DNA (deoxyribonucleic acid) preserving fluid suitable for multiple types of microbial samples and application of DNA preserving fluid

Abstract

The invention discloses DNA (deoxyribonucleic acid) preserving fluid suitable for multiple types of microbial samples and application, and belongs to the technical field of microbial sample preservation. The DNA preserving fluid is suitable for various types of microorganism samples, including all solid, liquid and trace microorganism samples well known by technicians in the field, and the DNA preserving fluid can preserve microorganisms of different types of samples for 28-30 days in a room temperature environment and can preserve microorganisms of different types of samples for 7-28 days in a 37 DEG C environment. The DNA preserving fluid is simple in preparation process and low in cost, meets the requirements of rapid and low-cost sample preservation in different environments, and is suitable for large-scale biological sample preservation.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial sample preservation, and in particular relates to a DNA preservation solution suitable for multiple types of microbial samples and its application. Background Art

[0002] As the importance of microbial research in the development of diseases becomes increasingly prominent, many common diseases such as diabetes, asthma, chronic diarrhea, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), obesity, and low immunity have been found to be closely related to the imbalance of microbial distribution balance. The composition of human microorganisms can be analyzed through sample collection, extraction of total microbial DNA, QPCR, 16SrRNA, metagenomic sequencing, and a series of bioinformatics, and the relationship between microbiota and human health and diseases can be further studied. However, the composition of microbial samples is very complex. After leaving the human body, the environment in which they are located has undergone great changes. Some bacteria die due to drastic changes in the environment, while some bacteria remain active and continue to metabolize and grow, which will have a great impact on the subsequent microbial data analysis. In addition, microbial samples contain a large amount of digestion residues and various nucleic acid degrading enzymes produced by bacteria, such as proteases, DNA enzymes (DNases) and RNA enzymes (RNases), and various PCR inhibitors (such as bile salts, bilirubin, humus, and pigments), making the cells and nucleic acids in microbial samples extremely vulnerable to damage and degradation. In addition, sample collection, transportation, storage, DNA extraction, sequencing and biometric analysis will interfere with microbial diversity and composition. Therefore, how to accurately and quickly obtain and preserve samples is of far-reaching significance for the study of human microorganisms.

[0003] Cryopreservation (-80°C) is considered the gold standard in most biological sample microbiological studies. However, due to the sampling environment and special equipment for preserving samples, the scope of application of cryopreservation is fixed, and the equipment investment and operating costs are high. The market demand for room temperature storage samples is increasing. At present, commercial and experimental stool sample preservation reagents have been developed. The microbial preservation solution provided by Chinese patent CN115466682 A provides a carrier for microbial preservation, which can effectively protect the activity of microorganisms and improve the survival rate and activity of bacteria; Chinese patent CN107988076A discloses a stool preservation solution and its application, which can stabilize the number, composition and abundance of microorganisms in stool samples, ensure the accuracy of sequencing analysis of human intestinal flora, and realize room temperature storage of stool samples; Chinese patent CN109750029A invents a non-frozen urine DNA preservation solution suitable for the detection of reproductive tract pathogens, which can preserve urine at room temperature and is suitable for extracting DNA for the detection of reproductive tract pathogens. The above-mentioned patented preservation solutions all have disadvantages such as single applicable sample type, complex preservation solution composition, and high cost, and have certain limitations in use.

[0004] To solve the above problems, the present invention provides a microbial DNA preservation solution that can effectively maintain the stability of microorganisms in solid microbial samples (feces), liquid microbial samples (saliva microorganisms) and trace microbial samples (skin microorganisms, oral mucosal microorganisms, dental plaque microorganisms, female reproductive tract microorganisms, etc.), reduce the degradation of microbial DNA nucleic acid, and realize the room temperature storage of different types of samples. The solution is applicable to a wide range of samples. Summary of the invention

[0005] One object of the present invention is to provide a DNA preservation solution for preserving multiple types of microbial samples and a preparation method thereof; a second object of the present invention is to provide the use of the DNA preservation solution; and a further object of the present invention is to provide a method for preserving microbial samples using the DNA preservation solution.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] In the first aspect of the present invention, the present invention provides a microbial sample DNA preservation solution, characterized in that each liter of the DNA preservation solution comprises the following components: 5-30% (v / v) glycerol, 0.02-0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.01-0.1 mol / L tris(hydroxymethyl)aminomethane, 0.01-0.05 mol / L nucleic acid stabilizer or nuclease inhibitor, hydrochloric acid to adjust the pH to 5.6-7.6, and the balance is water.

[0008] The nucleic acid stabilizer is selected from one or a combination of two or more of sodium pyrophosphate, sodium tripolyphosphate, sodium ascorbate, sodium hexametaphosphate, trisodium phosphate, and sodium citrate.

