CN111631758A - Structure and method for preserving sampling material in gastrointestinal tract - Google Patents
Structure and method for preserving sampling material in gastrointestinal tract Download PDFInfo
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Abstract
The invention discloses a storage structure and a storage method of a sampling material in gastrointestinal tracts, wherein the storage structure comprises a sampling capsule, a wrapping structure is arranged in the sampling capsule, a medicament is wrapped in the wrapping structure, the inner side wall of the sampling capsule is pushed to a central position by a support structure, and the wrapping structure is composed of a water-absorbing decomposition material, a semi-permeable membrane material or a waterproof material. The invention puts the sampling capsule into a simulated gastrointestinal tract device, and the experimental results show that the flora varieties and structures of the samples sampled by the drug storage sample group, the frozen storage sample group and the control group are basically consistent with those of the samples directly sampled and refrigerated by a refrigerator, thereby proving the storage effect of the sampling capsule.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a structure and a method for preserving sampling materials in gastrointestinal tracts.
Background
The intestinal tract is the main organ for digestion and absorption of human body, and the intestinal tract of human body is parasitized with a large number of microorganisms including bacteria, mycovirus \ bacteriophage \ protozoa and the like, wherein 99% of the bacteria account for about 10 trillion and are called as intestinal flora. The number of genes of the intestinal flora is about 100 times that of the human body, and therefore is also called "second genome" of the human body. The intestinal flora can not only influence the digestion and absorption of human body to protein, lipid, carbohydrate and other nutrients, but also synthesize various vitamins necessary for the growth and development of human body, regulate the immunity of the organism and resist the risk of infection and autoimmune diseases. In recent years, with the development of second-generation sequencing technologies and the increasing sophistication of bioinformatic analysis methods, it has been demonstrated that intestinal microorganisms are closely related to the occurrence and development of various diseases.
The development of second generation sequencing technology and bioinformatic analysis method are represented by 16SrDNA technology, and 16SrDNA is present in all bacterial chromosomal genes and can encode a DNA sequence corresponding to 16SrRNA, and the sequence comprises 9 hypervariable regions and 10 conserved regions. The conserved regions reflect the genetic relationship between biological species, while the hypervariable regions reflect the species-to-species differences. The sequence of the hypervariable region is amplified by designing a primer, then the second generation sequencing is carried out, and the strain is further distinguished by applying a bioinformatics analysis method. The method can be applied to the identification and analysis of intestinal microorganisms.
Since the human intestinal tract provides an optimum growth environment for microorganisms, once the microorganisms are sampled, the microorganisms are separated from the human intestinal tract environment, the growth rate and the death rate of most microorganisms are greatly changed, and the microbial population condition of the gastrointestinal tract can not be accurately reflected. The preservation method which is generally used at present can be used for low-temperature freezing preservation and medicament treatment, but the existing methods can be used in vitro and have great difficulty for the treatment in the digestive tract of human or other animals. Particularly, the capsules after sampling in the intestinal tract are a real and urgent challenge to keep the activity or the flora quantity of the contents in the digestive tract unchanged.
In view of the relative stability of DNA itself, if the agent directly destroys the cells and destroys or inhibits the activity of DNase, the DNA of the content will be fixed and preserved, and 2 formulations are proposed for the DNA preservation solution of feces samples at normal temperature and the preparation method and the application thereof in the patent of Yangzhi et al (application No. 201910101292.0) at Zhejiang university, and the effect of preserving DNA at normal temperature is good. A DNA preservative solution is prepared by dissolving 20-50mM Tris-HCl buffer solution, 10-50mM disodium ethylenediaminetetraacetate (EDTA-2Na), 100-150mM NaCl and 0.1% -0.5% Sodium Dodecyl Sulfate (SDS) in sterile water, and adjusting the pH to 8.0. Or a non-patent protection existing formula, 25mM Tris-buffer, 140mM/L sodium chloride (NaCl), 45mM/L disodium ethylene diamine tetraacetate (EDTA-2Na), 0.02 percent of Kathon preservative and 0.5 percent of Sodium Dodecyl Sulfate (SDS) by mass fraction. Patent of Donggao Times Gene science and technology Co., Ltd (201711370058.5): ethanol is used as a fixing agent, sodium citrate is used as a fixing auxiliary agent, EDTA-disodium is used as an anticoagulant, Tris-HCl is used as a buffer solution, NaCl is used as an ionic strength maintaining agent, and sodium dodecyl sulfate is matched.
