CN114806884A - Normal-temperature long-acting microorganism preserving fluid and preparation method, kit and application thereof - Google Patents
Normal-temperature long-acting microorganism preserving fluid and preparation method, kit and application thereof Download PDFInfo
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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
- C12N1/04—Preserving or maintaining viable microorganisms
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a normal-temperature long-acting microorganism preserving fluid, a preparation method thereof, a kit and application. The normal-temperature long-acting microorganism preserving fluid provided by the invention consists of a protease inhibitor, a chelating agent, a denaturant, a stabilizer, a biological buffer solution and water. The microorganism can be efficiently preserved for 14 days at room temperature without obvious change, and the room-temperature long-acting microorganism preserving fluid provided by the invention does not need to be additionally added with preservatives, such as sodium azide, Kathon preservative and the like, so that the microorganism preserving effect is better.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a normal-temperature long-acting microorganism preserving fluid, a preparation method, a kit and application thereof.
Background
At present, the microorganism preservation in clinical samples or animal experiments is usually performed by adopting a low-temperature freezing preservation mode. However, in practical application, great inconvenience exists, and in most cases, collected microbial human or animal samples cannot be immediately transferred to a deep low-temperature refrigerator or liquid nitrogen, and the propagation of microbes in the samples is easily caused under long-time room-temperature storage, so that the detection result is influenced.
Disclosure of Invention
Therefore, a room-temperature long-acting microorganism preservation solution, a preparation method thereof, a kit and application thereof are needed to be provided.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
firstly, the invention provides a normal-temperature long-acting microorganism preserving fluid, which consists of a protease inhibitor, a chelating agent, a denaturant, a stabilizer, a biological buffer solution and water; the protease inhibitor is 1-200mM guanidine isothiocyanate, the chelating agent is 1-200mM disodium ethylene diamine tetraacetate, the denaturant is 1-100mM sodium dodecyl sulfate and 10% -50% ethanol by volume fraction, the stabilizer is 1% -10% dimethyl sulfoxide and 0.1% -2% Tween-20 by volume fraction, and the biological buffer solution is 1-100mM tris (hydroxymethyl) aminomethane hydrochloride and 1-500mM sodium chloride.
Among them, guanidinium isothiocyanate acts as a protease inhibitor.
Disodium edetate is an important complexing/chelating agent which reacts with calcium to form a chelate, thereby efficiently preventing biological or enzymatic reactions, and is often used as an inhibitor of nucleases and proteases.
The ethanol and the sodium dodecyl sulfate are used as denaturants, so that organic substances such as proteins in the microorganism sample can be denatured, the bacteriostatic effect is achieved, and the abundance of the microorganisms is further prevented from changing.
Sodium chloride and tris hydrochloride were used as biological buffers.
Dimethyl sulfoxide is often used as a permeable cytoprotective agent, and even if a low concentration of dimethyl sulfoxide has an inhibitory effect on cell growth, the addition of dimethyl sulfoxide to the preservation solution can avoid changes in abundance and structural characteristics of microorganisms.
Tween-20 is a nonionic surfactant, and has emulsifying, dispersing, solubilizing, and stabilizing effects.
Preferably, the normal-temperature long-acting microorganism preservation solution is prepared from 20mM of guanidine isothiocyanate, 25mM of disodium ethylene diamine tetraacetate, 10mM of tris (hydroxymethyl) aminomethane hydrochloride, 17mM of sodium dodecyl sulfate, 154mM of sodium chloride and the following components in percentage by volume: 50% of ethanol, 0.5% of Tween-20 and 4% of dimethyl sulfoxide.
Preferably, the pH of the normal-temperature long-acting microorganism preservation solution is 8.0.
Further, the application of the normal-temperature long-acting microorganism preservation solution in microorganism sample preservation is provided.