[0009] The nuclease inhibitor is selected from one or a combination of two or more of diethyl pyrophosphate (DEPC), guanidine isothiocyanate, and urea.

[0010] Preferably, each liter of DNA preservation solution consists of the following components: 5-30% (v / v) glycerol, 0.02-0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.01-0.1 mol / L tris(hydroxymethyl)aminomethane, 0.01-0.05 mol / L nucleic acid stabilizer, hydrochloric acid to adjust the pH to 5.6-7.6, and the balance is water; the nucleic acid stabilizer is selected from one of sodium pyrophosphate, sodium tripolyphosphate, and sodium ascorbate.

[0011] More preferably, each liter of DNA preservation solution consists of the following components: 5-15% (v / v) glycerol, 0.05-0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.06-0.1 mol / L tris(hydroxymethyl)aminomethane, 0.01-0.03 mol / L nucleic acid stabilizer, hydrochloric acid to adjust the pH to 7.0-7.5, and the balance is water; the nucleic acid stabilizer is selected from sodium pyrophosphate.

[0012] In a specific embodiment of the present invention, each liter of DNA preservation solution is composed of the following components: 5% (v / v) glycerol, 0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.1 mol / L tris(hydroxymethyl)aminomethane, 0.03 mol / L sodium pyrophosphate, hydrochloric acid to adjust the pH to 7.5, and the balance is water.

[0013] In a specific embodiment of the present invention, each liter of DNA preservation solution is composed of the following components: 10% (v / v) glycerol, 0.05 mol / L disodium ethylenediaminetetraacetic acid, 0.06 mol / L tris(hydroxymethyl)aminomethane, 0.01 mol / L sodium pyrophosphate, hydrochloric acid to adjust the pH to 7.1, and the balance is water.

[0014] In a specific embodiment of the present invention, each liter of DNA preservation solution is composed of the following components: 15% (v / v) glycerol, 0.1 mol / L disodium ethylenediaminetetraacetic acid, 0.08 mol / L tris(hydroxymethyl)aminomethane, 0.02 mol / L sodium pyrophosphate, hydrochloric acid to adjust the pH to 7.1, and the balance is water.

[0015] In a second aspect of the present invention, the present invention provides a method for preparing a microbial sample DNA preservation solution, characterized in that the method comprises the following steps:

[0016] (1) Based on 1 liter of DNA preservation solution, take 50-300 ml of glycerol, 0.02-0.2 mol of disodium ethylenediaminetetraacetate, 0.01-0.1 mol of tris(hydroxymethyl)aminomethane, and 0.01-0.05 mol of sodium pyrophosphate;

[0017] (2) dissolving sodium pyrophosphate in a portion of sterile water, filtering the solution through a microfiltration membrane with a pore size of 0.22 μm, and setting aside;

[0018] (3) Take some sterile water to dissolve disodium ethylenediaminetetraacetate and tris(hydroxymethylaminomethane), mix with glycerol, sterilize in a high-pressure steam autoclave, cool to room temperature, add the sodium pyrophosphate solution in step (2), and use sterile water to make up the volume to 1 L.

[0019] Preferably, in step (3), the sterilization pressure of the high-pressure steam sterilizer is 103.4-104.4 kPa, the temperature is 121.3-122.0° C., and the sterilization time is 15-20 min.

[0020] In the most preferred embodiment of the present invention, the sterilization pressure of the high-pressure steam sterilizer is 103.4 kPa, the temperature is 121.3° C., and the sterilization time is 15 min.

[0021] In a third aspect of the present invention, the present invention provides a use of a DNA preservation solution for preserving microbial samples under non-freezing conditions.

[0022] The non-freezing condition is selected from the storage condition with a temperature > 0°C.

[0023] In a preferred embodiment of the present invention, the non-freezing condition is selected from a storage condition with a temperature of 25-37°C.

[0024] The microbial sample is selected from solid microbial samples, liquid microbial samples and trace microbial samples well known to those skilled in the art.

[0025] In some embodiments of the present invention, the solid microbial sample is selected from a stool sample.

[0026] In some embodiments of the present invention, the liquid microbial sample is selected from a blood sample, a saliva sample, a urine sample or a cerebrospinal fluid sample.

[0027] In some embodiments of the present invention, the trace sample is selected from a skin tissue sample, an oral mucosa sample, a dental plaque sample or a reproductive tract secretion sample.

[0028] In a fourth aspect of the present invention, a method for preserving a microbial sample is provided, characterized in that the DNA preservation solution provided by the present invention is mixed with the microbial sample in a volume ratio of (1-5):1 and preserved.

[0029] Preferably, it is stored for 28-30 days at room temperature and for 7-28 days at 37°C.

