WO2023228205A1

WO2023228205A1 - Saliva preservation compositions and processes thereof

Info

Publication number: WO2023228205A1
Application number: PCT/IN2023/050488
Authority: WO
Inventors: Markkandan KESAVAN; Balasubramanian ELAVARASI
Original assignee: Oneomics Private Limited
Priority date: 2022-05-26
Filing date: 2023-05-23
Publication date: 2023-11-30

Abstract

The present invention discloses a buffer composition for storage and preservation of biological samples such as saliva and cheek swabs. More particularly, the present invention discloses a cost-effective saliva preservation buffer composition for long- term storage and preservation of samples at room temperature. The present invention also discloses a process of preparation of said Saliva preservation buffer composition.

Description

SALIVA PRESERVATION COMPOSITIONS AND PROCESSES THEREOF

CROSS REFERENCE TO THE RELATED APPLICATION:

This application claims the priority to, and benefit of Indian Patent Application No. 202141054882 filed on May 26, 2022; the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION:

The present invention relates to a buffer composition for storage and preservation of biological samples such as saliva and cheek swabs. More particularly, the present invention relates to a cost-effective saliva preservation buffer composition for longterm storage and preservation of samples at room temperature. The present invention also relates to the process of preparation of said Saliva preservation buffer composition.

BACKGROUND OF THE INVENTION:

Nucleic acids often play a central role in routine procedures in molecular biology, forensic analysis, and diagnostics. The quality and quantity of nucleic acid, for such testing and procedure are directly affected by the stability and integrity of the sample collected, stored, and preserved. This particularly is a huge challenge in mass collection of samples due to various factors, including the non-availability of skilled practitioner, requirement of high degree of carefulness in invasive collection methods, presence of fragile biological elements in the samples, and various environmental factors.

Nucleic acids are generally used for PCR or qPCR for clinical diagnosis. The desired nucleic acid samples are generally extracted from anticoagulated blood samples. However, challenges like coagulation of blood and lack of skilled professionals for sample collection causes inconvenience in the efficient nucleic acid extraction from the blood samples. In addition to being an invasive method of sample collection, blood sample preservation adds to the complexity of nucleic acids sampling and testing.

Alternatively, saliva also serves as a source of nucleic acids and can easily be collected without any wound. It is a suitable and preferred mode of sample collection for large-scale sampling. Saliva has components such as mucosal cells, sugar, water, microbes, albumin, globulin, mucins, and digestive enzymes, which creates a hurdle in the preservation of saliva for a longer period.

In addition to the problems of preparing collection and preservation buffer compositions for nucleic acid samples obtained from humans, there remains an ongoing need to specifically improve methods for recovery of nucleic acids from the stored samples. For example, Mucins are the high molecular weight glycosylated multimeric proteins, which form a major part of a protective biofilm on the surface of epithelial cells and provide a barrier to particulate matters and microorganisms. Existing solutions use reagents that reduce disulfides as well as viscosity of mucin. However, such reagents have short shelf life which are limited to one to two days only.

Cheek swab is another source of nucleic acids-containing cells. Comparatively it is non-invasive than withdrawing blood from humans. The collection method also requires simpler training compared to that of the blood collection. Once collected, the recovery time of the usable nucleic acids can be extended by either direct drying of the swab or by wiping onto filter paper. However, as the inside of the mouth is not sterile, the microorganisms present in the buccal cavity can reduce the quality of the nucleic acids collected within a short period of time.

The patent CN102919218B assigned to Hubei Weidajian Gene Technology Co. Ltd. discloses a composition for preservation of human saliva and a preparation method thereof. The composition comprises of Tris-HCL with pH being 6 to 8.5, EDTA, NaOAc, cane sugar, N-acetyl-5-methoxytryptamine, propylparaben, diazonium imidazolidinyl urea, and protease K. However, this solution uses many chemicals or ingredients and is also not cost-effective. Further, this composition is able to provide stability only for upto 4 months at room temperature.

