CN109735600A - A kind of genital tract flora nucleic acid preservation formula of liquid, method of preparation and use - Google Patents
A kind of genital tract flora nucleic acid preservation formula of liquid, method of preparation and use Download PDFInfo
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Abstract
The present invention relates to genital tract flora nucleic acid preservation formula of liquid, including following components: the sodium chloride that ethylenediamine tetra-acetic acid that Tris-HCl buffer that concentration is 0.01-1M, concentration are 0.01-1M, concentration are 0.01-0.5M, concentration is the sodium citrate of 0.01-0.5M and the dehydrated alcohol of 10-30%；It is 8.0-10.0 with the pH value for saving liquid is postponed；According to above-mentioned recipe configuration and for collecting genital tract sample; with microorganism and its nucleic acid can be protected in genital tract from degradation; microbial diversity is protected, is conducive to the transport of genital tract sample collection, is conducive to the features such as downstream gene detection; facilitate the research of genital tract bacterial diversity, and using genital tract flora as the genetic test application of target spot.
Description
Technical field
The present invention relates to microorganisms technical field, more specifically to a kind of genital tract flora nucleic acid preservation formula of liquid,
Method of preparation and use.
Background technique
Bacterium inhabites at human body almost each position, and quantity number is with trillion, up to 1014, about human body cell sum
10 times.There are nearly thousand kinds of bacteriums in human gastrointestinal tract at a rough estimate.Compared with intestinal flora, vaginal flora type is simpler
It is single, about 40 kinds of Pseudomonas may be present on the side wall mucous membrane of Women of Childbearing Age vagina surrounding, a strain more than 100 weighs 20 grams.Including cream
Bacillus, vagina atropic Podbielniak bacterium, megacoccus, corynebacteria, Escherichia coli, Veillonella and Jia Dena bacterium etc..These vaginas are thin
Bacterium can be roughly classified into " beneficial bacterium " and " conditioned pathogen " two major classes.Most bacterium in healthy Women of childbearing age vaginal flora quantity
Kind (being in the ascendance) is lactobacillus, it is the beneficial bacterium for maintaining microecology in vaginas balance, to rejection condition pathogenic bacteria
Intrusion and the various vagina infections of prevention play an important role.If certain factors cause beneficial bacterium quantity to reduce, can cause
A certain or several conditioned pathogen abnormality proliferations, cause Dysbiosis, are easy to appear various vagina infections.Therefore, with life
Flora is grown as target spot, its composed structure and changing rule is detected with means such as high-flux sequence, genetic chips,
Become a kind of and measures human health status or even for the new method of the adjoint diagnosis of specified disease.
After genital tract flora sample completes acquisition, the methods of extracted nucleic acid, genetic test and bioinformatic analysis,
Whole microbial DNA information therein are obtained, the composition information of whole microbe species in genital tract can be obtained.But
During acquisition and transport, microorganism and its nucleic acid composition in genital tract sample are influenced by following factors: first, it is raw
It is largely anaerobic bacteria in growing, due to the change of oxygen content in environment, these bacteriums may quickly stop growing simultaneously dead
It dies, leads to its cell wall rupture, nucleolysis；Second, the change of temperature will also result in the change of different bacterium growth rate, make
It obtains genital tract flora composition to change, certain thermally sensitive microbe species death are decomposed, and nucleolysis disappears.And it is normal
(- 80 DEG C) of the preserving type of rule, i.e. ultralow temperature preservations are not particularly suited for conventional sampling at home and transport, and certain customers is caused to abandon
Detection or testing result do not conform to the actual conditions.Currently, being badly in need of development stability, effective method, at normal temperature in genital tract sample
Microorganism and its nucleic acid protected, guarantee genital tract bacteria detection result reliability.
Summary of the invention
The technical problem to be solved in the present invention is that in view of the above drawbacks of the prior art, providing a kind of genital tract flora
Nucleic acid preservation formula of liquid；
Additionally providing a kind of genital tract flora nucleic acid preservation liquid and preparation method thereof and a kind of genital tract flora nucleic acid preservation liquid makes
Use method.
The technical solution adopted by the present invention to solve the technical problems is:
Construct a kind of genital tract flora nucleic acid preservation formula of liquid, wherein including following components: concentration is 0.01-1M's
The sodium chloride that ethylenediamine tetra-acetic acid that Tris-HCl buffer, concentration are 0.01-1M, concentration are 0.01-0.5M, concentration are
The sodium citrate of 0.01-0.5M and the dehydrated alcohol of 10-30%.
