A composition for stabilizing genetic information material of a biological sample

Abstract

The present invention discloses a composition for stabilizing biological sample genetic information material, which comprises a protein denaturant, a bacteriostatic agent and a dispersant selected from at least one of alcohol ether or polyether or a combination thereof, the alcohol ether or polyether has a structure consisting of one polyhydric alcohol or two or more polyhydric alcohols forming ether bonds with each other, and is water-miscible, and the composition essentially does not contain a substance with a flash point lower than 60 DEG C.

Description

Technical Field

[0001] This invention relates to a composition and preparation of genetic information material for stabilizing biological samples, which can be used as a room temperature preservation solution for mammalian fecal samples. It relates to the room temperature preservation of fecal samples and the protection of the composition and structure of intestinal microorganisms in feces, and particularly to the preservation and transportation of fecal samples under general household conditions. Background Technology

[0002] The human gut is home to a vast and diverse array of microorganisms, often referred to by scientists as the "second genome." As scientific research deepens, increasing evidence suggests a close link between the gut microbiota and various aspects of human health, such as digestion and absorption, endocrine function, tumor formation and development, immune responses, and the nervous system. Numerous population studies have shown significant differences in gut microbiota among individuals with different physiological and disease states. Therefore, detecting key indicators of the gut microbiota can guide daily life and aid in clinical diagnosis and treatment.

[0003] Most current scientific research on human gut microbiota primarily involves the collection and analysis of fecal samples. However, the accuracy of gene sequencing, a crucial method for analyzing gut microbiota species and functions, heavily relies on the preservation of the microbial genomes within the fecal sample.

[0004] The human gut microbiota is mainly composed of bacteria, with a complex population structure. Bacteria with different oxygen tolerances will experience varying degrees of growth inhibition or promotion after feces are exposed to oxygen outside the body, resulting in changes in the microbial composition. To ensure the accuracy of results based on gene detection technology, it is necessary to perform special processing on the collected fecal samples within a short period of time to maintain the stability of information such as microbial composition and abundance in the samples.

[0005] Currently, the academically accepted standard method for preserving fecal samples is to freeze them in a -80°C freezer or dry ice as soon as possible after collection according to the operating procedures (Consensus on Standardized Sample Banks for Digestive Tract Microbiota, Yang Yunsheng, *Journal of Translational Medicine*, August 2018, Vol. 7, No. 4). However, this method requires high-level conditions, such as a large ultra-low temperature freezer or sufficient dry ice at the collection site, and is not suitable for ordinary households.

[0006] Most commercially available fecal preservation solutions contain high concentrations of organic solvents such as methanol, ethanol, and isopropanol (CN106596211A) and are used in combination with antimicrobial agents to maintain the stability of microbial composition and abundance in samples. However, the use of high concentrations of organic alcohol solvents often results in low flash points and imposes specific transportation restrictions; some contain guanidine isothiocyanate (CN109371015A), which has a certain degree of toxicity. Some preservation solutions cannot completely kill microorganisms, posing a biohazard risk to preservation and transportation (A novel affordable reagent for room temperatures, storage, and transport of fecal samples for metagenomic analyses. Han et al. Microbiome (2018) 6:43). Some preservation solutions contain high concentrations of chelating agents, which, if not properly removed, can inhibit downstream enzymatic reactions (US 2017 / 0166955A1).

[0007] Therefore, developing a safe and effective fecal sample preservation solution that meets routine transportation conditions still has significant commercial application value and will help popularize gut microbiota analysis and testing in the consumer market. Summary of the Invention

[0008] In this application, any of the following terms shall be interpreted in accordance with their meanings.

[0009] "Patient" refers to mammals, including humans.

[0010] "Pharmaceutical acceptable" substances are those substances that, within the bounds of normal medical judgment, are suitable for contact with a patient's tissues without causing undue toxicity, irritation, allergic reactions, etc., have a reasonable risk-benefit ratio, and are effective for their intended use.

[0011] "Inert" substances refer to substances that can affect the bioavailability of drugs but do not have pharmaceutical activity in other respects.

