CN106596211A - Stool sample preservation liquid, preparation method and application thereof - Google Patents
Stool sample preservation liquid, preparation method and application thereof Download PDFInfo
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- 239000003795 chemical substances by application Substances 0.000 claims abstract description 24
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- 230000002550 fecal effect Effects 0.000 claims description 137
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- 238000000034 method Methods 0.000 claims description 32
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a stool sample preservation liquid, a preparation method and application thereof. Specifically, the stool sample preservation liquid contains: 45 vol%-60 vol% of a fixing agent; 0.1 mass%-15 mass% of a fixing agent adjuvant; 0.01 mass%-1.5 mass% of an anticoagulant; 10 vol%-15 vol% of a buffer solution; 0.01 mass%-1 mass% of an ionic strength maintenance agent; 0.01 vol%-0.5 vol% of a nonionic detergent; and the balance water. The stool sample preservation liquid provided by the invention has the characteristics of easy preparation and low cost, can preserve stool samples at room temperature, also can effectively inhibit DNA degradation and cell damage of stool samples, maintain cell shape, and can preserve samples for a long time, and the preserved samples have high qualification rate during reutilization.
Description
Technical field
The present invention relates to fecal sample preserves liquid and its preparation method and application.
Background technology
The non-damage sample such as excrement, hair, saliva, urine is widely used in medical jurisprudence, wild animal research, clinical research,
Wherein fecal sample is the effective means of clinical examination and generally investigates the most frequently used, with simple and easy to do, with low cost, noninvasive, suitable
The characteristics of closing large-scale crowd examination.With the development of molecular biology, the generation development of tumour is attributed to associated gene mutation,
And the cast-off cells in excrement include the mutator close with relationship between colorectal cancer, genetic test in excrement is expected to become screening and examines
The new method of the early screening of disconnected gastroenteric tumor.It is reported that, u s company develop faeces DNA (Stool-basedDNA,
SDNA) screening test, for detecting the DNA marks related to colon cancer.10000 clinical testing datas are displayed in knot
It is 92% from the point of view of in the sensitivity of intestinal cancer detection, the sensitivity for detecting precancerous polyp is 42%, the inspection of the polyp big more than 2 centimetres
It is 66% to survey sensitivity, and the specificity of all clinical datas is 87%.
Additionally, there are a large amount of microbe colonies in excrement, gut flora composition is the result with the long-term common evolutionary of host, and
Health and disease such as obesity, diabetes, IBD with host is closely related, the total genome (enteron aisle of enteron aisle in excrement
Metagenomics) research become current hot research field.
But, excrement composition is sufficiently complex, containing substantial amounts of digestion is remaining and bacteriogenic various nucleolysis enzyme such as protease,
DNA enzymatic (DNases) and RNase (RNases), various PCR inhibitors (such as cholate, bilirubin, humus and pigment),
So that the cell and nucleic acid in fecal sample is highly prone to destroy and degrades.And current fecal sample store method, when can preserve
Between it is short, sample is low using qualification rate once again, and preservation condition is harsh, high cost.
Thus, current fecal sample liquid and store method still has much room for improvement.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.For this purpose, it is an object of the present invention to carrying
Go out a kind of holding time length, sample and preserve means using qualification rate height, the convenient fecal sample of preservation once again.
It should be noted that the present invention is completed based on the following discovery of inventor：
So far, domestic and international researcher has developed the method that various excrement are preserved, and principle includes：1) low temperature reduces various digestive enzymes
Activity, K cryogenic treatment is freezing, is typically stored at -20 DEG C；2) dehydrating the various biochemical reactions of suppression has silica gel to do
Dry, silica drying, microwave drying etc.；3) preserve the presence or absence of the conventional water-ethanol of liquid, 70% ethanol, dimethyl sulfoxide (DMSO),
Dimethyl sulfoxide saturated salt solution (DETs) etc. suppresses digestive enzyme and microbial activity.These store method compositions are simple, act on single,
As time went on preservation effect is not obvious.
Inventor attempted the common method including more than of various store methods in long-term fecal sample gene studies, and effect is not
Ideal, Cord blood causes strange land to collect sample to be restricted, and sample transport cost is greatly increased, and some are through optimization as (preserved
Liquid transport, preservation liquid add low temperature to preserve for a long time) short run effect is also possible that but sample placement finds that and still have substantially for 1-3 month
Degraded, amount of DNA deficiency brings very big puzzlement to subject study, also promotes inventor to carry out kinds of experiments exploration, to carrying
For a kind of normal temperature, simple, cheap and long-acting fecal sample store method.
According to an aspect of the present invention, the invention provides a kind of fecal sample preserves liquid.Embodiments in accordance with the present invention, should
Fecal sample preserves liquid and includes：The fixative of 45 volume %-60 volumes %；The fixative adjuvant of 0.1 mass %-15 mass %；
The anti-coagulants of 0.01 mass %-1.5 mass %；The buffer solution of 10 volume %-15 volumes %；The ion of 0.01 mass %-1 mass %
Intensity maintains agent；The nonionic detergent of 0.01 volume %-0.5 volume %；And balance of water, wherein, the fixative is
Selected from least one of methyl alcohol, ethanol, ethylene glycol and isopropanol, the fixed adjuvant be selected from acetic acid and acetate at least
One kind, the anti-coagulants is at least one of the water soluble salt selected from ethylenediamine tetra-acetic acid and ethylenediamine tetra-acetic acid, the buffer solution
It is selected from phosphate buffer, acetate buffer, ACES buffer solutions, ADA buffer solutions, BIS-TRIS buffer solutions, MES
At least one of buffer solution and PIPES buffer solutions, it is chlorate that the ionic strength maintains agent, and the nonionic detergent is choosing
From at least one of polysorbas20, NP-40 and TritonX-100.
