WO2016029020A1

WO2016029020A1 - Devices, solutions and methods for sample collection related applications

Info

Publication number: WO2016029020A1
Application number: PCT/US2015/046119
Authority: WO
Inventors: Youssef Biadillah; Stephen Andrews
Original assignee: Abogen, Inc.
Priority date: 2014-08-20
Filing date: 2015-08-20
Publication date: 2016-02-25

Abstract

A bodily fluid sample collection device for the collection of naturally expressed bodily fluids (e.g. saliva), comprises a composition for extracting and preserving nucleic acids in the collected sample. In some embodiments, the composition is effective in extracting and preserving nucleic acids from a cell organelle. The organelle may be a mitochondrion. In some embodiments, the composition combines multiple lysing agents. In some embodiments, the composition comprises at least one ionic detergent, at least one non-ionic detergent, and a salt.

Description

DEVICES, SOLUTIONS AND METHODS FOR SAMPLE COLLECTION

RELATED APPLICATIONS

Field of the Disclosure

The disclosure relates to devices, solutions and methods for collecting samples of bodily fiuids or other substances, including hazardous and/or toxic substances, and in particular, a naturally expressed bodily fluid (e.g., saliva, urine). Additionally or alternatively, the disclosure relates generally to functional genetics and/or to the isolation and preservation of DNA from such bodily fluids, for later genetic studies (for example). Additionally or alternatively, the disclosure relates generally to such features suitable for home use.

Background

WO 2003/104251 (DNA Genotek Inc.) describes a composition and method for preserving and extracting nucleic acids from a collected sample of saliva. The composition includes a chelating agent, a denaturing agent, buffers to maintain the pH of the composition within ranges desirable for DNA and/or RNA. The composition may also include a reducing agent and/or antimicrobial agent. That document also describes a saliva sample collection container having a chamber containing the composition, and a technique for releasing the composition by disestablishment of a separating barrier when a cap is fitted to the container.

While the concept of a self-contained collection and preservation device has potential for many different genetic testing applications without requiring a donor to physically visit a laboratory, such potential may be severely limited by the efficacy of the composition, and the quality of the preserved solution. Some non-limiting aspects of the invention may seek to at least mitigate such issues.

Reference is made herein to an improved sample collection device described in WO 2012/177656. The content of this application is incorporated herein by reference as if reproduced here in its entirety. Summary of the Disclosure

The following presents a simplified summary of the disclosure in order to provide a basic, non-limiting, understanding of some aspects of the disclosure.

In one aspect, a (e.g. portable) collection device is described that is suitable for use by a sample donor, for example, without having to visit a laboratory. The sample collection device comprises a container for receiving a naturally expressed bodily fluid, for example, saliva or urine. The device further comprises a chamber containing a composition for extracting and preserving nucleic acids in the collected sample, wherein the composition is effective in extracting and preserving nucleic acids from a cell organelle. The organelle may be a mitochondrion (although the same principles may apply to other organelles). Additionally or alternatively, in one aspect, a composition is described for use in a sample collection device for collecting a sample of a naturally expressed bodily fluid, and extracting and preserving nucleic acids in the collected sample, wherein the composition is effective in extracting and preserving nucleic acids from a cell organelle. The organelle may be a mitochondrion (although the same principles may apply to other organelles).

The nucleic acids may be DNA and/or RNA, as desired.

Mitochondrial DNA may have very different characteristics from nuclear DNA:

(i) Mitochondrial DNA is very small compared to nuclear DNA. It has the smallest chromosome encoding of only 36 genes, consists of 16600bp.

(ii) Mitochondrial DNA is also relatively sparse in concentration compared to nuclear DNA. From about 100 to about 10,000 separate copies of mitochondrial DNA may be present per cell.

(iii) Mitochondrial DNA is inherited only from the mother. (iv) Mitochondrial DNA is circular.

(v) Mitrochrondrial DNA is very susceptible to mutation. This is one factor that may make mitochrondrial DNA of interest for research and analysis, because it is more capable of being mutated or marked than nuclear DNA. For example, it is believed that certain mutation may be a useful indicator or neurological disease that may be difficult (or impossible) to detect from nuclear DNA.

However, it is not known hitherto to extract and preserve mitochondrial DNA with a sample collection device of the type disclosed herein. The above characteristics make mitochondrial DNA difficult to extract and preserve. Firstly, the susceptibility to mutation means that mitochondrial DNA is particularly susceptible to damage and mutation by the conventional process used to lyse sample cells to extract the DNA. Secondly, the sparse concentration, and small size, of mitochondrial DNA exacerbates the difficulty of obtaining a sufficient quantity of undamaged mitochondrial DNA to provide a quality sample for downstream analysis.

In some embodiments, the composition may be configured to lyse the cell organelle (e.g. mitochondrion). Example compositions are described later.

Additionally or alternatively to either of the above, in one aspect, a composition is described for use in a sample collection device for collecting a sample of a naturally expressed bodily fluid, and extracting and preserving nucleic acids in the collected sample, wherein (i) the composition is significantly more concentrated in lysing and/or preservation agents than a conventional composition, and/or (ii) the composition has a volume not substantially larger than the volume of the bodily fluid sample for which the composition is intended.

In some embodiments, the composition may have a volume smaller than the volume of the bodily fluid sample for which the composition is intended.

By using a composition volume not substantially larger than the volume of the bodily fluid sample for which the composition is intended, the volume increase when mixing the composition with the collected sample, can be equally small. Providing a relatively small volume reduces the impact of the volume increase on the concentration of nucleic acids in the resulting mixture and/or reduces the dilution effect of the composition volume in terms of DNA concentration per unit volume. For example, in one embodiment, the ratio of a volume of a composition, to a volume of a collected bodily fluid sample may be about 1 : 1. With such a ratio, the concentration of extracted and preserved nucleic acids per unit volume, is halved because the net volume doubles. Thus the concentration can remain relatively high. It is believed that by using a smaller volume of composition, the impact of the volume increase can be even further reduced. For example, if the volume of the composition is about half the volume of the bodily fluid sample, it is believed that the concentration of extracted and preserved nucleic acids per unit volume may be two- thirds the original, instead of half. The concentration per unit volume is thereby enhanced by approximately 33% compared to the 1 : 1 embodiment.

