CN107723240A - A kind of microorganism in skin, oral cavity, genital tract sample preserves reagent and its preparation method and application - Google Patents
A kind of microorganism in skin, oral cavity, genital tract sample preserves reagent and its preparation method and application Download PDFInfo
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- CN107723240A CN107723240A CN201610662637.6A CN201610662637A CN107723240A CN 107723240 A CN107723240 A CN 107723240A CN 201610662637 A CN201610662637 A CN 201610662637A CN 107723240 A CN107723240 A CN 107723240A
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- 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
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Abstract
The invention discloses the microorganism in a kind of skin, oral cavity, genital tract sample to preserve reagent, including：PH buffer Tris HCl；Ionic strength maintains agent NaCl；Protein denaturant lauryl sodium sulfate；Protein degradation agent Proteinase K；Bacteriostatic agent ethylenediamine tetra-acetic acid；Albumen enzymatic protective reagent trehalose.In addition, the invention also discloses the preparation method and application that the microorganism preserves reagent.Reagent of the present invention need not freeze, you can have the function that stable wherein microorganism and its composition.Under room temperature even more high temperature conditionss (37 DEG C), the holding time is up to 14 days as long as.So that the preservation of sample, transport become to be more prone to, cost of transportation can be effectively reduced, the protection for sample is also more safe and simple, is easily achieved.
Description
Technical field
It is micro- in more particularly to a kind of skin, oral cavity, genital tract sample the invention belongs to Molecular Ecology of Microbiology field
Biology preserves reagent and its preparation method and application.Present invention can apply to standard PCR amplification, Real Time PCR, high flux
Carry out microbial diversity detection etc..
Background technology
With the development of science and technology and people's living standards continue to improve, increasing people recognize oral cavity, skin
Skin, genital tract microorganism have important influence to health.With sequencing technologies maturation and cost decline, increasingly
More individuals and unit regard oral cavity, skin, genital tract microorganism detection as one of health check-ups.The collection of sample and
Preservation is to detect oral cavity, skin, the first step of genital tract microorganism, and a crucial step.Correctly collection and preservation sample, can
With more truly react human oral cavity, skin, genital tract microorganism diversity.
At present for oral cavity, skin, the multi-purpose collection swab collection sample of genital tract microorganism.Because microorganism has tanacity
Life ability, after sample collection, the microorganism in sample still can be grown using the nutriment of sample itself
Breeding.If sample is placed in the conventional environments such as interior for a long time, the type and quantity of the microorganism in sample can change,
Human oral cavity, skin, the diversity of genital tract microorganism can not truly be reflected.Current optimal sample preservation, be by
The sample collected is immediately placed in freezen protective in liquid nitrogen or -80 DEG C of refrigerators.Within a period of time, in liquid nitrogen or -80 DEG C of refrigerators
Significant change will not occur for the sample in environment, the species and quantity of its microorganism.But in many hospitals, laboratory, family
It can not realize and preserve sample with liquid nitrogen or -80 DEG C of refrigerators.
Because sequenator is costly, most hospitals, laboratory do not have sequenator, and it is public that current most samples are delivered to detection soon
Department is detected.In transportation, low temperature environment is realized using dry ice.Often 1g sample needs 3-4 kilograms of even 10 public affairs
Dry ice more than jin is sent, and the cost of transport can greatly improve.
The content of the invention
One of the technical problem to be solved in the present invention is in the microorganism in a kind of skin, oral cavity, genital tract sample is provided
Reagent is preserved, without freezing, you can have the function that stable wherein microorganism and its composition.In room temperature even more high temperature conditionss
Under (37 DEG C), the holding time be up to 14 days as long as.So that the preservation of sample, transport become to be more prone to, fortune can be effectively reduced
Defeated cost, protection for sample is also more safe and simple, is easily achieved.
The second technical problem to be solved by the present invention is in the microorganism in above-mentioned skin, oral cavity, genital tract sample is provided
Preserve the preparation method of reagent.
The third technical problem to be solved by the present invention is in the microorganism in above-mentioned skin, oral cavity, genital tract sample is provided
Preserve the application of reagent.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
In one aspect of the invention, there is provided the microorganism in a kind of skin, oral cavity, genital tract sample preserves reagent, including：
PH buffer Tris-HCl；Ionic strength maintains agent NaCl；Protein denaturant lauryl sodium sulfate (Sodium dodecyl
Sulfate, SDS)；Protein degradation agent Proteinase K；Bacteriostatic agent ethylenediamine tetra-acetic acid (Ethylene Diamine
Tetraacetic Acid, EDTA)；Albumen enzymatic protective reagent trehalose.