It is surprisingly found by the inventors that, using the fecal sample of the present invention preserve liquid energy effectively suppress in fecal sample DNA degradation and
Cell damage, maintain cellular morphology, it is ensured that sample quality so that preserve fecal sample amplifying nucleic acid and cell can reach into
The needs of one step research.Additionally, embodiments in accordance with the present invention, the fecal sample of the present invention preserves liquid and prepares easy, low cost,
Fecal sample can be preserved under normal temperature, and it is long to the Sample preservation time, sample is high using qualification rate once again after preservation.
In addition, fecal sample according to the above embodiment of the present invention preserves liquid can also have following additional technical characteristic：
Embodiments in accordance with the present invention, the pH value of the buffer solution is 7.4-8.0.Thereby, it is possible to effectively maintain fecal sample pH
It is stable, maintain cellular morphology.
Some embodiments of the invention, it is 7.4-8.0 that the fecal sample preserves the pH value of liquid.Thus, for preserving excrement
Just during sample, the not degradable fractures of DNA in sample.
Some specific examples of the invention, it is at least one selected from sodium chloride and potassium chloride that the ionic strength maintains agent.
Some preferred embodiments of the invention, the anti-coagulants is disodium ethylene diamine tetraacetate.
Some preferred embodiments of the invention, the content of the fixative is 50 volume %-60 volumes %.Thus, it is fixed
Albumen, suppress DNA degradation effect it is good.
Some preferred embodiments of the invention, the content of the anti-coagulants is 0.1 mass %-1.5 mass %.Thus, resist
Solidifying effect is good, and low cost.
Some preferred embodiments of the invention, it is 0.5 mass %-1 mass % that the ionic strength maintains the content of agent.By
This, cellular morphology maintains effect good.
Some embodiments of the invention, the fecal sample of the present invention preserves liquid and includes：The methyl alcohol of 50 volume %-60 volumes %；
The acetic acid of 0.1 mass %-15 mass %；The disodium ethylene diamine tetraacetate of 0.1 mass %-1.5 mass %；10 volume %-15 volumes %
Phosphate buffer；The sodium chloride of 0.5 mass %-1 mass %；The Tween-20 of 0.01 volume %-0.5 volume %；And surplus
For water.Thus, fecal sample preserves the preservation effect protrusion of liquid.
A specific example of the invention, the fecal sample of 1000ml preserves liquid and includes：
500ml methyl alcohol；
3.5g acetic acid；
1g disodium ethylene diamine tetraacetates；
100ml phosphate buffers；
10g sodium chloride；
0.5ml Tween-20s；And
399.5ml water.
Some embodiments of the invention, the fecal sample of 1000ml preserves liquid and includes：
450ml ethanol；
1g acetic acid；
3g disodium ethylene diamine tetraacetates；
100ml acetate buffers；
1g potassium chloride；
0.1ml Tween-20s；And
449.9ml water.
Other embodiments of the invention, the fecal sample of 1000ml preserves liquid and includes：
550ml ethylene glycol；
2.5g acetic acid；
7g disodium ethylene diamine tetraacetates；
150ml PIPES buffer solutions；
5g sodium chloride；
2.5ml NP-40；And
297.5ml deionized water.
According to a further aspect in the invention, present invention also offers a kind of prepare the method that fecal sample preserves liquid.According to the present invention
Embodiment, the method comprises the following steps：
1) fecal sample as described above preserves the formula offer raw material of liquid；
2) add ionic strength to maintain agent, cushioning liquid and anti-coagulants in deionized water, and mix each composition dissolving, with
Just mixture is obtained；
3) carry out pH value to adjust to pH by the mixture is 7.4-8.0；
4) fixative, fixative adjuvant and nonionic detergent are added in the mixture adjusted through pH value, after mixing
Sterilizing, to obtain the fecal sample liquid is preserved.
Fecal sample thereby, it is possible to efficiently prepare the present invention preserves liquid, and low cost, and the fecal sample of acquisition is preserved
Liquid quality is good, can for a long time preserve effective for the normal temperature of fecal sample, and sample DNA degraded and cell damage are few after preservation,
Sample is high using qualification rate once again.
In accordance with a further aspect of the present invention, present invention also offers foregoing fecal sample preservation liquid is in fecal sample is preserved
Purposes.Embodiments in accordance with the present invention, using the fecal sample of the present invention liquid is preserved, and effectively fecal sample can be carried out
Normal temperature is preserved for a long time, and preserves low cost, and effect is good, and sample is high using qualification rate once again.
According to another aspect of the invention, present invention also offers a kind of method for preserving fecal sample.Enforcement of the invention
Example, the method includes：Foregoing fecal sample is preserved into liquid and fecal sample according to volume ratio 1-3：1 ratio is mixed,
Sealing at room temperature is kept in dark place.Inventor has found, using the method for the preservation fecal sample of the present invention, can be effectively realized
The normal temperature of fecal sample is preserved for a long time, and it is simple to operate, low cost is preserved, effect is good, and the holding time is long, and sample is sharp once again
It is high with qualification rate.
Some preferred embodiments of the invention, it is 1.5 that the fecal sample preserves liquid and the volume ratio of the fecal sample：1.
Thus, low cost is preserved, and preservation effect is projected.
Furthermore, it is necessary to explanation, fecal sample according to embodiments of the present invention preserves liquid and store method has following advantages
At least one：
1) fecal sample of the invention preservation liquid energy enough suppresses degradation of the bacteriogenic a large amount of digestive ferments to DNA in excrement,
Simultaneously cellular morphology can also be maintained, beneficial to cell separation morphological observation.
