CN105385599A - Coprophilous fungus preservation solution and method for preserving coprophilous fungi - Google Patents
Coprophilous fungus preservation solution and method for preserving coprophilous fungi Download PDFInfo
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- CN105385599A CN105385599A CN201511009229.2A CN201511009229A CN105385599A CN 105385599 A CN105385599 A CN 105385599A CN 201511009229 A CN201511009229 A CN 201511009229A CN 105385599 A CN105385599 A CN 105385599A
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- caprophyl
- coprophilous
- preservation solution
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The invention discloses a coprophilous fungus preservation solution and a method for preserving coprophilous fungi by adopting the preservation solution. The coprophilous fungus preservation solution is prepared from, by weight, 0.9% of sodium chloride, 0.5%-2.0% of vitamin C, 20%-50% of glycerinum and the balance sterile purified water, and the pH value of the preservation solution ranges from 7.0 to 7.4. According to the coprophilous fungi preservation solution and the method for preserving the coprophilous fungi, the coprophilous fungi can be preserved for up to three months under the simple preservation condition, and it is guaranteed that the microorganism composition is still similar to that of the preservation solution which is initially separated and extracted; from the angle of the formula composition, the advantages of being low in cost, easy to prepare and the like are achieved, and therefore development of coproghilous fungus treatment is facilitated; from the results of specific implementation cases, the coprophilous fungi can be kept basically invariable for up to three months under the conventional preservation condition, and therefore a main guarantee is supplied to coprophilous fungus transplantation standardization.
Description
Technical field
The invention belongs to medical detection field, particularly a kind of caprophyl conserving liquid and adopt this conserving liquid to preserve the method for caprophyl.
Background technology
The structural imbalance of intestinal microflora is the important factor of bringing out various chronic disease, recent domestic research finds by " caprophyl transplanting " (fecalmicrobiotatransplantation, FMT), namely with the intestinal microflora from Healthy People, recover the balance of Bacterial community in chronic disease human body, rebuild the intestinal microecology with normal configuration, the rehabilitation of flora function can be promoted, thus reach the result for the treatment of of enteron aisle and the outer disease of enteron aisle.So far, all there is employing caprophyl implantation method successfully to cure or alleviate Clestridium difficile both at home and abroad and infect the diseases related clinical report of the gi tract such as (CDI), inflammatory bowel.2013, FMT was indexed in the standard care guide of recurrent CDI by U.S. FDA.Although the treatment clinical effectiveness of " caprophyl transplanting " is remarkable, it is to be solved that its promotion and application in medical field still have problems to have, the standardized construcion of such as FMT.Wherein, the preservation of caprophyl liquid is the steps necessary in FMT.After flora leaves enteron aisle, residing for it, namely environment changes, and its activity and composition may be affected.In order to ensure the result for the treatment of of clinical transplantation, a kind of effective store method and means need be adopted to maintain activity and the composition of intestinal microflora.
Refrigeration is generally adopted to be used for directly preserving human excrement and urine at present clinically, namely when frozen in-20 DEG C or lower temperature immediately after feces collection, because bacterium metabolic rate under low-temperature condition reduces, therefore can maintain the formation of bacterium in ight soil within for some time, thus reach the object stablized its microorganism and form.When needs carry out FMT operation, frozen product are obtained caprophyl by separation method again, this not only adds difficulty and the workload of caprophyl separation, also need larger storage space.In addition, caprophyl, as " medicine " in a kind of methods for the treatment of, can not, directly in clinical middle application, need first frozen ight soil to extract rear use more on the contrary, such therapeutic process can increase the dependency of operation and equipment undoubtedly, can hinder the development of caprophyl transplantation treatment method.Meanwhile, a lot of medical institutions of China do not have the ability be separated caprophyl.Therefore, although a kind of ight soil conserving liquid (CN104480012A) of patent documentation reports utilize the material such as edta salt and Trisodium Citrate, fecal sample can be preserved one week under sealing normal temperature using the conserving liquid that water and ethanol are prepared as solvent, but this method is unsuitable for preserving the caprophyl in caprophyl transplanting.
The clinical phosphate buffered saline buffer (PBS) that often adopts in caprophyl migration process mixes with donor caprophyl at present, due to the characteristic of fluid of mixed solution, adopt clinically and to spray under scope or in bowel lavage process, short for duration of contact with intestinal walls, affect caprophyl field planting effect.Caprophyl liquid is preserved as needed, then need additionally to add glycerine, too much step easily increases the chance of exogenous infection.