Preferably, described pH buffer Tris-HCl concentration is 1~50mM, pH value is 7.0~8.0.
Preferably, it is 50~500mM that described ionic strength, which maintains agent NaCl concentration,.
Preferably, described protein denaturant lauryl sodium sulfate (Sodium dodecyl sulfate, SDS)
Concentration is 0.1%~1%.
Preferably, the concentration of protein degradation agent Proteinase K is 50~100 μ g/mL.
Preferably, the concentration that the bacteriostatic agent is ethylenediamine tetra-acetic acid (EDTA) is 1~100mM.
Preferably, the concentration of albumen enzymatic protective reagent trehalose is 1~100mM.
In another aspect of this invention, there is provided the microorganism in the skin, oral cavity, genital tract sample preserves the preparation of reagent
Method, comprise the following steps：
(1) agent NaCl, bacteriostatic agent ethylenediamine tetra-acetic acid is maintained to mix the pH buffer Tris-HCl, ionic strength；
(2) in above-mentioned mix reagent, the protein denaturant lauryl sodium sulfate, albumen enzyme protection are sequentially added
Agent, trehalose protein degradation agent Proteinase K, it is sufficiently mixed uniformly.
In another aspect of this invention, there is provided a kind of mentioned microorganism preserves reagent in skin, oral cavity, genital tract sample
Microorganism store method in application.
Preferably, the microorganism store method in the skin, oral cavity, genital tract sample is：By skin, oral cavity, life
Grow the microorganism that sample is added described in item to preserve in reagent, preserve at ambient temperature.
Preferably, described save as at ambient temperature preserves under 4 DEG C~37 DEG C environment.
Preferably, the skin, oral cavity, the swab quantity (individual) and the volume of microorganism preservation reagent of genital tract sample
(mL) than being 2:1~1:10.
Preferably, the preservation for up to 14 days.
Compared with prior art, the beneficial effects of the present invention are：
(1) in skin of the invention, oral cavity, genital tract sample microorganism preservation reagent, without freezing, you can reach steady
The effect of fixed wherein microorganism and its composition, room temperature condition can be preserved up to 14 days, so that the preservation of sample, transport become
It is more prone to, can effectively reduces cost of transportation, the protection for sample is also more safe and simple, is easily achieved.
(2) contain bacteriostatic agent EDTA in the present invention, can effectively prevent the growth of non-sample microorganism.
(3) present invention adds protein degradation agent Proteinase K, the microorganism in sample can be digested, splits its cell
Solution, the amount of the genomic DNA in the reset condition of sample is farthest kept, and then ensure the wherein species of microorganism, ratio
Example, quantity do not change；Albumen enzymatic protective reagent trehalose is also added into the present invention simultaneously, to ensure the work of Proteinase K
Property.
(4) the pH buffer Tris-HCl in the present invention, there is provided necessary ion buffering capacity, be more beneficial for directly carrying out
Follow-up DNA extraction, protein denaturant lauryl sodium sulfate (Sodium dodecyl sulfate, SDS), is extracted in DNA
During, opened after making protein denaturation with DNA points, the efficiency of enhancing genome extracting.
(5) ionic strength in the present invention maintains agent NaCl, maintains certain ionic strength, is carried for follow-up genome
For necessary condition.
(6) present invention can apply to standard PCR amplification, Real Time PCR, high flux to carry out microbial diversity detection
Etc. multiple platforms.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description：
Fig. 1 be directly freeze and preserve in the embodiment of the present invention 5 liquid preserve 14 days skin, oral cavity, in genital tract sample
Bacteria total DNA extraction effect figure；Wherein, Fig. 1 (A) represents dermatological specimens, and Fig. 1 (B) represents oral cavity sample, and Fig. 1 (C) represents reproduction
Road sample；Wherein, M is Marker DL9000, and applied sample amount 3uL, bright band is 30ng/ μ L, and remaining band is 10ng/ μ L.Base
Because a group DNA applied sample amounts are similarly 3 μ L, electrophoresis result band brightness is shown, genomic DNA concentration is about 10-30ng/ μ L.