[0030] The technical solution provided by the present invention has the following technical advantages:

[0031] A DNA preservation solution is prepared according to the formula and preparation method provided by the present invention. The preservation solution and a microbial sample are mixed in a ratio of 1:1 to 5:1. The preservation solution can quickly penetrate into the microorganisms in the sample at room temperature, so that the microorganisms in the sample are in a semi-dissolved state and stop growing; at the same time, the enzymes in the microorganisms are quickly inactivated, the rate of DNA degradation is reduced, the quality and integrity of DNA are maintained, and the changes in the abundance of microorganisms in the sample are controlled, thereby achieving the purpose of transporting or storing the sample at room temperature.

[0032] The DNA preservation solution provided by the present invention is suitable for various types of microbial samples, including all solid, liquid and trace microbial samples familiar to those skilled in the art, and is suitable for sample preservation in a non-frozen environment, such as at room temperature, it can preserve microorganisms of different types of samples for 28-30 days, and at 37°C, it can preserve microorganisms of different types of samples for 7-28 days. The preservation solution provided by the present invention can maintain the quality and integrity of sample DNA under non-freezing conditions, and control the changes in the abundance of sample microorganisms, thereby achieving the purpose of transporting or storing samples at room temperature. The preparation process of the preservation solution is simple and low-cost, which meets the needs of fast and low-cost sample preservation in different environments, and is suitable for large-scale biological sample preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Statistical results of DNA concentration in fecal samples with different treatments.

[0034] Figure 2 Statistical results of DNA absorbance in fecal samples with different treatments.

[0035] Figure 3 Preservation of stool samples; A: DNA integrity of different treatments, from left to right: fresh samples, samples stored in preservation solution (-20, 25, 37°C) for 0, 7, 30, and 60 days, M stands for Maker; B: Microbial taxonomic composition (genus) of samples with different treatments, X represents fresh stool samples, letter B represents stool samples stored in preservation solution, and the numbers after the letters represent the number of storage days and temperature, respectively; for example, B725: represents stool samples stored in 25°C preservation solution for 7 days, the same naming method below; C: Alpha diversity index of microorganisms in samples with different treatments; D: Beta diversity of microorganisms in samples with different treatments.

[0036] Figure 4 Oral sample preservation; A: DNA integrity of different treatments, from left to right, samples stored in preservation solution (-20, 25, 37℃) for 0, 7, 30, 60 days, M is Maker; B: Microbial taxonomic composition (genus) of samples with different treatments, K represents oral samples stored in preservation solution, J represents oral samples stored in competitor preservation solution; the numbers after the letters represent the number of storage days and temperature, respectively; for example, S725: represents oral samples stored in 25℃ preservation solution for 7 days, the naming method is the same below; C: Alpha diversity index of microorganisms in samples with different treatments; D: Beta diversity of microorganisms in samples with different treatments.

[0037] Figure 5Skin sample preservation; A: DNA integrity of different treatments, from left to right: samples stored in preservation solution (-20, 25, 37°C) for 0, 7, 30, and 60 days, M stands for Maker; B: Microbial taxonomic composition (genus) of samples with different treatments, P represents skin samples stored in preservation solution, and J represents skin samples stored in competitor preservation solution; the numbers after the letters represent the number of storage days and temperature, respectively; for example, P725: represents a skin sample stored in 25°C preservation solution for 7 days, and the naming method is the same below; C: Microbial Alpha diversity index of samples with different treatments; D: Microbial Beta diversity of samples with different treatments.

[0038] Figure 6 Preservation of reproductive tract samples; A: DNA integrity of different treatments, from left to right, samples stored in preservation solution (-20, 25, 37℃) for 0, 7, 30, and 60 days, M is Maker; B: Microbial taxonomic composition (genus) of samples with different treatments, S represents reproductive tract samples stored in preservation solution, and J represents reproductive tract samples stored in competitor preservation solution; the numbers after the letters represent the number of storage days and temperature, respectively; for example, S725: represents reproductive tract samples stored in 25℃ preservation solution for 7 days, the naming method is the same below; C: Alpha diversity index of microorganisms in samples with different treatments; D: Beta diversity of microorganisms in samples with different treatments.

[0039] Figure 7 Preservation of saliva samples; A: DNA integrity of different treatments, from left to right: samples stored in preservation solution (-20, 25, 37°C) for 0, 7, 30, and 60 days, M stands for Maker; B: Microbial taxonomic composition (genus) of samples with different treatments, T represents saliva samples stored in preservation solution, and the numbers after the letters represent the number of storage days and temperature, respectively; for example, T725: represents a saliva sample stored in 25°C preservation solution for 7 days, and the naming method is the same below; C: Alpha diversity index of microorganisms in samples with different treatments; D: Beta diversity of microorganisms in samples with different treatments.