[0012] The terms “substantially” and “approximately” indicate the inherent degree of uncertainty that can be caused by any quantitative comparison, numerical value, measurement, or other method of representation. These terms are also used herein to indicate that the representation of quantities may deviate to a certain extent from the stated reference value, but without causing a change in the fundamental function of the object being referred to.

[0013] The terms “substantially none” and “substantially not containing” refer to the components discussed in amounts of up to 10%, preferably up to 5%, more preferably up to 1%, more preferably up to 0.5%, more preferably up to 0.1%, more preferably up to 0.05%, more preferably up to 0.03%, more preferably up to 0.02%, and most preferably up to 0.01% by weight of the composition.

[0014] Those skilled in the art will understand that "about" can vary to some extent in the context in which the term is used. If the use of a term is unclear to those skilled in the art, "about" will mean up to 20% plus or minus a particular term, taking into account the context in which it is used. When the term "about" is used to describe the value or endpoint of a range, it should be understood that the invention includes the specific value or endpoint referenced. Regardless of whether the numerical value or endpoint of a range in the invention uses "about," the numerical value or endpoint of a range includes two implementations: one modified by "about" and the other not modified by "about." It should also be understood that each endpoint value of a range is meaningful both in combination with another endpoint value and independently of another endpoint value.

[0015] Any references to temperature ranges, pH ranges, weight (mass) ranges, molecular weight ranges, percentage ranges, etc., in this specification, whether expressed using the terms "range" or "various ranges," include the specified endpoints and the points between the two endpoints.

[0016] The technical problem to be solved by the present invention is a composition of stable biological sample genetic information material that meets the conditions of normal transportation. Specifically, it is a fecal sample preservation solution that basically does not contain unsafe substances or unsafe additives such as those with low flash point or toxicity to humans.

[0017] On one hand, this application relates to a composition for stabilizing genetic information material in mammalian biological samples. In one or more specific embodiments of this application, it is preferred to stabilize genetic information material in human biological samples, and more preferably to stabilize genetic information material contained in biological samples from human excretions and / or excrement. The genetic information material may be deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) from humans and / or microorganisms.

[0018] In one or more embodiments of this application, the composition for stabilizing the genetic information material of mammalian biological samples comprises a protein denaturant, an antibacterial agent, and a dispersant.

[0019] In one or more embodiments of this application, the protein denaturing agent is preferably a chelating agent, which can be selected from CDTA, EDTA, etc., such as EDTA·4Na, EDTA·2Na, etc.; CDTA refers to 1,2-cyclohexanediaminetetraacetic acid, such as CDTA·4Na, CDTA·2Na, etc. In addition to chelating agents, the protein denaturing agent can also be a high-concentration salt, a high-concentration heavy metal salt, a reducing agent, guanidine hydrochloride, urea, and / or a protease, etc.

[0020] In one or more embodiments of this application, the antibacterial agent may be selected from triclosan, triclocarban, diclosan (HP-100), p-chloroxylenol, etc.

[0021] In one or more embodiments of this application, the dispersant is selected from alcohol ethers, which are readily soluble in water and have a flash point above 60°C; preferably, the alcohol ether is miscible with water. The term "alcohol ether" as used in this invention refers to an ether containing a hydroxyl group.

[0022] In one or more embodiments of this application, the selected alcohol ether structure is formed by at least one hydroxyl group of one polyol or two or more polyols forming an ether bond with at least one monohydric alcohol, wherein the alcohol ether contains at least one hydroxyl group. Preferably, the molecular weight of the alcohol ether is less than 2000.

[0023] In one or more embodiments of this application, the dispersant is selected from at least one of alcohol ethers or polyethers or a combination thereof, wherein the alcohol ether or polyether has a structural formula consisting of a polyol or two or more polyols forming an ether bond with each other, is readily soluble in water, preferably miscible with water, and has a flash point higher than 60°C.