Fig. 2 be directly freeze and preserve in the embodiment of the present invention 6 liquid preserve 14 days skin, oral cavity, in genital tract sample
Bacterial 16 S rRNA standard PCR amplification design sketch；Wherein, Fig. 2 (A) represents dermatological specimens, and Fig. 2 (B) represents oral cavity sample, figure
2 (C) represent genital tract sample；Wherein, Marker DL2000, applied sample amount 3uL, bright band 30ng/uL, remaining band are
10ng/uL。
Fig. 3 be directly freeze and preserve in the embodiment of the present invention 7 liquid preserve 14 days skin, oral cavity, in genital tract sample
Bacterial 16 S rRNA Real Time PCR amplification curve diagrams；Wherein, Fig. 3 (A) represents dermatological specimens, and Fig. 3 (B) represents oral cavity
Sample, Fig. 3 (C) represent genital tract sample.
Fig. 4 be directly freeze and preserve in the embodiment of the present invention 9 liquid preserve 14 days skin, oral cavity, in genital tract sample
Bacterium Alpha diversity compares figure；Wherein, Fig. 4 (A) represents dermatological specimens, and Fig. 4 (B) represents oral cavity sample, and Fig. 4 (C) represents life
Grow sample.
Fig. 5 be in the embodiment of the present invention 9 door and belong to it is each level on, directly freeze and preserve liquid preservation 14 days skin,
Bacteria abundance ratio of components is relatively schemed in oral cavity, genital tract sample；Wherein, Fig. 5 (A) represents dermatological specimens phylum levels, Fig. 5 (B)
Dermatological specimens genus levels are represented, Fig. 5 (C) represents oral cavity sample phylum levels, and Fig. 5 (D) represents oral cavity sample genus water
Flat, Fig. 5 (E) represents genital tract sample phylum levels, and Fig. 5 (F) represents genital tract sample genus levels.
Embodiment
Following examples are only illustrative of the invention and is not intended to limit the scope of the invention.It is unreceipted specific in embodiment
The experimental method of condition, generally according to normal condition, or according to the condition proposed by manufacturer.
Below by taking skin, oral cavity, genital tract sample as an example, to the embodiment of the microorganism preservation reagent in swab sample
It is described in detail.Following example is merely to illustrate the present invention.Unreceipted specific technology or condition in example, according to ability
The technical conditions of document description in domain are carried out according to product description.
Embodiment 1：Skin, the preparation in oral cavity, genital tract swab quasi-microorganism preservation reagent.
Final concentration of 1mM is configured with deionized water, pH buffer Tris-HCl that pH value is 7.0, concentration are 50mM ions
The bacteriostatic agent ethylenediamine tetra-acetic acid (EDTA) that intensity maintains agent NaCl, concentration is 1mM；In above-mentioned mix reagent, sequentially add
Mass percent concentration be 0.1% protein denaturant lauryl sodium sulfate (Sodium dodecyl sulfate, SDS),
Albumen enzymatic protective reagent trehalose that concentration is 1mM, the protein degradation agent Proteinase K that concentration is 50 μ g/mL, are stirred continuously, fill
Divide and be well mixed, be settled to 100mL, that is, be made.
Embodiment 2：Skin, the preparation in oral cavity, genital tract swab quasi-microorganism preservation reagent.
Configure final concentration of 50mM with deionized water, pH buffer Tris-HCl that pH value is 8.0, concentration be 500mM from
The bacteriostatic agent ethylenediamine tetra-acetic acid (EDTA) that sub- intensity maintains agent NaCl, concentration is 100mM；In above-mentioned mix reagent, successively
Add mass percent concentration be 1% protein denaturant lauryl sodium sulfate (Sodium dodecyl sulfate,
SDS albumen enzymatic protective reagent trehalose that), concentration is 100mM, the protein degradation agent Proteinase K that concentration is 100 μ g/mL, constantly
Stirring, it is sufficiently mixed uniformly, is settled to 100mL, that is, is made.
Embodiment 3：Skin, the preparation in oral cavity, genital tract swab quasi-microorganism preservation reagent.
