A sputum sample microbial flora nucleic acid DNA preservation solution

Abstract

The present invention relates to a sputum sample microbial flora and nucleic acid DNA preservation solution, including the following components by mass: 0.5-2 parts of trishydroxymethylaminomethane, 0.2-1 parts of mucus digestive agent, 3-6 parts of N-octylpyridinium bromide, 20-50 parts of fixative, 0.6-1.5 parts of auxiliary fixative, 1-2 parts of metal ion chelating agent, 0.5-3 parts of inorganic salt, the metal ion chelating agent includes at least 50wt% of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; the auxiliary fixative is selected from at least one of sodium citrate and potassium citrate. The sputum sample microbial flora and nucleic acid DNA preservation solution using the technical solution of the present invention has the following advantages, can be stored and transported at room temperature for a long time, and is easy to operate; the sputum sample can be effectively liquefied by immersing the sputum sample in the protective solution, and the structural composition of the microorganism is stabilized at room temperature, protecting the diversity of its flora structure and DNA stability.

Description

Technical Field

[0001] The invention relates to a biological sample preservation solution, in particular to a sputum sample microbial flora and nucleic acid DNA preservation solution at room temperature. Background Art

[0002] Sputum testing includes microscopic examination, smear examination, pathogen examination and high-throughput NGS methods, which can assist in the diagnosis or diagnosis of certain respiratory diseases. Pathogen isolation and culture is the "gold standard" for determining pathogens of lower respiratory tract infections and is a routine method for clinical laboratory departments to detect respiratory secretions. The high-throughput NGS method can identify both culturable pathogens and unculturable pathogens, and can also analyze the diversity of upper respiratory tract flora to assist clinical diagnosis. With the development of molecular biology technology, molecular biology detection methods such as tNGS, mNGS, and PCR can qualitatively diagnose pathogen infection, with the advantages of rapidity, sensitivity, and strong specificity. This method requires the extraction of nucleic acid from sputum for testing. However, there is a large amount of mucus in sputum, and the cell components in it are not easy to separate, which poses great difficulties in nucleic acid extraction. If the sputum sample cannot be tested immediately after collection, it may be contaminated or bacterial growth, microbial death, nucleic acid degradation, etc. may lead to inaccurate test results due to a long time, and the composition and abundance of microorganisms in the original sample will also change and cannot reflect the real situation. The collected samples are generally stored at -20°C or lower, which will cause great inconvenience to the transportation of samples. Microorganisms of lung infection can be analyzed through nucleic acid testing of sputum samples, but in some parts of my country, due to poor local medical conditions, some samples need to be transported to hospitals with testing capabilities for testing. Or during long-term transportation on complex terrain and rugged mountain roads, the bacteria in the sputum samples will rupture, resulting in the release of DNA. Therefore, the stability of DNA in the preservation solution is of great significance for the analysis of microbial flora polymorphism in sputum samples and the detection of key microorganisms. Therefore, it is very important to liquefy and homogenize the samples before sputum nucleic acid extraction, prevent nucleic acid degradation and transport the samples.

[0003] The human respiratory flora constitutes a complex microbial community, and different types of microorganisms perform their own degradation functions on nutrients, including fiber degradation (Ruminococcaceae), glycolysis (Enterococcus family), mucus degradation (Bacteroidetes family), etc. The diversity of the body's flora is positively correlated with the stability of the ecosystem, that is, the higher the diversity of flora species, the more stable the microbial community. The respiratory microbiome is an important part of respiratory health and is related to susceptibility to infection. Sputum samples coughed up or induced by patients with respiratory diseases are the best substitutes for the lower respiratory tract microbial community in airway diseases. They can be easily obtained in a patient-friendly way, and changes in the diversity of sputum flora structure can reflect changes in the abundance and diversity of lung microbial communities. Changes in the diversity of respiratory microbiome, including the invasion of foreign pathogens or the reduction of probiotics, may lead to the destruction of immune balance, leading to disease activation, susceptibility to new infections or interference with disease treatment. Studies have shown that the composition of sputum bacterial microbiome can be used to predict the course of non-tuberculous mycobacterial lung disease (NTM-LD) and may be used to develop precision therapy to improve the symptoms of non-tuberculous mycobacterial lung disease by regulating the composition of respiratory microorganisms. Therefore, in addition to preserving the integrity of nucleic acids, the diversity and integrity of sputum microbial flora are also required.