2) existing fecal sample preservation liquid composition is all more single, and what is had is only suitable for short time preservation, and such as absolute ethyl alcohol is preserved,
Excrement is dehydrated, and disperses heterogeneity, contacts incomplete with solution；What is had needs deepfreeze, all harsh to instrument and traffic condition to want
Ask, though sample breakdown has slowed down, the Quality Down of fecal sample, the holding time is not long.And utilize the excrement of the present invention
Just Sample preservation liquid normal temperature can preserve sample, its various composition beneficial to excrement preserve liquid in homogenize, enable excrement with it is molten
Liquid is fully contacted, and the holding time is long, and each composition does not result in impact to subsequent extracted nucleic acid and enzymatic reaction.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become apparent from the description below,
Or recognized by the practice of the present invention.
Description of the drawings
The present invention above-mentioned and/or additional aspect and advantage will be apparent from from the description with reference to accompanying drawings below to embodiment and
It is easy to understand, wherein：
Fig. 1 is the OD values method ratio that fecal sample DNA extracted amounts change with the holding time under different preservation conditions in embodiment 4
Relatively result；
Fig. 2 is the gel electrophoresis that fecal sample DNA extracted amounts change with the holding time under different preservation conditions in embodiment 4
Comparative result；
Fig. 3 is that the DNA extracted amounts of people in fecal sample in embodiment 5 compare with the real time fluorescent quantitative method that the holding time changes
As a result；And
Fig. 4 is the observation comparative result (40x) that epithelial cell form changes with the holding time in fecal sample in embodiment 6.
Specific embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining the present invention, and
It is not considered as limiting the invention.
According to an aspect of the present invention, the invention provides a kind of fecal sample preserves liquid.Embodiments in accordance with the present invention, should
Fecal sample preserves liquid and includes：The fixative of 45 volume %-60 volumes %；The fixative adjuvant of 0.1 mass %-15 mass %；
The anti-coagulants of 0.01 mass %-1.5 mass %；The buffer solution of 10 volume %-15 volumes %；The ion of 0.01 mass %-1 mass %
Intensity maintains agent；The nonionic detergent of 0.01 volume %-0.5 volume %；And balance of water, wherein, the fixative is
Selected from least one of methyl alcohol, ethanol, ethylene glycol and isopropanol, the fixed adjuvant be selected from acetic acid and acetate at least
One kind, the anti-coagulants is at least one of the water soluble salt selected from ethylenediamine tetra-acetic acid and ethylenediamine tetra-acetic acid, the buffer solution
It is selected from phosphate buffer, acetate buffer, ACES buffer solutions, ADA buffer solutions, BIS-TRIS buffer solutions, MES
At least one of buffer solution and PIPES buffer solutions, it is chlorate that the ionic strength maintains agent, and the nonionic detergent is choosing
From at least one of polysorbas20, NP-40 and TritonX-100.
It is surprisingly found by the inventors that, preserve liquid energy using the fecal sample of the present invention and effectively suppress in a long time in fecal sample
DNA degradation and cell damage, maintain cellular morphology, it is ensured that sample quality so that preserve fecal sample amplifying nucleic acid and
Cell can reach the needs of further research.Additionally, embodiments in accordance with the present invention, the fecal sample preservation liquid preparation of the present invention
Easily, low cost, can preserve fecal sample under normal temperature, and long to the Sample preservation time, and sample is once again using qualified after preservation
Rate is high.
It should be noted that the fecal sample in the present invention is preserved in liquid, primarily serving the purpose of for fixative is maintained in fecal sample
DNA and protein integrity so that albumen enzyme denaturation, prevent degradation of the enzyme to albumen, fixed eucaryotic cell structure.Inventor
It was found that, fecal sample is preserved in liquid and fixes agent content below 45 volumes %, though can ankyrin, effect is poor, can cause
DNA degradation；And fixed agent content can then cause cell gross distortion to condense more than 60 volumes %, subsequent samples are affected to utilize
When observation and interpretation to cellular morphology, while so that DNA is extracted increases difficulty.Some preferred embodiments of the invention,
The content of the fixative is 50 volume %-60 volumes %.Thus, ankyrin, suppress DNA degradation effect it is good.
The effect that adjuvant is fixed in fecal sample preservation liquid is so that cell expansion, reduces contraction of the fixative to cell.
But, more than when 15 mass % cell transition can expand, cellular morphology can be impacted for fixed auxiliary agent content.
Preserve in liquid in the fecal sample of the present invention, anti-coagulants primarily serves the purpose of removal complexation heavy metal ion, prevents excrement sample
Cell clumping in this.Some preferred embodiments of the invention, the anti-coagulants is disodium ethylene diamine tetraacetate.Additionally, existing
Have more containing anti-coagulants in technology, often up to 15%-50% (w/w), however, it is found by the inventors that, actual fecal sample preserves liquid
The consumption of middle anti-coagulants reduces to 0.01%-1.5% (w/w), during preferred 0.1-1.5% (w/w), on the basis of cost is reduced,
Still ensure that same anticoagulant effect.This is because the cell number in fecal specimens is less, floating preteins is less, then floating preteins
With reference to conjunction heavy metal ion it is also few.Thus, some preferred embodiments of the invention, the content of the anti-coagulants is 0.1
Quality %-1.5 mass %.
Preserve in liquid in the fecal sample of the present invention, what ionic strength maintained agent primarily serves the purpose of holding Premeabilisation of cells pressure, remains thin
Born of the same parents' form, prevents cell rupture from causing DNA to be released in preservation liquid.Some specific examples of the invention, the ion is strong
It is at least one selected from sodium chloride and potassium chloride that degree maintains agent.Some preferred embodiments of the invention, the ionic strength
The content for maintaining agent is 0.5 mass %-1 mass %.