Using an increased concentration of lysing and/or preservation agents can compensate for the reduced volume of the composition.

Additionally or alternatively, using an increased concentration of lysing and/or preservation agents can provide for rapid lysing and/or preservation action, and/or lysing of a cell organelle within the cell, either or both of which can reduce the time during which mutation of organelle DNA (e.g., mitochondrial DNA) may occur.

Additionally or alternatively to any of the above aspects, a composition is described for use in a sample collection device for collecting a sample of a naturally expressed bodily fluid, and extracting and preserving nucleic acids in the collected sample, wherein the composition comprises: at least one lysing agent, and/or at least one preservation agent. Optionally, the composition may comprise at least two lysing agents, optionally at least three lysing agents.

Additionally or alternatively, the composition may optionally comprise as the at least one preservation agent, at least one chemical inhibitor and/or denaturing agent, for blocking proteins and/or DNAases. Optionally, the composition may comprise at least two such chemical inhibitors and/or denaturing agents, optionally at least three such chemical inhibitors and/or denaturing agents. Details of example compositions are further explained in the detailed description.

Additionally or alternatively to any of the above aspects, in some embodiments, a composition is described comprising one or any combination of two, or all three, of: at least one lysing agent for liberating nucleic acids from a sample cell, the at least one lysing agent comprising at least one, optionally at least two; optionally at least three, selected from: sodium dodecyl sulfate (SDS); Triton; Triton-X; Triton 100; Triton-X 100; deoxycholate (e.g. sodium deoxycholate); cholate (e.g. sodium cholate); sodium lauroyl sarcosinate (and/or sarkosyl); maltoside (e.g. n-dodecyl- -D- maltopyranoside and/or DDM); glycoside (e.g. Digitonin); Tween (e.g. Tween 20 and/or Tween 80); 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (and/or CHAPS); nonyl phenoxypolyethoxylethanol (and/or Tergitol-type NP-40 and/or NP-40); sodium chloride (NaCl); lithium chloride (LiCl); potassium chloride (KC1); or a derivative (e.g. a commercial derivative) of any of the aforementioned. a buffer agent comprising tris and/or ethylenediaminetetraacetic acid (EDTA); at least one chemical inhibitor and/or denaturing agent, for blocking proteins and/or DNAases, the at least one chemical inhibitor and/or denaturing agent comprising at least one, optionally at least two, optionally at least three, selected from: 2-mercaptoethanol; Ca²⁺ ions; ethylene glycol tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); sodium dodecyl sulfate (SDS); iodoacetate; urea.

Additionally or alternatively to any of the above aspects, in some embodiments, a composition is described comprising: at least one ionic surfactant (or ionic detergent); at least one non-ionic surfactant (or non-ionic detergent); and a salt. The composition may be a solution for lysing a cell and/or an organelle.

It is believed that the combination of ionic and non-ionic surfactants may have a synergistic stabilizing effect on micelle formation, and make micelle formation more tolerant to high salt concentration. This can avoid undesirable precipitation of one or more of the surfactants even in relatively high surfactant concentrations and/or high salt concentration. Enabling a solution to have both high micellular surfactant concentration(s) and high salt concentrations may enhance the cell and/or organelle lysing capabilities of the solution.

Examples of ionic surfactants or detergents include any one or more of: sodium dodecyl sulfate (SDS); deoxycholate (e.g. sodium deoxycholate); cholate (e.g. sodium cholate); sodium lauroyl sarcosinate (and/or sarkosyl); 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (and/or CHAPS); a derivative (e.g. a commercial derivative) of any of the aforementioned.

In some embodiments, ionic surfactants (or detergents) may include zwitterionic surfactants (or detergents), of which CHAPS is an example. In other embodiments, ionic surfactants (or detergents) may optionally not include zwitterionic surfactants; for example, ionic surfactants (or detergents) may include anionic or cationic surfactants (or detergents).

Examples of non-ionic surfactants or detergents include any one or more of: Triton; Triton-X; Triton 100; Triton-X 100; maltoside (e.g. n-dodecyl- -D-maltopyranoside and/or DDM); glycoside (e.g. Digitonin); Tween (e.g. Tween 20 and/or Tween 80); 3-[(3-cholamidopropyl)dimethylammonio]- 1 -propanesulfonate (and/or CHAPS); nonyl phenoxypolyethoxylethanol (and/or Tergitol-type NP-40 and/or NP-40); a derivative (e.g. a commercial derivative) of any of the aforementioned.

Examples of salt include any one of more of: sodium chloride (NaCl); lithium chloride (LiCl); potassium chloride (KC1).

Additional aspects of the disclosure are defined in the claims. Any independent aspect or claim of the disclosure is specifically envisaged to be combinable with any one or more other independent aspects or claims of the disclosure, whether or not such relation is present in the current claims. Any dependent aspect or claim of the disclosure is specifically envisaged to be combinable with and/or made dependent on any other independent or dependent aspect or claim of the disclosure, whether or not such relation is present in the current claims.

Although the various aspects of the disclosure have been highlighted above and/or in the claims, this does not limit the scope of the invention. Protection is claimed for any novel feature and/or idea described herein and/or illustrated in the drawings whether or not emphasis has been placed thereon.

Brief Description of the Drawings

Non-limiting embodiments are described below, by way of example only, with reference to the accompanying drawings, in which:-

Fig. 1 is a schematic section through a first example sample collection device;

Fig. 2 is a schematic section through a second example sample collection device;

Fig. 3 is a schematic graph illustrating a variation in sample concentration, per unit volume, in dependence on volume of a preservative composition that is added.