[0004] CN111926059A discloses a sputum microbial nucleic acid preservation solution, comprising the following components calculated by mass: 15-30% nuclease inhibitor; 1.5-5% pH stabilizer; 0.8-1.5% chelating agent; 0.05-0.15% mucolytic agent; 0.1-0.5% sodium lauryl sarcosinate; 10-20% isopropanol; 2-15% glycerol; the balance is sterile water. CN111218444A discloses a sputum preservation solution, comprising the following components in the following amounts: 118g-236g / L guanidine thiocyanate, 0.01-0.5M 8-hydroxyquinoline, 1-10 g / L sodium lauryl sarcosinate, 1-10mM ethylenediaminetetraacetic acid, 1-10g / L potassium chloride, 1%-10% v / v glycerol, 5-20% v / v isopropanol. CN111020001A discloses a saliva preservation solution, comprising the following components: EDTA 1-10mmol / L, Tris-HCl 1-50mmol / L, sodium chloride 10-100mmol / L, sodium sorbate 0.1-3% (w / v), sodium diacetate 0.1-3% (w / v), proclin300 0.01-0.1% (v / v), TWEEN20 0.1-1% (v / v), NP400.1-1% (w / v), SDS 0.1-2% (w / v). However, the preservation solution of the above patent needs to be used at low temperature. CN111944876A discloses a preservation solution for sputum, wherein each 1000ML of the preservation solution comprises the following components: 0.5-3.5g dithiothreitol, 5.0-30g tris(hydroxymethyl)aminomethane, 0.1-10.5g sodium chloride, 0.01-2.0g phosphorus trichloride, 0.01-2.5g disodium hydrogen phosphate, 0.01-2.5g potassium dihydrogen phosphate, 0.2-3.5g guanidine isothiocyanate, and the balance is nucleic acid-free water, and the pH value is 5.5-8.5. The patent optimizes the preservation solution formula and increases the insulation temperature, but still requires 2-8°C insulation, and can only be stored for 2-3 days at 2-8°C, and can only be stored for more than 10 days at ultra-low temperature (-70°C), which still has inconvenience in use. Moreover, the above protection solution contains toxic components of guanidine isothiocyanate, which is harmful to health and has safety transportation risks.

[0005] CN116287120A discloses a preservation solution that can be stored at room temperature and can preserve human cell nucleic acid DNA and RNA in saliva samples at a high temperature of 60°C, comprising the following components: 1,2-cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylpiperazine-2-ethanesulfonic acid, SDS, sodium chloride, ethanol and tween-20; in the preservation solution, the molar concentration of the 1,2-cyclohexanediaminetetraacetic acid is 0.1-10M, the molar concentration of the hydroxyethylpiperazine-2-ethanesulfonic acid is 0.01-1M, the percentage of the SDS is 0.1%-20% v / v, the percentage of the sodium chloride is 0.1%-1% w / v, the percentage of the ethanol is 5%-80% v / v, the molar concentration of the tween-20 is 0.5-1M, and the pH of the preservation solution is 6.0-10.0. The patented preservation solution can stably preserve nucleic acids in saliva at 60°C for 7 days.

[0006] CN112438253A discloses a saliva preservation solution, which includes a cell stabilizer, a nuclease inhibitor, a surfactant, a protein denaturant and a preservative; the preservative is one or a combination of 2-methyl-4-isothiazoline-3-one and 5-chloro-2-methyl-4-isothiazoline-3-one. The patent increases the preservation temperature of the preservation solution to room temperature by adding a preservative.

[0007] CN106561631A discloses a saliva preservation solution, comprising: a DNA stabilizer, wherein the DNA stabilizer comprises disodium ethylenediaminetetraacetic acid and tartaric acid; a microbial inhibitor, wherein the microbial inhibitor comprises penicillin, streptomycin and geneticin (G418); and a buffer, comprising a TE buffer; wherein, per liter of the saliva preservation solution, disodium ethylenediaminetetraacetic acid (EDTA·2Na) is 1mM to 10mM, tartaric acid is 26.65mM to 66.63mM, penicillin is 50U / mL to 200U / mL, streptomycin is 86.00mM to 343.9mM, geneticin (G418) is 0.144mM to 0.577mM, and tris-HCl (Tris-HCl) in the TE buffer is 5mM to 20mM. The patent maintains the cell morphology by adding microbial inhibitors, prevents lysis, and inhibits the growth of microorganisms, and can be preserved for a long time at room temperature. However, the cells need to be lysed to obtain the nucleic acids in the cells, which increases the time and procedures of the extraction.

