CN115486438A

CN115486438A - Composition for preventing biological sample genetic information substance from degrading and use method thereof

Info

Publication number: CN115486438A
Application number: CN202111097464.5A
Authority: CN
Inventors: 丁晓颖; 马宇航; 赫慧琛; 马磊; 刘标
Original assignee: Shanghai Juben Biotechnology Co ltd; Shanghai First Peoples Hospital
Current assignee: Shanghai Juben Biotechnology Co ltd; Shanghai First Peoples Hospital
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2022-12-20

Abstract

The invention discloses a composition for preventing genetic information substances of biological samples from degrading and a using method thereof. The composition provided by the invention can condense the protective liquid which does not reach the freezing point temperature of the protective liquid system, so that the fluidity of the protective liquid is greatly reduced, and the colony composition of a fecal sample is further prevented from changing; the protection liquid system recovers the fluidity at the test temperature, so that the test of an instrument is facilitated; and the composition of the protective solution does not have negative influence on the test result of the instrument.

Description

Composition for preventing biological sample genetic information substance from degrading and use method thereof

Technical Field

The invention relates to a composition for preventing genetic information substances of biological samples from being degraded and a using method thereof, which can be used as a normal-temperature protective solution for fecal samples of mammals, and relates to normal-temperature storage of the fecal samples and protection of intestinal microbial composition structures in the fecal samples, in particular to storage and transportation of the fecal samples under common household conditions.

Background

The human gut harbors a large number and variety of microorganisms, known to scientists as the "second genome" of humans. With the progress of scientific research, there is a growing body of evidence that the intestinal flora is closely related to various aspects of human health, such as digestive absorption, endocrine secretion, tumor formation and development, immune response, nervous system, and the like. A large amount of population experimental data show that people with different physiological and disease states often have obvious difference between intestinal flora. Therefore, people can be guided to daily life or assisted with clinical diagnosis and treatment by detecting some key indexes of the intestinal flora.

Most of the current scientific research on human intestinal microorganisms mainly comprises the collection of fecal samples for detection and analysis. The gene sequencing is used as an important detection means for analyzing the species and functions of the intestinal flora, and the accuracy of the detection result of the gene sequencing is seriously dependent on the preservation condition of the microbial genome in the fecal sample.

Human intestinal microorganisms are mainly composed of bacteria and have complex population structures. After the excrement is exposed to oxygen in vitro, the bacteria with different oxygen tolerance capacities can be quickly inhibited or promoted by growth to different degrees, and the microbial composition is changed. In order to ensure the accuracy of the results based on the genetic testing technology, it is necessary to perform special treatment on the collected fecal samples in a short time and maintain the information such as the composition and abundance of microorganisms in the samples to be stable and unchanged.

Currently, the standard methods for stool sample preservation recognized by the academia are: feces are collected according to the operating specifications and then put into a refrigerator at minus 80 ℃ or dry ice for cryopreservation as soon as possible (consensus on digestive tract microecological standardized sample banks, yangyuan, J.transmutation medicine, vol.8, 8, 2018, vol.7, no. 4). However, the implementation of the method requires high conditions, such as a large ultra-low temperature refrigerator or enough dry ice required by a collection field, and is not suitable for general families.

Therefore, the developed excrement sample protection solution is safe and effective, has huge commercial application value and helps the popularization of intestinal microorganism analysis tests at the consumption end.

Disclosure of Invention

In the present application, the following terms are included and should be construed with reference to the following meanings.

"patient" refers to a mammal, including a human.

"pharmaceutically acceptable" substances are those which are, within the scope of normal medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, and effective for their intended use.

"inert" material refers to a material that affects the bioavailability of a drug, but otherwise has no pharmaceutical activity.

The terms "substantially" and "approximately" mean the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The use of these terms herein also indicates that the quantitative representation may vary from the stated reference without resulting in a change in the basic function of the subject to which it is related.

The terms "substantially free", "substantially free" and "comprising" mean that the component in question is present in an amount of at most 10%, preferably at most 5%, more preferably at most 1%, more preferably at most 0.5%, more preferably at most 0.1%, more preferably at most 0.05%, more preferably at most 0.03%, more preferably at most 0.02%, and most preferably at most 0.01%, based on the composition (weight percentage).