Fig. 4 is a schematic representation of overall DNA quality, measured by gel electrophoresis, in saliva samples preserved by three different preservative solutions, A, B and C, as part of a comparative experiment. Detailed Description of Preferred Embodiments

Referring to Figs. 1 and 2, a sample collection device 10 is depicted of a type that may be suitable for home use, or at least without the need to visit a laboratory. The sample collection device 10 may, for example, be portable and/or be provided to a user at home (for example, via a postal or courier delivery), for the user to donate a sample of a naturally expressed bodily fluid. The bodily fluid may, for example, be saliva or urine. The following description focuses on saliva, but it will be appreciated that the same principles may be applied to urine, or to another naturally expressed bodily fluid. In the illustrated examples, a composition 12 is provided for extracting and/or preserving nucleic acids in the collected bodily fluid sample. In use, the composition is intended to be mixed with the collected bodily fluid sample, for example, upon closing the device to seal it closed. The device 10 may then be returned to the sender, or sent to a collection agent or to a laboratory for analysis, for example, genetic analysis.

A function of the composition 12 may be to extract nucleic acids from the bodily fluid sample, and/or to preserve the extracted nucleic acids (or the bodily fluid sample). The nucleic acids may be DNA and/or R A.

In some embodiments, the composition 12 may be effective to lyse a cell organelle (for example, a mitochondrion) and/or preserve extracted organelle nucleic acids.

In some embodiments, the composition 12 may avoid or reduce, mutation, damage or other degradation, of DNA in the collected sample, thereby providing sufficient time for the device to be returned for analysis. The composition 12 may be effective to preserve extracted nucleic acids at room temperature or a period of at least a week, optionally at least two weeks, optionally at least three weeks, optionally at least a month, or even longer.

The composition 12 may be a liquid. Further details of examples of the composition are described later below.

The composition 12 may optionally be stored in an internal chamber 14 of the device, and released to mix with the collected bodily fluid sample when, for example, the user seals the device closed or performs some other manipulation of the device 10. Figs. 1 and 2 illustrate two alternative examples of collection device 10. The device 10 may generally comprise a first body (e.g. tube) 16 having a mouth 18 and defining a collection region 20 for the naturally expressed bodily fluid introduced into the device through the mouth 18. The device 10 may further comprise a second body (e.g. closure) 22 attachable to or over the mouth 18 to seal the device 10 closed after the donated sample has been introduced. In the example of Fig. 1, the chamber 14 containing the composition 12 is provided in the second body (e.g. closure) 22. In the example of Fig. 2, the chamber 14 containing the composition is provided in the first body (e.g. tube) 16. In either example, the chamber 14 is configured to be opened by a mechanism (shown schematically at 24), to communicate with the collection region 20. The opening mechanism 24 may be responsive to fitting the second body (e.g. closure) 22 to the first body (e.g. tube) 16, or to some other manual manipulation of the device 10. Various mechanism 24 are envisaged, including but not limited to one or more selected from: release of an internal closure or cap, opening of a tap, rupture of a frangible wall, removal or displacement of an internal cover, relative rotation of a cap or nut.

Optionally, further details of exemplary constructions of device 10 and opening mechanisms 24 are provided in the aforementioned WO 2012/177656 incorporated herein by reference.

The device 10 may be configured for collection of a predetermined sample volume "Vs" of the naturally expressed bodily fluid. The device may, for example, include a visual fill scale, or a fill line (e.g. indicated at 26) or other indicia, to indicate when the predetermined sample volume Vs has been attained. In some embodiments, the sample collection space may have a size that is equal to the predetermined volume Vs, or the sample collection space may be larger in volume. By way of example only, the predetermined volume Vs may in some embodiments be at least about 1ml, or at least about 2ml, or at least about 3ml, or at least about 4ml, or at least about 5ml, or more. By way of example only, the predetermined volume Vs may in some embodiments be not more than about 5ml, or not more than about 4ml, or not more than about 3ml, or not more than about 2ml, or not more than about 1ml. By way of example only, in some embodiments, the predetermined volume Vs may in some embodiments be from about 1ml to about 5ml, optionally from about 1ml to about 4ml, optionally from about 1ml to about 3ml, optionally from about 1ml to about 2ml. By way of example only, the predetermined volume Vs may in some embodiments be about 1ml, or about 2ml, or about 3ml, or about 4ml, or about 5ml. In some embodiments, the volume "Vc" of the composition 12 may be not substantially larger than, or optionally smaller than, the predetermined sample volume Vs. By way of example only, the composition volume Vc may in some embodiments, be not more than about 100% of Vs, or not more than about 90% of Vs, or not more than about 80% of Vs, or not more than about 70% of Vs, or not more than about 60% of Vs, or not more than about 50% of Vs, or not more than about 40% of Vs, or not more than about 30% of Vs, or not more than about 20% of Vs. By way of example only, the composition volume Vc may in some embodiments, be about 100% of Vs, or about 90% of Vs, or about 80% of Vs, or about 70% of Vs, or about 60% of Vs, or about 50% of Vs, or about 40% of Vs, or about 30% of Vs, or about 20% of Vs. By way of example only, the composition volume Vc may in some embodiments be about a third of Vs, or about a quarter of Vs. The composition volume Vc may be same size as the interior space of the chamber 14 in order to fill the chamber 14, or the composition volume Vc may smaller, partly filling the chamber 14.

By making the composition volume Vc not substantially larger than, or optionally smaller than, the predetermined sample collection volume Vs, the volume increase when mixing the composition 12 with the collected sample, can be equally small. The volume increase may be equivalent to dilution of the sample in terms of DNA concentration per unit volume. Fig. 3 illustrates schematically the impact of dilution on DNA sample concentration per unit volume, depending on the composition volume Vc. The horizontal axis represents a ratio R of sample volume Vs to composition volume Vc, over a range of ratios R=Vs/Vc from 1/1 to 5/1 (or R = from 1 to 5). The vertical axis represents the fraction of the DNA concentration per unit volume after mixing with the composition volume, compared to the original sample volume. The value varies as the function Vs/(Vs+Vc), and is equivalent to dilution by the composition volume.