Some of the typical saliva preservation compositions in the prior art contains cell stabilizer, nuclease inhibitor, surfactant, protein denaturant, microbial inhibitor, and buffering liquid. Table- 1 below enumerates various chemicals in a composition and their respective functions:

Table- 1: Chemicals and their functions in a saliva preservation composition

This combination of ingredients in the above composition can prove to be costly and may pose health hazard. Therefore, none of the existing solutions in the prior art provides a better composition or a cost-effective solution.

Hence, there is a need for the inclusion of a cost-effective, stable reducing agent into the preservation buffer composition, such that nucleic acids can be conveniently recovered from it, especially after extended periods of time in the presence of oxygen at neutral or mildly alkaline pH. In the past 25 years, salivary diagnostics have emerged as a significant tool in biomedical research and clinical diagnostics. Saliva collection kits with preservation solution has good potential value in Singapore, South Korea, Japan, and Taiwan and many other countries, as part of personal genome service. Saliva collection kits can be used in hospitals, where they can fast track the sample collection and provide patient ease and satisfaction. Clinical diagnostics in India is emerging and hence saliva collection kit with preservation buffer composition has huge demand or value in terms of medical genomics in order to identify cancer biomarkers and genetic rare diseases.

Therefore, there is a need for a composition that caters to a fast-track, efficient, cost-effective, and non-invasive sample collection kit that proves convenient for patients including old age patients, without having to visit the hospital.

OBJECTS OF THE INVENTION:

The primary object of the present invention is to provide a buffer composition for preserving biological samples such as saliva and cheek swabs.

Another object of the present invention is to provide buffer compositions for the long-term preservation of nucleic acid containing cells in saliva samples at room temperature.

Another object of the present invention is to provide saliva preservation buffer compositions that are cost-effective.

Another object of the present invention is to provide compositions that are compatible with most of the nucleic acid isolation methods.

Another object of the present invention is to provide cost-effective nucleic acid preserving compositions that provides high yield of high-quality nucleic acids. A further object of the present invention is to provide the compositions for storing samples, from which nucleic acids can be isolated and used for high throughput next-generation sequencing (NGS) applications such as Whole Genome Sequencing and Whole Exome Sequencing.

Yet another object of the present invention is to provide a process for the preparation of the saliva preservation buffer compositions.

SUMMARY OF THE INVENTION:

Accordingly, the present invention provides buffer compositions wherein the compositions can preserve the biological samples such as saliva and cheek swabs, obtained through non-invasive methods. Particularly present invention discloses cost-effective saliva preservation buffer compositions that are compatible with most of the nucleic acid isolation methods. The nucleic acid obtained has high yield and can efficiently be used for high-throughput next-generation sequencing (NGS) applications such as whole genome sequencing and whole exome sequencing.

In one aspect the present invention discloses saliva preservation composition, comprising: a) a protein denaturant at concentration of 0.1% to 10%; b) a cell stabilizer at a concentration of 0.1% to 10%; and c) a microbial inhibitor at a concentration of 1.0% to 20%; wherein pH of the composition is in the range of 7.0 to 9.0.

In the said composition, the protein denaturant is present at a concentration of 0.2% to 5%.

The protein denaturant of said composition is selected from Guanidine hydrochloride, Urea, or CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate) . The preferred protein denaturant in the said composition is Guanidine hydrochloride.

In the said composition, the cell stabilizer is present at a concentration of 0.2% to 5%.

The cell stabilizer of said composition is selected from Sodium benzoate, Potassium benzoate, Sodium sorbate, Potassium sorbate, Tartaric acid, or Sodium dodecyl sulphate.

The preferred cell stabilizer in said composition is Sodium benzoate.

In the said composition, the microbial inhibitor is present at a concentration of 2% to 20%.

The microbial inhibitor in said composition is selected from alcohols comprising ethyl alcohol, isopropyl alcohol or n-propanol or antibiotics.

The preferred microbial inhibitor in said composition is ethyl alcohol.

The protein denaturant and microbial inhibitor are present in trace amounts.