Genital tract flora nucleic acid preservation formula of liquid of the present invention, which is characterized in that be with the pH value for saving liquid is postponed
8.0-10.0。
A kind of genital tract flora nucleic acid preservation liquid and preparation method thereof, according to above-mentioned genital tract flora nucleic acid preservation formula of liquid,
Its implementation is as follows:
Step 1: weighing required Tris according to the above formula ratio, with salt acid for adjusting pH to 5.0-10.0, Tris-HCl is made
Buffer；
Step 2: the desired amount of ethylenediamine tetra-acetic acid is dissolved in aseptic deionized water, with salt acid for adjusting pH to 5.0-10.0,
EDTA solution is made；
Step 3: the desired amount of sodium chloride and sodium citrate are dissolved in aseptic deionized water, and be added Tris-HCl solution,
The mixing of EDTA solution adjusts pH value to 8.0-10.0, sterilization treatment with NaOH；
Step 4: above-mentioned solution is added in the desired amount of dehydrated alcohol, miillpore filter degerming is crossed.
Genital tract flora nucleic acid preservation liquid and preparation method thereof of the present invention, wherein further include step 5: by prepared
It saves liquid to dispense to probe tube, room temperature is strictly sealed.
Genital tract flora nucleic acid preservation liquid and preparation method thereof of the present invention, wherein in the third step, sterilization treatment in
121 DEG C of high pressure sterilization 30min.
A kind of genital tract flora nucleic acid preservation liquid application method, according to above-mentioned genital tract flora nucleic acid preservation liquid preparation side
Method, which is characterized in that implementation method is as follows: to equipped with according to the genital tract flora nucleic acid preservation liquid and preparation method thereof preparation
In the probe tube for saving liquid, cotton swab head or cotton swab after genital tract sampling is added, sealing is protected from light immediately, controls temperature at 40 DEG C
Within save and/or transport.
The beneficial effects of the present invention are: according to above-mentioned recipe configuration and for collecting genital tract sample, having can be protected
Microorganism and its nucleic acid are from degradation in genital tract, under protecting microbial diversity, transporting, be conducive to conducive to genital tract sample collection
The features such as swimming genetic test facilitates the research of genital tract bacterial diversity, and using genital tract flora as the genetic test of target spot
Using.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below in conjunction with attached drawing and reality
Applying example, the invention will be further described, and the accompanying drawings in the following description is only section Example of the invention, for this field
For those of ordinary skill, without creative efforts, it can also be obtained according to these attached drawings other accompanying drawings:
Fig. 1 is the genital tract flora nucleic acid preservation liquid and preparation method thereof flow chart of present pre-ferred embodiments；
Fig. 2 is that the DNA of the 5 volunteer's genital tract samples saved through 5 kinds of preserving types extracts result comparison schematic diagram；
Fig. 3 is that the biodiversity index of the 5 volunteer's genital tract samples saved through 5 kinds of preserving types compares signal
Figure；
Fig. 4 is that signal is compared in the microorganism principal component analysis of the 5 volunteer's genital tract samples saved through 5 kinds of preserving types
Figure.
Specific embodiment
In order to keep the purposes, technical schemes and advantages of the embodiment of the present invention clearer, implement below in conjunction with the present invention
Technical solution in example carries out clear, complete description, it is clear that and described embodiment is section Example of the invention, and
It is not all of embodiment.Based on the embodiment of the present invention, those of ordinary skill in the art are not before making the creative labor
Every other embodiment obtained is put, protection scope of the present invention is belonged to.
The genital tract flora nucleic acid preservation formula of liquid of present pre-ferred embodiments, including following components: concentration 0.01-1M
Tris-HCl buffer, concentration be 0.01-1M ethylenediamine tetra-acetic acid, concentration be 0.01-0.5M sodium chloride, concentration is
The sodium citrate of 0.01-0.5M and the dehydrated alcohol of 10-30%.
It preferably, is 8.0-10.0 with the pH value for saving liquid is postponed；
According to above-mentioned recipe configuration and for collecting genital tract sample, microorganism and its nucleic acid in genital tract can be protected by having
From degradation, protection microbial diversity is conducive to the transport of genital tract sample collection, is conducive to the features such as downstream gene detection, helps
It is studied in genital tract bacterial diversity, and using genital tract flora as the genetic test application of target spot.