In some embodiments disclosed herein, the ratio Vs/Vc may be about 1/1 (or R= ). It can be seen in Fig. 3 that such a composition volume Vc reduces the DNA concentration per unit volume to 50% of the original amount, which is eminently satisfactory. However, it is believed that by using a smaller composition volume Vc (equivalent to a larger ratio of sample volume Vs to composition volume Vc), the concentration of DNA per unit volume can be increased compared to the 1/1 ratio embodiment(s). For example, using a composition volume of half the sample volume, equivalent to a sample volume Vs to composition volume Vc ratio of 2/1 (R=2), it is believed that the concentration of DNA can be increased, for example, by up to 66% of the original amount, which is a 30% improvement compared to 1/1 ratio embodiment(s). Using a composition volume Vc of one-third of the sample volume Vs, equivalent to a sample volume Vs to composition volume Vc ratio of 3/1 (R=3), it is believed that the concentration of DNA can be increased, for example, by up to 75% of the original amount, which is a 50% improvement compared to the 1/1 ratio embodiment(s). Using a composition volume Vc of one-quarter of the sample volume Vs, equivalent to a sample volume Vs to composition volume Vc ratio of 4/1 (R=4), it is believed that the concentration of DNA can be increased, for example, by up to 80% of the original amount, which is a 60% improvement compared to the 1/1 ratio embodiment(s).

Achieving a desirably high DNA concentration per unit volume may provide important advantages for downstream analysis. Firstly, modern automated testing procedures tend to use only small sample volumes. Ensuring that even a small volume of the sample, after extraction and preservation, contains a good concentration of the DNA is important to be able to benefit from automated testing. Secondly, in a collected sample, mitochondrial DNA is much sparser in concentration compared to much more preponderant nuclear DNA. Improving the overall concentration of DNA per unit volume helps increases the sensitivity of automated testing to such sparse DNA. It may contribute to enabling detection of mitochondrial DNA, using automated testing of sample collected in a relatively simple collection device, which is understood not to be possible using devices that are currently available commercially.

In one specific example, the predetermined sample volume Vs may be about 2ml, and the composition volume Vc may be about 2ml. In another specific example, the predetermined sample volume Vs may be about 1ml, and the composition volume Vc may be about lml. In another specific example, the predetermined sample volume Vs may be about lml, and the composition volume Vc may be about 250μ1. Additionally or alternatively to any of the above, and by way of example only, the composition volume Vc may in some embodiments be from about ΙΟΟ Ι to about 1ml inclusive (or optionally less than 1ml). Additionally or alternatively, the composition volume Vc may optionally be at least about ΙΟΟμΙ, optionally at least about 250μ1, optionally at least about 500μ1. Additionally or alternatively, the composition volume may optionally be about 1ml, optionally less than 1ml, optionally less than about 750μ1, optionally less than about 500μ1. By way of example only, the composition volume Vc may in some embodiments be about ΙΟΟμΙ, or about 250μ1, or about 500μ1, or about 750μ1.

The concentrations of the active components of the composition 12 may be varied as appropriate to compensate for using a small composition volume Vc. For example, when using a composition volume Vc of about half that of the benchmark, the concentrations of the active components of the composition 12 may be doubled or more compared to concentrations of active components of a benchmark composition 12. Varying the compositions of the active ingredients can provide at least the same (or better) extraction and preservation capabilities despite the smaller composition volume Vc. More detailed explanation of component concentrations is discussed later.

Additionally or alternatively to the above features of reducing dilution by using a small composition volume Vc (e.g. not substantially larger than, or optionally smaller than, the sample volume Vs), further features of the present disclosure may relate to the constituents of the composition 12. These features relating to the constituents may be used irrespective of the ratio of the sample volume Vs to the composition volume Vc, although the ratio may be useful to determining suitable concentrations of the constituents in the composition. In some embodiments, the composition may comprise any one or more selected from:

(i) at least one cell and/or organelle lysing agent for lysing cells and/or organelles to liberate nucleic acids therefrom. Suitable lysing agents may include one or more surfactants; and/or one or more detergents; and/or one or more salts. An example surfactant may be sodium dodecyl sulfate (SDS), although other surfactants may be used additionally or alternatively. Example detergents may be Triton (e.g. Triton 100; Triton-X; Triton-X 100); and/or deoycholate (e.g. sodium deoxycholate), although other detergents may be used additionally or alternatively. Example salts may be selected from: sodium chloride (NaCl); and/or lithium chloride (LiCl); and/or potassium chloride (KC1), although other salts may be used additionally or alternatively. Additional surfactants and/or detergents are further described below. For the avoidance of doubt, where the context permits, the terms "surfactant" and "detergent" may be used interchangeably, and are intended to be read interchangeably. Also, where surfactants or detergents are mentioned by name, the scope is intended to cover equally any derivative (e.g. a commercial derivative).

The surfactant and/or detergent can solubilize cellular and membrane components to break the membrane. The salt can regulate the osmolarity.

In some embodiments, at least two lysing agents (e.g. selected from the above) are used in combination, optionally at least three lysing agents (e.g. selected from the above) are used in combination.

In some embodiments, the salt concentration (in the composition 12 and/or the combination of composition 12 when mixed with the sample) may be at least 250mM, optionally greater than 250mM. This salt concentration may be the aggregate salt concentration, or the concentration of at least one or more major salt components. In some embodiments, this salt concentration may be at least 300mM, optionally in the range of 300-750mM. Such a concentration is higher than would be conventionally used, and may contribute to an organelle lysing ability of the composition 12.