[0024] In one or more embodiments of this application, the alcohol ether is preferably one of ethylene glycol methyl ether, ethylene glycol ethyl ether, diethylene glycol methyl ether, triethylene glycol methyl ether, tetraethylene glycol methyl ether, pentaethylene glycol methyl ether, propylene glycol methyl ether, dipropylene glycol methyl ether, tripropylene glycol methyl ether, tetrapropylene glycol methyl ether, pentapropylene glycol methyl ether, polyglycerol ether, polyethylene glycol, and polypropylene glycol, or a combination thereof. The polyglycerol ether, polyethylene glycol, or polypropylene glycol has a chemical formula containing one or more hydroxyl groups and a molecular weight of less than 2000, preferably less than 1000.

[0025] In one or more embodiments of this application, such alcohol ether is further preferably diethylene glycol ethyl ether, triethylene glycol methyl ether, triethylene glycol ethyl ether, or tripropylene glycol methyl ether.

[0026] In one or more embodiments of this application, the selected alcohol ether has a flash point higher than 60°C.

[0027] In one or more embodiments of this application, the alcohol ether is readily soluble in water, and preferably miscible with water; the preferred alcohol ether is selected from diethylene glycol ethyl ether, triethylene glycol methyl ether, triethylene glycol ethyl ether, and tripropylene glycol methyl ether.

[0028] In one or more embodiments of this application, the dispersant content in the composition is 1-50% by weight; preferably 5-40%; more preferably 10-30%; even more preferably 10-25%; and most preferably 12-25%.

[0029] In one or more embodiments of this application, a cell permeability modifier and a solubilizer may be further included.

[0030] In one or more embodiments of this application, the cell permeability modifier is selected from one or more of anionic surfactants (such as SDS) and nonionic surfactants (such as Tween-20, Triton X-100), preferably from one or more of the following reagents: polyethylene glycol monooctylphenyl ether (e.g., Triton X-100), polysorbates (e.g., polysorbate-20, Tween 20, polysorbate-80, Tween 80), sodium dodecylbenzenesulfonate, sodium dodecyl sulfate, sodium dodecyl sulfate, etc.; the cosolvent is selected from one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and N-methylpyrrolidone (NMP).

[0031] In one or more embodiments of this application, the flash point of the composition is higher than 60°C.

[0032] In one or more embodiments of this application, the dispersant is substantially free of substances with a flash point below 60°C, preferably the dispersant is substantially free of substances with a flash point below 60°C; preferably, the composition is substantially free of substances with a flash point below 60°C, and more preferably the composition is substantially free of substances with a flash point below 60°C, such as methanol, ethanol, n-propanol or isopropanol and other lower alcohols.

[0033] In one or more embodiments of this application, the dispersant contains substantially no substances toxic to humans, preferably not including substances toxic to humans, such as guanidine isothiocyanate. More preferably, in one or more embodiments of this application, the composition contains substantially no substances toxic to humans, and even more preferably not including substances toxic to humans, such as guanidine isothiocyanate. Attached Figure Description

[0034] Figure 1The effect of different concentrations of triethylene glycol methyl ether on DNA extraction yield (with the DNA yield of the control group being 1, and the average value of the results of the same treatment groups for volunteers A, B, and C being taken);

[0035] Figure 2 Agarose gel electrophoresis results of DNA from different treatment groups of volunteer A using triethylene glycol methyl ether as solvent;

[0036] Figure 3 The effect of different concentrations of triethylene glycol ethyl ether on DNA extraction yield;

[0037] Figure 4 The agarose gel electrophoresis results of DNA from different treatment groups using triethylene glycol ethyl ether as solvent;

[0038] Figure 5 PCoA analysis results for different volunteers under different treatment conditions;

[0039] Figure 6 For volunteer A, the Shannon index is used for each treatment group.

[0040] Figure 7 The relative abundance of species at the species level in each treatment group of volunteer A (different gray blocks represent the proportion of different genera of microorganisms).

[0041] PCoA Analysis: Principal Coordinate Analysis Method Based on Bray-Curtis Distance.

[0042] Shannon Index: A species diversity assessment indicator based on ASV. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments.

[0044] Unless otherwise specified, the concentration units used in this application and specific embodiments have the following meanings: percentage concentration refers to weight percentage concentration; M and mM refer to molar concentration, i.e., mol / L or mmol / L; ppm refers to the concentration expressed as the mass of solute as a percentage of the total mass of the solution, i.e., parts per million concentration.