Summary of the invention
The object of the invention is the defect for prior art, providing can available protecting intestinal microflora activity and abundance.Thus ensure the conserving liquid of caprophyl transplantation effect, and above-mentioned conserving liquid is adopted to preserve the method for caprophyl.
To achieve these goals, the present invention is by the following technical solutions: a kind of caprophyl conserving liquid, comprise following component by weight percentage: the sodium-chlor of 0.9%, the vitamins C of 0.5% ~ 2.0%, the glycerine of 20% ~ 50%, all the other are axenic purification water, and the pH value of described conserving liquid is 7.0 ~ 7.4.
Conserving liquid preserves a method for caprophyl, comprises the following steps:
(1) will collect the caprophyl obtained after ight soil is separated, the caprophyl 100mL conserving liquid that every 50g ight soil obtains is resuspended, then-80 DEG C of frozen preservations;
(2), when using, first frozen for caprophyl thing is thawed in ice bath, be then diluted in the stroke-physiological saline solution of 250mL, transplant for caprophyl.
The present invention relates to a kind of formula of caprophyl conserving liquid, according to the described preparation caprophyl conserving liquid of formula.By the fresh excreta gathered, through stirring, the step such as centrifugal, resuspended, filtration directly adopts caprophyl conserving liquid to preserve, or directly adopt caprophyl separation system to be separated and obtain caprophyl, then direct caprophyl is placed in conserving liquid, less than-80 DEG C frozen, can reach the effect that stable wherein microorganism is formed, the shelf time reaches 3 months.Use this formula conserving liquid, under corresponding preservation condition, the result for the treatment of that the quantity of microorganism in caprophyl and abundance do not affect follow-up caprophyl and transplant can be ensured, transplant stdn for caprophyl and provide possibility.
Caprophyl conserving liquid in the present invention protects activity and the abundance of intestinal microflora well, thus ensure that caprophyl transplantation effect, also simplify caprophyl graft procedure simultaneously, appears on the market clinically at present without analogous products described in the invention or method.
The present invention's formula under simple preservation condition, can reach the preservation of 3 months to caprophyl, ensure that the microorganism that its microorganism is formed when still extracting to initial separation forms similar.Form angle from formula, it is low that the present invention has cost, prepares the features such as simple, and this will contribute to the development of caprophyl treatment; From the result of concrete case study on implementation, formula of the present invention, can realize under conventional storage conditions, and it is substantially constant that maintenance caprophyl reaches 3 months, and this transplants stdn for caprophyl and provides main guarantee.
Accompanying drawing explanation
Fig. 1 is the DNA cloning results contrast after the fecal sample of 4 volunteers in embodiment does 3 kinds of different treatment.
Fig. 2 is that the bacterium richness figure before and after the faecal samples caprophyl liquid of 3 volunteers in embodiment is preserved changes.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
A kind of caprophyl conserving liquid, comprise following component by weight percentage: the sodium-chlor of 0.9%, the vitamins C of 0.5%, the glycerine of 50%, all the other are axenic purification water, and the pH value of conserving liquid is 7.4.
To collect the caprophyl obtained after ight soil is separated, the caprophyl 100mL conserving liquid that every 50g ight soil obtains is resuspended, then-80 DEG C of frozen preservations.
During use, first frozen for caprophyl thing is thawed in ice bath, be then diluted in the stroke-physiological saline solution of 250mL, transplant for caprophyl.
Acquire the fecal sample of 4 volunteers, and utilize caprophyl separation system to be separated the caprophyl obtaining correspondence.The caprophyl of each volunteer is divided into 3 parts, does 3 kinds of different treatment respectively:
(1) process immediately: portion is separated the caprophyl obtained and is dispersed in stroke-physiological saline solution, every 50g ight soil is separated the caprophyl obtained and is scattered in 100mL physiological saline.Get 300 μ L, carry out DNA extraction immediately, the process such as rrna 16SRNA gene the 4th variable region amplification.
(2) caprophyl conserving liquid-80 DEG C is preserved 3 months: portion is separated the caprophyl obtained and is dispersed in caprophyl conserving liquid, every 50g ight soil is separated the caprophyl obtained and is scattered in 100mL conserving liquid, and-80 DEG C frozen 3 months.The process such as then ice bath gets 300 μ L after thawing, and carries out DNA extraction, rrna 16SRNA gene the 4th variable region amplification.