A kind of genital tract flora nucleic acid preservation liquid and preparation method thereof, according to above-mentioned genital tract flora nucleic acid preservation formula of liquid,
As shown in Figure 1, its implementation is as follows:
S01: weighing required Tris according to the above formula ratio, and with salt acid for adjusting pH to 5.0-10.0, it is slow that Tris-HCl is made
Fliud flushing；
S02: being dissolved in aseptic deionized water for the desired amount of ethylenediamine tetra-acetic acid, with salt acid for adjusting pH to 5.0-10.0, system
Obtain EDTA solution；
S03: being dissolved in aseptic deionized water for the desired amount of sodium chloride and sodium citrate, and be added Tris-HCl solution,
The mixing of EDTA solution adjusts pH value to 8.0-10.0, sterilization treatment with NaOH；
S04: above-mentioned solution is added in the desired amount of dehydrated alcohol, crosses miillpore filter degerming.
Preferably, further include S05: prepared preservation liquid being dispensed to probe tube, room temperature is strictly sealed.
Preferably, in third step, sterilization treatment is in 121 DEG C of high pressure sterilization 30min.
A kind of genital tract flora nucleic acid preservation liquid application method, according to above-mentioned genital tract flora nucleic acid preservation liquid preparation side
Method, which is characterized in that implementation method is as follows: to the preservation for filling valid genital tract flora nucleic acid preservation liquid and preparation method thereof preparation
In the probe tube of liquid, cotton swab head or cotton swab after genital tract sampling is added, sealing is protected from light immediately, controls temperature within 40 DEG C
It saves and/or transports.
Experimental data, referring to fig. 2-4:
1. acquiring the genital tract sample of 5 volunteers, preservation and nucleic acid extraction are carried out according to following 5 kinds of conditions respectively:
(1) extract immediately after sampling: 2 cotton swabs of acquisition or cotton swab genital tract sample carry out DNA extraction immediately；
(2) the direct cryopreservation of one week: 2 cotton swabs of acquisition or cotton swab genital tract sample are frozen immediately in -80 DEG C,
DNA extraction is carried out after saving 7 days；
(3) presses this preservation formula of liquid sample combination room temperature and cryopreservation one week: 2 cotton swabs of acquisition or cotton swab are raw
Grow sample, be immediately placed in it is isometric save liquid, be mixed by inversion, saved 3 days in 40 DEG C, then at -80 DEG C save 4 days after carry out
DNA is extracted；
(4) is pressed this preservation formula of liquid sample cryopreservation one week: 2 cotton swabs of acquisition or cotton swab genital tract sample are stood
It is put into isometric preservation liquid, is mixed by inversion, carries out DNA extraction after saving 7 days in 40 DEG C；
(5) after sampling, preservation liquid is not added, directly after 40 degree are placed 7 days, carries out DNA extraction；
2. genital tract sample process and nucleic acid extraction
(1) processing for the genital tract sample that is saved
The genital tract sample of fresh genital tract sample, the genital tract sample of direct cryopreservation and direct 40 degree of preservations:
Take 2 cotton swabs or cotton swab genital tract sample in centrifuge tube, addition 1.4mL Buffer ASL (DNA Stool
Kit, Qiagen, Germany), it shakes in vortex instrument to full and uniform；
It is stored in the genital tract sample saved in liquid: mixing is fullyd shake into whole Zhi Baocun pipe in vortex instrument, takes 300-
500 μ L mixtures, addition 1mL Buffer ASL (DNA Stool Kit, Qiagen, Germany), in vortex instrument
It is upper to shake to full and uniform；
(2) 5min is incubated in .70 DEG C of constant-temperature metal bath or water-bath；
(3) sufficient vortex 15 seconds, high speed centrifugation 1 minute, precipitated impurities；
(4) draws 1.2mL supernatant, discards precipitating；
(5) to each sample tube (supernatant) be added 1 tablet of InhibitEX Tablet tablet (DNA Stool
Kit, Qiagen, Germany), sufficient vortex 1 minute, dissolves tablet sufficiently immediately.Room temperature is incubated for 1 minute；
(6) high speed centrifugation 3 minutes draw whole supernatants, discard precipitating；
(7) to be added in each new blank pipe the above-mentioned supernatant solution of 200 μ L and 15 μ L Proteinase Ks (DNA
Stool Kit, Qiagen, Germany), it is uniformly mixed；200 μ L BufferAL are added, are vortexed again for being uniformly mixed；
(8) 10min is incubated in .70 DEG C of constant-temperature metal bath or water-bath；
(9) 500 μ L dehydrated alcohols are added in, and concussion mixes and micro- centrifugation；
(10) by the above mixed liquor be slowly added to QIAamp centrifugal column (DNA Stool Kit,Qiagen,
Germany), 1min is centrifuged with 20000rcf revolving speed, discards raffinate；
(11) opening QIAamp centrifugal column, 500 μ L Buffer AW1 of addition (DNA Stool Kit,