In some embodiments, it is believed beneficial to use a non-ionic surfactant and/or detergent (e.g. Triton) in combination with an ionic surfactant and/or detergent (e.g. SDS). The ionic and non-ionic surfactants and/or detergents may buffer each other, and stabilize the aggregation of the surfactants to form micelles desirably, even in a relatively high salt concentration (for example, as in the preceding paragraph). For example, Triton may have a stabilizing effect on the SDS, preventing or reducing risk of precipitation of the SDS that might otherwise occur in a relatively high salt concentration. Additionally or alternatively, for example, SDS may stabilize the formation of Triton micelles, even in the presence of the relatively high salt concentration.

It is believed that the stabilizing effects of the combination of ionic and non-ionic surfactants and/or detergents may be explained by the following, although this is not intended to limit the scope of the present disclosure in any way. In some embodiments, the compositions may include both (i) surfactant/detergent and (ii) salt components. Detergents are surfactants and they are used to disrupts cell membranes, thus assist with cell lysis and facilitate the release of intracellular materials in a soluble form. Detergents break protein-protein, protein-lipid, and lipid-lipid associations. Further, detergents denature proteins and various other macromolecules and prevent the non-specific binding of immunochemical assays and protein crystallization.

Surfactants/detergents dissolved in solution may tend to aggregate to form so-called micelles. All detergents at a specific concentration will form micelles, this concentration is referred to as the critical micelle concentration (CMC). Once CMC has been reached the detergent monomers form micelles. At concentrations higher than the CMC both micelles and monomers exist in solution alongside other non- micellar phases, which may not be soluble in water.

An ionic surfactant/detergent (e.g. SDS) forms ionic micelles. When these micelles (if alone) are exposed to high salt concentrations, the micelles break and the SDS precipitates out of the solution and does not have the desired effect within the solution.

Triton X is another detergent, but whereas SDS is ionic, triton-x is non-ionic. When both SDS and triton are dissolved in the solution the micelle population is one of both ionic (SDS) and non-ionic (triton) micelles and considered a mixed micelle population. This mixed micelle population has a much higher tolerance to salt concentrations and the non-ionic micelles buffers against the effects of salt on the ionic micelles. Temperature, pH and salt concentration can each affect the CMC of a solution (see below for detailed explanation of each). Therefore, altering any of the above mentioned may lead to the formation of a precipitate. The concentration of detergent required to form the micelles is specific. Example buffers disclosed herein contain additional sodium in the form of NaCl, which decreases the critical micelle concentrations, thus exceeding the CMC dramatically and risking precipitation the SDS to precipitate were it not for the presence of the complementary ionic/non-ionic surfactant.

The combination of SDS and Triton X is synergistic in terms of lowering the CMC and leading to the formation of larger mixed - ionic/non-ionic micelles. Concentrations and ratios of the two detergents play a role in determining the stability of the micelle. Therefore, these mixed detergent micelles may be more stable in a high salt buffer, thus preventing any precipitation of SDS. The combination of SDS and Triton-X is likely forming larger, mixed micelles with enhanced stability, as well as greater water molecule association (due to the NaCl concentration), and together contributing to the lack of SDS precipitation. Synergistically, the presence of NaCl may also effect the micelle formation of triton- X, by reducing the CMC, increasing the size of micelles formed, and a greater number of water molecules non-specifically bonded with the micelle. It is believed that this can also be buffered by a mixed micelle population (ie addition of ionic micelles) in accordance with the principles disclosed herein.

Other detergents that may be used and do not alter biological activity include: 2% Tween-20, NP40, deoxycholate 0.1-1%.

Below are two non-limiting tables of example detergents: Table 1 lists the categories of detergents and Table 2 lists example applications of each detergent as described herein. Table 1 :

Table 2:

(ii) a buffer agent, for example, comprising or consisting of Tris and/or ethylenediammetetraacetic acid (EDTA). The combination Tris and EDTA can buffer the solution and eliminate magnesium (inhibits DNAases). The combination can provide a basic DNA storage buffer. The EDTA can degrade the RNA (if desired).

In some embodiments, the pH of the sample and composition, after mixing together, may be slightly basic, for example, from about 7.4 to about 8. The pH of the composition 12 alone (e.g. prior to mixing with the sample) may be higher. The pH of the composition 12 alone may be at least about 8, optionally greater than 8, for example, about 8.3 (iil) at least one chemical inhibitor and/or denaturing agent to block proteins and/or DNAases. Suitable inhibitors and/or denaturing agents may include one or more selected from: 2-mercaptoethanol; Ca²⁺ ions; ethylene glycol tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); sodium dodecyl sulfate (SDS); iodoacetate; urea.

As mentioned above, EDTA is a chelating agent of divalent cations such as Mg²⁺ which is a co factor for DNAase nucleases). In some embodiments, the EDTA serves simultaneously as a buffer agent and a denaturing agent. (iv) Proteinase K for eliminating proteins. Proteinase K may be more effective in combination with SDS. For example, Proteinase K may be up to about ten times more effective on proteins that have been denatured, and SDS is effective as a protein denaturant. Proteinase K may avoid being inhibited by SDS or EDTA or other chemicals and protein inhibitors in solution. In some examples, where long term stability of Proteinase K might be considered an issue, this can generally be overcome by increasing the amount of Proteinase K in the composition 12, thereby pre- compensating for expected partial loss over time. By way of example only, the amount of Proteinase K in the composition 12 and/or in the combination of the composition 12 and sample after mixing together, may be between about 30 and 70mg/ml, optionally between about 40 and 60mg/ml, optionally about 50mg/ml.