The preferred pH of the present composition is 7.9.

The preferred buffer composition comprises of Guanidine hydrochloride, sodium benzoate and ethyl alcohol.

According to the present invention the saliva preservation composition is in liquid form. According to the present invention, the nucleic acid from the nucleic acid containing cells present in saliva is stable in the said composition at room temperature and for at least a year.

A saliva preservation composition of the present invention comprises: a) Guanidine hydrochloride at a concentration of 0.2 to 5% w/v; b) Sodium benzoate at a concentration of 0.2 to 5% w/v; and c) ethyl alcohol at a concentration of 2 to 20% v/v; wherein pH of the composition is in the range of 7.0 to 9.0.

In another aspect present invention discloses a process for preparation of saliva preservation composition, comprising the steps of: a. weighing and dissolving protein denaturant, cell stabilizer and microbial inhibitor in double distilled water; b. adjusting pH of obtained solution of step (a) to 7.0-9.0; c. making up the obtained solution of step (b) to respected volume; and d. followed by autoclaving or filter sterilizing to obtain saliva preservation composition.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1: Experimental Representation of Qubit Assay Analysis.

Figure 2: Agarose gel electrophoresis of 7 years old child’s DNA samples, wherein the individual gel lane is represented as Lane L: 1 Kb Ladder; Lane 1: 0^th day; Lane 2: 1^st day; Lane 3: 15^th day; Lane 4: 30^th day; Lane 5: 60^th day; Lane 6: 90^th day; Lane 7: 120^th Day; Lane 8: 180^th day; Lane 9: 240^th day and Lane 10: 360^th day.

Figure 3: Agarose gel electrophoresis of 30 years old Male DNA samples, wherein the individual gel lane is represented as Lane L: 1 Kb Ladder; Lane 1: 0^th day; Lane 2: 1^st day; Lane 3: 15^th day; Lane 4: 30^th day; Lane 5: 60^th day; Lane 6: 90^th day; Lane 7: 120^th Day; Lane 8: 180^th day; Lane 9: 240^th day and Lane 10: 360^th day.

Figure 4: Agarose gel electrophoresis of 35 years old Female DNA samples, wherein the individual gel lane is represented as Lane L: 1 Kb Ladder; Lane 1: 0^th day; Lane 2: 1^st day; Lane 3: 15^th day; Lane 4: 30^th day; Lane 5: 60^th day; Lane 6: 90^th day; Lane 7: 120^th Day; Lane 8: 180^th day; Lane 9: 240^th day and Lane 10: 360^th day.

DETAILED DESCRIPTION OF THE INVENTION:

Embodiments described herein can be understood more readily by reference to the following detailed description, examples, and drawings. Elements, apparatus, and methods described herein are merely illustrative of the principles of the present invention and are not limited to the specific embodiments presented in the detailed description, examples, and drawings. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which the methods belong. Although any compositions or methods similar or equivalent to those described herein can also be used in the practice or testing of the embodiments of the present invention, representative illustrative methods and compositions are now described.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.

It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Before providing the compositions, processes and methods of the present disclosure are described in detail, it is to be understood that the invention is not limited to particular embodiment/s and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiment/s only and is not intended to be limiting the scope of the invention.

Disclosed herein is the detailed description of the present invention pertaining to the preservation buffer compositions and method of preparations thereof, for storage and preservation of human biological samples such as saliva and cheek swabs. The present invention discloses a cost-effective composition capable of storage and preservation of nucleic acid-containing cells specifically in the saliva samples, which can be stored at room temperature for at least a year or more.

Accordingly in one aspect the present invention provides a buffer composition for preservation of biological samples such as saliva and cheek swabs.

The said preservation buffer compositions of the present invention comprise of protein denaturants along with microbial inhibitors, cell stabilizers and antibiotics.

Particularly present invention discloses a saliva preservation composition, comprising: a) a protein denaturant; b) a cell stabilizer; and c) a microbial inhibitor,

In one embodiment, the concentration of protein denaturant present in the composition is in the range of 0.1% to 10%, preferably 0.2% - 5%.