[0040] Figure 8 Preservation of dental plaque samples; A: DNA integrity of different treatments, from left to right, samples stored in preservation solution (-20, 25, 37℃) for 0, 7, 30, and 60 days, M is Maker; B: Microbial taxonomic composition (genus) of samples with different treatments, Y represents dental plaque samples stored in preservation solution, and J represents dental plaque samples stored in competitor preservation solution; the numbers after the letters represent the number of storage days and temperature, respectively; for example, Y725: represents dental plaque samples stored in 25℃ preservation solution for 7 days, the naming method is the same below; C: Microbial Alpha diversity index of samples with different treatments; D: Microbial Beta diversity of samples with different treatments. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1 Microbial sample DNA preservation solution

[0043] Each liter of DNA preservation solution is composed of the following components: 10% (v / v) glycerol, 0.05 mol / L disodium ethylenediaminetetraacetic acid, 0.06 mol / L tris(hydroxymethyl)aminomethane, 0.01 mol / L sodium pyrophosphate, hydrochloric acid to adjust the pH to 7.1, and the balance is water.

[0044] Preparation method: Dissolve sodium pyrophosphate in some sterile water, filter it with a microfiltration membrane with a pore size of 0.22 μm, and set aside; Dissolve disodium ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane in some sterile water, mix with glycerol, and place in a high-pressure steam sterilizer at a pressure of 103.4 kPa (1.05 kg / cm 2 ), sterilize at 121.3°C for 15 min, cool to room temperature, add sodium pyrophosphate solution, adjust pH to 7.1 with hydrochloric acid, and make up the volume to 1 L with sterile water.

[0045] Example 2 Microbial sample DNA preservation solution

[0046] Each liter of DNA preservation solution is composed of the following components: 15% (v / v) glycerol, 0.1 mol / L disodium ethylenediaminetetraacetic acid, 0.08 mol / L tris(hydroxymethyl)aminomethane, 0.02 mol / L sodium pyrophosphate, hydrochloric acid to adjust the pH to 7.1, and the balance is water.

[0047] Preparation method: Dissolve sodium pyrophosphate in some sterile water, filter it with a microfiltration membrane with a pore size of 0.22 μm, and set aside; Dissolve disodium ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane in some sterile water, mix with glycerol, and place in a high-pressure steam sterilizer at a pressure of 103.4 kPa (1.05 kg / cm 2 ), sterilize at 121.3°C for 15 min, cool to room temperature, add sodium pyrophosphate solution, adjust pH to 7.1 with hydrochloric acid, and make up the volume to 1 L with sterile water.

[0048] Example 3 Microbial sample DNA preservation solution

[0049] Each liter of DNA preservation solution is composed of the following components: 5% (v / v) glycerol, 0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.1 mol / L tris(hydroxymethyl)aminomethane, 0.03 mol / L sodium pyrophosphate, hydrochloric acid to adjust the pH to 7.5, and the balance is water.

[0050] Preparation method: Dissolve sodium pyrophosphate in some sterile water, filter it with a microfiltration membrane with a pore size of 0.22 μm, and set aside; Dissolve disodium ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane in some sterile water, mix with glycerol, and place in a high-pressure steam sterilizer at a pressure of 103.4 kPa (1.05 kg / cm 2 ), sterilize at 121.3°C for 15 min, cool to room temperature, add sodium pyrophosphate solution, adjust pH to 7.5 with hydrochloric acid, and make up the volume to 1 L with sterile water.

[0051] Example 4 Microbial sample DNA preservation solution

[0052] The preparation method and raw materials are the same as those in Example 1, except that sodium pyrophosphate is replaced with sodium tripolyphosphate of the same molar mass to prepare preservation solution 4.

[0053] Example 5 Microbial sample DNA preservation solution

[0054] The preparation method and raw materials are the same as those in Example 1, except that sodium pyrophosphate is replaced with sodium ascorbate of the same molar mass to prepare preservation solution 5.

[0055] Example 6 Microbial Sample DNA Preservation Solution

[0056] The preparation method and raw materials are the same as those in Example 1, except that sodium pyrophosphate is replaced with diethyl pyrophosphate (DEPC) of the same molar mass to prepare preservation solution 6.

[0057] Example 7 Microbial Sample DNA Preservation Solution

[0058] The preparation method and raw materials are the same as those in Example 1, except that sodium pyrophosphate is replaced with guanidine isothiocyanate of the same molar mass to prepare preservation solution 7.

[0059] Fresh feces were mixed with sterile water and preservation solutions 1-7 in a ratio of 1:3 to form fresh sample + sterile water group, fresh sample + preservation solution group 1, fresh sample + preservation solution group 2, fresh sample + preservation solution group 3, fresh sample + preservation solution group 4, fresh sample + preservation solution group 5, fresh sample + preservation solution group 6, and fresh sample + preservation solution group 7. Three replicates were set up in parallel for each group. Among them, the fresh sample + sterile water group was stored at -20℃ for 7 days, and the other treatment groups were stored at 25℃ for 7 days. After storage for 7 days, 500μl of the mixed solution was taken and centrifuged at 12500 rpm for 4min for DNA extraction. DNA extraction was extracted using a commercial fecal nucleic acid extraction kit. The test method was referred to the instruction manual. The DNA concentration and quality were detected using a spectrophotometer. The test results are shown in Table 1.