[0045] Example 1:

[0046] Table 1 Formulation Table for Example 1

[0047]

[0048] The preparation method of Example 1 is as follows:

[0049] 1. Take 2L of deionized water, filter it through a 0.22μm filter membrane, and sterilize it at 121℃ for 30min to obtain sterile water.

[0050] 2. Weigh 57.03g of EDTA·4Na powder (purity >98%) and dissolve it in sterile water, then make up to 500ml to obtain a 300mM EDTA·4Na aqueous solution.

[0051] 3. Weigh 1g of triclosan powder and dissolve it in 100ml of DMSO to obtain a 1% triclosan solution.

[0052] 4. Add 100 ml of triethylene glycol methyl ether (purity >98%), 5 ml of Triton X-100 (purity >98%), and 5 ml of 1% triclosan solution to the 500 ml EDTA·4Na aqueous solution obtained in the previous step.

[0053] 5. Dilute the mixture obtained in the previous step to 1L with sterile water.

[0054] 6. Filter the mixed solution after the previous step is brought to volume using a 0.22μm filter membrane and dispense it into the matching fecal sample collector.

[0055] 7. During sample collection, 4 ml of preservative solution can protect a maximum of 1 g of fecal sample. Collect an appropriate weight of fecal sample into the sampler, and shake to thoroughly mix the fecal sample with the preservative solution to achieve the effect of protecting the microbial DNA in the feces.

[0056] The final formulation of Example 1 is shown in Table 1.

[0057] Example 2:

[0058] Referring to the preparation method of Example 1, the amount of each reagent added was adapted and configured according to the formula in Table 2 to form Example 2.

[0059] Table 2 Formulation Table for Example 2

[0060]

[0061] Example 3:

[0062] Referring to the preparation method of Example 1, the amount of each reagent added was adapted and configured according to the formula in Table 3 to form Example 3.

[0063] Table 3 Formulation Table for Example 3

[0064]

[0065] Example 4:

[0066] Referring to the preparation method of Example 1, the amount of each reagent added was adapted and configured according to the formula in Table 4 to form Example 4.

[0067] Table 4 Formulation Table for Example 4

[0068]

[0069] Example 5:

[0070] Referring to the preparation method of Example 1, the amount of each reagent added was adapted and configured according to the formula in Table 5 to form Example 5.

[0071] Table 5 Formulation Table for Example 5

[0072]

[0073] Examples 6-9:

[0074] Referring to the preparation method of Example 1, the amount of each reagent added was adapted and configured according to the formulation in Table 6 to form Examples 6-9, and the corresponding protective liquid samples were designated as P1-4.

[0075] Table 6 Formulation Table for Examples 6-9

[0076] Example 6 Example 7 Example 8 Example 9 Types of protective fluid P1 P2 P3 P4 Triethylene glycol methyl ether concentration 5％ 10％ 25％ 50％ EDTA·4Na 150mM 150mM 150mM 150mM Triton X-100 0.5％ 0.5％ 0.5％ 0.5％ Triclosan 50ppm 50ppm 50ppm 50ppm DMSO 0.5％ 0.5％ 0.5％ 0.5％

[0077] Example 10:

[0078] This study investigated the effect of readily soluble or water-miscible alcohol ether solvents at high concentrations on the extraction and purification of microbial DNA from fecal samples. The specific experimental procedure was as follows: different types of alcohol ether solvents were prepared into 50% aqueous solutions, mixed thoroughly with the fecal samples, and allowed to stand for 2 days before extracting and detecting fecal microbial DNA.

[0079] The yield of DNA extracted directly from a unit weight of fresh fecal sample is 100% (unless otherwise specified, this standard applies to other examples involving DNA yield); OD260 / 230 is the absorbance value measurement result, used to evaluate the residual impurities such as salts in the DNA sample, and is generally around 2.0.

[0080] Table 7. Effects of alcohol and ether solvents on the extraction and purification of microbial DNA from fecal samples.

[0081]

[0082] After standing for two days, the alcohol ether solvents were fully absorbed into the fecal samples. Different types of solvents had varying effects on the microbial DNA extraction yield and impurity removal. As shown in Table 7, triethylene glycol methyl ether had the least impact, followed by triethylene glycol ethyl ether. Next, the optimal concentrations of the two preferred alcohol ether solvents were explored, and other components were adjusted.