Configure final concentration of 20mM with deionized water, pH buffer Tris-HCl that pH value is 7.5, concentration be 100mM from
The bacteriostatic agent ethylenediamine tetra-acetic acid (EDTA) that sub- intensity maintains agent NaCl, concentration is 60mM；In above-mentioned mix reagent, add respectively
Enter mass percent concentration be 0.6% protein denaturant lauryl sodium sulfate (Sodium dodecyl sulfate,
SDS albumen enzymatic protective reagent trehalose that), concentration is 80mM, the protein degradation agent Proteinase K that concentration is 70 μ g/mL, are constantly stirred
Mix, be sufficiently mixed uniformly, be settled to 100mL, that is, be made.
Embodiment 4：Skin, the collection in oral cavity, genital tract swab sample, directly freeze and 37 DEG C preserve the processing of 14 days.
Two volunteers are chosen, are sampled using aseptic cotton carrier.Skin, oral cavity, genital tract sample (3 swab cottons are taken respectively
Signed-off sample sheet) preserved in 5-10mL microorganisms of the present invention in reagent, after concussion mixes, by each type of sample of each volunteer
Divide 4 pipes (1 pipe/1mL) respectively, wherein 2 pipes freeze in -80 DEG C immediately, as parallel laboratory test and positive control, remaining 2 pipes are placed in 37
In DEG C water-bath, constant temperature preserves 14 days.Freeze after sampling in -80 DEG C of refrigerators, extracted for subsequent gene group immediately.
Embodiment 5：Bacteria total DNA preservation effect is tested in skin, oral cavity, genital tract swab sample.
The method that Proteinase K cracks is taken to carry out genome extracting to the sample of embodiment 4, electrophoresis detection carries out Quality Control,
As a result as shown in figure 1, the concentration of genomic DNA is about 10-30ng/ μ L.
Embodiment 6：Bacterial 16 S rRNA gene standard PCR amplifications effect is real in skin, oral cavity, genital tract swab sample
Test.
Using the extracting genomic DNA of embodiment 5 as template, performing PCR is entered to the V1-V2 hypervariable regions of its 16S rRNA gene
Amplification, PCR results are as shown in Figure 2.
Electrophoresis result is visible, directly freezes sample and 37 DEG C of samples for preserving 14 days pass through standard PCR amplification, testing result
No significant difference.
Embodiment 7：Bacterial 16 S rRNA gene Real Time PCR amplifications effect in skin, oral cavity, genital tract swab sample
Fruit is tested.
The standard plasmid of the V1-V2 hypervariable regions of 16S rRNA genes is built, draws standard curve, while to directly freezing
Real Time PCR quantitative experiments are carried out with 37 DEG C of samples for preserving 14 days, amplification curve is as shown in figure 3, freeze sample conduct
Control (1 reaction), 37 DEG C of samples for preserving 14 days are in contrast (2 parallel reactions).
Experimental result shows that for 37 DEG C of samples for preserving 14 days compared with the sample directly frozen, amplification is not notable
Change, Ct values are basically identical, and negative control Ct values do not influence substantially 27 or so on pattern detection.37 DEG C preserve 14 days
Sample sample Real-time PCR amplification the later stage, the DNA copy number in it has reached plateau, with directly freezing sample
In DNA copy number it is suitable.The quantity of genomic DNA is with directly freezing in sample in 37 DEG C of samples for preserving 14 days of this explanation
DNA quantity is essentially identical.
Embodiment 8：Bacterial 16 S rRNA gene V1-V2 areas library construction in skin, oral cavity, genital tract swab sample
Using the extracting genomic DNA of embodiment 5 as template, performing PCR is entered to the V1-V2 hypervariable regions of its 16S rRNA gene
Amplification, while add joint needed for sequencing.PCR primer is reclaimed, quantified, equimolar than mix, Quality Control it is qualified after, use
Illumina MiSeq platforms, library is sequenced.
Embodiment 9：High-flux sequence and relevant biological information and statistical analysis.
The sequencing initial data that embodiment 8 is obtained, carry out sequence assembly, Quality Control and bioinformatics and statistics credit
Analysis：
(1) the alpha diversity analysis based on OTU
Alpha diversity (Alpha diversity, alpha diversity) is the analysis to species diversity in single sample, point
It is as shown in Figure 4 to analyse result.Shannon index have reacted the diversity (diversity) of sample.By alpha diversity statistical result
It can be seen that identical sample preserves 14 days results and the sample directly frozen without significant difference (P in liquid is preserved>0.05) this, is illustrated
Preserve reagent has preferable preservation effect for Phylogenetic diversity of bacteria.