In another embodiment, the concentration of cell stabilizer in the present composition is in the range of 0.1% to 10%, preferably 0.2% - 5%.

In yet another embodiment, the concentration of microbial inhibitor in the present composition is in the range of 1% to 20%, preferably 2% - 20%.

In one embodiment of the present invention, the protein denaturant is selected from Guanidine hydrochloride, Urea, or CHAPS (3-[(3-cholamidopropyl) dimethylammonio] - 1 -propanesulfonate) . The protein denaturants used in the present invention helps in inhibiting or denaturing lytic proteins involved in the enzymatic degradation of nucleic acids, e.g., nucleases.

In one of the preferred embodiments, the protein denaturant is Guanidine hydrochloride. The preferred concentration range of Guanidine hydrochloride is 0.2 to 5% w/v.

In another embodiment the cell stabilizer used in the present composition is selected from Sodium benzoate, Potassium benzoate, Sodium sorbate, Potassium sorbate, tartaric acid, or sodium dodecyl sulphate.

The cell stabilizers used in the present invention retains the stability of nucleic acid containing cells in the biological samples.

In one of the preferred embodiments, the cell stabilizer is Sodium benzoate. The preferred concentration range of Sodium benzoate is 0.2 to 5% w/v.

In yet another embodiment, the microbial inhibitor of the present composition is selected from alcohol such as ethyl alcohol, isopropyl alcohol or n-propanol or antibiotics. The preferred microbial inhibitor is ethyl alcohol.

The microbial inhibitors used in the present invention reduce the rate of microbial growth in the preserved biological sample stored in the composition of the present invention. Accordingly in present invention, use of microbial inhibitor in the present composition results in reduction in the rate of growth of microorganisms by at least by 95%.

In another preferred embodiment, the microbial inhibitor is ethyl alcohol. The preferred concentration range of ethyl alcohol is 2 to 20% v/v. The present invention uses protein denaturant and microbial inhibitor in trace amounts.

In one embodiment the pH of the compositions ranges between 7.0 and 9.0. This enables the compositions to be equally effective for DNA and RNA isolations.

In one of the preferred embodiments, the pH of the composition is 7.9.

In another preferred embodiment, the saliva preservation composition is in liquid form.

Accordingly, the present invention discloses a saliva preservation composition, comprising: a) Guanidine hydrochloride at a concentration of 0.2 to 5% w/v; b) Sodium benzoate at a concentration of 0.2 to 5% w/v; and c) Ethyl alcohol at a concentration of 2 to 20% v/v; wherein pH of the composition is in the range of 7.0 to 9.0.

In another aspect present invention discloses a process for preparation of saliva preservation composition.

Accordingly, a process for preparation of saliva preservation composition, comprising the steps of: a. weighing and dissolving protein denaturant, cell stabilizer and microbial inhibitor in double distilled water; b. adjusting pH of obtained solution of step (a) to 7.0-9.0; c. making up the obtained solution of step (b) to respected volume; and d. followed by autoclaving or filter sterilizing to obtain saliva preservation composition. Nucleic acids, both DNA and RNA, isolated from the preserved samples can be used for high throughput next-generation sequencing (NGS) applications. The preserved samples are compatible with most of the nucleic acid isolation methods and give high nucleic acids yield.

The compositions provided in the present invention stabilizes buccal cells and white blood cells found in saliva at room temperature for at least a year. The present composition can stabilize and maintain the integrity of the nucleic acids in the stored cells. Additionally, since the nucleic acid containing cells are extracted by non-invasive methods (from saliva and from the cheek swabs), the risk of bloodborne infection is also decreased. The composition of the present invention is compatible with most of the commonly used nucleic acid isolation methods.

The composition as disclosed in the present invention can be used in saliva collection, saliva microbiome study, oral cancer diagnostic, personal genome services and clinical diagnosis using whole exome sequencing. The compositions as disclosed in the present invention are also a right fit for nucleic acid-based analysis in forensics, law enforcement, military, human medicine, clinical testing, diagnostics, veterinary medicine, and related scientific research.

