human saliva microbiome extraction and analysis

Saliva, exclusively and predominantly excreted in the mouth is hydrous in nature and excreted by diverse salivary organs found in humans and animals. In humans, water being the sole constituent of saliva measures at (99. 5%), with other substances at the remaining 0. 5% (Fejerskov and Kidd, 2007). These substances include but are not limited to amylase, electrolytes, epithelial cells, lipase, lysozyme, mucus, secretory IgA, white blood cells, etc. (Fejerskov and Kidd, 2007).

Enzymes formed in saliva has been implicated in many vital functions in the body, such as breakdown of fat and starch present in diets, breakdown of food residue trapped in the dental nook, prevention of teeth decay caused by bacteria, and maintaining oral hygiene (Fejerskov and Kidd, 2007). Deficiency in saliva secretion will lead to a notable spread (occurrence and recurrence) of oral diseases including dental caries, gingivitis, periodontitis (Fejerskov and Kidd, 2007). In addition, saliva stands as a lubricant, adding moisture to food and allowing for deglutition, prevents dehydration of the mucosal wall of the oral cavity, prevents mechanical trauma during eating, swallowing and talking/speaking (Edgar et al. , 2004).

In the absence or reduced production of saliva otherwise called xerostomia, mouth soreness is implicated and food glues to the walls of the mouth (Fejerskov and Kidd, 2007). Saliva formation in hale and hearty individuals in a day is estimated between 0. 75 to 1. 5litres.

This is during the active period, while this amount reduces drastically at night (while sleeping) to almost nothing (Edgar et al. , 2004). The sub-mandibular gland is accountable for 70 – 75% of saliva formed, the parotid gland releases about 20 – 25%, the remaining glands (sublingual gland inclusive) produces minute amount and adds up to the total measure of saliva formed (Edgar et al. , 2004). Saliva has been shown to maintain the mouth’s pH. Saliva’s concentration is spiked in the presence of diverse ions. Some saliva proteins hinders precipitation, hence, forming salts.

The presenting ions serves as buffer that helps to retain the acidic level of the mouth at a specific range, usually pH 6. 2 – 7. 4, thus keeps from degrading, the minerals found in the dental hard tissues. (Edgar et al. , 2004). The two nervous systems (sympathetic and parasympathetic) stimulates saliva formation. A thicker saliva is formed following sympathetic stimulation while water-like saliva is formed following parasympathetic stimulation. While respiration is facilitated by saliva formed from sympathetic stimulation, digestion on the other hand is facilitated by parasympathetic stimulation. Sialagogue (a drug that promotes the formation of saliva) is used in pharmacology to stimulate the formation of saliva and in reverse antisialagogues can suppress saliva production. (Edgar et al. , 2004).

Saliva as a diagnostic tool

Saliva is the hydrous solution excreted in the mouth of humans. Although saliva is easily accessible and collection of saliva sample is non invasive, it was previously underutilized as a tool for diagnosis.

Microbiome analysis

Microbiome analysis in human is the study of total microorganisms associated with human bodies (whether inside or on the body). The purpose of human microbiome studies is to figure out the part played by microbes (nature and function) in relation to disease and well-being. Thus, analysis of saliva microbiome is the study of microorganisms found in saliva and/or in the gut (Vesty et al. , 2017). Some studies shows that altering the microorganisms found in the gut, for example, correlates with intestinal and extraintestinal disease.

Microorganisms has proven to be an attractive target for developing biomarkers for disease detection, management, and potential therapeutic applications, even though only a few mechanisms are known (Claesson et al. , 2017). Latest and powerful techniques (Bioinformatics tools) such as Next-generation sequencing, 16S rRNA sequencing and Shotgun metagenomics has been implicated to observe changes to pathogens at a genome level and the make-up of microorganisms, also, facilitating investigation on the affiliation between microorganisms and the resulting diseases (Hao et al. , 2017).

DNA Extraction

Extraction of DNA, also referred to as isolation of DNA is the process of decontaminating DNA from samples using different methods combined, these include chemical and physical methods. In 1869, Friedeich Miescher did the first DNA isolation (Dahm, 2008). Extraction of DNA has become a frequent practice in molecular science as well as forensic studies. Different methods of extraction leads to different yield (quantity) and purity (quality) of DNA extracted. There are basic standards which should be met by any DNA extraction method irrespective of the sample with which DNA is being isolated, these include

• effective/productive extraction
• adequate amount of extracted DNA to be used for downstream processes
• decontamination
• extracted DNA’s quality and quantity

Conventional/common methods adopted for the extraction of DNA includes

• Organic
• Salting out
• Magnetic
• Silica
• Exchange of anion
• Cesium chloride density gradient, and
• Chelex 100 resin

Recently, different companies have come up with development of different kits for the purpose of DNA extraction, these kits uses chemical methods as well as some techniques characterized by the above methods. It is noteworthy that to select the right kit saves time on optimization of kits and likewise, procedures for DNA extraction. This commercial kits however differ in their results (extracted DNA; quantity and quality) as detection in PCR sensitivity has revealed considerable disparity between the kits (Yoshikawa et al. , 2011). There are some factors to consider before a kit can be selected for DNA extraction, such includes