[0008] Due to the differences in flora structure and DNA preservation environment, the above patents do not consider the diversity and integrity of flora preservation while maintaining DNA integrity, and often DNA integrity and flora integrity are compromised. During the preservation process, the types and proportions of flora will change significantly, and some flora may even disappear, resulting in the loss of the ability to detect and evaluate the course of respiratory diseases through sputum flora structure and immune assessment. Summary of the invention

[0009] Based on the above background, in order to overcome the problem that the existing sputum preservation solution is difficult to preserve both the flora structure and DNA well at the same time. The existing technology is relatively poor in practicality for sputum samples, and has the following problems: it is difficult to preserve at room temperature, and it is difficult to take into account both the inhibition of nuclease activity and liquefaction treatment at the same time; the antibacterial effect is poor, and microorganisms are easy to breed; the preservation time is not long, etc. The present invention provides a sputum sample microbial flora and nucleic acid preservation solution, which can effectively liquefy the sputum sample at room temperature, and can also effectively inhibit the activity of nuclease, protect the stability of DNA and microbial flora structure in the sputum sample, does not contain toxic components such as guanidine isothiocyanate, has a long preservation time, is suitable for transportation at room temperature, etc., and has important application value.

[0010] The technical solution adopted by the present invention is as follows:

[0011] A sputum sample microbial flora nucleic acid DNA preservation solution comprises the following components in parts by weight: 0.5-2 parts of tris(hydroxymethyl)aminomethane, 0.2-1 parts of a mucus digestant, 3-6 parts of N-octylpyridinium bromide, 20-50 parts of a fixative, 0.6-1.5 parts of an auxiliary fixative, 1-2 parts of a chelating agent, and 0.5-3 parts of an inorganic salt, wherein the chelating agent comprises at least 50 wt% of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and the auxiliary fixative is selected from at least one of sodium citrate and potassium citrate.

[0012] Furthermore, the sputum sample microbial flora nucleic acid DNA preservation solution includes the following components in parts by weight: 1.2-1.8 parts of trishydroxymethylaminomethane, 0.5-0.8 parts of mucus digestant, 4-5 parts of N-octylpyridinium bromide, 0.7-1.5 parts of inorganic salt, 30-40 parts of fixative, 0.8-1.3 parts of auxiliary fixative, and 1.3-1.8 parts of chelating agent.

[0013] Furthermore, the mucus digester is selected from one or more of DTT (dithiothreitol), N-acetyl-L-cysteine, and TCEP; preferably TCEP. Compared with other mucus digesters, TCEP can destroy the intermolecular disulfide bonds and intercellular filaments of mucin, thereby achieving the purpose of digesting mucus.

[0014] Furthermore, the inorganic salt is selected from at least one of sodium chloride, potassium chloride, lithium chloride, sodium sulfate and sodium bicarbonate; preferably sodium chloride, which can better regulate the osmotic pressure.

[0015] Furthermore, the fixative is selected from one or more of ethanol, isopropanol, and paraformaldehyde, preferably ethanol. The amount of the fixative used in the preservation solution is the largest and has a better fixation effect, preferably ethanol.

[0016] Furthermore, the auxiliary fixing agent is selected from one of sodium citrate and potassium citrate.