The formula and the using method of the three excrement storage liquids are particularly suitable for storing the sample taken out from the gastrointestinal tract internal sampling capsule besides being very suitable for storing the in-vitro excrement sample at normal temperature, and the sample is naturally discharged along with the gastrointestinal tract peristalsis, so that the time is generally 6-48H or even longer; if a medicament for promoting gastrointestinal peristalsis is used, the real microbial survival state of the gastrointestinal tract is difficult to simulate, and under the condition that the gastrointestinal tract is stored for a long time, the state of the microbes during sampling is difficult to maintain. Therefore, the reagent can keep the DNA in the sample stable, and then metagenomic detection is carried out on the DNA, so that analysis becomes important. The above 2 patents give a good method, but gastrointestinal sampling, unlike in vitro sampling, is considered safe and practical, avoiding the use of potentially harmful substances to the human body, such as: guanidine thiocyanate, dimethyl sulfoxide (DMSO), beta-mercaptoethanol, and the like. We propose solutions to this patent based on the formulation of several patents.
In addition, the material sampled from the gastrointestinal tract not only includes gastrointestinal microorganisms, but also various local biological active substances such as protein, carbohydrate, lipid and the like, including hormone and the like in the body, and the substances also need to be stored after being obtained and can be analyzed. In the united states, pluronittes corporation (CN109843182A), numerous content retention agents are mentioned including the use of gums, surfactants, antibiotics, proteases, and the like. However, it does not take into account which preservative formulation is most effective, nor does it take into account which organization and which formulation would be more effective in a single-use sampling capsule without electronic control equipment.
Aiming at DNA, protein, carbohydrate, lipid and other bioactive small molecular compounds in a sample and even some active cells after the sampling of a digestive tract sampling capsule, a structure and a method capable of preserving the sampled digestive tract contents at normal temperature (human or animal body temperature) are provided.
Disclosure of Invention
The present invention is directed to a structure and method for preserving a sampling material in the gastrointestinal tract, which solves the above-mentioned problems of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme: sample material is in the structure of preserving of intestines and stomach, including the sample capsule, the inside parcel column structure that is provided with of sample capsule, and the parcel has the medicament in the parcel column structure.
Preferably, the sampling capsule interior is the interior side wall of the capsule, pushed to a central location with a support structure, or the like.
Preferably, the wrapping structure is composed of a water-absorbing decomposition material, a semi-permeable membrane material or a water-impermeable material, and the medicament is one or a mixture of more of Tris (hydroxymethyl) aminomethane (Tris-HCl), disodium ethylene diamine tetraacetate (EDTA-2Na), sodium chloride (NaCl), Sodium Dodecyl Sulfate (SDS), a kathon preservative, ethanol or sodium citrate.
Preferably, the wrapping structure is made of a material easy to dissipate heat, and ammonium nitrate or other substances capable of absorbing water or reducing temperature by reaction are wrapped inside the wrapping structure.
A method for preserving a sampled material in the gastrointestinal tract, the method comprising the steps of:
A. according to several different formulas, the solid components in the formula are fully mixed, and are sealed into one or more small bags by water-absorbing material, and then are dispersed in the capsule shell and/or on the capsule shell wall in the modes of adsorption, wrapping and the like;
B. the capsule shell is made of a material with certain softness, and the contents can be fully stirred under the extrusion of gastrointestinal movement;
C. the sampled material can dissolve various adsorption and coating substances due to moisture, and the dissolved substances act on the sampled material to realize the fixation and preservation of DNA of the sampled material.