In some embodiments, the aggregate salt concentration may be less than about 3000mM, optionally less than about 2000mM, optionally less than about lOOOmM, optionally less than about 750mM, optionally less than about 500mM. The salt concentration may be additive amongst all salt components in the composition 12 (including EDTA, for example). An overly high total salt concentration may restrict the ability to isolate DNA in later processing of the collected and preserved sample for genetic analysis. The salt concentration may be that referred to when mixed with saliva, or that of the composition in isolation. One specific example of the composition 12 may include Tris, SDS, Triton, NaCl and EDTA. Optionally, the composition may include one or more selected from: Proteinase K, sodium deoxycholate, urea. In one example, the relative concentrations of the constituents, determined in the combination of both the composition 12 and the saliva sample mixed together, may be as in Table 3 below:

Table 3:

The concentrations may be varied, individually and independently, by up to 5%, optionally up to 10%, optionally up to 20%>, within the scope of this specific example.

As mentioned above, the concentrations are desired concentrations in the solution containing both the composition 12 and the saliva sample. The concentrations of the constituents in the composition 12 alone may depend on the ratio R of the sample volume Vs to composition volume Vc (R=Vs/Vc) as described hereinbefore. The concentrations may vary according to a concentration multiplier of (R+l) or ((Vs/Vc)+1). General values, and some specific examples are illustrated in Table 4 below: Table 4:

Again, the concentrations may be varied, individually and independently, by up to 5%, optionally up to 10%, optionally up to 20%, within the scope of these specific examples.

The present disclosure also contemplates, within its scope:

(a) Varying the percentage of SDS, including down to 0% (omitting SDS).

(b) Varying the percentage of triton, including down to 0% (omitting Triton).

(c) Adding KCl and/or LiCl (salt that maintains ionic strength of the buffer), either as a substitute for the NaCl (omitting NaCl), or in combination with NaCl as a complement.

(d) Varying the individual and/or aggregate salt concentration(s).

(e) Adding Proteinase K, for example in a quantity as described above. (f) Adding 0.1 - 1% of sodium deoxycholate, which is a biological detergent capable of solubilizing membrane and cellular components, and may increase organelle lysis. The sodium deoxycholate may be used to substitute SDS and/or Triton, or used in combination with both. The relative percentages of SDS and/or Triton may also be varied. (For example, the range quoted above may refer to the composition when mixed with saliva. The concentration may be increased in the composition alone, in accordance with the factor R+l described herein.)

(g) Adding urea. Urea may increase protein denaturation and increase cell lysis. The percentages of SDS and/or triton, and/or other denaturing agents may be varied.

(h) using one or more other ionic and/or non-ionic surfactants and/or detergents in addition to, or as an alternative to, SDS and/or triton. If desired, according to an intended application, the composition 12 may further comprise ethanol. Alternatively, if desired, the composition 12 may be substantially free of ethanol.

In some embodiments, the composition 12 is effective in extracting and preserving nucleic acids from a cell organelle. The organelle may, for example, be a mitochondrion.

Extracting and preserving mitochondrial DNA is known to be difficult. Mitochondrial DNA is highly susceptible to mutation by conventional lysing techniques, and is very sparse in concentration compared to nuclear DNA. This results in such DNA being difficult to extract and preserve undamaged in sufficient quantity for downstream analysis, especially using a sample collection device of the type for home use. In a laboratory environment different from a sample collection device for home use, the general wisdom for obtaining mitochondrial DNA is to lyse cells very slowly and delicately under controlled conditions that do not present significant environmental impact, so as reduce the risk of damage to the mitochondrial DNA. Some embodiments of the present disclosure present a different approach. By using multiple lysing agents in combination, each in the high range of suggested use, for example, at least two lysing agents, optionally at least three lysing agents, or more, it is believed that not just cell membranes, but also organelle membranes may be lysed to liberate quickly nucleic acids from a cell organelle, thereby reducing the time during which lysing damage may occur.

Additionally or alternatively, by using multiple chemical inhibitor and/or denaturing agents in combination, each in the high range of suggested use, for example at least two such agents, optionally at least three such agents, it is believed that the extracted nucleic acids can be protected quickly from damage by surrounding environment materials, thereby reducing the amount of organelle DNA that is damaged.

Both of the above techniques provide surprising advantages compared to the conventional laboratory wisdom of a slow and delicate approach under controlled conditions that do not present significant environmental impact on organelle DNA. Using the above techniques in combination can provide surprisingly efficacy.

The results of experimental testing, described below, fully support the surprising efficacy of the techniques described herein. The experimental testing involved enrichment of mitochondrial DNA from whole saliva DNA samples preserved by different preservation solutions, and comparison of the end-yield of mitochondrial DNA (mDNA). The same testing was done using three different preservation solutions to compare the efficacy of each, namely: (A) pure water ; (B) a preservation solution according to the present disclosure, namely that of Table 4 (example 1) above, but without the optional components; and (C) a leading commercially available DNA preservation solution.

The specific methodology used was:

- 6ml samples of saliva were collected from 4 individuals and split equally into

3 x 2mls

2mls of a respective preservation solution A, B or C (explained above) was added to a 2ml sample from each individual

Samples were stored at room temperature for 3 days and DNA then isolated DNA quantity and purity was determined using nanodrop spectrometry in triplicate (results in Tables 5 and 6 below) and DNA quality was further analyzed by gel electrophoresis (results in drawings Fig. 4)

The DNA samples were then enriched for mDNA using Qiagen NREPLI-g® Mitochondrial DNA Kit in triplicate per each sample

The Enriched DNA was purified and quantified using nanodrop spectrometry to assess the end-yield of mDNA (Table 7 below).

Table 5 below indicates the total DNA quantity for each preservation solution, which was effectively the same for each (referring to the measurement standard deviation). This confirms that the later comparison is not distorted by different overall quantities of DNA in the samples prior to enrichment.

Table 5 - DNA Yield

Table 6 below indicates the DNA purity for each preservation solution, which was also effectively the same for each (referring to the measurement standard deviation). This confirms that the later comparison is not distorted by different purities of DNA.

Table 6 - DNA Purity

Fig. 4 of the drawings illustrates the DNA quality for each preservation solution (prior to mDNA enrichment). The left column is an index column. The second, third and fourth columns represent the samples using preservation solutions A, B, C, respectively. It can be seen that there is very little high quality DNA for the sample preserved with preservation solution A, namely water. High quality DNA only remains for preservative solutions B and C.