Qiagen, Germany), 1min is centrifuged with 20000rcf revolving speed, discards raffinate；
(12) opening QIAamp centrifugal column, 500 μ LbufferAW2 of addition (DNA Stool Kit,
Qiagen, Germany), 1min is centrifuged with 20000rcf revolving speed, discards raffinate；Repeated centrifugation is primary；
(13) QIAamp centrifugal column is put into new 2mL EPP and managed by, dries 2min at room temperature；
(14) be added 100 μ L Buffer ATE (DNA Stool Kit, Qiagen, Germany), in room
Temperature is incubated for 3min, is centrifuged 1min with 20000rcf revolving speed, eluted dna collects DNA solution；
Above 5 kinds of modes save genital tract sample and extract DNA result it is as shown in Figure 2.Extracted target DNA master tape
In 15Kb or so, if there is obvious light tone band in the position, and band shows successfully to be extracted the genital tract sample clearly without traction
Interior microbial DNA, DNA molecular integrality are not affected by destruction.The result shows that immediately in -80 degree after extracting and sample immediately
The extractable complete DNA out of the sample standard deviation frozen；The sample standard deviation that this preservation liquid is stored under the conditions of two kinds is extractable out
Complete DNA；And serious degradation has occurred in the sample of direct 40 degree of placements 7 days, DNA, without obvious main band, only in glue figure
There is lesser segment in lower section.Result explanation, this preservation liquid can be protected effectively under the two kinds of temperature preservation conditions tested
Microbial DNA is protected, extraction effect is good；
3. ribosomes 16S rRNA gene V3-V4 area's fragment amplification and sequencing
(1) reagent: High fidelity PCR polymerase system (Kapa Biosystems)
(2) primer sequence:
Upstream primer (341F): 5 '-ACTCCTACGGGAGGCAGCAG
Downstream primer (806R): 5 '-GGACTACHVGGGTWTCTAAT
(3) operating procedure: setting PCR total system is 30 μ L, including 15 μ L 2KAPA HiFi HotStartReadyMix
(Kapa Biosystems), each 0.2 μM of upstream and downstream primer, DNA profiling 30-50ng.It is carried out according to following PCR reaction condition anti-
It answers:
(4) segment obtained after amplification progress agarose gel electrophoresis is cut into glue purification, is sequenced using IlluminaMiSeq
Platform is sequenced with PE300 mode.
4. sequencing result is analyzed and is compared
(1) pass through above-mentioned experiment, we obtain the genital tract samples of 5 volunteers to measure after saving under the conditions of 4 kinds
Bacterial diversity sequencing result.By bioinformatic analysis, we have obtained the microorganism Alpha- multiplicity of each sample
Property analysis as a result, its result species richness (i.e. species number, with activity classification unit, Operational Taxonomic
Units, OTU number) and diversity indices (Shannon diversity index, ShannonDiversityIndex) Lai Daibiao.Such as Fig. 3
It is shown, the results showed that, the 1-4 sample of each volunteer, it may be assumed that immediately extract and sample after immediately in -80 degree freeze sample, point
The sample of this preservation liquid is not stored under the conditions of two kinds, flora richness (figure a) is consistent with diversity (figure b)；And each will
No. 5 samples of hope person, it may be assumed that before the sample of direct 40 degree of placements 7 days after sampling, flora richness and diversity are significantly lower than
No. 1-4 each sample is stated, is prompted due to DNA degradation, apparent decline has occurred in the species diversity that can be detected.The result is said
Bright, this preservation liquid can effectively reflect the species diversity information of flora under the two kinds of temperature preservation conditions tested.
(2) by bioinformatic analysis, the weighting UniFrac distance between different samples, and principal component point are calculated
It analyses (Principal Coordinates Analysis, PCoA), is compared.PCoA institute based on weighting UniFrac distance
The sample room correlation of display is as shown in Figure 4.Scheme the reproduction that upper each point represents 1 volunteer that a kind of preserving type obtains
The composition information of road flora.Two point distances are closer, and the composed structure for representing the two floras is more similar, and vice versa.Fig. 4's
The result shows that the 1-4 sample of each volunteer, it may be assumed that immediately extract and sample after immediately in -80 degree freeze sample, respectively at
The sample of this preservation liquid is stored under the conditions of two kinds, flora species composition is more like, and No. 5 samples of each volunteer, it may be assumed that takes
The sample of direct 40 degree of placements 7 days, flora species composition and appeal 1-4 differences between samples are larger after sample.Particularly, in 1-4
In st volunteer, under the conditions of observing to save two kinds of liquid preservation, flora species composition and the sample flora object extracted immediately
Kind composition is increasingly similar.