Preferably, the formulation in step A is Tris-buffer, 10-50mM disodium ethylene diamine tetraacetate (EDTA-2Na), 100-150mM sodium chloride (NaCl) and 0.1% -0.5% Sodium Dodecyl Sulfate (SDS) by mass or by using ethanol, sodium citrate, EDTA-disodium, Tris-HCl, NaCl and sodium dodecyl sulfate or conventional experimental formulation, and the water-absorbing material is mainly polysaccharides and proteins, including but not limited to celluloses, gelatin, agarose, arabic gum, xanthan gum, resins and the like, and the small bag can be strung by linear material fixed on the capsule wall with certain interval, and the capsule wall is made of heat-insulating material, including polyurethane foam, polystyrene board, phenolic foam sponge, Polyethylene, polystyrene foam, glass wool or the like, and the wrapping part is made of a water-absorbing material which controls the reaction or participates in the reaction.
Preferably, the storage mode in step C is refrigeration storage, and the refrigeration storage is chemical slow endothermic reaction, mainly for the storage structures of other bioactive substances, proteins, tumor cells and the like, by using endothermic chemical reaction or physical change, preferably one reactant is water, or salts in gastrointestinal tract and food, meanwhile, the material for endothermic reaction is placed in the package in the middle of the capsule shell or in a plurality of packages, and the material for endothermic reaction reacts or dissolves the water absorbing wall layer under extrusion, and then reacts.
Compared with the prior art, the invention has the following beneficial effects:
through experiments, samples preserved by using DNA preservation medicines or samples preserved by using a chemical reaction refrigeration method are obtained, after the samples are preserved for 8 hours, the samples are directly sampled and then are refrigerated for 8 hours in a refrigerator, and the flora type and the structure are basically consistent, so that the preservation effect of the preservation method in vivo is stable and reliable, and the preservation level of the refrigerator can be achieved. The method has extremely important significance for the detection and examination of the sampled material in the later period of sampling, and the material still can not be timely checked in the intestinal tract, and the stability of the flora species and the structure is preserved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The storage structure of the sampling material in the gastrointestinal tract comprises a sampling capsule, a wrapping structure is arranged in the sampling capsule, a medicament is wrapped in the wrapping structure, the inner side wall of the sampling capsule refers to the inner side wall of the capsule, the sampling capsule is pushed to a central position by a support structure and the like, the wrapping structure is composed of a water absorption decomposition material, a semi-permeable membrane material or a waterproof material, the medicament is one or a mixture of more of Tris (Tris-HCl), disodium ethylene diamine tetraacetate (EDTA-2Na), sodium chloride (NaCl), Sodium Dodecyl Sulfate (SDS), a kathon preservative, ethanol or sodium citrate, the wrapping structure is also made of an easy heat dissipation material, ammonium nitrate is wrapped in the wrapping structure, or other substances which absorb water or react to reduce the temperature.
A method for preserving a sampled material in the gastrointestinal tract, the method comprising the steps of:
A. according to several different formulas, the solid components in the formula are fully mixed, and are sealed into one or more small bags by water-absorbing material, and then are dispersed in the capsule shell and/or on the capsule shell wall in the modes of adsorption, wrapping and the like;
B. the capsule shell is made of a material with certain softness, and the contents can be fully stirred under the extrusion of gastrointestinal movement;
C. the sampled material can dissolve various adsorption and coating substances due to moisture, and the dissolved substances act on the sampled material to realize the fixation and preservation of DNA of the sampled material.