[0017] Further, the metal ion chelator is selected from at least one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), EDTA, and EDTA·2Na, provided that the metal ion chelator includes at least 50wt% of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, preferably includes more than 60wt% of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and more preferably includes more than 70wt% of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0018] The inventor unexpectedly found that a certain proportion of N-octylpyridinium bromide and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid in the preservation solution can simultaneously play an excellent preservation role for the microbial flora and DNA in sputum under the action of ethanol fixative, and can preserve the DNA intact at room temperature for 14 days, and the structural diversity of the flora is not damaged. The inventor analyzed the reason and found that it may be because N-Octylpyridinium bromide (N-octylpyridinium bromide) has good surface activity and antibacterial and preservative properties, which enables the sample to maintain the original microbial abundance and nucleic acid quality. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid can form a coordination geometry with metal cations, and has a strong chelating force for calcium, magnesium, iron and other transition metal ions. It chelates metal ions in sputum, inhibits the activity of nucleases in the system, and prevents the nucleic acid in the sputum sample from being degraded. Absolute ethanol can maintain the morphology of cells by dehydration, and sodium citrate stabilizes its morphology. The above combination can effectively maintain the number and structure of microbial flora after TCEP liquefaction of sputum at room temperature, including a variety of bacteria including Gram-positive and Gram-negative bacteria, and can also maintain the stability of nucleic acid DNA at the same time. The sputum sample processed by the present invention can not only directly detect the DNA in the sputum sample, but also evaluate the flora structure at the same time, greatly simplifying the extraction of respiratory disease flora samples obtained separately, and can carry out auxiliary detection of respiratory diseases based on flora structure analysis and course evaluation while detecting viral nucleic acid detection. The present invention uses N-octylpyridinium bromide, and conventional antibacterial agents do not need to be added.

[0019] The inventors have also tried N-butylpyridinium bromide and N-dodecylpyridinium bromide, but they did not perform as well as N-octylpyridinium bromide, indicating that the length of the alkyl chain in N-alkylpyridinium bromide has an important influence on the preservation effect.

[0020] The present invention also provides a method for preparing the above-mentioned sputum sample microbial flora nucleic acid DNA preservation solution, comprising the following steps:

[0021] Weigh appropriate amounts of tris(hydroxymethyl)aminomethane, mucus digestant, N-octylpyridinium bromide, fixative, auxiliary fixative, chelating agent, and inorganic salt, make up to volume with sterile deionized water, sterilize, and obtain sputum sample microbial flora and nucleic acid DNA preservation solution.

[0022] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0023] The present invention achieves simultaneous protection of microbial flora and DNA in sputum samples at room temperature by rationally combining the components of the preservation solution, especially the combination of ethanol with N-octylpyridinium bromide and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, without toxic components such as guanidine isothiocyanate. When the sputum sample is in the state of the protection solution of the present invention, the nucleic acid and microbial flora structure of the sputum sample can be simultaneously detected. Not only can pathogens such as viral DNA be analyzed, but also the diversity of microbial flora can be detected without resampling, and the course of respiratory diseases and immune assessment can be performed. Similar to literature reports, the composition of sputum bacterial microbial structure can be used to predict the course of non-tuberculous mycobacterial lung disease (NTM-LD), and may be used to develop precision treatment to improve the symptoms of non-tuberculous mycobacterial lung disease by regulating the composition of respiratory microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The relative abundance of bacteria at the phylum classification level in Example 1;

[0025] Figure 2 The relative abundance of bacteria at the phylum classification level in Example 2;

[0026] Figure 3 The relative abundance of bacteria at the phylum classification level in Example 3;

[0027] Figure 4 The relative abundance of bacteria at the phylum classification level in comparative example 1;

[0028] Figure 5 The relative abundance of bacteria at the phylum classification level in comparative example 2;

[0029] Figure 6 The relative abundance of bacteria at the phylum level was stored at -80℃. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Unless otherwise specified, the experimental raw materials, reagents and materials used in the following examples are all common commercially available products.

[0032] Example 1

[0033] Weigh 1.5 g of tris(hydroxymethyl)aminomethane, 0.6 g of TCEP, 1.1 g of sodium chloride, 1.2 g of sodium citrate, 1.3 g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 0.3 g of EDTA, 4 g of N-octylpyridinium bromide, and 40 g of anhydrous ethanol, add to sterile deionized water, mix and dissolve, and make up to 100 mL with sterile deionized water; use a 0.22 μm sterilizing filter membrane to filter and sterilize to obtain sputum preservation solution.

[0034] Example 2

[0035] Weigh 1.8 g of tris(hydroxymethyl)aminomethane, 0.8 g of TCEP, 1.5 g of sodium chloride, 1.3 g of sodium citrate, 1.2 g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 0.5 g of EDTA, 4 g of N-octylpyridinium bromide, and 50 g of anhydrous ethanol, add to sterile deionized water, mix and dissolve, and make up to 100 mL with sterile deionized water; use a 0.22 μm sterilizing filter membrane to filter and sterilize to obtain sputum preservation solution.