One of ordinary skill in the art will understand "about" and will vary to some extent in the context in which the term is used. If the use of a term is not clear to one of ordinary skill in the art, in view of the context in which it is used, "about" will mean up to plus or minus 20% of the particular term. When the term "about" is used to describe a value or an end-point of a range, it is understood that the invention includes the particular value or end-point referenced. Whether or not "about" is used with respect to a value or an endpoint of a range in the present invention, the value or endpoint of the range includes two embodiments: one modified with "about" and the other not modified with "about". It will be further understood that the endpoints of each of the ranges are significant both in combination with the other endpoint, and independently of the other endpoint.

Any reference in this specification to temperature ranges, pH ranges, weight (mass) ranges, molecular weight ranges, percentage ranges, and the like, whether expressed in terms of "range" or "ranges" or not, is intended to include the endpoints specified, and points between the endpoints.

After the feces are collected, deep cryopreservation of the sample is usually required in order to maintain the original colony composition of the collected feces to the maximum. However, such a test requirement cannot be satisfied in a scene of home use or the like, and therefore, techniques such as a protective solution capable of suppressing bacterial growth have been developed in succession, and changes in colony composition have been reduced as much as possible by refrigeration.

The invention provides a protective solution formula which can solidify a feces sample below a certain storage temperature or greatly reduce the fluidity of the feces sample and restore the feces sample to flow dynamics above a certain use temperature.

The formula of the protective solution provided by the invention can condense the protective solution which does not reach the freezing point temperature of the protective solution system, so that the fluidity of the protective solution is greatly reduced, and the colony composition of a fecal sample is further prevented from changing; the protection liquid system recovers the fluidity at the test temperature, so that the test of an instrument is facilitated; and the composition of the protective solution does not have negative influence on the test result of the instrument.

In order to solve the technical problems, the invention provides a formula for preventing the genetic information substance of a biological sample from degrading and a using method thereof.

In one aspect, the present invention provides a temperature-sensitive composition (protective solution formulation) for preventing the degradation of genetic information in a biological sample, which can maintain the fluidity (fluid state) of a water sample liquid at a relatively high temperature (e.g., higher than 20 ℃) and can change into a gel-like substance in an opaque state at a relatively low temperature (e.g., 0 to 20 ℃), and which can adjust the temperature change point between the fluid state and the gel state of a hydrogel in a wider range.

First, the formulation of the present invention forms a hydrogel that has clear fluid-like properties. Next, in the present invention, an ionic surfactant, preferably an anionic surfactant, is added to the hydrogel protective solution. The invention introduces the anionic surfactant as a 'heat sensitive response component' into a gel grid of the hydrogel protective solution, when the temperature of the hydrogel protective solution is at or below the Krafft point, the anionic surfactant can be separated or dissolved in the gel grid, and the separated anionic surfactant can lead the hydrogel protective solution to be coagulated to form gel, so that the hydrogel protective solution is changed in two states of fluid and gel, and the purpose of controlling the fluidity of the hydrogel by temperature is realized. Third, the present disclosure can change the temperature change point of the fluidity of the hydrogel by adjusting the ratio of water and the organic solvent, adjusting the concentration and kind of the anionic surfactant, and the like. In the present invention, an organic solvent which is miscible with water at an arbitrary ratio is preferred, and therefore the adjustment range of the temperature change point of the hydrogel fluidity is wider.

In one or more embodiments of the present application, the present invention provides a temperature-sensitive composition for preventing the degradation of genetic information material of a biological sample, comprising: comprises a protein denaturant, a bacteriostatic agent, an anionic surfactant and a dispersing agent.

In one or more embodiments of the present application, the protein denaturing agent is preferably a chelating agent, which may be selected from CDTA, EDTA, and the like, e.g., EDTA 4Na, EDTA 2Na, and the like; CDTA means 1, 2-cyclohexanediaminetetraacetic acid, for example, CDTA 4Na, CDTA 2Na and the like. In addition to the chelating agent, the protein denaturant may be a high concentration salt, a high concentration heavy metal salt, a reducing agent, a guanidine hydrochloride, urea, and/or a protease, and the like.

In one or more embodiments herein, the bacteriostatic agent may be selected from triclosan, triclocarban, dichlorohenne (HP-100), parachloroxylenol, and the like.