Advantages of the present saliva preservation composition:

The present invention provides a saliva preservation composition that has several advantages over the existing prior arts:

1. Cost - effective composition: The composition in the present invention is cost effective with fewer ingredients making it a robust and cheaper saliva preservation composition such as over the known prior arts. 2. Long shelf life: The buffer composition of the present invention can preserve biological samples such as saliva for at least a year or beyond at room temperature, making it a highly stable sample preserving composition.

3. Composition compatibility: The preserved samples in the present buffer composition are compatible with most of the nucleic acid isolation methods and give high nucleic acids yields even after long term storage.

4. Quality retention of sample: The nucleic acids, both DNA and RNA, isolated from the preserved samples can be used for high throughput nextgeneration sequencing (NGS) applications, providing that the present composition can retain the quality and stability of nucleic acid stored.

5. Non-invasive method: The present composition can store the nucleic acidcontaining cells obtained from saliva making this process non-invasive without having to create a wound, thereby avoiding the risk of blood-borne infections during sample collection.

The present invention has good market potential and is industrially applicable.

Certain specific aspects and embodiments of the present invention will be explained in detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the invention in any manner.

EXAMPLES

All the biological materials used herein in this invention were solely for the purposes of testing the effectivity and efficacy of the preservation compositions. None of the biological material is part of any of the compositions or the invention and is merely used as a source of nucleic acids to demonstrate the effectiveness. Example 1: Generalized Composition for Saliva preservation and their role:

The liquid saliva preservation compositions are prepared with the various concentration of the major ingredients as exemplified in table-2.

The table-2 also illustrates the role of various ingredients in the composition.

Table 2: Ingredients in Saliva preservation composition and their roles

Example 2: Process of Preparation of the Composition for Saliva preservation:

The ingredients in the said composition namely Guanidine hydrochloride as Protein denaturant, Sodium benzoate as Cell stabilizer and Ethyl alcohol as Microbial inhibitor were weighed and dissolved in double distilled water, followed by adjusting the pH to 7.0-9.0.

The obtained solution is made up to respected volume and then autoclaved or filter sterilized to obtain final liquid composition for Saliva preservation.

Example 3: Compositions for Saliva preservation:

Different compositions 1-5 were prepared as described below in Tables 3:

Using the various concentrations of the ingredients as mentioned in Table-2, various preservation compositions (1 to 5) were prepared. Table-3 below enumerates the various compositions. Table-3: Saliva preservation compositions

These compositions are made by carefully mixing the said chemical components or ingredients in particular concentration at pH 7.9 as described in above process.

Each of the compositions were used to preserve the saliva samples. Further the nucleic acid was isolated from the saliva samples preserved in the present composition. The isolated nucleic acid was quantified using Qubit Assay.

Example 4: Nucleic acid Quantification using Qubit Assay:

Qubit assay utilizes target selective dyes that emit fluorescence when bound to DNA, RNA or protein, unlike UV absorbance which can overestimate sample concentration due to contaminants in the sample. Fluorescence measurements in Qubit assay are much more sensitive and accurate than UV absorbance, giving accurate measurements with significantly less noise.

Qubit Assay Analysis was performed as shown in Figure 1.

Standards were used, and l-20pE samples (1-5 compositions of Example 3) were individually mixed with 180-199pE of Qubit working solution to form final volume of 200 pF each and Qubit assay was carried out, results of which is presented in the Table-4 below: Table-4: Qubit Assay Results for different compositions

The average concentration of nucleic acid quantified using the Qubit Assay was found to be much higher than the minimum required DNA concentration of 0.01 ng/qL (10 pg/pL) for performing PCR reactions. The molecular weight of the DNA thus obtained was also found to be high.

The result of the above assay confirms that the composition of the present invention provides efficient preservation of the nucleic acid containing cells obtained from saliva. The preserved samples in said composition are compatible with most of the nucleic acid isolation methods and give high nucleic acids yield.