(3) do not add conserving liquid-80 DEG C to preserve 3 months: portion is separated the caprophyl obtained and is dispersed in stroke-physiological saline solution, every 50g ight soil is separated the caprophyl obtained and is scattered in 100mL physiological saline, and-80 DEG C frozen 3 months.The process such as then ice bath gets 300 μ L after thawing, and carries out DNA extraction, rrna 16SRNA gene the 4th variable region amplification.
The fecal sample of 4 volunteers carries out DNA extraction, and the step of rrna 16SRNA gene the 4th variable region amplification process is as follows:
(1) DNA extraction:
300 μ L caprophyl solution are placed in the 1.5mL centrifuge tube containing 0.1mm silicon ball; Then, add the PBS containing 50 μ L N,O-Diacetylmuramidases (10mg/ml), 6 μ L mutanolysin (25000U/mL) and 3 μ L lysostaphins, fully the rear 37 DEG C of incubations of mixing 30 minutes; Then, add 10 μ L Proteinase Ks (20mg/ml), 100 μ L concentration be 10% SDS (Sodium dodecylbenzene sulfonate) and 20 μ L ribonuclease As (20mg/ml), vortex concussion fully rear 55 DEG C of incubations of mixing 45 minutes; Subsequently, mill pearl homogenizer lysing cell is adopted; Last by specification uses the faeces DNA of ZYMOResearch company to extract test kit extraction purification DNA.
(2) rrna 16SRNA gene the 4th variable region amplification:
Reagent (Takara company):
Primer: upstream: 27F-59-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGG
CTCAG-39 and downstream 338R-59-GCCTCCCTCGCGCCATCAGNNNNNNNNCATGC
TGCCTCCCGTAGGAG-T-39; .TaqDNA polysaccharase; .MgCl2; 2.5mmol/LdNTPMixture; ddH
2o; 10xExTaqBuffer (not containing magnesium ion); DNA profiling.
Operation steps:
Each PCR reaction system is 25 μ L, comprising: 15.75 μ LddH
2o, 2.5 μ L10xExTaqBuffer, 2 μ LdNTPMixture, 1.5 μ LMgCl
2, 0.25 μ LTaqDNA polysaccharase, upstream primer and each 0.5 μ L of downstream primer, 2 μ L template DNAs.
Divide after installing the PCR reaction system of each 25 μ L in each EP pipe, according to following PCR program amplified reaction:
(step1)：94℃，2min；(step2)：92℃，30s；(step3)：52℃，30s；(step4)：72℃，45s；(step5)：gotostep2，29cycles；(step6)：72℃，5min；(step7)：4℃，forever；(step8)：end
Increase the fragment obtained, and is sent to order-checking company and carries out high-flux sequence, carries out subsequent bio information analysis, obtain flora result after obtaining sequence.
DNA cloning results contrast after the fecal sample of 4 volunteers does 3 kinds of different treatment is shown in Fig. 1.In figure: 1. volunteer 1 sample processes immediately; 2. volunteer 1 sample retention liquid-80 DEG C is preserved 3 months; 3. direct-80 DEG C of volunteer 1 sample is preserved 3 months; 4. volunteer 2 sample processes immediately; 5. volunteer 2 sample retention liquid-80 DEG C is preserved 3 months; 6. direct-80 DEG C of volunteer 2 sample is preserved 3 months; 7. volunteer 3 sample processes immediately; 8. volunteer 3 sample retention liquid-80 DEG C is preserved 3 months; 9. direct-80 DEG C of volunteer 3 sample is preserved 3 months; 10. volunteer 4 sample processes immediately; 11. volunteer 4 sample retention liquid-80 DEG C are preserved 3 months; Direct-80 DEG C of 12. volunteer 4 samples are preserved 3 months; M:Marker.
The sample of 3 volunteer's faecal samples caprophyl liquid preservations processes, and send order-checking company to carry out high-flux sequence, and analyzed by bioinformatics method, bacterium richness compares sees Fig. 2.Can find out in figure: fecal bacteria composition is similar, and often kind of flora Plantago fengdouensis is not obvious, illustrate that caprophyl conserving liquid can ensure that caprophyl keeps good activity and flora composition under-80 DEG C of conditions, for caprophyl transplanting work from now on provides safeguard.
As mentioned above, although represented with reference to specific preferred embodiment and described the present invention, it shall not be construed as the restriction to the present invention self.Under the spirit and scope of the present invention prerequisite not departing from claims definition, various change can be made in the form and details to it.