Preserve in liquid in the fecal sample of the present invention, buffer solution can voluntarily adjust the self-dissolving by-product of cell bacterial release in fecal sample
The caused pH changes of thing institute, maintain pH to stablize, and maintain cellular morphology, while the DNA in reduction sample is too high due to pH
Or too low natural degradation crack conditions.Embodiments in accordance with the present invention, the pH value of the buffer solution is 7.4-8.0.According to this
Some embodiments of invention, it is 7.4-8.0 that the fecal sample preserves the pH value of liquid.
Preserve in liquid in the fecal sample of the present invention, the mucus for primarily serving the purpose of dissociation excrement of nonionic detergent increases excrement
The dispersion rate of sample.
Some embodiments of the invention, the fecal sample of the present invention preserves liquid and includes：The methyl alcohol of 50 volume %-60 volumes %；
The acetic acid of 0.1 mass %-15 mass %；The disodium ethylene diamine tetraacetate of 0.1 mass %-1.5 mass %；10 volume %-15 volumes %
Phosphate buffer；The sodium chloride of 0.5 mass %-1 mass %；The Tween-20 of 0.01 volume %-0.5 volume %；And surplus
For water.Thus, fecal sample preserves the preservation effect protrusion of liquid.
A specific example of the invention, the fecal sample of 1000ml preserves liquid and includes：
500ml methyl alcohol；
3.5g acetic acid；
1g disodium ethylene diamine tetraacetates；
100ml phosphate buffers；
10g sodium chloride；
0.5ml Tween-20s；And
399.5ml water.
A specific example of the invention, the fecal sample of 1000ml preserves liquid and includes：
450ml ethanol；
1g acetic acid；
3g disodium ethylene diamine tetraacetates；
100ml acetate buffers；
1g potassium chloride；
0.1ml Tween-20s；And
449.9ml water.
A specific example of the invention, the fecal sample of 1000ml preserves liquid and includes：
550ml ethylene glycol；
2.5g acetic acid；
7g disodium ethylene diamine tetraacetates；
150ml PIPES buffer solutions；
5g sodium chloride；
2.5ml NP-40；And
297.5ml deionized water.
According to a further aspect in the invention, present invention also offers a kind of prepare the method that fecal sample preserves liquid.According to the present invention
Embodiment, the method comprises the following steps：
1) fecal sample as described above preserves the formula offer raw material of liquid；
2) add ionic strength to maintain agent, cushioning liquid and anti-coagulants in deionized water, and mix each composition dissolving, with
Just mixture is obtained；
3) carry out pH value to adjust to pH by the mixture is 7.4-8.0；
4) fixative, fixative adjuvant and nonionic detergent are added in the mixture adjusted through pH value, after mixing
Sterilizing, to obtain the fecal sample liquid is preserved.
Fecal sample thereby, it is possible to efficiently prepare the present invention preserves liquid, and low cost, and the fecal sample of acquisition is preserved
Liquid quality is good, can for a long time preserve effective for the normal temperature of fecal sample, and sample DNA degraded and cell damage are few after preservation,
Sample is high using qualification rate once again.
In accordance with a further aspect of the present invention, present invention also offers foregoing fecal sample preservation liquid is in fecal sample is preserved
Purposes.Thus, liquid is preserved using the fecal sample of the present invention, effectively normal temperature can be carried out to fecal sample and be preserved for a long time,
And low cost is preserved, effect is good, and sample is high using qualification rate once again.
According to another aspect of the invention, present invention also offers a kind of method for preserving fecal sample.Enforcement of the invention
Example, the method includes：Foregoing fecal sample is preserved into liquid and fecal sample according to volume ratio 1-3：1 ratio is mixed,
Sealing at room temperature is kept in dark place.Embodiments in accordance with the present invention, using the method for the preservation fecal sample of the present invention, Neng Gouyou
Effect ground is realized preserving the normal temperature of fecal sample for a long time, and simple to operate, preserves low cost, and effect is good, and the holding time is long, sample
This is high using qualification rate once again.
Some preferred embodiments of the invention, it is 1.5 that the fecal sample preserves liquid and the volume ratio of the fecal sample：1.
Thus, low cost is preserved, and preservation effect is projected.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that the following examples
The present invention is merely to illustrate, and be should not be taken as limiting the scope of the invention.Unreceipted particular technique or condition, press in embodiment
Carry out according to the technology or condition described by document in the art or according to product description.Agents useful for same or the unreceipted life of instrument
Produce manufacturer person, be can pass through city available from conventional products.
Conventional method：
The fecal sample being related in embodiments below preserves the preparation method of liquid, generally comprises following steps：
1) fecal sample as described above preserves the formula offer raw material of liquid；
2) add ionic strength to maintain agent, cushioning liquid and anti-coagulants in deionized water, and mix each composition dissolving, with
Just mixture is obtained；
3) carry out pH value to adjust to pH by the mixture is 7.4-8.0；
4) fixative, fixative adjuvant and nonionic detergent are added in the mixture adjusted through pH value, after mixing
Sterilizing, to obtain the fecal sample liquid is preserved.
Embodiment 1
According to foregoing conventional method, according to the formula shown in following table, prepare fecal sample and preserve liquid.It is specific as follows：
Ionized water (Milli-Q ultra-pure waters) is removed, adds ionic strength to maintain agent, cushioning liquid and anti-coagulants, pH is adjusted
To after 8.0, fixative, fixative adjuvant and nonionic detergent are added, sterilized after mixing, obtained final product.