[0036] Example 3

[0037] Weigh 1.2 g of tris(hydroxymethyl)aminomethane, 0.5 g of TCEP, 0.7 g of sodium chloride, 0.8 g of sodium citrate, 1.1 g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 0.6 g of EDTA, 5 g of N-octylpyridinium bromide, and 40 g of anhydrous ethanol, add sterile deionized water to mix and dissolve, and make up to 100 mL with sterile deionized water; use a 0.22 μm sterilizing filter membrane to filter and sterilize to obtain sputum preservation solution.

[0038] Comparative Example 1

[0039] The rest is the same as Example 1, except that N-octylpyridinium bromide is not added, and an equal mass of SDS is used instead.

[0040] Comparative Example 2

[0041] The other parts are the same as those in Example 1, except that 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid is not added, and an equal mass of EDTA is used instead.

[0042] Application Examples

[0043] The preservation solution of the above-mentioned embodiment and comparative example was tested for its performance of DNA preservation performance: sputum of 6 healthy volunteers was selected, and 30 minutes before collecting saliva samples, the subjects rinsed their mouths with clean water and kept their oral cavity clean. After cleaning the oral cavity, they did not eat, smoke, chew gum, drink water, etc., and put the sputum into a clean and disinfected sputum box immediately after coughing up, and samples of 6 subjects were obtained respectively, among which samples No. 1-5 were added with the preservation solution of embodiments 1-3, comparative examples 1 and 2 according to a volume ratio of 1:2.5, and sample No. 6 was added with the same volume of sterile deionized water, and each sample No. 1-6 was divided into 4 parts on average, and the sputum samples added with the preservation solution of the embodiment and comparative example (samples No. 1-5) were placed at room temperature for 0 days, 3 days, 7 days, and 14 days respectively; sample No. 6 was the control group, and the storage temperature was minus 80°C, and it was placed for 0 days, 3 days, 7 days, and 14 days respectively. The total amount and purity of nucleic acid were tested after purification in each time period, and the 16SrDNA high-throughput DNA sequencing method was used to identify the bacterial species and diversity in the sputum.

[0044] The kit uses a magnetic bead bacterial extraction kit and performs nucleic acid extraction testing according to the following steps:

[0045] 1) Collect the bacteria by centrifugation at 10,000 g for 5 minutes, discard the waste liquid, and add 500 µl Buffer TL to resuspend the pellet.

[0046] 2) Incubate at 90°C for 20 minutes, then centrifuge at 13,000 g for 3 minutes.

[0047] 3) Transfer 300 µL of supernatant to a new centrifuge tube, add 20 µL of magnetic beads and 500 µL of magnetic bead binding solution (2M guanidine hydrochloride, 30% isopropanol mixture). Invert and mix 10-15 times, leave at room temperature for 5 minutes, vortex several times. Transfer to a magnetic stand and adsorb for 2 minutes, then discard the solution by pouring or aspirating.

[0048] 3) Add 500 µl of rinse buffer, vortex for 10 seconds, transfer to a magnetic rack and adsorb for 1 minute, then discard the solution by decantation or aspiration.

[0049] 4) Add 750 µl of 75% ethanol, vortex for 10 seconds, transfer to a magnetic rack and adsorb for 1 minute, then discard the solution by decantation or aspiration.

[0050] 5) Repeat step 4) once.

[0051] 6) Collect the droplets on the tube wall by centrifugation, transfer to a magnetic rack, absorb the residual liquid, and air dry for 5 minutes.

[0052] 7) Add 50µl of elution buffer (10mM Tris buffer, 1mM EDTA) and vortex to disperse the beads. Incubate at 55ºC for 10 minutes. If vortexing is not necessary, vortex 2-3 times to accelerate the dissolution of DNA.

[0053] 8) Transfer to a magnetic rack and adsorb for 5 minutes, then transfer the DNA to a new centrifuge tube.

[0054] The extracted nucleic acid was tested for concentration and purity using a nanodrop ultra-micro spectrophotometer, with the concentration unit being ng / ul. Purity uses A260 / A280 as an indicator. A260 (nm) is the absorption wavelength of the highest absorption peak of nucleic acid, and A280 (nm) is the absorption wavelength of the highest absorption peak of protein and phenolic substances. The ratio of the two, A260 / A280, can be used to evaluate the purity of nucleic acid samples: the A260 / A280 ratio of pure DNA is 1.8-2.0. If the ratio is lower than 1.8, it indicates contamination by protein (aromatic) or phenolic substances, and the sample needs to be purified; if the ratio is higher than 2.0, it indicates that it contains RNA or small fragments of nucleic acid or is degraded. The results are shown in Table 1 below:

[0055] Table 1: The concentration and purity of nucleic acid DNA at different times under different storage solutions

[0056] .