Further, a preparation method of the normal-temperature long-acting microorganism preservation solution is provided, wherein 1-200mM of guanidinium isothiocyanate, 1-200mM of disodium ethylene diamine tetraacetate, 1-100mM of tris (hydroxymethyl) aminomethane hydrochloride, 1-100mM of sodium dodecyl sulfate, 1-500mM of sodium chloride are dissolved in double distilled water, and the preparation method comprises the following steps: 10 to 50 percent of ethanol, 0.1 to 2 percent of Tween-20 and 1 to 10 percent of dimethyl sulfoxide, and adjusting the pH.
Preferably, 20mM guanidine isothiocyanate, 25mM disodium ethylenediaminetetraacetate, 10mM tris hydrochloride, 17mM sodium dodecylsulfate, 154mM sodium chloride, in volume fraction: 50% ethanol, 0.5% Tween-20 and 4% dimethyl sulfoxide, and adjusting pH.
Preferably, the pH is adjusted to 8.0.
Further, a kit for preserving a microorganism sample is also provided, and the kit comprises the normal-temperature long-acting microorganism preserving fluid.
Further, the application of the kit for preserving the microbial sample in the preservation of the microbial sample is also provided.
Based on the technical scheme, the invention has the following beneficial effects:
the normal-temperature long-acting microorganism preserving fluid provided by the invention can be used for preserving microorganisms at normal temperature, can be stored for a long time and is simple and convenient to operate. Is a microorganism preserving fluid which can be efficiently preserved for 14 days at room temperature. At present, most of the normal-temperature preservation solution can be preserved for only 1 week. The preservation solution only contains the commonly used biochemical reagents, and the cost is relatively low. The preservative solution does not need to be additionally added with preservatives, such as sodium azide, kathon preservative and the like.
Drawings
FIG. 112 shows the results of the electrophoretic detection of the samples;
FIG. 212 results of Beta diversity analysis of samples.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
All materials, reagents and the like in the following examples are commercially available unless otherwise specified.
Example 1
The components and contents of the normal-temperature long-acting microorganism preservation solution in the embodiment are as follows:
1mM of guanidinium isothiocyanate, 1mM of disodium ethylene diamine tetraacetate (EDTA-Na2), 1mM of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 1mM of Sodium Dodecyl Sulfate (SDS), 1mM of sodium chloride, 10% of ethanol (volume fraction), 0.1% of Tween-20 (volume fraction) and 1% of dimethyl sulfoxide (volume fraction), and the balance of water.
The preparation method of the normal-temperature long-acting microbial preservation solution in the embodiment is as follows:
the pH was adjusted to 8.0 by dissolving 1mM guanidine isothiocyanate, 1mM disodium ethylenediaminetetraacetate (EDTA-Na2), 1mM Tris-hydroxymethyl aminomethane hydrochloride (Tris-HCl), 1mM Sodium Dodecyl Sulfate (SDS), 1mM sodium chloride, 10% ethanol (volume fraction), 0.1% Tween-20 (volume fraction) and 1% dimethylsulfoxide (volume fraction) in double distilled water.
Example 2
The components and contents of the normal-temperature long-acting microorganism preservation solution in the embodiment are as follows:
200mM of guanidinium isothiocyanate, 200mM of disodium ethylene diamine tetraacetate (EDTA-Na2), 100mM of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 100mM of Sodium Dodecyl Sulfate (SDS), 500mM of sodium chloride, 50% of ethanol (volume fraction), 2% of Tween-20 (volume fraction) and 10% of dimethyl sulfoxide (volume fraction), and the balance of water.
The preparation method of the normal-temperature long-acting microbial preservation solution in the embodiment is as follows:
200mM of guanidinium isothiocyanate, 200mM of disodium ethylenediaminetetraacetate (EDTA-Na2), 100mM of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 100mM of Sodium Dodecyl Sulfate (SDS), 500mM of sodium chloride, 50% of ethanol (volume fraction), 2% of Tween-20 (volume fraction) and 10% of dimethylsulfoxide (volume fraction) were dissolved in double distilled water, and the pH was adjusted to 8.0.