[0060] Table 1 DNA concentration and quality of fecal samples treated with different methods

[0061]

[0062] From Table 1, Figure 1-2 The statistical results show that there is no significant difference in DNA concentration and quality between the feces samples stored at room temperature for 7 days in the preservation solutions 1-3 provided by the present invention and the feces samples stored at -20°C for 7 days. This indicates that the preservation solution for microbial sample preservation provided by the present invention is consistent with the effect of the current gold standard, and the preservation solution provided by the present invention can be used for sample preservation at room temperature, with low storage cost and convenient transportation.

[0063] In the above preservation solutions, sodium pyrophosphate, sodium tripolyphosphate and sodium ascorbate are all nucleic acid stabilizers, which provide salt ions and precipitate after binding with DNA. They also have emulsification, dispersibility, prevent fat oxidation, improve protein binding, and have the ability to remove contaminants from different types of samples. By comparing the nucleic acid stabilization effects of preservation solution 1 and preservation solutions 4 and 5, it can be found that the use of sodium pyrophosphate as a nucleic acid stabilizer has the best effect.

[0064] When the nucleic acid stabilizer of the present invention is replaced with commonly used nuclease inhibitors in the art, such as DEPC and guanidine isothiocyanate, by comparing the preservation effects, it can be found that the preservation effect of nuclease inhibitors on stool samples is poor. The inventor analyzed the reasons and believed that DEPC denatures proteins by binding to the imidazole ring of histidine, the active group of RNase, thereby inhibiting the activity of the enzyme. It is a strong but incomplete RNase inhibitor and is carcinogenic; guanidine isothiocyanate mainly inhibits nucleases released by cells from degrading DNA by lysing cells and denaturing proteins in lysed cells, but guanidine salts are unstable in light and heat, and the action time is limited, so the long-term preservation effect is not good.

[0065] In the following application experiments, the inventors used preservation solution 1 to preserve stool samples, oral mucosa samples, skin samples, reproductive tract samples, saliva samples and dental plaque samples, tested the DNA integrity of biological samples after preservation, and analyzed the changes in the abundance of microorganisms in the samples. The specific experimental process and results are as follows:

[0066] Application Example 1: Stool Sample Storage

[0067] 1.1 Feces preservation solution system

[0068] Mix fresh feces with preservation solution 1 in a ratio of 1:3, take 3 portions at different storage time points (0 day, 7 days, 30 days, 60 days) under different temperature conditions (-20, 25, 37°C), take 500 μl after expiration, centrifuge at 12500 rpm for 4 min for DNA extraction; DNA is extracted using a commercial fecal nucleic acid extraction kit, and the experimental method refers to the instruction manual.

[0069] 1.2 Analysis of microbial DNA quality and integrity

[0070] The DNA concentration and quality were detected by spectrophotometer, and the DNA integrity was tested by agarose gel electrophoresis.

[0071] 1.3 Analysis of microbial abundance

[0072] The genomic DNA of the samples stored under the above-mentioned different conditions and the DNA of the samples stored at -80°C without adding preservation solution were taken for 16s amplicon sequencing (V3 / V4 region) to analyze the changes in the abundance of microorganisms in the samples.

[0073] 1.4 Storage results of storage fluid (feces)

[0074] DNA quality of stool samples stored in preservation solution 1 at different temperatures (-20, 25, 37°C) for 0, 7, 30, and 60 days ( Figure 3 A) No significant difference, the DNA yield of the sample did not deviate from the yield of the control group. The composition distribution of each sample at the genus level is visualized as above Figure 3 As shown in B, the bar graph shows that there is no significant difference in genus and species among the fecal samples stored in the preservation solution for 7 days, 30 days, and 60 days at different temperatures (-20, 25, and 37°C). In order to comprehensively evaluate the alpha diversity of the microbial community, Chao1 and Observed species indices were used to characterize the richness. The results showed that ( Figure 3 C): There was no significant difference in the microbial Alpha diversity of fecal samples stored in the preservation solution for 7 days, 30 days, and 60 days compared with the control under different temperatures (-20, 25, and 37°C). Intergroup difference analysis showed that ( Figure 3 D): There was no significant difference between each treatment and the initial control sample.

[0075] Application Example 2: Oral Sample Preservation

[0076] 2.1 Oral sample preservation solution system

[0077] Mix the fresh oral swab sample with 1 mL of preservation solution 1, take 3 portions at different storage time points (0 days, 7 days, 30 days, 60 days) under different temperature conditions (-20, 25, 37°C), and after expiration, take 600 μl and centrifuge at 12500 rpm for 4 min for DNA extraction; DNA is extracted using a commercial nucleic acid extraction kit, and the experimental method refers to the instruction manual.