The formula in the step A is Tris (hydroxymethyl) aminomethane (Tris-HCl) buffer solution, 10-50mM disodium ethylene diamine tetraacetate (EDTA-2Na), 100-150mM sodium chloride (NaCl) and 0.1% -0.5% Sodium Dodecyl Sulfate (SDS) by mass fraction or by using ethanol, sodium citrate, EDTA-disodium, Tris-HCl, NaCl and sodium dodecyl sulfate or a conventional experimental formula, the water-absorbing material is mainly polysaccharides and proteins and comprises cellulose, gelatin, agarose, Arabic gum, xanthan gum, resins and the like, meanwhile, the small bag can be stringed by linear materials which are fixed on the wall with a certain interval, and the capsule shell wall is made of heat-insulating materials and comprises foamed polyurethane, polystyrene foam plastic, a polyphenyl board, phenolic foam rubber-plastic sponge, polyethylene foam, Polystyrene foam or glass wool, and the wrapping part is made of water-absorbing materials which control the reaction or participate in the reaction.
The preservation mode in the step C is refrigeration preservation, the refrigeration preservation is chemical slow endothermic reaction, the heat absorption chemical reaction or physical change is mainly used for preserving structures of other biological active substances, proteins, tumor cells and the like, preferably, one reactant is water or salt in gastrointestinal tracts and food, and meanwhile, the material for the heat absorption reaction is placed in a package in the middle of the capsule shell or in a plurality of packages, and the material can react or dissolve a water absorption wall layer under extrusion and then react.
Experimental verification and concrete steps: the sampling capsules were placed in a simulated gastrointestinal device, which was selected from the TIM (tno Intestinal model) (TIM is a multi-unit computer-controlled in vitro digestion simulation system developed by Minekus and Havenaar, university of wakening, netherlands) for drug dynamic testing. The system is divided into two parts, a stomach, small intestine simulator (TIM-1) and a large intestine simulator (TIM-2). The test uses a large intestine simulator (TIM-2) with the most complex intestinal flora as a detection device.
Sampling into 1, 2 and 3 groups corresponding to a drug storage sample group, a frozen storage sample group and a control group respectively, wherein each group comprises five capsules; the control group was prepared by sampling both capsules simultaneously, sampling from the simulator and placing the samples in sterile Ep tubes, and storing in a freezer at-80 ℃ until use. The drug preservation sample group and the freeze preservation sample group are respectively taken out after staying in the simulated large intestine for 8 hours. Finally, we performed total DNA extraction using DNA extraction Kit (QIAamp PowerFecal DNA Kit, Qiagen, Germany) for three groups of samples. The 16SrRNA gene hypervariable region V4 was PCR amplified using universal primers (338F-ACTCCTACGGGAGGCAGGCAGCA, 806R-GGACTAC-HVGGGTWTCTAAT) for the 16SrRNA gene of bacteria, to which a linker sequence was added. Constructing a DNA library, sending a product to gene sequencing, and inputting a sequencing result into a Genbank for comparison.
The results of the average contrast indexes of the intestinal microbial distribution and the flora abundance of the large intestine are shown in the following table:
the results of the relative abundance of the drug storage sample group and the freeze storage sample group with the control group are both (P is less than 0.05) through single-factor variance analysis, which shows that the difference between the species structure of the intestinal flora of the drug storage sample, the freeze storage sample and the control group is not significant. The flora types and structures of the samples sampled from the drug preservation sample group, the frozen preservation sample group and the control group are basically consistent with those of the samples directly sampled and refrigerated in a refrigerator.
The test results show that the method for preserving the samples by the drugs and the method for preserving the samples by freezing have good preservation effect.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The structure for preserving the sampling material in the gastrointestinal tract comprises a sampling capsule, and is characterized in that: the inside parcel column structure that is provided with of sample capsule, and the parcel has the medicament in the parcel column structure.
2. A containment structure for a sampling material in the gastrointestinal tract according to claim 1, wherein: the interior of the sampling capsule refers to the inner side wall of the capsule, pushed to a central position by a support structure and the like.