Table 1
| Ingredient names | Addition |
| Methyl alcohol | 500ml |
| Acetic acid | 3.5g |
| Disodium ethylene diamine tetraacetate | 1g |
| Phosphate buffer | 100ml |
| Sodium chloride | 10g |
| Tween-20 | 0.5ml |
| Deionized water | 399.5ml |
Wherein, the formula of phosphate buffer is：Sodium dihydrogen phosphate (NaH is added in 100ml deionized waters2PO4·H2O) 0.9g,
Disodium hydrogen phosphate (Na2HPO4·7H2O)24.97g。
Embodiment 2
Method with reference to described in embodiment 1, according to the formula shown in table 2 below, prepares fecal sample and preserves liquid.Wherein, acetic acid
The formula of salt buffer is：12.89g acetic acid (CH are added in 100ml deionized waters3COOH) 0.32g, sodium acetate
(CH3COONa)。
Table 2
| Ingredient names | Addition |
| Ethanol | 450ml |
| Acetic acid | 1g |
| Disodium ethylene diamine tetraacetate | 3g |
| Acetate buffer | 100ml |
| Potassium chloride | 1g |
| Tween-20 | 0.1ml |
| Deionized water | 449.9ml |
Embodiment 3
Method with reference to described in embodiment 1, according to the formula shown in table 3 below, prepares fecal sample and preserves liquid.Wherein, PIPES
The formula of buffer solution is：The ethyl sulfonic acids (PIPES) of 3.02g piperazines -1,4- two are added in 150ml deionized waters.
Table 3
| Ingredient names | Addition |
| Ethylene glycol | 550ml |
| Acetic acid | 2.5g |
| Disodium ethylene diamine tetraacetate | 7g |
| PIPES buffer solutions | 150ml |
| Sodium chloride | 5g |
| NP-40 | 2.5ml |
| Deionized water | 297.5ml |
Embodiment 4
A Healthy People feces volume about 10g excrement is fitted in the sterile faeces acquisition cup of 60ml (amount of about 1/4 acquisition cup),
And parallel 8 part sample is equally divided into, and then carry out excrement and preserve confirmatory experiment, it is specific as follows：
Experiment sets 5 treatment groups：Absolute ethyl alcohol group, 1 group of liquid of preservation, 2 groups of liquid of preservation, refrigeration group, room temperature group.Wherein,
Absolute ethyl alcohol group, using the absolute ethyl alcohol of 1.5 times of volumes of fecal sample room temperature preservation is carried out；1 group of liquid is preserved, using excrement
(its proportioning is the DETs buffer solutions of 1.5 times of volumes of sample：20%DMSO, 0.25M EDTA-Na2) carry out room temperature guarantor
Deposit；2 groups of liquid is preserved, the fecal sample of the invention prepared using the embodiment 1 of 1.5 times of volumes of fecal sample is preserved
Liquid carries out room temperature preservation；Refrigeration group, only by fecal sample carry out -20 DEG C it is stored refrigerated；Room temperature group, is only carried out fecal sample
Room temperature preservation.Also, absolute ethyl alcohol group, 1 group of liquid of preservation and 2 groups of liquid of preservation are all provided with 2 repetitions (i.e. 2 samples).
For each treatment group, in the different time points for preserving 0h, 24h, 72h, faeces DNA is extracted respectively, then utilize
OD values method and gel electrophoresis respectively process the DNA mass of sample.
Wherein, faeces DNA sample extraction adopts QIAGEN companies QIAamp DNA Stool Mini Kit kits, often
Secondary to take 200mg excrement, extraction step is as follows：
1) sample：Go sharp pipette tips to draw 1.5ml homogeneous samples, supernatant is removed in 1200rpm centrifugations, take 180-220mg excrement in
In 2ml centrifuge tubes, place on ice；
2) digest：1.6ml Buffer ASL are added, is shaken until sample is into homogeneous；12000rpm is centrifuged 1min, takes 1.4ml
Supernatant is to new 2ml centrifuge tubes；Often pipe adds 1 InhibitEX Tablet, and 1min is to leaching completely for concussion, then stands 1min,
12000rpm is centrifuged 3min, and supernatant is gone to into new 1.5ml centrifuge tubes, 12000rpm centrifugation 3min；Take the μ l of supernatant 600
To 2ml centrifuge tubes, 25 μ l proteinase K are added, after mixing, add 600 μ l Buffer AL, shake 15s；70 DEG C of water-baths
10min is short from 600 microlitres of absolute ethyl alcohols of addition are mixed；
3) post absorption is crossed：Taking 600 μ l adds mark QIAamp spincolumn pipes, 12000rpm centrifugation 1min to remove lower liquid,
Step 5) liquid at twice cross post, remove centrifugal liquid；
4) rinse：Plus 500 μ l Buffer AW1,12000rpm centrifugation 1min, remove centrifugal liquid；Plus 500 μ l Buffer AW2,
12000rpm is centrifuged 3min, goes centrifugal liquid, then 12000rpm skies to get rid of 1min；
5) elute：As for new 1.5ml centrifuge tubes, carefully uncap QIAamp spincolumn centrifuge tubes the 3min that dries in the air, and adds
In post central authorities, room temperature places 3min, 12000rpm centrifugation 1min to 50 μ l Buffer AE；Repeat 50 μ lElution Buffer of addition
Wash-out, removes post, and it is the DNA for extracting that gained liquid is centrifuged, and -20 degree are preserved, and mark time and sample name.
The step of OD value methods is：Take the employing of 1 μ lDNA solutionUltraviolet specrophotometer carries out UV absorbance detection,
Elution Buffer do blank, and readout units are ng/ μ l.8 parts of sample different time points OD values the results are shown in Table 4 and Fig. 1.