[0078] 2.2 Analysis of microbial DNA quality and integrity

[0079] The DNA concentration and quality were detected by spectrophotometer, and the DNA integrity was tested by agarose gel electrophoresis.

[0080] 2.3 Analysis of microbial abundance

[0081] The genomic DNA of the samples stored under the above-mentioned different conditions and the DNA of the samples stored at -80°C without adding preservation solution were taken for 16s amplicon sequencing (V3 / V4 region) to analyze the changes in the abundance of microorganisms in the samples.

[0082] 2.4 Results of preservation fluid (oral sample)

[0083] The DNA integrity of oral samples stored in the preservation solution at different temperatures (-20, 25, 37°C) for 0, 7, 30, and 60 days ( Figure 4 A), there is no significant difference in concentration, and the DNA yield of the sample does not deviate from the yield of the control group. The composition distribution of each sample at the phylum and genus classification level is visualized as above Figure 4 As shown in B, the bar graph shows that there is no significant difference in phylum and genus species in oral samples stored in the preservation solution for 7 days, 30 days, and 60 days under different temperatures (-20, 25, and 37°C). In order to comprehensively evaluate the alpha diversity of the microbial community, Chao1 and Observed species indices were used to characterize the richness. The results showed that ( Figure 4 C): There was no significant difference in the microbial Alpha diversity of oral samples stored in the preservation solution for 7 days, 30 days, and 60 days compared with the control under different temperatures (-20, 25, and 37°C). Inter-group difference analysis showed that ( Figure 4 D): There are significant differences among the treatments, but no significant differences between the treatments and the initial control sample.

[0084] Application Example 3: Skin Sample Preservation

[0085] 3.1 Skin microbial sample preservation solution system

[0086] Mix the fresh skin swab sample with 1 mL of preservation solution 1, take 3 portions at different storage time points (0 days, 7 days, 30 days, 60 days) under different temperature conditions (-20, 25, 37°C), and after expiration, take 500 μl and centrifuge at 12500 rpm for 4 min for DNA extraction; DNA is extracted using a commercial nucleic acid extraction kit, and the experimental method refers to the instruction manual.

[0087] 3.2 Analysis of microbial DNA quality and integrity

[0088] The DNA concentration and quality were detected by spectrophotometer, and the DNA integrity was tested by agarose gel electrophoresis.

[0089] 3.3 Analysis of microbial abundance

[0090] The genomic DNA of the samples stored under the above-mentioned different conditions and the DNA of the samples stored at -80°C without adding preservation solution were taken for 16s amplicon sequencing (V3 / V4 region) to analyze the changes in the abundance of microorganisms in the samples.

[0091] 3.4 Results of storage fluid (skin microorganisms)

[0092] The DNA integrity of skin samples stored in the preservation solution at different temperatures (-20, 25, 37°C) for 0, 7, 30, and 60 days ( Figure 5 A), there is no significant difference in concentration, the sample DNA yield does not deviate from the control group ((Micro-Base Bio, catalog number GDS-01A)), and there is no difference with the results of competing products. However, the concentration and quality of microbial DNA in skin samples are low. Subsequent experiments can increase the sampling volume to improve the concentration and quality of microbial DNA in skin samples. The composition distribution of each sample at the phylum and genus classification level is visualized as above Figure 5 As shown in B, the bar graph shows that: there is no significant difference in phylum and genus species in skin samples stored in the preservation solution for 7 days, 30 days, and 60 days under different temperatures (-20, 25, and 37°C). In order to more comprehensively evaluate the alpha diversity of the microbial community, Chao1 and Observed species indices were used to characterize the richness. The results showed that ( Figure 5 C): The Alpha diversity of microorganisms in skin samples stored in the preservation solution for 7 days, 30 days, and 60 days had no significant difference from that in the control group. The inter-group difference analysis showed that ( Figure 5 D): There are significant differences among the treatments, but no significant differences between the treatments and the initial control sample.

[0093] Application Example 4: Preservation of reproductive tract samples

[0094] 4.1 Reproductive tract microbial sample preservation solution system

[0095] Mix the fresh genital microbial swab sample with 1 mL of preservation solution 1, take 3 portions at different storage time points (0 day, 7 days, 30 days, 60 days) under different temperature conditions (-20, 25, 37°C), take 500 μl after expiration, centrifuge at 12500 rpm for 4 min for DNA extraction; DNA is extracted using a commercial nucleic acid extraction kit, and the experimental method refers to the instruction manual.

[0096] 4.2 Analysis of microbial DNA quality and integrity

[0097] The DNA concentration and quality were detected by spectrophotometer, and the DNA integrity was tested by agarose gel electrophoresis.

