Preserving solution for preserving host and gram-negative bacterium nucleic acid in faeces sample

Abstract

The invention provides a preserving fluid for preserving host and gram-negative bacterium nucleic acid in an excrement sample, and particularly discloses an excrement preserving fluid capable of effectively splitting and releasing host intestinal cast-off cell DNA and gram-negative bacterium DNA and splitting gram-positive bacterium DNA less. Therefore, the Gram-negative pathogenic bacteria can be used for screening various Gram-negative pathogenic bacteria and can also be used for primary screening and general survey of diseases such as colorectal cancer of a host, and interference of nucleic acid of Gram-positive bacteria on detection is remarkably reduced.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a preservation solution for preserving host and Gram-negative bacteria nucleic acids in feces samples and a preparation method thereof. Background Art

[0002] The intestine is the largest immune organ in the human body, with an intestinal mucosal area of ​​400m 2 , about the size of a tennis court, 70% of the body's immune cells and IgA immunoglobulins are distributed in the intestines, which are a very important line of defense for the body against diseases. Due to bad living and eating habits, the incidence of colorectal cancer is increasing.

[0003] Now, with the advancement of molecular biology and detection technology, people have better means to study the relationship between the intestine and human health and diseases. Fecal samples are an important source of samples for intestinal disease research and intestinal clinical testing. A certain amount of exfoliated cells enter the feces every day, and the nucleic acids of these exfoliated cells contain cancer signals. Because it has many advantages such as being non-invasive, not requiring dietary restrictions, and not requiring special equipment, it is often used in the initial screening of diseases such as colorectal cancer. Compared with chemical or immunological fecal occult blood tests, full colonoscopy and other methods, it can better achieve the goal of "early detection, early diagnosis, and early treatment."

[0004] Feces also contain a large number of intestinal microorganisms, many of which are Gram-negative bacteria that are closely related to human diseases. Escherichia coli can reduce the harm of protein decomposition products to the human body, synthesize vitamins needed by the human body and colicin with bactericidal effects. It is an internationally recognized indicator of health monitoring and is an indispensable test object in food hygiene and safety testing. However, when the number of E. coli is too large, gastrointestinal symptoms such as nausea, vomiting, abdominal distension, abdominal pain, diarrhea, etc. are prone to occur. In severe cases, dehydration, electrolyte disorders and even shock may occur due to a large amount of water loss. Salmonella is a common foodborne pathogen. People infected with Salmonella or the feces of carriers contaminate food and can cause food poisoning. According to statistics, food poisoning caused by Salmonella often ranks first among bacterial food poisoning in countries around the world. Its typical symptoms include fever, nausea, vomiting, diarrhea and abdominal cramps, which usually improve within 72 hours after the fever. Infants, the elderly, and patients with low immune function may develop severe and life-threatening bacteremia due to the entry of Salmonella into the blood, and a few may also be complicated by meningitis or osteomyelitis. The endotoxin of Campylobacter jejuni can invade the mucosa of the small intestine and large intestine to cause acute enteritis, and can also cause outbreaks of diarrhea or collective food poisoning. Campylobacter jejuni has a wide range of infection and is a pathogen that infects both humans and animals. People are generally susceptible, and the incidence rate decreases with age. It is considered to be the main cause of bacterial diarrhea in humans. Vibrio cholerae is the pathogen of human cholera, which is one of the ancient and widespread virulent infectious diseases. It has caused many pandemics in the world, mainly manifested by severe vomiting, diarrhea, dehydration, and a high mortality rate. It is an international quarantine infectious disease. Vibrio cholerae produces enterotoxin, which is a severe diarrheal toxin. The toxin acts on the intestinal wall to promote the extreme secretion of intestinal mucosal cells, thereby causing excessive excretion of water and salt, leading to severe dehydration and collapse; and then causing metabolic acidosis and acute renal failure. Helicobacter pylori is a spiral-shaped, slightly anaerobic bacterium that has very strict requirements for growth conditions. It is the only known microorganism that can survive in the human stomach. Helicobacter pylori (infection) is one of the most common bacterial pathogens on the list of Class I carcinogens. This bacterial infection first causes chronic gastritis, and then leads to gastric ulcers and gastric atrophy, and in severe cases, it develops into gastric cancer. According to statistics, the incidence of atrophic gastritis and gastric cancer is high in people who are first infected with Helicobacter pylori at an early age, and the mortality rate of Helicobacter pylori infection and gastric cancer is parallel.