[0057] From the results in Table 1, it can be seen that the samples in Examples 1, 2, and 3 treated with the preservation solution of the present invention and stored at -80°C are stored at room temperature for 0 days, 3 days, 7 days, and 14 days, and the concentration of the obtained nucleic acid remains basically stable, without obvious degradation, and the purity remains between 1.8-2.0. The nucleic acid concentration and nucleic acid purity of the samples of Comparative Examples 1 and 2 decreased significantly at 7 and 14 days. It can be seen that the sputum sample microbial flora nucleic acid preservation solution provided by the present invention can better maintain the stability of nucleic acids in sputum, the total amount of nucleic acid decreases slowly, and the nucleic acid purity is also well maintained, reaching a level close to that of storage at minus 80°C. Among them, the effect of Example 1 is better than that of Example 2 and Example 3.

[0058] Application Example 2

[0059] In order to verify the test of bacterial community integrity and diversity, the extracted DNA samples were sequenced in the 16s rRNA gene V4 region. After obtaining the sequencing data, bioinformatics analysis was performed to calculate the bacterial phyla to which the bacteria in each sample belonged and compared. The relative abundance of bacteria at the phylum level is shown in the attached figure. Figure 1-6 As shown, Figure 1 , 2, 3, 4, 5, and 6 are schematic diagrams of the proportion of bacterial phyla levels in four different storage time periods of Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and the control group stored at -80°C for 0 days, 3 days, 7 days, and 14 days. The results show that the proportion of bacterial phyla levels in the storage solution of the present invention stored at room temperature for 14 days and at -80°C is close to the result of storage for 0 day.

[0060] Based on the DNA extraction concentration, the change in the total amount of nucleic acid DNA, the bacterial richness analysis and the differences and similarities in the flora composition at the phylum level, it can be concluded that the preservation solution formula of the present invention has a preservation effect on the diversity of microbial flora in sputum that is closer to the 0-day preservation treatment, and the protection of nucleic acid DNA is also significantly better than the preservation scheme of the comparative example.

[0061] The above results show that in the examples, whether it is sample extraction, storage after extraction, or gate level analysis results after sequencing, Example 1 is slightly better than Examples 2 and 3. Finally, Example 1 of the present invention is the most preferred implementation scheme, but the preservation solutions of Examples 2 and 3 can also meet the better preservation requirements and are also within the protection scope of the present invention.

Claims

1. A sputum sample microbial flora nucleic acid DNA preservation solution, characterized in that: The invention comprises the following components in parts by weight: 1.2-1.8 parts of tris(hydroxymethyl)aminomethane, 0.5-0.8 parts of a mucus digestant, 4-5 parts of N-octylpyridinium bromide, 40-50 parts of a fixative, 0.8-1.3 parts of an auxiliary fixative, 1.6-1.7 parts of a chelating agent, and 0.7-1.5 parts of an inorganic salt, wherein the chelating agent is selected from at least one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, EDTA, and EDTA·2Na, and the chelating agent is selected from at least one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, EDTA, and EDTA·2Na. The agent includes at least 60wt% of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; the auxiliary fixative is selected from at least one of sodium citrate and potassium citrate; the mucus digester is selected from one or more of DTT, N-acetyl-L-cysteine, and TCEP; the inorganic salt is selected from at least one of sodium chloride, potassium chloride, lithium chloride, sodium sulfate, and sodium bicarbonate; the fixative is selected from one or more of ethanol, isopropanol, and paraformaldehyde.

2. The sputum sample microbial flora nucleic acid DNA preservation solution according to claim 1, characterized in that: The chelating agent comprises more than 70 wt % of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

3. The method for preparing the sputum sample microbial flora nucleic acid DNA preservation solution according to claim 1 or 2, characterized in that: The following steps are involved: Weigh appropriate masses of tris(hydroxymethyl)aminomethane, mucus digestant, N-octylpyridinium bromide, fixative, auxiliary fixative, chelating agent, and inorganic salt, make up to volume with sterile deionized water, sterilize, and obtain sputum sample microbial flora nucleic acid DNA preservation solution.

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