In one or more embodiments herein, the dispersant is selected from alcohol ethers, which are selected to be readily soluble in water; the preferred alcohol ethers are miscible with water. The term "alcohol ether" as used herein refers to an ether containing a hydroxyl group.

In one or more embodiments of the present application, the dispersant is selected from at least one of alcohol ethers or polyethers, or a combination thereof, the alcohol ethers or polyethers being of a structure consisting of one polyol or of two or more polyols forming ether linkages with each other, and being readily soluble in water, preferably miscible with water.

In one or more embodiments of the present application, the alcohol ether structure comprises at least one hydroxyl group of a polyol or of two or more polyols forming ether linkages with each other, wherein at least one hydroxyl group forms an ether linkage with at least one monohydric alcohol, and is readily soluble in water, preferably miscible with water.

In one or more embodiments of the present application, the alcohol ether is preferably selected from one of ethylene glycol methyl ether, ethylene glycol ethyl ether, diethylene glycol methyl ether, diethylene glycol ethyl ether, triethylene glycol methyl ether, triethylene glycol ethyl 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, triethylene glycol butyl ether, polyglyceryl ether, polyethylene glycol, and polypropylene glycol, or a combination thereof. The chemical formula of the polyglycerol ether, the polyethylene glycol or the polypropylene glycol contains one or more hydroxyl groups, and the molecular weight is less than 2000, preferably less than 1000.

In one or more embodiments herein, such alcohol ethers are further preferably triethylene glycol methyl ether, triethylene glycol ethyl ether, triethylene glycol propyl ether, triethylene glycol butyl ether, diethylene glycol butyl ether, triethylene glycol pentyl ether, or analogs thereof.

In one or more embodiments of the present application, the anionic surfactant is preferably one or a combination of sodium or potassium salts of alkylbenzene sulfonic acid, alkyl sulfuric acid, hydrocarbyl carboxylic acid, and the like. The sodium or potassium salt of the alkylbenzene sulfonic acid may be selected from sodium (or potassium) dodecylbenzene sulfonate, the sodium or potassium salt of the alkylsulfonic acid may be selected from sodium (or potassium) dodecylbenzene sulfonate, and the sodium or potassium salt of the alkylsulfuric acid may be selected from sodium (or potassium) dodecylbenzene sulfate; the hydrocarbyl carboxylic acid may be selected from stearic acid, palmitic acid, lauric acid, and the like among alkyl carboxylic acids, or may be selected from oleic acid among alkylene carboxylic acids. The anionic surfactant is preferably one or a combination of sodium dodecylbenzene sulfonate, sodium dodecyl sulfate and sodium laurate.

In one or more embodiments herein, the anionic surfactant is present in the composition in an amount of 0.1 to 8% by weight; preferably 0.5 to 5%; further preferably 0.7 to 3%; more preferably 0.8 to 2%; most preferably 0.95-1.05%.

In one or more embodiments herein, the dispersant is present in the composition in an amount of 1 to 50% by weight; preferably 5 to 40%; further preferably 10 to 30%; more preferably 10-25%; most preferably 12-25%.

In one or more embodiments of the present application, substantially no substance toxic to the human body is added to the dispersant, and preferably, substantially no substance toxic to the human body, such as substantially no guanidine isothiocyanate, is contained in the dispersant. Preferably, in one or more embodiments of the present application, the composition does not substantially contain a substance toxic to the human body, and more preferably, the composition does not substantially contain a substance toxic to the human body, for example, substantially does not contain guanidine isothiocyanate or the like.

In one aspect, the present invention also provides methods of using one or more of the aforementioned compositions for preventing the degradation of genetic information material of a biological sample.

The one or more compositions for preventing the genetic information substances of the biological samples from being degraded are matched with the heat preservation container to preserve the collected biological samples, preferably human excretion and/or excretion substance samples, in the environment lower than the Krafft point (for example, 0-20 ℃), a user can place the samples into the container for preservation after collecting the samples by self, and the samples are sent to a detection mechanism for detection, so that the risk of deterioration of the substances to be detected caused in the process from sample collection to detection is reduced to the greatest extent.