Table 4
The step of gel electrophoresis is：After extracting DNA, using RNAase enzymic digestion 1h, then 1% Ago-Gel, 160V
Electrophoresis 45min, dyeing observation, the electrophoresis result of 8 parts of sample different times is Fig. 2.In fig. 2, A figures are 8 parts parallel
Sample 0h extracts DNA electrophoretograms, and B is the DNA electrophoretograms for preserving each sample after 24h, and C figures are to preserve various kinds after 72h
The DNA electrophoretograms of this extraction.And as shown in Fig. 2 the band in each figure is respectively from left to right：Absolute ethyl alcohol group sample 1,
Absolute ethyl alcohol group sample 2, preservation 1 group of sample 3 of liquid, preservation 1 group of sample 4 of liquid, preservation 2 groups of samples 5 of liquid, 2 groups of liquid of preservation
Sample 6, refrigeration group sample 7, room temperature group sample 8；M represents Mark, maximum segment 12K in each figure.
The result of Fig. 1, Fig. 2 shows：0h () being preserved after Sampling hold immediately and extracting DNA, each treatment group OD value is surveyed
Obtain concentration and electrophoretic band shows difference less, band is single, and master tape, in more than 12K, is human gene group DNA, there is egg
White and RNA is removed not thoroughly, measures OD values bigger than normal；After 24h is placed, the sample DNA bar of refrigeration group and room temperature group
With substantially decrease, degraded is serious, and the DNA band brightness deteriorations of other treatment groups (i.e. under other conditions), concentration reduction,
Illustrate there is degraded；After preserving 72h, sample breakdown speed is all relative in each treatment group slows down, and room temperature places sample DNA bar
With faint, RNA and the miscellaneous band of albumen decrease, and the detection of OD values and electrophoresis detection result show and preserve in liquid 2 group 24 hours
Degradation speed is most fast, degrades afterwards very slow, and DNA concentration tends towards stability, and (i.e. embodiment 1 is prepared to preserve 2 groups of liquid
The present invention fecal sample preserve liquid) in DNA change always less, preservation effect is obvious.
Also, it was found that, sample after collection as (different disposal group) under different preservation conditions after, absolute ethyl alcohol
In group, absolute ethyl alcohol preserves fecal sample dehydration deflation and is difficult sampling, and sampling error is larger；In refrigeration group, sample, table are refrigerated
Face cooling is fast, generally wants 2-3h sample interiors just to freeze；In room temperature group, extremely there is biochemical reaction in the sample that room temperature is placed,
Darken, produce pungent stench, have a strong impact on operation；The sample preserved in 2 groups of liquid is easier to be dispersed into homogeneous, color
Change is little, without special odor.
Further, the fecal sample for preparing to embodiment 2-3 preserves liquid and carries out above-mentioned experimental verification, similarly, as a result shows
Show that, relative to other treatment groups, the fecal sample of the present invention preserves the sample DNA mass change minimum that liquid is preserved, preservation effect
It is best.
Embodiment 5
A Healthy People feces volume about 10g excrement is fitted in the sterile faeces acquisition cup of 60ml (amount of about 1/4 acquisition cup),
And parallel 4 part sample is equally divided into, then carry out excrement and preserve confirmatory experiment, with the fecal sample of the further detection present invention
The ageing of liquid is preserved, it is specific as follows：
Experiment sets 2 process：1 is processed, the sheet that sample 1-3 is prepared using the embodiment 2 of 1.5 times of volumes of fecal sample
The fecal sample of invention preserves liquid room temperature preservation；2 are processed, sample 4 adopts the DETs buffer solutions of 1.5 times of volumes of fecal sample
(its proportioning is：20%DMSO, 0.25M EDTA-Na2) room temperature preservation.Then, for the sample of each process, protecting
The DNA in sample is extracted in the 1st, 3,7,15,60 days for depositing, with the conservative gene β-action of people as reference, using glimmering
The changes of contents situation of people DNA in Fluorescent Quantitative PCR comparative sample.
Wherein, faeces DNA sample extraction adopts QIAGEN companies QIAamp DNA Stool Mini Kit kits, often
Secondary to take 200mg excrement, extraction step is as described in example 3 above.
Quantitative fluorescent PCR is quantitative using conservative gene β-action, and using primer β-action-F and β-action-R, (purpose is produced
Thing 175bp), SYBR fluorescence quantitative kits (Takara companies), the μ l of reaction system 25, the μ l of upstream and downstream primer consumption 0.5
(10 μM/L of concentration), annealing temperature is 64 DEG C, 45 circulations, and negative control is H2O, positive control behaviour ags cell
It is DNA, as a result takes amplification curve CT values, concrete outcome is shown in Fig. 3.
Wherein, primer sequence is as follows：
β-actin-F：
5’-TGGTGATGGAGGAGGCTCAGCAAGT-3’(SEQ ID NO：1)；
β-actin-R：
5’-AGCCAATGGGACCTGCTCCTCCCTTGA-3’(SEQ ID NO：2).
The fluorescent quantitation result of Fig. 3 confirmed the holding time to 60 days, and the fecal sample of the present invention preserves the fecal sample that liquid is preserved
Less, prolonged effect is much better than DETs buffer solutions normal temperature and preserves (sample 4) effect the change in concentration of people DNA in 1-3
Really, and comfort level even more better than absolute ethyl alcohol and deepfreeze.
Further, the excrement for preparing to embodiment 1,3 preserves liquid and carries out above-mentioned experimental verification, similarly, as a result shows
When holding time was to 60 days, relative to other treatment groups, the sample DNA change in concentration of the excrement preservation liquid preservation of the present invention is most
Little, preservation effect is best.