Example 3
The components and contents of the normal-temperature long-acting microorganism preservation solution in the embodiment are as follows:
20mM of guanidinium isothiocyanate, 25mM of disodium ethylene diamine tetraacetate (EDTA-Na2), 10mM of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 17mM of Sodium Dodecyl Sulfate (SDS), 154mM of sodium chloride, 50% of ethanol (volume fraction), 0.5% of Tween-20 (volume fraction) and 4% of dimethyl sulfoxide (volume fraction), and the balance of water.
The preparation method of the normal-temperature long-acting microbial preservation solution in the embodiment is as follows:
the pH was adjusted to 8.0 by dissolving 20mM guanidine isothiocyanate, 25mM disodium ethylenediaminetetraacetate (EDTA-Na2), 10mM Tris-hydroxymethyl aminomethane hydrochloride (Tris-HCl), 17mM Sodium Dodecyl Sulfate (SDS), 154mM sodium chloride, 50% ethanol (volume fraction), 0.5% Tween-20 (volume fraction) and 4% dimethylsulfoxide (volume fraction) in double distilled water.
Example 4
In this example, the preservation effect was examined using the long-acting normal-temperature microbial preservation solution provided in example 3.
1. Collecting samples:
fecal samples were collected from 3 volunteers, each volunteer sample was divided into 4 aliquots for 4 different treatments:
(1) immediate processing (Day 1): at least 0.3g of fecal sample was collected and mixed well with 1mL of microbial stock solution. Microbial DNA was extracted and sequenced from the 16SrDNA V3-V4 region.
(2) Storage at ambient temperature for 4 days (Day 4): at least 0.3g of a fecal sample is collected, mixed with 1mL of a microbial preservation solution uniformly, and stored for 4 days at normal temperature. Microbial DNA was then extracted and sequenced from the 16SrDNA V3-V4 region.
(3) Storage at ambient temperature for 7 days (Day 7): at least 0.3g of a fecal sample is collected, mixed with 1mL of a microbial preservation solution uniformly, and stored for 7 days at normal temperature. Microbial DNA was then extracted and sequenced from the 16SrDNA V3-V4 region.
(4) Storage at ambient temperature for 14 days (Day 14): at least 0.3g of a fecal sample is collected, mixed with 1mL of a microbial preservation solution uniformly, and stored for 14 days at normal temperature. Microbial DNA was then extracted and sequenced from the 16SrDNA V3-V4 region.
2. Extracting sample microorganism DNA:
transferring the sample to a 2.0mL centrifuge tube, pouring the grinding beads into the sample tube, and adding 700. mu.L of SL 2; add 150. mu.L of Enhancer SX and mix by vortexing. And (3) placing the sample tube into a shaking and mixing instrument, and shaking at 70 ℃, 1000-1200rpm for 10min to promote sample cracking. The lysed sample was centrifuged at 12000rpm for 2min, and approximately 700. mu.L of the supernatant was transferred to a new 2.0mL inlet centrifuge tube, 150. mu.L of SL3 was added, the mixture was inverted and incubated at 4 ℃ for 5 min. And centrifuging again. After the inhibitor was removed by filtration, 250. mu.L of Buffer SB was added to the filtrate, and the mixture was gently aspirated and mixed to carry out DNA binding reaction.
And then cleaning and drying the silicon substrate membrane, starting a vacuum pump, adjusting the vacuum degree to the maximum value, continuously pumping for 10-15min to ensure that the column membrane is completely dried, and sucking residual washing liquid at the lower end of the column on water absorption paper.