Table 7 below illustrates the amount of DNA remaining after mDNA enrichment, thereby providing comparative results of mDNA yield. The yield differs greatly between the three samples. Preservation solution A (water) did not yield any DNA, while solution B preserved samples yielded maximal DNA with maximal efficiency of amplification due to high amount of mitochondrial DNA in the total DNA sample. Solution C preserved samples did not yield the minimal that shows the amplification was working efficiently because of a low percentage of mitochondrial DNA in the total DNA sample; as explained below, it is expected that some solution C preserved samples might not even be viable for downstream processing.

Table 7 - mDNA Enrichment Yield

The experimental test results confirm that there is no difference in amount of DNA isolated, nor purity of the isolated DNA, between preservation solutions including just water. Additionally, this experiment confirms that without a true preservation solution, DNA is quickly degraded (e.g. as illustrated in water) and of low molecular weight and quality; thusly, not appropriate for downstream applications such as Next Gen sequencing (NGS).

Most importantly, this experiment shows that there is a high overall % of mDNA in only the solution B preserved samples (in accordance with the present disclosure). mDNA preserved with just water cannot be enriched. The samples preserved with the commercially available solution C yielded less enriched mDNA due to a lower % of mitochondrial DNA in the total DNA sample. The quality of the enrichment is of critical importance for downstream analysis such as NGS sequencing. The ideal yield from this kit that shows optimal amplification activity is about 5ug. Only solution B preserved samples approached, or reached (or even exceeded, within the standard deviation) 5ug enriched mitochondrial DNA. All of the solution B preserved samples exceeded the threshold of enrichment believed to be the minimum appropriate for downstream application, thus meaning that all of the solution B samples were viable for downstream analysis. In contrast, the mDNA yield from the commercially available preservation solution C was far lower than 5ug. Some samples preserved with the commercially available preservation solution C may even be below the threshold of enrichment believed to be the minimum appropriate for downstream application; therefore, some samples preserved with the commercially available preservation solution C are expected to be non viable.

The compositions described herein may find use in many different applications for lysing cellular material to extract cellular components. The preferred embodiments are described in the context of DNA extraction, for example, organelle DNA extraction, but the composition is not limited only to such use.

It will be appreciated that the foregoing description is merely illustrative of preferred embodiments of the invention, and that many improvements, alternatives and/or equivalents may be used.

Claims

1. A composition for use in a sample collection device for collecting a sample of a naturally expressed bodily fluid sample, the composition effective to extract and preserve nucleic acids in the collected sample, wherein the composition is effective in extracting and preserving nucleic acids from a cell organelle.

2. The composition of claim 1, wherein the organelle is a mitochondrion.

3. The composition of claim 1„ wherein the nucleic acids comprise DNA.

4. The composition of claim 1„ wherein the composition comprises at least one lysing agent for lysing a cell organelle.

5. The composition of claim 4, wherein the composition comprises at least two, optionally at least three, lysing agents.

6. The composition of claim 1, wherein the composition and/or a lysing agent comprises at least one selected from: a surfactant; a detergent; a salt.

7. The composition of claim 1, wherein the composition and/or a lysing agent comprises at least one, optionally at least two, optionally at least three, selected from, including derivatives thereof: sodium dodecyl sulfate (SDS); Triton; Triton-X; Triton 100; Triton-X 100; deoxycholate (e.g. sodium deoxycholate); cholate (e.g. sodium cholate); sodium lauroyl sarcosinate (and/or sarkosyl); maltoside (e.g. n-dodecyl- -D- maltopyranoside and/or DDM); glycoside (e.g. Digitonin); Tween (e.g. Tween 20 and/or Tween 80); 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (and/or CHAPS); nonyl phenoxypolyethoxylethanol (and/or Tergitol-type NP-40 and/or NP-40); sodium chloride (NaCl); lithium chloride (LiCl); potassium chloride (KC1).

8. The composition of any claim 1, wherein the composition comprises in combination (i) Triton or a derivative thereof, (ii) SDS or a derivative thereof, and (iii) a salt.

9. The composition of claim 1, wherein the composition comprises at least one chemical inhibitor and/or denaturing agent, for blocking proteins and/or DNAases.

10. The composition of claim 1, wherein the composition comprises at least one, optionally at least two, optionally at least three, selected from: 2-mercaptoethanol; Ca²⁺ ions; ethylene glycol tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); sodium dodecyl sulfate (SDS); iodoacetate; urea.

11. A composition for use in a sample collection device for collecting a sample of a naturally expressed bodily fluid sample, the composition effective to extract and preserve nucleic acids in the collected sample, wherein the composition comprises at least one or any combination of two or all three of:

at least one lysing agent for liberating nucleic acids from a sample cell, the at least one lysing agent comprising at least one, optionally at least two; optionally at least three, selected from, including derivatives thereof: sodium dodecyl sulfate (SDS); Triton; Triton-X; Triton 100; Triton-X 100; deoxycholate (e.g. sodium deoxycholate); cholate (e.g. sodium cholate); sodium lauroyl sarcosinate (and/or sarkosyl); maltoside (e.g. n-dodecyl- -D-maltopyranoside and/or DDM); glycoside (e.g. Digitonin); Tween (e.g. Tween 20 and/or Tween 80); 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (and/or CHAPS); nonyl phenoxypolyethoxylethanol (and/or Tergitol-type NP-40 and/or NP-40); sodium chloride (NaCl); lithium chloride (LiCl); potassium chloride (KC1);

a buffer agent comprising tris and/or ethylenediaminetetraacetic acid (EDTA); at least one chemical inhibitor and/or denaturing agent, for blocking proteins and/or DNAases, the at least one chemical inhibitor and/or denaturing agent comprising at least one, optionally at least two, optionally at least three, selected from: 2-mercaptoethanol; Ca²⁺ ions; ethylene glycol tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); sodium dodecyl sulfate (SDS); iodoacetate; urea.