[0098] 4.3 Microbial abundance analysis

[0099] The genomic DNA of the samples stored under the above-mentioned different conditions and the DNA of the samples stored at -80°C without adding preservation solution were taken for 16s amplicon sequencing (V3 / V4 region) to analyze the changes in the abundance of microorganisms in the samples.

[0100] 4.4 Results of preservation fluid (genital tract microbial samples)

[0101] The DNA integrity of the reproductive tract samples stored in the preservation solution at different temperatures (-20, 25, 37°C) for 0, 7, 30, and 60 days ( Figure 6 A), there is no significant difference in concentration, the sample DNA yield does not deviate from the control group yield, and there is no difference with the competitor's results. The composition distribution of each sample at the phylum and genus classification level is visualized as follows Figure 6 As shown in B, the bar graph shows that there is no significant difference in phylum and genus species in the reproductive tract samples stored in the preservation solution for 7 days, 30 days, and 60 days under different temperatures (-20, 25, and 37°C). In order to more comprehensively evaluate the alpha diversity of the microbial community, Chao1 and Observed species indices were used to characterize the richness. The results showed that ( Figure 6 C): There was no significant difference in the microbial Alpha diversity of the reproductive tract samples stored in the preservation solution for 7 days, 30 days, and 60 days compared with the control under different temperatures (-20, 25, and 37°C). The inter-group difference analysis showed that ( Figure 6 D): There are significant differences among the treatments, but no significant differences between the treatments and the initial control sample.

[0102] Application Example 5: Saliva Sample Storage

[0103] 5.1 Saliva microbial sample preservation solution system

[0104] Mix the fresh saliva microbial sample with preservation solution 1 in a ratio of 1:2, take 3 portions at different storage time points (0 day, 7 days, 30 days, 60 days) under different temperature conditions (-20, 25, 37°C), take 500 μl after expiration, centrifuge at 12500 rpm for 4 min for DNA extraction; DNA is extracted using a commercial nucleic acid extraction kit, and the experimental method refers to the instruction manual.

[0105] 5.2 Analysis of microbial DNA quality and integrity

[0106] The DNA concentration and quality were detected by spectrophotometer, and the DNA integrity was tested by agarose gel electrophoresis.

[0107] 5.3 Microbial abundance analysis

[0108] The genomic DNA of the samples stored under the above-mentioned different conditions and the DNA of the samples stored at -80°C without adding preservation solution were taken for 16s amplicon sequencing (V3 / V4 region) to analyze the changes in the abundance of microorganisms in the samples.

[0109] 5.4 Results of preservation fluid (saliva microbial sample)

[0110] The DNA integrity of saliva samples stored in the preservation solution at different temperatures (-20, 25, 37°C) for 0, 7, 30, and 60 days ( Figure 7 A), there is no significant difference in concentration, the sample DNA yield does not deviate from the control group yield, and there is no difference with the competitor's results. The composition distribution of each sample at the phylum and genus classification level is visualized as follows Figure 7 As shown in B, the bar graph shows that there is no significant difference in phylum and genus in saliva samples stored in the preservation solution for 7 days, 30 days, and 60 days under different temperatures (-20, 25, and 37°C). Among them, the proportion of some genera in the samples stored in the preservation solution for 7 days at 25 and 37°C changed greatly from that in the initial samples. Further analysis showed that the above-mentioned differences in the proportion of genus level came from the repeated samples. In order to more comprehensively evaluate the alpha diversity of the microbial community, Chao1 and Observed species indices were used to characterize the richness. The results showed that ( Figure 7 C): There was no significant difference in the microbial Alpha diversity of saliva samples stored in the preservation solution for 7 days, 30 days, and 60 days compared with the control under different temperatures (-20, 25, and 37°C). The inter-group difference analysis showed that ( Figure 7 D): There are significant differences among the treatments, but no significant differences between the treatments and the initial control sample.

[0111] Application Example 6: Dental Plaque Sample Preservation

[0112] 6.1 Dental plaque microorganism sample preservation solution system

[0113] Mix the fresh dental plaque swab sample with 1 mL of preservation solution 1, take 3 portions at different storage time points (0 day, 7 days, 30 days, 60 days) under different temperature conditions (-20, 25, 37°C), and after expiration, take 500 μl and centrifuge at 12500 rpm for 4 min for DNA extraction; DNA is extracted using a commercial nucleic acid extraction kit, and the experimental method refers to the instruction manual.

[0114] 6.2 Analysis of microbial DNA quality and integrity

[0115] The DNA concentration and quality were detected by spectrophotometer, and the DNA integrity was tested by agarose gel electrophoresis.

[0116] 6.3 Microbial abundance analysis

[0117] The genomic DNA of the samples stored under the above-mentioned different conditions and the DNA of the samples stored at -80°C without adding preservation solution were taken for 16s amplicon sequencing (V3 / V4 region) to analyze the changes in the abundance of microorganisms in the samples.