[0005] However, once the feces are excreted from the body, the environment changes from an anaerobic state to an aerobic state. Air, especially oxygen, is a toxic environment for anaerobic bacteria, killing 50% of anaerobic organisms within 4-5 minutes and 95-97% of anaerobic organisms after only 20 minutes, which will cause the death of a large number of microorganisms. In addition, the composition of fecal samples is complex, containing a large amount of proteases, DNA enzymes (DNase), RNA enzymes (RNases) and various PCR inhibitors (such as polysaccharides, bile salts, humus, bile acid, pigments, etc.), making the cells and nucleic acids in fecal samples extremely vulnerable to damage and degradation. Therefore, it is necessary to take protective measures for fecal samples. The current commonly used preservation method is to freeze the fecal samples immediately after collecting them in a low temperature environment, such as -20°C or lower, to reduce the activity of the enzyme and reduce the degradation of nucleic acids. If it needs to be transported to a testing agency, dry ice or liquid nitrogen is often required as a means of maintaining low temperature conditions, which will greatly increase the cost and is not suitable for large health screening. Therefore, there is a need for a fecal sample nucleic acid preservation solution that can effectively preserve fecal samples for a long time at room temperature.

[0006] At present, some patents have provided preservation solutions that can store and transport fecal samples at room temperature, but most of them contain alcohols (methanol, ethanol, propanol, etc.) for fixing cells. For example, patent CN106255753B discloses methods, compositions and kits for stabilizing complex biological samples such as human and microbial deoxyribonucleic acid (DNA) in feces, but it uses ethanol to fix cells. Ethanol, as a volatile and flammable substance, will put forward higher requirements for packaging and transportation, increasing the cost of the product. Patent CN106596211A discloses fecal sample preservation solution and its preparation method and application, which are easy to prepare, low in cost, can store fecal samples at room temperature, and can effectively inhibit DNA degradation and cell damage in fecal samples, maintain cell morphology, and store samples for a long time. After storage, the qualified rate of samples reused is high. However, the invention does not actively release nucleic acids in fecal samples. In this way, the tester needs to use a pipette tip minus the tip to take a homogenized sample, and solid feces can be obtained by centrifugation before adding the lysis solution for extraction. This makes the operation process too long and complicated, and poses a psychological challenge to the personnel. The preservation solution of patent CN107937391A contains high concentrations of guanidine thiocyanate and surfactants. This preservation solution uses a high concentration of the chaotropic salt guanidine thiocyanate to release nucleic acids. However, there are too many microorganisms in the feces. If all the microbial nucleic acids are released, the host cell nucleic acids will be covered, which is not conducive to the subsequent detection of host nucleic acids. The preservation reagent of patent CN113005173A can effectively lyse human exfoliated cells in feces, and does not contain toxic components such as guanidine thiocyanate, thereby providing a method for directly extracting and detecting fecal genomes from fecal supernatants, which can effectively avoid interference from fecal impurities, and there is no need to precipitate the fecal sample again before detection. However, this preservation solution can only be used for the detection of human exfoliated cells, and cannot detect pathogenic microorganisms.

[0007] Therefore, it is of great significance to develop a fecal nucleic acid preservation solution that can store and transport fecal samples at room temperature and can effectively lyse and release host cells and Gram-negative bacterial nucleic acids. Summary of the invention

[0008] The purpose of the present invention is to provide a stool nucleic acid preservation solution that can store and transport stool samples at room temperature. After the preservation solution is mixed with the stool sample, it can quickly penetrate into the exfoliated cells and microbial cells in the stool, so that the host cells and Gram-negative bacteria in the stool are lysed and inactivated, and the nucleic acid is released, and the nucleic acid of Gram-positive bacteria is lysed as little as possible. At the same time, the preservation solution can inhibit the activity of nucleases in the sample, stabilize the state of DNA, and can preserve the nucleic acid of the stool sample for a long time at room temperature.

[0009] In a first aspect of the present invention, a stool sample nucleic acid preservation solution is provided, wherein the preservation solution contains a chaotropic agent, a chelating agent, a buffer, an inorganic salt and an antibacterial agent.

[0010] In another preferred embodiment, the pH of the preservation solution is 7.0-9.0.

