Emett Jason, David Roxanne, McDaniel Jaydene, McDaniel Steven, Kingsley Karl
Department of Clinical Sciences, University of Nevada, Las Vegas-School of Dental Medicine, 1700 W. Charleston Blvd, Las Vegas, NV 89106, USA.
DMD-Department of Advanced Education in Pediatric Dentistry, University of Nevada, Las Vegas-School of Dental Medicine, 1700 W. Charleston Blvd, Las Vegas, NV 89106, USA.
Methods Protoc. 2020 Jul 9;3(3):48. doi: 10.3390/mps3030048.
(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 ( = 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 ( = 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.
(1) 引言：由于唾液具有非侵入性，人们已采用多种方法从唾液样本中分离和收集DNA以进行微生物筛查。许多口腔微生物也存在于口腔生物膜中，口腔生物膜可能代表着显著不同的微生物成分，这些成分可能对口腔健康和疾病产生影响，包括龋齿和牙周疾病。此外，同一患者口腔内不同部位的生物膜可能存在差异。很少有研究评估从唾液中分离的DNA与特定部位生物膜中DNA的差异，几乎没有研究涉及儿科患者的此类分析。(2) 方法：确定了一个现有的样本库，其中包含先前从儿科患者（n = 47）采集的纸尖衍生生物膜、龈沟液（GCF）和非刺激性唾液样本。从生物膜部位（舌、上颌颊磨牙、下颌舌侧切牙）分离DNA，并使用苯酚：氯仿萃取法对GCF和唾液进行DNA定量比较。使用NanoDrop 2000分光光度计在A230 nm、A260 nm和A280 nm处读取吸光度，进行定量和定性分析。(3) 结果：这些数据表明成功从所有患者样本中分离出DNA，非刺激性唾液中的DNA浓度最高（4264.1 ng/μL），GCF中的DNA浓度最低（1771.5 ng/μL）。男性与女性患者以及少数族裔与非少数族裔患者之间未观察到差异。此外，成人样本中获得的DNA总体浓度略高于儿科样本，但差异不显著（p = 0.2827）。实时定量qPCR筛查显示，所有评估样本中所含细菌和人类DNA的数量和质量足以进行高于检测限（ΔRn = 0.01）的分子筛查。(4) 结论：目前有多种方法可用于口腔唾液及特定部位的采样和筛查，但包括纸尖采样和非刺激性唾液采集在内的简单低成本方法的验证和比较表明，这些方法和方案可为分子筛查及其他比较应用提供足够的DNA质量和数量。此外，尽管患者样本之间的异质性将是一个持续且一致的特征，但能从口腔不同部位提供相似且一致DNA的标准化方法可能为筛查和分子分析提供所需的一致性。