In the DNA elution step, the "MICROTUBE RACK" pad was first inserted into the vacuum chamber with its notched side facing up, and then the RACK of Tube Strips was placed on the pad. Covering the vacuum box cover; the NucleoSpin Soil Binding Plate is placed in the Plate groove on the cover again, 80 mu L of Buffer SE preheated to 70 ℃ is added to the center of the column membrane, and the elution effect is promoted by standing for 5min at room temperature. Finally the vacuum pump (-0.2to-0.6bar) was turned on until all liquid had flowed through the plate. The DNA was transferred to a new 1.5mL inlet centrifuge tube for microbial diversity pooling sequencing.
3. Library construction and sequencing:
a two-step library building method is adopted, DNA is used as a template in the first step, a primer with a joint is designed for carrying out PCR, and a PCR product in the first step is used as a template for carrying out PCR in the second step. The purpose of the primer adapter is to facilitate the addition of barcode/index during the second library construction.
(1) First, a target region PCR reaction is performed. The reaction system is as follows:
| name of reagent | Dosage of |
| Genomic DNA | 50ng±20％ |
| *Vn F(10μM) | 0.3 |
| *Vn R(10μM) | 0.3 |
| KOD FX Neo Buffer | 5 |
| dNTP(2mM each) | 2 |
| KOD FX Neo | 0.2 |
| ddH 2 O | Make up to 10 μ l |
And (3) PCR reaction conditions:
95℃5min；
25 cycles of 95 ℃ 30sec, 50 ℃ 30sec, 72 ℃ 40 sec;
72℃7min；
4℃∞。
(2)Solexa PCR
| name of reagent | Concentration of | Dosage of |
| PCR purified product of target region | - | 5μl |
| *MPPI-a | 2μM | 2.5μl |
| *MPPI-b | 2μM | 2.5μl |
| 2×Q5 HF MM | 10μl | |
| Total | 20μl |
Reaction conditions are as follows:
98℃30sec；
98℃10sec；
30sec at 65 ℃ and 30sec at 72 ℃ for 10 cycles;
72℃5min。
(3) quantification, sample mixing and purification
And mixing the PCR products in the steps according to the electrophoresis quantitative result in a mass ratio of 1: 1. After mixing, column purification was performed using an OMEGA DNA purification column.
(4) Cut rubber recovery and on-machine sequencing
After electrophoresis on a 1.8% agarose gel at 120V for 40min, the desired fragment was excised and recovered. The sequencing library formed by purification, quantification and homogenization is subjected to library quality inspection, and the library qualified by quality inspection is sequenced by Illumina Novaseq 6000. An original image data file obtained by high-throughput sequencing (such as a sequencing platform of Illumina Novaseq and the like) is analyzed and converted into an original sequencing sequence (Sequenced Reads) through Base recognition (Base Calling), and a result is stored in a FASTQ (fq for short) file format, wherein the sequence information of the sequencing sequence (Reads) and corresponding sequencing quality information are contained.
4. Microbial diversity data analysis:
(1) performing quality control on the original sequencing sequence, including low-quality filtration and length filtration to obtain a high-quality sequence;
(2) clustering/denoising the high-quality sequence, dividing OTUs/ASVs (hereinafter collectively referred to as Feature), and obtaining species classification according to the sequence composition of the Feature;
(3) performing taxonomic analysis on the sample at each classification level (genus of compendia) based on the Feature (Feature) analysis result;
(4) the diversity of species in a single sample is researched through Alpha diversity analysis, and Ace, Chao1, Shannon and Simpson indexes of each sample are counted;
(5) the difference sizes of different samples in terms of species diversity (colony composition and structure) were compared by Beta diversity analysis.
5. As a result:
as shown in FIG. 1, PCR electrophoresis analysis shows that 12 samples are qualified, the quality meets the requirement of library construction, and the total amount meets the requirement of library construction for 2 times and more.
The results in Table 1 indicate that the percentage of the bases with the mass value of more than or equal to 20 in the total number of bases exceeds 98%, and the sequencing result is qualified.
TABLE 1 statistics of sample sequencing data processing results
The results in Table 2 suggest that Alpha diversity in the microbial samples remained consistent for the 3 volunteer samples under the 4 treatment conditions.