In one or more embodiments of the present application, the insulated container may be a container similar to a thermos cup, in which an ice pack or ice cubes may be placed; the sampling container can be a double-layer container filled with liquid with higher specific heat capacity in the middle layer, the double-layer container can be stored in a refrigerator firstly, and after a user collects a sample by himself, the user places the sampling container into the double-layer container for storage.

In one or more embodiments of the present application, a user places a self-collected sample above the Krafft point of the formulation (i.e., ambient temperature) into a sampling container containing a composition that prevents the genetic information material of a biological sample from degrading; mixing uniformly; the user lowers the temperature of the sampling container below the Krafft point of the formula to enable the composition formula to form a gel; the temperature of the sampling vessel is maintained below the Krafft point of the composition formulation through the sample testing stage.

In another aspect, the present invention provides the use of one or more of the aforementioned compositions for preventing the degradation of genetic information material from a biological sample for stabilizing the genetic information material from a mammalian biological sample.

In one or more embodiments of the present application, it is preferable to stabilize genetic information material of a human biological sample, and it is further preferable to stabilize excretion and/or discharge material from the human body as genetic information material contained in the biological sample, and the genetic information material may be deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) or the like derived from a human and/or a microorganism.

In one or more embodiments of the present application, the one or more temperature-sensitive hydrogel protective solutions described above can stabilize genetic information material in a biological sample from a mammal.

The beneficial effects of the invention are as follows:

1. the temperature-sensitive hydrogel composition for preventing the genetic information substance of the biological sample from degrading has thermal sensitivity responsiveness, and the response temperature can be adjusted in a wide range, and can be used as a temperature response component of a protective solution.

2. The temperature-sensitive hydrogel composition for preventing the genetic information substance of the biological sample from being degraded takes an anionic surfactant as a matrix material, and a three-dimensional network structure formed by chemical crosslinking is stable and uniform in pores; the groups on the gel skeleton can not react with other molecules in the system, so that the formula of the hydrogel for preventing the genetic information substance of the biological sample from being degraded has reversibility of temperature response, and the formula can be repeatedly used.

3. According to the invention, the anionic surfactant is used as a temperature response component, and can be uniformly precipitated or dissolved in the matrix gel, and the anionic surfactant existing in a large amount in a solvent in a micelle form can be macroscopically expressed as the change of the transparency and the fluidity of the obtained gel to form a gel-like substance, so that the reduction of the fluidity of the protection liquid is realized, and the change of the colony composition of the excrement sample is further prevented.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis of DNA of fecal flora of example 3 at 10 deg.C (below the Krafft point) and 20 deg.C (above the Krafft point);

FIG. 2 shows the results of DNA agarose gel electrophoresis of fecal flora at 3 ℃ in example 1 (formulation 1) and comparative example 1 (formulation 2).

Detailed Description

The present invention will be further described with reference to the following specific examples.

Unless otherwise specified, the concentration units used in the present application and in the detailed description have the meaning: the percentage concentration is weight percentage concentration; m and mM refer to molar concentration, namely mol/L or mmol/L; ppm refers to the concentration of solute in parts per million based on the mass of the entire solution, i.e., parts per million concentration.

Example 1 (formulation 1):

table 1 example 1 formulation table

The preparation method of the embodiment 1 specifically comprises the following steps:

1. 2L of deionized water is taken, filtered by a 0.22 mu m filter membrane and sterilized for 30min at 121 ℃ to obtain sterile water.

2. 2g of EDTA-4 Na powder (purity > 98%) is weighed and dissolved in sterile water to a constant volume of 500ml, and an EDTA-4 Na aqueous solution is obtained.

3. 10g of sodium dodecylbenzenesulfonate was added to 500ml of the aqueous EDTA.4 Na solution obtained in the above step.

5. The mixed solution obtained in the above step was made to a volume of 1L with sterile water.

6. And (3) filtering the mixed solution with the constant volume in the previous step by using a 0.22-micron filter membrane, and subpackaging the mixed solution into a matched excrement sample collector.

7. At the time of sample collection, 4ml of protective solution protects a maximum of 1g of fecal sample. Collecting excrement samples with proper weight to a collector, and fully mixing excrement with the protective solution by shaking to realize the effect of protecting the DNA of microorganisms in the excrement.