12. The composition of claim 1 or 11 , further comprising a pH buffer.

13. The composition of claim 12, wherein the buffer is or comprises Tris.

14. The composition of claim 1 or 11, wherein the composition has a pH of greater than about 8.

15. The composition of claim 1 or 11, wherein the composition further comprises Proteinase K.

16. The composition of claim 1 or 11, wherein the composition has an aggregate salt concentration that is greater than at least one selected from: 249mM; 250mM; 275mM; 300mM; 325mM; 350mM.

17. The composition of claim 1 or 11, wherein the composition has an aggregate salt concentration that is not greater than at least one selected from: 300mM; 2000mM; lOOOmM; 750mM; 500mM.

18. The composition of claim 1 or 11, wherein the composition has a volume that is not greater than at least one selected from: 5ml; 4ml; 3ml; 2ml; lml; 750μ1; 500μ1; 250μ1; ΙΟΟμΙ.

19. The composition according to claim 1 or 11, comprising Tris, SDS, Triton, NaCl and EDTA.

20. The composition according to claim 19, further comprising one or more selected from: Proteinase K, sodium deoxycholate, urea.

21. The composition according to claim 19, wherein the concentrations of constituents in the composition are defined by:

Tris: about (R+l)* 10mM

SDS: about (R+l)* l% (v/v) Triton: about (R+l)* l% (v/v)

NaCl: greater than or equal to about (R+l)*250mM

EDTA: (R+l)*5mM

where:

R is a ratio Vs/V ;

Vs is a predetermined volume of a sample Vs with which the concentration is intended to be mixed in use, and

Vc is a volume of the composition.

22. A composition for lysing cellular material the composition comprising: at least one ionic surfactant (or ionic detergent); at least one non-ionic surfactant (or non- ionic detergent); and a salt.

23. The composition of claim 22, wherein the at least one ionic surfactant or detergent is selected as any one or more of: sodium dodecyl sulfate (SDS); deoxycholate (e.g. sodium deoxycholate); cholate (e.g. sodium cholate); sodium lauroyl sarcosinate (and/or sarkosyl); 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate (and/or CHAPS); a derivative of any of the aforementioned.

24. The composition of claim 22, wherein the at least one non-ionic surfactant or detergent is selected as any one or more of: Triton; Triton-X; Triton 100; Triton-X 100; maltoside (e.g. n-dodecyl- -D-maltopyranoside and/or DDM); glycoside (e.g. Digitonin); Tween (e.g. Tween 20 and/or Tween 80); 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (and/or CHAPS); nonyl phenoxypolyethoxylethanol (and/or Tergitol-type NP-40 and/or NP-40); a derivative of any of the aforementioned.

25. The composition of claim 22, wherein the salt is any one or more selected from: sodium chloride (NaCl); lithium chloride (LiCl); potassium chloride (KC1).

26. The composition of claim 22, wherein the composition has an aggregate salt concentration that is greater than at least one selected from: 249mM; 250mM; 275mM; 300mM; 325mM; 350mM.

27. The composition of claim 22, wherein the ionic and non-ionic surfactants or detergents form a mixed micelle population and/or mixed micelles.

28. The composition of claim 1, 11 or 22, further comprising ethanol.

29. The composition of claim 1, 11 or 22, wherein the composition is substantially ethanol free.

30. A bodily fluid sample collection device for the collection of naturally expressed bodily fluids comprising a container for receiving a naturally expressed bodily fluid, and a chamber containing a composition for extracting and preserving nucleic acids in the collected sample, wherein the composition is as defined according to claim 1, 11 or 22.

31. A bodily fluid sample collection device for the collection of a predetermined first volume of a naturally expressed bodily fluid, comprising a container for receiving a sample of the naturally expressed bodily fluid, and a chamber containing a second volume of a composition for extracting and preserving nucleic acids in the collected sample, wherein the second volume is not substantially more than, optionally smaller than, the first volume.

32. The bodily fluid sample device of claim 31, wherein the second volume is defined by at least one selected from: not more than about 100% of the first volume; not more than about 90% of the first volume; not more than about 80% of the first volume; not more than about 70% of the first volume; not more than about 60% of the first volume; not more than about 50% of the first volume; not more than about 40% of the first volume; not more than about 30% of the first volume; not more than about 20%) of the first volume; about 90%> of the first volume; about 80%> of the first volume; about 70%> of the first volume; about 60%> of the first volume; about 50%> of the first volume; about 40%> of the first volume; about 30%> of the first volume; about 20% of the first volume; about a third of the first volume; about a quarter of the first volume.

33. The device of claim 31, wherein the first volume is defined by at least one of: from about 0.5ml to about 2.25ml; from about 0.75ml to about 2.25ml, from about lml to about 2ml; from about 1ml to about 1.5ml; about 1ml.

34. The device of claim 31, wherein the first volume is about lml, and the second volume is about 250μ1.

35. A bodily fluid sample collection device for the collection of a sample of a naturally expressed bodily fluid, comprising a container for receiving a sample of the naturally expressed bodily fluid, and a chamber containing a composition for extracting and preserving nucleic acids in the collected sample, wherein the volume of the composition is defined by at least one selected from: from about ΙΟΟ Ι to about lml; at least about ΙΟΟμΙ and less than lml; from about 250μ1 to about 750μ1; at least about ΙΟΟμΙ; at least about 250μ1; at least about 500μ1; less than lml; less than about 750μ1; less than about 500μ1; about ΙΟΟμΙ; about 250μ1; about 500μ1; or about 750μ1.

36. Use in a sample collection device, of a composition as defined in any of claims 1 to 29, for extracting and preserving nucleic acids in a sample of a naturally expressed bodily fluid.

37. Use of proteinase K in a process for the extraction and preservation of mitochondrial DNA using a sample collection device for home use.