3. A containment structure for a sampling material in the gastrointestinal tract according to claim 1, wherein: the wrapping structure is composed of a water absorption decomposition material, a semi-permeable membrane material or an impermeable material, and the medicament is one or a mixture of more of Tris (hydroxymethyl) aminomethane (Tris-HCl), disodium ethylene diamine tetraacetate (EDTA-2Na), sodium chloride (NaCl), Sodium Dodecyl Sulfate (SDS), a kathon preservative, ethanol or sodium citrate.
4. A containment structure for a sampling material in the gastrointestinal tract according to claim 1, wherein: the wrapped structure is made of a material easy to dissipate heat, and ammonium nitrate or other substances capable of absorbing water or reducing temperature through reaction are wrapped inside the wrapped structure.
5. A method for preserving a sampling material in the gastrointestinal tract, comprising: the method comprises the following steps:
A. according to several different formulas, the solid components in the formula are fully mixed, and are sealed into one or more small bags by water-absorbing material, and then are dispersed in the capsule shell and/or on the capsule shell wall in the modes of adsorption, wrapping and the like;
B. the capsule shell is made of a material with certain softness, and the contents can be fully stirred under the extrusion of gastrointestinal movement;
C. the sampled material can dissolve various adsorption and coating substances due to moisture, and the dissolved substances act on the sampled material to realize the fixation and preservation of DNA of the sampled material.
6. A method of preserving a sampling material in the gastrointestinal tract according to claim 5, wherein: the formula in the step A is Tris (hydroxymethyl) aminomethane (Tris-HCl) buffer solution, 10-50mM disodium ethylene diamine tetraacetate (EDTA-2Na), 100-150mM sodium chloride (NaCl) and 0.1% -0.5% Sodium Dodecyl Sulfate (SDS) by mass fraction or a conventional experiment DNA storage formula, the water-absorbing material mainly comprises polysaccharides and proteins and comprises, but is not limited to, celluloses, gelatin, agarose, Arabic gum, xanthan gum, resins and the like, meanwhile, the small bag can be stringed by linear materials, the linear materials are fixed on the capsule wall with a certain interval, and the capsule shell wall is made of heat-insulating materials comprising foamed polyurethane, polystyrene rubber plastic foam, polystyrene board, phenolic foam sponge, Polyethylene, polystyrene foam, glass wool or the like, and the wrapping part is made of a water-absorbing material which controls the reaction or participates in the reaction.
7. A method of preserving a sampling material in the gastrointestinal tract according to claim 5, wherein: the preservation mode in the step C is refrigeration preservation, the refrigeration preservation is chemical slow endothermic reaction, the heat absorption chemical reaction or physical change is mainly used for preserving structures of other biological active substances, proteins, tumor cells and the like, preferably, one reactant is water or salt in gastrointestinal tracts and food, and meanwhile, the material for the heat absorption reaction is placed in a package in the middle of a capsule shell or a plurality of packages, and the material can react or dissolve a water absorption wall layer under extrusion and then react.
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| CN202010518866.7A CN111631758A (en) | 2020-06-09 | 2020-06-09 | Structure and method for preserving sampling material in gastrointestinal tract |
| PCT/CN2021/080033 WO2021180133A1 (en) | 2020-03-11 | 2021-03-10 | Biological response control state capsule, sampling and cryopreservation device and method for intestinal contents, and preservation structure and method for sampled material in gastrointestinal tract |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021180133A1 (en) * | 2020-03-11 | 2021-09-16 | 郑州味千生物技术有限公司 | Biological response control state capsule, sampling and cryopreservation device and method for intestinal contents, and preservation structure and method for sampled material in gastrointestinal tract |
| CN115486438A (en) * | 2021-09-18 | 2022-12-20 | 上海市第一人民医院 | Composition for preventing biological sample genetic information substance from degrading and use method thereof |
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| CN115486438A (en) * | 2021-09-18 | 2022-12-20 | 上海市第一人民医院 | Composition for preventing biological sample genetic information substance from degrading and use method thereof |
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