The cellular morphology of embodiment 6 preserves confirmatory experiment
A Healthy People feces volume about 20g excrement is fitted in the sterile faeces acquisition cup of 60ml (amount of about 1/3 acquisition cup),
Add the fecal sample of the invention that the embodiment 3 of 1.5 times of volumes is prepared to preserve liquid, add about 5.0x105Individual human mouth
Epithelial cell (mixes to prepare the test fecal sample containing a certain amount of cast-off cells), mixes to excrement and homogenizes, shady and cool room
Temperature stands.Then, the cast-off cells collected and observe in sample, it is specific as follows：
1) cell separation
What is preserved SCSR is used respectively within the 1st, 7,15 daysTMIn Fecal Cell Isolation Kit kit separating feces samples
Cell, comprises the following steps that：
Early stage rough segmentation：Go sharp pipette tips to draw 4 times, take the sample about 3ml that homogenizes and (containing about former excrement 1g, add by 20g excrement
30ml preserves the conversion of liquid equal proportion) in polybag as 330 μm of nylon wires, add the PBS of 10ml precoolings, external force to squeeze
Bag pressing causes sample further to refine, and will go in the 50ml centrifuge tubes of outfit that (this pipe upper end is can by the suspension of nylon wire
The 40 μm of dusting covers won), dusting cover is removed, filtrate in centrifuge tube is mended to 25ml with precooling PBS；
Centrifugation subdivision：Centrifugation bottom of the tube be slowly added to 10ml separating liquids (putting in advance to room temperature), it is to avoid bubble and with good pipe in
Filtrate mixes, it is seen that the interface is clear of two solution；200g room temperatures are centrifuged 10min, slowly suck supernatant, stay 3ml or so extraction raffinates
Suspension bottom cell；
Wash cell：Cell suspension adds precooling PBS (PH=7.2) to 40ml, mixes, 900g centrifugations 10min at 4 DEG C,
Supernatant is sucked, 15ml PBS (PH=7.2) come again, and carefully remove supernatant, stay the resuspended bottom cell of 2.5ml extraction raffinates, suspension
To on ice.
2) cell dyeing observation
Follow the steps below cell dyeing observation：
Smear, takes 1 μ l cell suspensions and prepares smear (push jack method), rapid to be dried；
Fixed, methyl alcohol fixed preparation 5-10min (separately has data to be 3-5min), distilled water rinsed clean (or natural drying)；
Dyeing, diluted staining solution (takes 9 parts of phosphate buffer, 1 part of Giemsa stain is sufficiently mixed), dye liquor is dropped in into tissue
Or on cell, drop dye 10-15min, distilled water rinsed clean can be taken advantage of wet with cover plate 20X, 40X microscopy when urgent, as a result see
Fig. 4.
As shown in figure 4, figure A is the preservation cellular morphology of the 1st day, it is the preservation cellular morphology of 7 days to scheme B, and figure C is guarantor
The cellular morphology of 15 days is deposited, periphery black particle mostly is Escherichia coli and excrement minute impurities；Figure D and figure E are oral epithelium
The initial form of cell, periphery black particle is impurity.
As shown in Figure 4：Figure D shows initial mouth epithelial cells dyeing core in aubergine, cytoplasm light red, some cells
Film is filled into irregular fold；Cell in fecal sample can be seen that with the increase of holding time by figure A-C, micro-
Microscopic observation to nuclear color gradually deepen, cellular morphology is affected by external influence, and change is little, and cell membrane is intact, very
It is easily discernible.In addition, the cell quantity that 1 μ l cell suspension smears are observed does not have rule, best once result is observed altogether
10 cells, with theoretical value (5.0 × 105Individual/30ml) * 3ml=10 are identical, and there is no the trend of reduction in 3 results, can
In being because sampling per se with cell number float very big and operating process loss it is different, or experiment number reason very little.
Further, the excrement for preparing to embodiment 1-2 preserves liquid and carries out above-mentioned experimental verification, similarly, as a result shows,
With the increase of holding time, cellular morphology receives external influence shadow to cell in the fecal sample that the excrement preservation liquid of the present invention is preserved
Ring change little, cell membrane is intact.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specific example ",
Or the description of " some examples " etc. means to combine specific features, structure, material or feature bag that the embodiment or example are described
In being contained at least one embodiment of the present invention or example.In this manual, the schematic representation of above-mentioned term is not necessarily referred to
Be identical embodiment or example.And, the specific features of description, structure, material or feature can be at any one
Or combine in an appropriate manner in multiple embodiments or example.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Without departing from this
Can these embodiments be carried out with various changes, modification, replacement and modification in the case of the principle and objective of invention, it is of the invention
Scope is limited by claim and its equivalent.
Claims (10)
1. a kind of fecal sample preserves liquid, it is characterised in that include：
The fixative of 45 volume %-60 volumes %；
The fixative adjuvant of 0.1 mass %-15 mass %；
The anti-coagulants of 0.01 mass %-1.5 mass %；
The buffer solution of 10 volume %-15 volumes %；
The ionic strength of 0.01 mass %-1 mass % maintains agent；
The nonionic detergent of 0.01 volume %-0.5 volume %；And
Balance of water,
Wherein,
The fixative is at least one selected from methyl alcohol, ethanol, ethylene glycol and isopropanol,
The fixed adjuvant is at least one selected from acetic acid and acetate,
The anti-coagulants is at least one of the water soluble salt selected from ethylenediamine tetra-acetic acid and ethylenediamine tetra-acetic acid,
The buffer solution is selected from phosphate buffer, acetate buffer, ACES buffer solutions, ADA buffer solutions, BIS-TRIS
At least one of buffer solution, MES buffer solutions and PIPES buffer solutions,
It is chlorate that the ionic strength maintains agent,
The nonionic detergent is at least one selected from polysorbas20, NP-40 and TritonX-100.
