomedical Sciences and Director of Student Research, University of Nevada, Las Vegas—School of Dental Medicine, 1001 Shadow Lane, Las Vegas, NV 89106, USA(1) Introduction: Due to the non-invasive nature of saliva, many methods have been used to isolate and collect DNA from saliva samples for microbial screening. Many oral microbes also inhabit the oral biofilm, which may represent significantly different microbial constituents that may contribute to oral health and disease, including caries and periodontal disorders. Moreover, the biofilm may vary within the same patient at different sites. Few studies have evaluated the comparison between DNA isolated from saliva and DNA from site-specific biofilm, with virtually no studies addressing this analysis among pediatric patients. (2) Methods: An existing repository of paper point derived biofilm, gingival crevicular fluid (GCF), and unstimulated saliva samples previously collected from pediatric patients (<i>n</i> = 47) was identified. DNA was isolated from biofilm sites (tongue, upper buccal molar, mandibular lingual incisor), and GCF and saliva were used for quantitative DNA comparison using a phenol:chloroform extraction. A quantitative and qualitative analysis was performed using the NanoDrop 2000 spectrophotometer using absorbance readings at A230 nm, A260 nm and A280 nm. (3) Results: These data demonstrated the successful isolation of DNA from all of the patient samples, with the highest concentrations observed among unstimulated saliva (4264.1 ng/μL) and the lowest derived from GCF (1771.5 ng/μL). No differences were observed between males and females or minorities and non-minority patients. In addition, comparison of the overall concentrations of DNA obtained from adult samples was slightly higher than, but not significantly different from, the concentrations obtained from pediatric samples (<i>p</i> = 0.2827). A real-time quantitative qPCR screening revealed that all of the samples evaluated harbored bacterial and human DNA of sufficient quantity and quality for a molecular screening greater than the limit of detection (ΔRn = 0.01). (4) Conclusions: Many methods are currently available to provide the sampling and screening of saliva and specific sites within the oral cavity, but the validation and comparison of simple and low-cost methods, that include paper point sampling and unstimulated saliva collection, may suggest these methods and protocols provide sufficient DNA quality and quantity for molecular screening and other comparison applications. In addition, although heterogeneity will be a constant and consistent feature between patient samples, standardized methods that provide similar and consistent DNA from various oral sites may provide needed consistency for screening and molecular analysis.https://www.mdpi.com/2409-9279/3/3/48saliva screeninggingival crevicular fluidoral biofilmpaper point samplingDNA isolation |
| spellingShingle | Jason Emett Roxanne David Jaydene McDaniel Steven McDaniel Karl Kingsley Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples Methods and Protocols saliva screening gingival crevicular fluid oral biofilm paper point sampling DNA isolation |
| title | Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples |
| title_full | Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples |
| title_fullStr | Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples |
| title_full_unstemmed | Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples |
| title_short | Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples |
| title_sort | comparison of dna extracted from pediatric saliva gingival crevicular fluid and site specific biofilm samples |
| topic | saliva screening gingival crevicular fluid oral biofilm paper point sampling DNA isolation |
| url | https://www.mdpi.com/2409-9279/3/3/48 |
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