(2) species taxonomy information carries out statistical analysis
(see Fig. 5) in door and each horizontal bacterium relative abundance pie graph of category, each pillar represents a sample one
Kind handling the average of lower 2 repetitions, different colours represent different species, species information such as legend, if what a certain bacterium accounted for
Ratio is more, then its shared ratio in pillar will be big.As seen from the figure, directly freeze and handled 14 days at 37 DEG C with preserving liquid
Sample, in the species composition horizontal from door and category, without significant difference, it was demonstrated that in the skin of the invention, oral cavity, swab sample
Microorganism preserve reagent, under room temperature or higher temperature (37 DEG C), can keep various microorganism groups in sample into ratio
Example, it is constant within 14 days.
The analysis formed based on Phylogenetic diversity of bacteria and Pseudomonas, learn that microorganism of the present invention preserves reagent, its effect
Processing is frozen no less than -80 DEG C.But the requirement of the preservation liquid to preservation condition is relatively low, room temperature preservation, and the holding time compared with
Long, up to 14 days.For the preservation of sample, condition of providing convenience is transported, laboratory great amount of samples extracts genomic DNA also more
It is convenient, drastically increase the operating efficiency in laboratory.
Claims (13)
1. the microorganism in a kind of skin, oral cavity, genital tract sample preserves reagent, it is characterised in that including：PH buffer
Tris-HCl；Ionic strength maintains agent NaCl；Protein denaturant lauryl sodium sulfate；Protein degradation agent Proteinase K；It is antibacterial
Agent ethylenediamine tetra-acetic acid；Albumen enzymatic protective reagent trehalose.
2. the microorganism in skin as claimed in claim 1, oral cavity, genital tract sample preserves reagent, it is characterised in that described
PH buffer Tris-HCl concentration is 1~50mM, and pH value is 7.0~8.0.
3. the microorganism in skin according to claim 1, oral cavity, genital tract sample preserves reagent, its special type is：Institute
The concentration for stating protein degradation agent Proteinase K is 50~100 μ g/mL.
4. the microorganism in skin according to claim 1, oral cavity, genital tract sample preserves reagent, its special type is：Institute
The concentration for stating albumen enzymatic protective reagent trehalose is 1~100mM.
5. the microorganism in skin according to claim 1, oral cavity, genital tract sample preserves reagent, its special type is：Institute
The mass percent concentration for stating protein denaturant lauryl sodium sulfate is 0.1%~1%.
6. the microorganism in skin according to claim 1, oral cavity, genital tract sample preserves reagent, it is characterised in that：Institute
The concentration for stating bacteriostatic agent ethylenediamine tetra-acetic acid is 1~100mM.
7. the microorganism in skin according to claim 1, oral cavity, genital tract sample preserves reagent, it is characterised in that：Institute
It is 50~500mM to state ionic strength and maintain agent NaCl concentration.
8. microorganism preserves reagent in a kind of skin according to any one of claim 1~7, oral cavity, genital tract sample
Preparation method, it is characterised in that comprise the following steps：
(1) agent NaCl, bacteriostatic agent ethylenediamine tetra-acetic acid is maintained to mix the pH buffer Tris-HCl, ionic strength；
(2) in above-mentioned mix reagent, the protein denaturant lauryl sodium sulfate, albumen enzymatic protective reagent marine alga are sequentially added
Sugar, protein degradation agent Proteinase K, it is sufficiently mixed uniformly.
9. the microorganism according to claim 1~7 any one preserves reagent in skin, oral cavity, genital tract sample
Application in microorganism store method.
10. application according to claim 9, it is characterised in that the microorganism in the skin, oral cavity, genital tract sample
Store method is：Skin, oral cavity, genital tract sample are added to the microorganism described in any one of claim 1~7 and preserve reagent
In, preserve at ambient temperature.
11. application according to claim 10, it is characterised in that：It is described to save as at ambient temperature at 4 DEG C~37 DEG C
Preserved under environment.
12. application according to claim 10, it is characterised in that the skin, oral cavity, the swab number of genital tract sample
The volume ratio that reagent is preserved with microorganism is 2:1~1:10.
13. application according to claim 9, it is characterised in that the preservation for up to 14 days.
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