[0083] Example 11:

[0084] The inhibitory effects of different concentrations of triclosan on the growth of common microorganisms in feces were tested.

[0085] Escherichia coli, Bifidobacterium longum, Lactobacillus rhamnosus, and Bacteroides fragilis were selected as representative strains from human fecal samples. The minimum inhibitory concentration (MIC) of the broad-spectrum bactericide triclosan was determined under anaerobic conditions, and the MIC value of triclosan was found to be 10 ppm.

[0086] An equal volume of fecal sample was mixed with a 100 ppm triclosan aqueous solution, and the precipitate was removed by low-speed centrifugation to obtain a fecal microbial suspension. The inhibition of fecal microbial growth was assessed using a plate-spreading method on a complete nutrient medium. No colonies were observed after 24 hours of anaerobic incubation, indicating that the presence of 50 ppm triclosan significantly inhibited microbial growth in the fecal sample.

[0087] Example 12:

[0088] The effect of triethylene glycol methyl ether concentration on fecal dispersion was tested.

[0089] Specific testing process:

[0090] A. Recruit 5 volunteers and collect 0.5g of fresh feces from each of them into a collection tube containing 4ml of different solutions.

[0091] B. Using a rotary mixer, the collection tube containing the fecal sample is inverted twice per second to simulate a real-world usage scenario.

[0092] C. Set the rotation mixer running time to 10 seconds and check the mixing effect after each run.

[0093] D. Record the number of runs required for each sample to be thoroughly mixed with the solution (without any non-dispersible particles larger than 5 mm) to evaluate the dispersion effect. Three replicates were set up for each volunteer sample, and the average number of runs was taken.

[0094] Table 8. Effect of triethylene glycol methyl ether concentration on fecal dispersion effect

[0095]

[0096] As shown in Table 8, triethylene glycol methyl ether solutions with concentrations of 5-25% can increase the dispersion rate of feces in aqueous solutions. However, with further increases in the concentration of triethylene glycol methyl ether, the dispersion is negatively affected due to the increased viscosity of the solution and the decreased solubility for aqueous substances. The results for triethylene glycol ethyl ether are similar, with the optimal dispersion concentration between 10% and 25%.

[0097] Example 13:

[0098] The effect of triethylene glycol methyl ether concentration on DNA protection and extraction yield.

[0099] Experimental procedure:

[0100] Take protective solutions P1-4 with different triethylene glycol methyl ether concentrations prepared in Examples 6-9.

[0101] Three volunteers (A, B, and C) were recruited. Fresh fecal samples were collected from each volunteer and placed into multiple collection tubes containing 4 ml of different solutions. After mixing, the samples were stored at room temperature for 1 day, 3 days, and 5 days (referred to as d1, d3, and d5 in the table and figures, respectively). Simultaneously, fresh fecal samples were collected from each volunteer for direct DNA extraction. The fecal sample processing groupings for each volunteer are shown in the table below (Table 9).

[0102] Table 9. Effects of triethylene glycol methyl ether concentration on DNA protection efficacy and extraction yield.

[0103]

[0104] Nucleic acid extraction experiments were performed on each fecal sample to obtain purified fecal microbial DNA. By comparing the DNA yield with that extracted directly from fresh samples, the effect of different concentrations of triethylene glycol methyl ether on the DNA extraction yield was determined (see appendix). Figure 1 ).

[0105] The results showed that prolonged soaking and preservation of fecal samples at triethylene glycol methyl ether concentrations of 50% and above affected the DNA extraction yield. This effect was acceptable when the triethylene glycol methyl ether concentration was in the range of 5%-25%.

[0106] DNA samples can be subjected to agarose gel electrophoresis to obtain DNA sample integrity test results. Taking volunteer A as an example, the DNA sample integrity is shown in the attached figure. Figure 2 As shown in the attached figure, M is the DL 15000 DNA Marker (brand: Vazyme, catalog number: MD103); Control is the untreated control group, which is the microbial DNA directly extracted from fresh fecal samples, containing the most accurate genetic information of real fecal microorganisms.