• Sample origin
• Preparation methods
• Intended use
• Humic contents
• Sample quantity
• Yield
• Automation
• Simplicity

Problem Statement

Researchers are recently giving notice to saliva as a tool for diagnosis because of its implication in oral microbial community and diseases affecting the whole body, it is also readily and effortlessly available and it is a non invasive body fluid. However, inefficiency in sensitive and defined techniques (guidelines and results) has deterred collection, transportation, storage and DNA purification from saliva samples from being standardized. This shortcoming has prevented the usage of saliva in microbiome related research. There are various kits (commercial) available for the purification of DNA derived from saliva samples. These commercial kits on their part generate significantly inconsistent results from the same samples/sample types depending on the kits used. Thus, this study is proposed to determine and document a baseline standard for collection, storage and DNA extraction protocols in the human saliva microbiome.

Aim

The target of this study is to carefully assess saliva microbiome profile (quantity and quality of purified DNA) based on selected methods of collecting samples, storage and processing (DNA extraction).

Objectives

1. To probe the repercussion of collection technique on saliva microbiome. Collection routine to be adopted include spit/drool, mouth rinse with water/saline, mouth rinse with commercial mouthwash.
2. To juxtapose the use of fresh sample against frozen and thawed sample.
3. To describe the ideal saliva proportion for analysis of saliva microbiome.
4. To explore the most productive extraction techniques for DNA in human saliva.

Materials

Saliva samples from healthy participants, lab consumables (including OMNIgene tubes), reagents (including those for conventional PCI method), 2 commercial DNA kits viz QIAAmp DNA Microbiome kit and QuickExtract DNA Extraction kit.

Methods

Systematic review and Pattern recognition

An extensive review of existing literature (offline, online, published and unpublished) will be carried out in a space of 3 to 4 months, using as many databases as available, this will help to identify the DNA extraction methods most commonly used, the availablecommercial kits as well as the most commonly used, and the outcome from all of these reviews taking note of the variations. A pattern recognition analysis (using computational techniques) will be done, whereby data collated from above will be explicitly differentiated showing the most effective DNA extraction method/protocol.

Precautions before sample collection adopted from Kumar et al. , 2014 and various other articles

• Participants must be healthy individuals with healthy oral cavity
• No history of drinking and smoking
• Should have been off the use of antibiotics for at least 3 months before sample collection
• Should not have dentures or any artificial attachment
• Samples will be taken 2hours after brushing and/or 1-2hrs after food and drink intake
• Rinse mouth 1hr before sample collection

Saliva sample collection adopted from Kumar et al. , 2014 and various other articles

• Saliva samples will be collected in the form of drool, spit, mouth rinse (with water) or (with mouthwash).
• The volume of saliva to be collected will depend on the method of DNA extraction adopted, not counting the foam, the volume of saliva should be at least 5ml.
• Before sample collection; the method of collection, holding temperatures and storage medium/method will be taken into consideration.
• In the case of high volume collection, saliva sample will be collected in a sterile Falcon tube with a capacity of at least 10-50ml, and then aliquot samples to be stored in reagents and/or tubes made available from DNA extraction kits under various temperatures and duration prior to DNA extraction(Please label all samples with appropriate code)

Mode of collection of saliva samples adopted from definition and various articles

• Spit: Force out saliva from the mouth into sterile tubes or reagent tubes provided
• Drool: Allow saliva to accumulate in the mouth, expectorate 5ml or more into specimen tubes every 60seconds
• Mouth rinse (water): Oral wash saliva samples are obtained using saline to rinse the mouth and expectorate into tubes. The tubes/samples are immediately centrifuged to harvest cell pellets and prevent cell lysis.
• Mouth wash (with 15%wt alcohol content): Swish and gargle vigorously with 10ml scoop mouthwash for 30secs and expel into specimen tubes.
• Parafilm chewing stimulated saliva: Saliva can also be collected using this method and used for the experiment.

Transport/Duration/Storage

Always transport samples on dry ice (very important) and store immediately at -20°C to -80°C within 10mins of collection/or according to Kits user’s manual.

DNA Extraction using Conventional PCI (Phenol-Chloroform based)Follow the instructions on any of the PCI method chosen.

DNA Extraction using QIAAmp DNA Microbiome kitSee and follow user’s manual.

DNA Extraction using QuickExtract DNA Extraction kitSee and follow user’s manual. NOTE: General Protocol adopted from Kumar et al. , 2014 (DNA Isolation using Kits)

• Isolate total DNA from the stored individual microbiome samples following the manufacturer’s instructions on the kits manual
• Add 750µl of the lysis solution to the samples (usually 100-200µl of saliva)
• Follow the subsequent processing steps to isolate DNA
• Use the isolated DNA immediately for PCR or store in standard Tris-EDTA buffer at 4°C
• Quantitate the isolated PCR DNA prior to PCR using a micro-spectrometer

Expected Outcome

At the conclusion of this study, we anticipate

• Acceptance of this study, leading to an establishment of baseline standard for collecting and purifying DNA from human saliva microbiome.