[0011] In another preferred embodiment, the chaotropic agent is selected from one or more of the following groups: sodium carbonate, lithium perchlorate, lithium thiocyanate, potassium thiocyanate, ammonium thiocyanate, and guanidine sulfate; preferably, the chaotropic agent is sodium carbonate.

[0012] In another preferred embodiment, the chelating agent is selected from one or more of: disodium ethylenediaminetetraacetate (EDTA-2Na), citric acid, ethylene glycol diethyl ether diaminetetraacetic acid (EGTA) and trisodium nitrilotriacetic acid monohydrate (NTA); preferably, the chelating agent is EDTA-2Na.

[0013] In another preferred embodiment, the buffer is selected from Tris, ethanolamine, triethanolamine, glycine, alanine, mannose and trisodium citrate, preferably Tris buffer.

[0014] In another preferred embodiment, the antibacterial agent is selected from the group consisting of Proclin 300, β-propiolactone (BPL), or a combination thereof, preferably Proclin 300.

[0015] In another preferred embodiment, the inorganic salt is selected from the group consisting of one or more of sodium chloride, potassium chloride, sodium fluoride, lithium chloride, and sodium iodide; preferably, the inorganic salt is sodium chloride.

[0016] In another preferred embodiment, the content of the chaotropic agent is 0.01-1M, preferably 0.05-0.5M, and more preferably 0.1-0.5M, based on the total amount of the stool sample nucleic acid preservation solution.

[0017] In another preferred embodiment, the content of the chelating agent is 0.01-2M, preferably 0.01-0.05M, based on the total amount of the stool sample nucleic acid preservation solution.

[0018] In another preferred embodiment, the content of the buffer is 0.01-2M, preferably 0.02-0.05M, based on the total amount of the stool sample nucleic acid preservation solution.

[0019] In another preferred embodiment, the content of the antibacterial agent is 1-100 ppm, preferably 8-15 ppm, based on the total amount of the stool sample nucleic acid preservation solution.

[0020] In another preferred embodiment, the content of the inorganic salt is 0.1-5M, preferably 0.2-0.5M, based on the total amount of the stool sample nucleic acid preservation solution.

[0021] The second aspect of the present invention provides the use of the stool sample nucleic acid preservation solution described in the first aspect of the present invention, which is used for (i) maintaining the stability of host exfoliated cells and microbial DNA in stool samples; and / or (ii) releasing the DNA of host exfoliated cells and Gram-negative bacteria in stool samples.

[0022] In another preferred embodiment, the feces comes from humans or non-human mammals.

[0023] In another preferred embodiment, the DNA includes microbial DNA or genomic DNA of human exfoliated cells (such as white blood cells).

[0024] A third aspect of the present invention provides a stool sample nucleic acid processing kit, the kit comprising the stool sample nucleic acid preservation solution according to the first aspect of the present invention; and

[0025] A label or instructions indicating that the kit is used to (i) maintain the stability of host exfoliated cells and microbial DNA in a stool sample; and / or (ii) release DNA of host exfoliated cells and Gram-negative bacteria in a stool sample.

[0026] In another preferred embodiment, the various components of the stool sample nucleic acid preservation solution are respectively located in different containers.

[0027] In another preferred embodiment, the various components of the stool sample nucleic acid preservation solution are located in the same container.

[0028] The fourth aspect of the present invention provides a use of the kit described in the third aspect of the present invention, for (i) maintaining the stability of host exfoliated cells and microbial DNA in fecal samples; and / or (ii) releasing the DNA of host exfoliated cells and Gram-negative bacteria in fecal samples.

[0029] A fifth aspect of the present invention provides a method for releasing DNA of host exfoliated cells and Gram-negative bacteria in a stool sample, comprising the steps of:

[0030] The stool sample is processed in the presence of the stool sample nucleic acid preservation solution described in the first aspect of the present invention.

[0031] In another preferred embodiment, the feces comes from humans or non-human mammals.

[0032] In another preferred embodiment, the method is for non-diagnostic purposes, for example, it is a method that only processes or detects tissues, body fluids or excrement that have been separated from the human or animal body to obtain information as an intermediate result, and its direct purpose is not to obtain diagnostic results or health conditions.

[0033] The sixth aspect of the present invention provides a method for preparing the stool preservation solution according to the first aspect of the present invention, comprising the steps of:

[0034] (1) Mix the required components according to the set ratio of chaotropic agent, chelating agent, buffer, antibacterial agent and inorganic salt, and make up to volume with deionized water;

[0035] (2) Filter to obtain the storage solution.