(3) pass through bioinformatic analysis and comparison, composition difference of the more different samples in Bacteriophyta categorization levels.
Bacterium relative abundance based on door level represents 1 volunteer that a kind of preserving type obtains as shown in figure 4, scheming upper each pillar
Genital tract flora information, the longitudinal axis represents the relative abundance of each Bacteriophyta in each sample.Fig. 4's the result shows that, it is each to volunteer
The 1-4 sample of person, it may be assumed that the sample that freezes immediately in -80 degree after extracting and sample immediately is stored under the conditions of two kinds
The sample of this preservation liquid, flora species composition is more like, and No. 5 samples of each volunteer, it may be assumed that direct 40 degree of placements after sampling
7 days samples, flora species composition and appeal 1-4 differences between samples are larger.
DNA extraction effect based on genital tract microorganism, diversity analysis, is detected detected bacterium richness
The composition of the Bacterial community, detected Bacteriophyta level that arrive, it may be concluded that genital tract flora nucleic acid of the present invention
It saves liquid and is used to save genital tract sample under condition of different temperatures, nucleic acid extraction effect and after acquiring fresh genital tract sample
The effect extracted nucleic acid immediately or extract nucleic acid after Cryopreservation immediately is consistent, and nucleic acid extraction effect is put better than direct room temperature
Postpone the effect for extracting nucleic acid.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Claims (6)
1. a kind of genital tract flora nucleic acid preservation formula of liquid, which is characterized in that including following components: concentration is 0.01-1M's
The sodium chloride that ethylenediamine tetra-acetic acid that Tris-HCl buffer, concentration are 0.01-1M, concentration are 0.01-0.5M, concentration are
The sodium citrate of 0.01-0.5M and the dehydrated alcohol of 10-30%.
2. genital tract flora nucleic acid preservation formula of liquid according to claim 1, which is characterized in that with the pH for postponing preservation liquid
Value is 8.0-10.0.
3. a kind of genital tract flora nucleic acid preservation liquid and preparation method thereof, genital tract flora core according to claim 1 to 2
Acid saves formula of liquid, which is characterized in that implementation method is as follows:
Step 1: weighing required Tris according to the above formula ratio, with salt acid for adjusting pH to 5.0-10.0, Tris-HCl buffering is made
Liquid；
Step 2: the desired amount of ethylenediamine tetra-acetic acid is dissolved in aseptic deionized water, with salt acid for adjusting pH to 5.0-10.0, it is made
EDTA solution；
Step 3: the desired amount of sodium chloride and sodium citrate are dissolved in aseptic deionized water, and Tris-HCl solution, EDTA is added
Solution mixing adjusts pH value to 8.0-10.0, sterilization treatment with NaOH；
Step 4: above-mentioned solution is added in the desired amount of dehydrated alcohol, miillpore filter degerming is crossed.
4. genital tract flora nucleic acid preservation liquid and preparation method thereof according to claim 3, which is characterized in that further include the 5th
Step: prepared preservation liquid is dispensed to probe tube, room temperature is strictly sealed.
5. genital tract flora nucleic acid preservation liquid and preparation method thereof according to claim 3 or 4, which is characterized in that the third
In step, sterilization treatment is in 121 DEG C of high pressure sterilization 30min.
6. a kind of genital tract flora nucleic acid preservation liquid application method, according to genital tract flora core as claimed in claim 3 to 5
Acid saves liquid and preparation method thereof, which is characterized in that implementation method is as follows: to equipped with according to the genital tract flora nucleic acid preservation liquid
In the probe tube of the preservation liquid of preparation method preparation, cotton swab head or cotton swab after genital tract sampling is added, sealing is protected from light immediately,
Control temperature is saved and/or is transported within 40 DEG C.
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| CN110129423A (en) * | 2019-05-27 | 2019-08-16 | 天益健康科学研究院（镇江）有限公司 | A method of utilizing high-throughput gene sequencing assessment genital tract flora health |
| 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 |
| 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 |
| CN112029824A (en) * | 2020-09-15 | 2020-12-04 | 北京康美天鸿生物科技有限公司 | Nucleic acid preservation solution universally used for multiple samples |
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