2. fecal sample according to claim 1 preserves liquid, it is characterised in that the pH value of the buffer solution is 7.4-8.0.
3. fecal sample according to claim 1 preserves liquid, it is characterised in that the fecal sample preserves the pH of liquid
It is worth for 7.4-8.0.
4. fecal sample according to claim 1 preserves liquid, it is characterised in that the ionic strength maintain agent be selected from
At least one of sodium chloride and potassium chloride,
Optionally, the anti-coagulants is disodium ethylene diamine tetraacetate.
5. fecal sample according to claim 1 preserves liquid, it is characterised in that the content of the fixative is 50 bodies
Product %-60 volumes %,
Optionally, the content of the anti-coagulants is 0.1 mass %-1.5 mass %,
Optionally, it is 0.5 mass %-1 mass % that the ionic strength maintains the content of agent.
6. fecal sample according to claim 5 preserves liquid, it is characterised in that include：
The methyl alcohol of 50 volume %-60 volumes %；
The acetic acid of 0.1 mass %-15 mass %；
The disodium ethylene diamine tetraacetate of 0.1 mass %-1.5 mass %；
The phosphate buffer of 10 volume %-15 volumes %；
The sodium chloride of 0.5 mass %-1 mass %；
The Tween-20 of 0.01 volume %-0.5 volume %；And
Balance of water,
Optionally, the fecal sample of 1000ml preserves liquid and includes：
500ml methyl alcohol；
3.5g acetic acid；
1g disodium ethylene diamine tetraacetates；
100ml phosphate buffers；
10g sodium chloride；
0.5ml Tween-20s；And
399.5ml water.
7. fecal sample according to claim 1 preserves liquid, it is characterised in that the fecal sample of 1000ml preserves liquid bag
Contain：
450ml ethanol；
1g acetic acid；
3g disodium ethylene diamine tetraacetates；
100ml acetate buffers；
1g potassium chloride；
0.1ml Tween-20s；And
449.9ml water,
Optionally, the fecal sample of 1000ml preserves liquid and includes：
550ml ethylene glycol；
2.5g acetic acid；
7g disodium ethylene diamine tetraacetates；
150mlPIPES buffer solutions；
5g sodium chloride；
2.5ml NP-40；And
297.5ml deionized water.
8. it is a kind of to prepare the method that fecal sample preserves liquid, it is characterised in that to comprise the following steps：
1) formula for preserving liquid according to the fecal sample described in any one of claim 1-7 provides raw material；
2) add ionic strength to maintain agent, cushioning liquid and anti-coagulants in deionized water, and mix each composition dissolving, with
Just mixture is obtained；
3) carry out pH value to adjust to pH by the mixture is 7.4-8.0；
4) fixative, fixative adjuvant and nonionic detergent are added in the mixture adjusted through pH value, after mixing
Sterilizing, to obtain the fecal sample liquid is preserved.
9. the fecal sample described in any one of claim 1-7 preserves purposes of the liquid in fecal sample is preserved.
10. it is a kind of preserve fecal sample method, it is characterised in that include：
Fecal sample described in any one of claim 1-7 is preserved into liquid and fecal sample according to volume ratio 1-3：1 ratio is mixed,
Sealing at room temperature is kept in dark place,
Optionally, it is 1.5 that the fecal sample preserves liquid and the volume ratio of the fecal sample：1.
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| CN201510671258.9A CN106596211A (en) | 2015-10-15 | 2015-10-15 | Stool sample preservation liquid, preparation method and application thereof |
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| CN201510671258.9A CN106596211A (en) | 2015-10-15 | 2015-10-15 | Stool sample preservation liquid, preparation method and application thereof |
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| CN201510671258.9A Pending CN106596211A (en) | 2015-10-15 | 2015-10-15 | Stool sample preservation liquid, preparation method and application thereof |
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| CN107988076A (en) * | 2017-12-19 | 2018-05-04 | 东莞博奥木华基因科技有限公司 | A kind of excrement preserves liquid and its application |
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| CN109517883A (en) * | 2018-12-05 | 2019-03-26 | 广州市达信智能科技有限公司 | The preservation liquid of cast-off cells in a kind of human faecal mass sample |
| CN109735600A (en) * | 2019-02-27 | 2019-05-10 | 深圳市金锐生物科技有限公司 | A kind of genital tract flora nucleic acid preservation formula of liquid, method of preparation and use |
| CN111826371A (en) * | 2019-04-15 | 2020-10-27 | 上海锐翌生物科技有限公司 | Excrement nucleic acid preservation solution and preparation method and application thereof |
| CN110272897A (en) * | 2019-06-26 | 2019-09-24 | 成都罗宁生物科技有限公司 | Intestinal contents Sample preservation liquid and preparation method |
| CN111197042A (en) * | 2019-12-19 | 2020-05-26 | 武汉艾米森生命科技有限公司 | Feces exfoliated cell nucleic acid preservation reagent and preparation method and application thereof |
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| CN113729008A (en) * | 2021-09-30 | 2021-12-03 | 苏州中科先进技术研究院有限公司 | Excrement storage liquid, preparation method and application thereof |
| CN114369537A (en) * | 2022-01-11 | 2022-04-19 | 广东量子天健医疗科技有限公司 | Excrement storage liquid and preparation method and application thereof |
| CN115486439A (en) * | 2022-09-23 | 2022-12-20 | 深圳微辰生命科技有限公司 | Feces preservation method and application of ethanol in preparation of feces preservation solution and short-chain fatty acid detection reagent or kit |
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Application publication date: 20170426 |