Comparison of DNA Extracted from Pediatric Saliva, Gingival Crevicular Fluid and Site-Specific Biofilm Samples

Abstract

<jats:p>(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 (n = 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 (p = 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.</jats:p>

Molecular Screening and Analysis Reveal Novel Oral Site-Specific Locations for the Cariogenic Pathogen Scardovia wiggsiae

“…DNA was isolated from 100 μL of saliva samples and from paper points that were placed into 100 μL of sterile 1X phosphate-buffered saline (PBS) and vortexed to release any bacteria or other microbial constituents. Each sample was then processed using the phenol: chloroform extraction method, as previously described [ 21 , 28 ]. In brief, 300 μL of TriZol was added to each sample and mixed to dissolve microbial membranes and cell walls.…”

“…Most recently, a series of site-specific sampling studies from this group have revealed differential oral locations for SW distribution among patient samples—a major step towards understanding the potential sites for colonization [ 27 , 28 ]. However, due to the limited number of patient samples available in these initial studies, a larger and more comprehensive microbial screening analysis was the overall objective of this current study.…”

Microbial Screening Reveals Oral Site-Specific Locations of the Periodontal Pathogen Selenomonas noxia

“…DNA was extracted from paper points by adding 100 µL of sterile 1X phosphate buffered saline (PBS) to each tube and vortexing the tube to release any collected materials. TRIzol from ThermoFisher (Fair Lawn, NJ, USA) was then added to each tube (300 µL) and the extraction method was completed according to the manufacturer protocol, as previously described [24]. In brief, 200 µL of chloroform was added to each tube and then centrifuged at 12,000× g (relative centrifugal force, RCF) for 15 min at 4 • C. The supernatant was removed and placed into a new micro centrifuge tube with 100 µL of isopropanol to precipitate the DNA.…”

Screening for Selenomonas Noxia and Akkermansia Muciniphila from the Oral Cavity of Pediatric Patients

“…Due to its non-invasive nature, saliva collection has been used to isolate and collect DNA from saliva samples for microbial screening [15 The original protocol for sample collection involved patients providing up to five mL of unstimulated saliva in sterile saliva tubes from each patient. As noted previously [18], the samples were stored in a freezer ( biomedical safety level 2 (BSL-2) laboratory for the purpose of longevity and analysis of the samples. The saliva samples were provided with random, non-repetitive numbers to remove any patient identification factors from the origin of the samples.…”

“…The DNA in the saliva was isolated using the phenol:chloroform extraction method with Invitrogen (TRIzol reagent) known to isolate high quality DNA/RNA from samples in fluids and tissues, as previously described [18]. analysis, 100 μL of saliva was removed μL of TRIzol reagent was added and mixed of chloroform added to the mixture after five minutes.…”