[0107] The results of agarose gel electrophoresis showed that different concentrations of triethylene glycol methyl ether had no significant effect on the protection of DNA integrity.

[0108] Examples 14-15:

[0109] Referring to the preparation method of Example 1, the amount of each reagent added was adapted and configured according to the formulation in Table 10 to form Examples 14 and 15, and the corresponding protective liquid samples were designated as A1 and A2.

[0110] Table 10 Formulation Tables for Examples 14 and 15

[0111] Example 14 Example 15 Types of protective fluid A1 A2 Triethylene glycol ethyl ether concentration 10％ 25％ EDTA·4Na 150mM 150mM Triton X-100 0.5％ 0.5％ Triclosan 50ppm 50ppm DMSO 0.5％ 0.5％

[0112] Example 16:

[0113] The effect of triethylene glycol ethyl ether concentration on DNA protection and extraction yield.

[0114] The experimental procedure is as described in Example 13.

[0115] Triethylene glycol ethyl ether has a slightly greater impact on DNA extraction yield than triethylene glycol methyl ether, but has no effect on the protection of DNA integrity. Different concentrations of triethylene glycol ethyl ether, in the presence of other reagents of the same composition and concentration, were mixed with fecal samples and allowed to stand for 1, 3, and 5 days before DNA extraction and detection (referred to as d1, d3, and d5 in the table and figures, respectively). The results of agarose gel electrophoresis are shown in the attached figure. Figure 4 In the case of two different concentrations of triethylene glycol ethyl ether as solvents, the integrity of microbial DNA in fecal samples was well preserved. In the attached figure, M is the DL 15000 DNA Marker (brand: Vazyme, catalog number: MD103); Control is the untreated control group, i.e., microbial DNA directly extracted from fresh fecal samples, containing the most accurate genetic information of real fecal microorganisms.

[0116] Example 17:

[0117] The fecal microbial DNA samples extracted in Example 13 were subjected to 16S V3V4 region gene sequencing to identify the types and proportions of microorganisms (bacteria) in each treatment group, thereby determining the effect of different concentrations of triethylene glycol methyl ether on the microbial composition (see appendix). Figure 5 Appendix Figure 6 Appendix Figure 7 Appendix Figure 5 This demonstrates that the fecal samples from each volunteer (A, B, C) retained their original individual microbial composition characteristics after treatment with the preservative solution. (Attached) Figure 6 This indicates that, taking volunteer A as an example, the volunteer's fecal sample maintained its original species diversity even after being treated with a preservative solution containing different concentrations of triethylene glycol methyl ether for different durations. (Attached) Figure 7 This demonstrates that, taking volunteer A as an example, the volunteer's fecal sample retained its original microbial composition even after treatment with a preservative solution containing different concentrations of triethylene glycol methyl ether for varying durations. This indicates that, under the test conditions, the preservative solution in the example effectively preserved the microbial DNA in the fecal sample.

[0118] Finally, it should be noted that the above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the technical scope disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A composition for stabilizing genetic information material of a biological sample, comprising a protein denaturant, a bacteriostatic agent, and a dispersing agent, characterized by: The dispersant is an alcohol ether, which is readily soluble in water and has a flash point higher than 60°C; the alcohol ether has a molecular weight lower than 2000; the alcohol ether is selected from the group consisting of triethylene glycol methyl ether, triethylene glycol ethyl ether, and tripropylene glycol methyl ether.

2. The composition of claim 1, wherein: The dispersant has a weight percentage content of 10-30%.

3. The composition of claim 1, wherein: The composition has a flash point higher than 60°C.

4. The composition of claim 1, wherein: Further comprising a cell permeability modifier and a cosolvent.

5. The composition of claim 4, wherein: The cell permeability modifier is selected from one or more of polyethylene glycol monooctyl phenyl ether, polysorbate-20, polysorbate-80, sodium dodecylsulfonate, and sodium dodecylsulfate; the cosolvent is selected from one or more of dimethyl sulfoxide, N,N-dimethylformamide, and N-methyl pyrrolidone.

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