The final formulation of example 1 is shown in table 1.

The Krafft point for example 1 is: about 5 deg.c.

Comparative example 1 (formulation 2):

referring to the preparation method of example 1, each reagent addition amount was adaptively modified according to the formulation of table 2 to prepare comparative example 1.

Table 2 formula table of comparative example 1

Example 2:

referring to the preparation method of example 1, each reagent addition amount was configured as example 2 with an adaptation to the formulation of table 3.

Table 3 example 2 formulation table

The Krafft point for example 2 is: about 10 deg.c.

Example 3:

referring to the preparation method of example 1, each reagent addition amount was configured as example 3 with an adaptation to the formulation of table 4.

Table 4 example 3 formulation table

The Krafft point for example 3 is: about 15 deg.c.

The test of the above example 2 and example 3 according to the method of the experiment of the effect of preventing degradation (preventing bacterial colony change) of example 1 proves that the actual degradation prevention effect of the example 2 and the example 3 is obviously superior to that of other non-anionic surfactant systems, and the degradation prevention effect of the example under the Krafft point is obviously superior to that of the example under the Krafft point, so that the temperature control system in the specific implementation mode of the invention has a significant enhancement effect on the degradation prevention (the improvement of the bacteriostatic effect to prevent bacterial colony change) by gelation.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the following descriptions are only illustrative and not restrictive, and that the scope of the present invention is not limited to the above embodiments: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A composition for preventing the genetic information substance of a biological sample from degrading, which comprises a protein denaturant, a bacteriostatic agent, an anionic surfactant and a dispersing agent, and is characterized in that: the dispersing agent is alcohol ether; the anionic surfactant is selected from one of sodium salt or potassium salt of alkyl benzene sulfonic acid, alkyl sulfuric acid, and hydrocarbyl carboxylic acid, or their combination.

2. The composition of claim 1, wherein: the alcohol ether structure formula is formed by that at least one hydroxyl group of one polyhydric alcohol or more than two polyhydric alcohols forming ether bonds with each other forms ether bonds with at least one monohydric alcohol, and then the alcohol ether at least contains one hydroxyl group.

3. The composition of claim 2, wherein: the alkyl benzene sulfonic acid is selected from dodecyl benzene sulfonic acid, the alkyl sulfonic acid is selected from dodecyl sulfonic acid, and the alkyl sulfuric acid is selected from dodecyl sulfuric acid; the hydrocarbyl carboxylic acid is selected from alkyl carboxylic acid or alkenyl carboxylic acid.

4. The composition of claim 3, wherein: the alcohol ether is selected from one of ethylene glycol methyl ether, ethylene glycol ethyl ether, diethylene glycol methyl ether, diethylene glycol ethyl ether, triethylene glycol methyl ether, triethylene glycol ethyl 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, triethylene glycol propyl ether, triethylene glycol butyl ether, diethylene glycol butyl ether, triethylene glycol pentyl ether, polyglycerol ether, polyethylene glycol and polypropylene glycol or a composition thereof.

5. The composition of claim 4, wherein: the weight percentage content of the dispersant is 1-50%, and the weight percentage content of the anionic surfactant is 0.1-8%.

6. The composition of claim 5, wherein: the alcohol ether is selected from one of diethylene glycol ethyl ether, triethylene glycol methyl ether, triethylene glycol ethyl ether, tripropylene glycol methyl ether, triethylene glycol ethyl ether, triethylene glycol propyl ether, triethylene glycol butyl ether, diethylene glycol butyl ether and triethylene glycol pentyl ether or a combination thereof.

7. The composition of claim 6, wherein: the weight percentage content of the dispersant is 5-40%, and the weight percentage content of the anionic surfactant is 0.5-5%.

8. The composition of claim 7, wherein: the weight percentage content of the dispersant is 10-30%, and the weight percentage content of the anionic surfactant is 0.8-2%.

9. A method of using the composition of any one of claims 1-8, wherein: (1) Placing the collected sample into a sampling container containing the composition; (2) Mixing a sample with the composition at a temperature above the Krafft point of the composition; (3) The temperature is reduced below the Krafft point of the composition for storage.

10. Use of a composition according to any one of claims 1 to 8 for the stabilization of genetic information material from a biological sample from a mammal.