[0118] 6.4 Results of preservation solution (dental plaque microorganism sample)

[0119] Dental plaque samples stored in the preservation solution at different temperatures (-20, 25, 37°C) for 0, 7, 30, and 60 days were intact in DNA. Figure 8 A), there is no significant difference in concentration, the sample DNA yield does not deviate from the control group yield, and there is no difference with the competitor's results. However, the concentration and quality of microbial DNA in dental plaque samples are low. Subsequent experiments can increase the sampling volume to improve the concentration and quality of microbial DNA in skin samples. The composition distribution of each sample at the phylum and genus classification level is visualized as above Figure 8 As shown in B, the bar graph shows that there is no significant difference in phylum and genus species in dental plaque samples stored in the preservation solution for 7 days, 30 days, and 60 days under different temperatures (-20, 25, and 37°C). In order to comprehensively evaluate the alpha diversity of the microbial community, Chao1 and Observed species indices were used to characterize the richness. The results showed that ( Figure 8 C): There was no significant difference in the microbial Alpha diversity of dental plaque samples stored in the preservation solution for 7 days, 30 days, and 60 days compared with the control under different temperatures (-20, 25, and 37°C). Inter-group difference analysis showed that ( Figure 8 D): There are significant differences among the treatments, but no significant differences between the treatments and the initial control sample.

[0120] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microbial sample DNA preservation solution, characterized in that: Each liter of the DNA preservation solution comprises the following components: 5-30% (v / v) glycerol, 0.02-0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.01-0.1 mol / L tris(hydroxymethyl)aminomethane, 0.01-0.05 mol / L nucleic acid stabilizer or nuclease inhibitor, hydrochloric acid to adjust pH to 5.6-7.6, and the balance is water.

2. The DNA preservation solution according to claim 1, characterized in that The nucleic acid stabilizer is selected from one or a combination of two or more of sodium pyrophosphate, sodium tripolyphosphate, sodium ascorbate, sodium hexametaphosphate, trisodium phosphate, and sodium citrate; the nuclease inhibitor is selected from one or a combination of two or more of diethyl pyrophosphate, guanidine isothiocyanate, and urea.

3. The DNA preservation solution according to claim 2, characterized in that Each liter of DNA preservation solution consists of the following components Composition: 5-30% (v / v) glycerol, 0.02-0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.01-0.1 mol / L tris(hydroxymethyl)aminomethane, 0.01-0.05 mol / L nucleic acid stabilizer, hydrochloric acid to adjust pH to 5.6-7.6, the balance is water; the nucleic acid stabilizer is selected from one of sodium pyrophosphate, sodium tripolyphosphate and sodium ascorbate.

4. The DNA preservation solution according to claim 2, characterized in that Each liter of DNA preservation solution consists of the following components Composition: 5-15% (v / v) glycerol, 0.05-0.2 mol / L disodium ethylenediaminetetraacetic acid, 0.06-0.1 mol / L tris(hydroxymethyl)aminomethane, 0.01-0.03 mol / L nucleic acid stabilizer, hydrochloric acid to adjust pH to 7.0-7.5, the balance is water; the nucleic acid stabilizer is selected from sodium pyrophosphate.

5. A method for preparing a microbial sample DNA preservation solution according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: (1) Based on 1 liter of DNA preservation solution, take 50-300 ml of glycerol, 0.02-0.2 mol of disodium ethylenediaminetetraacetate, 0.01-0.1 mol of tris(hydroxymethyl)aminomethane, and 0.01-0.05 mol of sodium pyrophosphate; (2) dissolving sodium pyrophosphate in a portion of sterile water, filtering the solution through a microfiltration membrane with a pore size of 0.22 μm, and setting aside for later use; (3) Take some sterile water to dissolve disodium ethylenediaminetetraacetate and tris(hydroxymethylaminomethane), mix with glycerol, sterilize in a high-pressure steam autoclave, cool to room temperature, add the sodium pyrophosphate solution in step (2), and use sterile water to make up the volume to 1 L.

6. Use of the microbial sample DNA preservation solution according to any one of claims 1 to 4 in preserving microbial samples under non-freezing conditions.

7. The use according to claim 6, characterized in that: The non-freezing condition is selected from the storage condition with a temperature > 0°C.

8. The use according to claim 6, characterized in that: The non-freezing condition is selected from the storage condition of the temperature of 25-37°C.

9. A method for preserving a microbial sample, characterized in that: The microbial sample DNA preservation solution according to any one of claims 1 to 4 is mixed with the microbial sample in a volume ratio of (1-5):1 and preserved.

10. The use according to claim 9, characterized in that: At room temperature, it can be stored for 28-30 days; at 37℃, it can be stored for 7-28 days.

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