After the Qubit Assay, the Saliva DNA was used for various downstream analysis like PCR, RT-PCR and 16S metagenome analysis.

Example 5: Stability study:

In order to study the stability of the various compositions as disclosed in example 3, the saliva samples of different human subjects were collected and stored. In this experiment the stability of the buffer composition 5 was evaluated. The saliva samples from individuals of different age groups (a 7-year-old child; a 30- year-old male, and a 35 -year-old female) were collected and stored at room temperature in the composition 5. DNA extraction from the stored samples was performed at regular time intervals (0^th day, 15^th day, 30^th day, 60^th day, 90^th day, 120^th day, 180^th, 240^th and 360^th day) and the extracted DNA was checked for integrity in 0.8% Agarose gel electrophoresis as shown in figures 2-4.

The results of the stability study confirm that the nucleic acid (DNA) obtained from human subjects of any age group remains stable for at least a year at room temperature when stored in the composition of the present invention. Thereby the composition of the present invention is a stable composition which provides longer shelf life to the biological samples at room temperature without effecting the integrity of the nucleic acid.

Claims

We claim:

1. A saliva preservation composition, comprising: a) a protein denaturant at concentration of 0.1% to 10%; b) a cell stabilizer at a concentration of 0.1% to 10%; and c) a microbial inhibitor at a concentration of 1.0% to 20%; wherein pH of the composition is in the range of 7.0 to 9.0.

2. The composition as claimed in claim 1, wherein the protein denaturant is present at a concentration of 0.2% to 5%.

3. The composition as claimed in claim 1, wherein the protein denaturant is selected from Guanidine hydrochloride, Urea, or CHAPS (3-[(3- cholamidopropyl)dimethylammonio] - 1 -propanesulfonate) .

4. The composition as claimed in claim 3, wherein the protein denaturant is Guanidine hydrochloride.

5. The composition as claimed in claim 1, wherein the cell stabilizer is present at a concentration of 0.2% to 5%.

6. The composition as claimed in claim 1 , wherein the cell stabilizer is selected from Sodium benzoate, Potassium benzoate, Sodium sorbate, Potassium sorbate, Tartaric acid, or Sodium dodecyl sulphate.

7. The composition as claimed in claim 6, wherein the cell stabilizer is Sodium benzoate.

8. The composition as claimed in claim 1, wherein the microbial inhibitor is present at a concentration of 2% to 20%. The composition as claimed in claim 1, wherein the microbial inhibitor is selected from alcohols comprising ethyl alcohol, isopropyl alcohol or n- propanol or antibiotics. The composition as claimed in claim 9, wherein the microbial inhibitor is ethyl alcohol. The composition as claimed in claim 1, wherein protein denaturant and microbial inhibitor are present in trace amounts. The composition as claimed in claim 1, wherein the pH of the composition is 7.9. The composition as claimed in claim 1, wherein the composition is in the liquid form. The composition as claimed in claim 1, wherein saliva comprises nucleic acid containing cells. The composition as claimed in claim 14, wherein said nucleic acid from the nucleic acid containing cells is stable in said composition at room temperature. The composition as claimed in claim 14, wherein said nucleic acid containing cell is stable in the composition for a year. A saliva preservation composition, comprising: a) Guanidine hydrochloride at a concentration of 0.2 to 5 % w/v; b) Sodium benzoate at a concentration of 0.2 to 5 % w/v; and c) ethyl alcohol at a concentration of 2 to 20 % v/v; wherein pH of the composition is in the range of 7.0 to 9.0. A process for preparation of saliva preservation composition, comprising the steps of: a. weighing and dissolving protein denaturant, cell stabilizer and microbial inhibitor in double distilled water; b. adjusting pH of obtained solution of step (a) to 7.0-9.0; c. making up the obtained solution of step (b) to respected volume; and d. autoclaving or filter sterilizing to obtain saliva preservation composition.