TABLE 2 Alpha diversity index statistics
As shown in fig. 2, 4 samples of the same volunteer were clustered in a consistent manner, and grouped into individuals, with small intra-group variation and heterogeneity. UPGMA analysis, i.e. non-weighted group mean method, can also be understood as hierarchical clustering of samples, which is a common method of cluster analysis. Based on four distance matrixes obtained by Beta diversity analysis, hierarchical clustering is carried out on samples by adopting a non-weighted pairwise averaging method (UPGMA) through an R language tool so as to judge the similarity of species composition among the samples. The sample hierarchical clustering tree is shown in FIG. 2: the closer the samples are, the shorter the branch length, indicating that the species composition of the two samples is more similar.
By combining the results, the normal-temperature long-acting microorganism preservation solution can effectively ensure that the abundance of the microorganisms in the sample is not obviously changed within 14 days.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. The normal-temperature long-acting microorganism preservation solution is characterized by consisting of a protease inhibitor, a chelating agent, a denaturant, a stabilizer, a biological buffer solution and water; the protease inhibitor is 1-200mM guanidine isothiocyanate, the chelating agent is 1-200mM disodium ethylene diamine tetraacetate, the denaturant is 1-100mM sodium dodecyl sulfate and 10% -50% ethanol by volume fraction, the stabilizer is 1% -10% dimethyl sulfoxide and 0.1% -2% Tween-20 by volume fraction, and the biological buffer solution is 1-100mM tris (hydroxymethyl) aminomethane hydrochloride and 1-500mM sodium chloride.
2. The normal-temperature long-acting microorganism preservation solution according to claim 1, wherein the normal-temperature long-acting microorganism preservation solution is prepared from 20mM of guanidinium isothiocyanate, 25mM of disodium ethylene diamine tetraacetate, 10mM of tris (hydroxymethyl) aminomethane hydrochloride, 17mM of sodium dodecyl sulfate, 154mM of sodium chloride, and the following components in percentage by volume: 50% of ethanol, 0.5% of Tween-20 and 4% of dimethyl sulfoxide.
3. The ambient-temperature long-acting microbe preservation solution according to claim 1 or 2, wherein the PH of the ambient-temperature long-acting microbe preservation solution is 8.0.
4. Use of a long-acting normal-temperature microbial preservation solution according to any one of claims 1 to 3 in preservation of a microbial sample.
5. A preparation method of a normal-temperature long-acting microorganism preservation solution is characterized in that double distilled water is used for dissolving 1-200mM of guanidinium isothiocyanate, 1-200mM of disodium ethylene diamine tetraacetate, 1-100mM of tris (hydroxymethyl) aminomethane hydrochloride, 1-100mM of sodium dodecyl sulfate, 1-500mM of sodium chloride, and the components are calculated according to volume fractions: 10 to 50 percent of ethanol, 0.1 to 2 percent of Tween-20 and 1 to 10 percent of dimethyl sulfoxide, and adjusting the pH.
6. The method according to claim 5, wherein 20mM of guanidinium isothiocyanate, 25mM of disodium ethylenediaminetetraacetate, 10mM of tris hydrochloride, 17mM of sodium dodecylsulfate, 154mM of sodium chloride, in terms of volume fraction: 50% ethanol, 0.5% Tween-20 and 4% dimethyl sulfoxide, and adjusting pH.
7. The method according to claim 5 or 6, wherein the pH is adjusted to 8.0.
8. A kit for preserving a microbial sample, comprising the ambient-temperature long-acting microbial preservation solution according to any one of claims 1 to 3.
9. Use of the kit for preservation of a microbial sample according to claim 8 in preservation of a microbial sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP4414458A1 (en) * | 2023-02-07 | 2024-08-14 | Senckenberg Gesellschaft Für Naturforschung | Collect & extractct solution for collection and storage of biological sample types for extraction of biomolecules |
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