Claims (2)
1. a caprophyl conserving liquid, is characterized in that comprising following component by weight percentage: the sodium-chlor of 0.9%, the vitamins C of 0.5% ~ 2.0%, the glycerine of 20% ~ 50%, and all the other are axenic purification water, and the pH value of described conserving liquid is 7.0 ~ 7.4.
2. caprophyl conserving liquid according to claim 1 preserves a method for caprophyl, it is characterized in that comprising the following steps:
(1) will collect the caprophyl obtained after ight soil is separated, the caprophyl 100mL conserving liquid that every 50g ight soil obtains is resuspended, then-80 DEG C of frozen preservations;
(2), when using, first frozen for caprophyl thing is thawed in ice bath, be then diluted in the stroke-physiological saline solution of 250mL, transplant for caprophyl.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695374A (en) * | 2016-04-21 | 2016-06-22 | 湖北中化东方肥料有限公司 | Bacillus diluent, bacillus diluent liquid preparation and liquid preparation storage method |
| CN106497790A (en) * | 2016-10-09 | 2017-03-15 | 江西省水产科学研究所 | A kind of method that tissue preserration liquid and its preparation separate aquatic biological bacterium with preservation field sampling |
| CN106665436A (en) * | 2017-01-26 | 2017-05-17 | 中国水产科学研究院黑龙江水产研究所 | Method of transplanting healthy fish intestinal feces and treating ill fishes |
| CN107034141A (en) * | 2017-06-08 | 2017-08-11 | 深圳微健康基因科技有限公司 | Gut flora fixer and preparation method thereof in human faecal mass |
| CN108192827A (en) * | 2017-12-29 | 2018-06-22 | 深圳谱元科技有限公司 | A kind of intestinal flora sample room-temperature extender and its preparation method and application |
| CN108308172A (en) * | 2017-12-26 | 2018-07-24 | 博朗（厦门）生物科技有限公司 | A kind of Sample storage liquid for parasitic ovum detection |
| CN108949572A (en) * | 2018-08-21 | 2018-12-07 | 卓源健康科技有限公司 | A kind of caprophyl freezen protective liquid and its store method |
| CN109294952A (en) * | 2018-10-23 | 2019-02-01 | 上海市第十人民医院 | A kind of preparation method of intestines bacteria composition |
| CN109706235A (en) * | 2019-01-29 | 2019-05-03 | 广州康昕瑞基因健康科技有限公司 | A kind of the detection and analysis method and its system of intestinal microflora |
| CN110692625A (en) * | 2019-10-24 | 2020-01-17 | 黄铁胜 | Environment-friendly fungus preservation solution and preparation method thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695374A (en) * | 2016-04-21 | 2016-06-22 | 湖北中化东方肥料有限公司 | Bacillus diluent, bacillus diluent liquid preparation and liquid preparation storage method |
| CN106497790A (en) * | 2016-10-09 | 2017-03-15 | 江西省水产科学研究所 | A kind of method that tissue preserration liquid and its preparation separate aquatic biological bacterium with preservation field sampling |
| CN106665436A (en) * | 2017-01-26 | 2017-05-17 | 中国水产科学研究院黑龙江水产研究所 | Method of transplanting healthy fish intestinal feces and treating ill fishes |
| CN107034141A (en) * | 2017-06-08 | 2017-08-11 | 深圳微健康基因科技有限公司 | Gut flora fixer and preparation method thereof in human faecal mass |
| CN108308172A (en) * | 2017-12-26 | 2018-07-24 | 博朗（厦门）生物科技有限公司 | A kind of Sample storage liquid for parasitic ovum detection |
| CN108192827A (en) * | 2017-12-29 | 2018-06-22 | 深圳谱元科技有限公司 | A kind of intestinal flora sample room-temperature extender and its preparation method and application |
| CN108949572A (en) * | 2018-08-21 | 2018-12-07 | 卓源健康科技有限公司 | A kind of caprophyl freezen protective liquid and its store method |
| CN109294952A (en) * | 2018-10-23 | 2019-02-01 | 上海市第十人民医院 | A kind of preparation method of intestines bacteria composition |
| CN109706235A (en) * | 2019-01-29 | 2019-05-03 | 广州康昕瑞基因健康科技有限公司 | A kind of the detection and analysis method and its system of intestinal microflora |
| CN110692625A (en) * | 2019-10-24 | 2020-01-17 | 黄铁胜 | Environment-friendly fungus preservation solution and preparation method thereof |
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