[0036] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0037] After long-term, extensive and in-depth research, through a large number of screenings and tests, the inventor unexpectedly discovered for the first time a stool preservation solution that can effectively lyse and release the host's intestinal exfoliated cell DNA and Gram-negative bacterial DNA, while less lysing Gram-positive bacterial DNA. It can be used for screening various Gram-negative pathogens, and can also be used for the initial screening of host diseases such as colorectal cancer, while significantly reducing the interference of Gram-positive bacterial nucleic acids on detection. On this basis, the inventor completed the present invention.

[0038] sample

[0039] As used herein, the terms "sample" and "specimen" are used interchangeably. In the present invention, the source of the sample is not particularly limited, and it can be collected from an organism. In a preferred embodiment, the "sample" is a stool sample. In addition, the method for collecting the sample is not particularly limited, and it can be collected by conventional methods.

[0040] Nucleic acid storage solution

[0041] In one embodiment of the present invention, a stool sample nucleic acid preservation solution is provided, comprising the following components: a chaotropic agent, a chelating agent, a buffer, an inorganic salt and an antibacterial agent, and the pH of the preservation solution is 7.0-9.0.

[0042] The chaotropic agent is configured to destroy the structure of macromolecules such as proteins and nucleic acids and denature them. The present invention unexpectedly found that certain specific chaotropic agents can effectively lyse host cells and Gram-negative bacteria, but the lysis effect on Gram-positive bacteria is significantly weaker. Exemplary chaotropic agents include sodium carbonate, lithium perchlorate, lithium thiocyanate, potassium thiocyanate, ammonium thiocyanate, guanidine sulfate, etc. The present invention preferably uses sodium carbonate as a chaotropic agent.

[0043] The chelating agent refers to an ion or molecule containing two or more electron-donating groups that can chelate with metals to form stable chemicals. Fecal samples contain a large amount of nucleases that can degrade cell and microbial nucleic acids. Therefore, it is necessary to make the nucleases ineffective. The activity of nucleases depends on Mg. 2+ Chelating agents such as EDTA-2Na, citric acid, EGTA, and NTA can effectively remove divalent metal ions from samples and inhibit most Mg-dependent 2+ The activity of the nuclease acting on the metal ion chelator is inhibited, the risk of nucleic acid degradation by the nuclease is reduced, and the stability of the nucleic acid is maintained. EDTA-2Na is preferably used as the metal ion chelator in the present invention.

[0044] The buffer can be an organic buffer solution and / or an inorganic buffer solution, wherein the organic solution can be Tris, ethanolamine, triethanolamine, glycine, alanine, mannose or trisodium citrate, etc., and the inorganic solution can be carbonate, bicarbonate, phosphate, hydrogen phosphate or borate. A given compound / buffer can buffer the pH of a solution only when its concentration is sufficient and the pH of the solution is close to (within about 1 pH unit) its pKa (acid dissociation constant). The present invention preferably uses Tris as a buffer. Tris buffer is a type of solution that can resist the influence of a small amount of strong acid and strong base from the outside world and maintain the pH of the system unchanged. It has a very high buffering capacity, high solubility in water, and is inert to many enzyme reactions, so it is of great significance in biochemical research. According to buffer theory, the effective buffering range of Tris is between 7.0-9.2.

[0045] The antibacterial agent is a substance that can inhibit bacterial growth. The antibacterial agent may not be able to kill bacteria, but it can inhibit the growth of bacteria, prevent excessive bacterial growth and endanger health. Common antibacterial agents include bile salts, bile acid, sodium selenite, potassium tellurite, sodium dodecyl sulfate, phenylethyl alcohol, cetyltrimethylammonium bromide, sodium tetrathiosulfonate, sodium azide, sodium sulfite, sodium propionate, thallium acetate, iodine solution, rhodamine acid, brilliant green, malachite green, crystal violet, penicillin G, gentamicin sulfate, amphotericin B, polymyxin B, polymyxin E, streptomycin, chloramphenicol, ProClin 300, cycloheximide, cephalosporin, bacitracin, vancomycin, novobiocin, trimethoprim (TMP), sulfapyridine, nystatin, oxytetracycline, etc. The present invention preferably uses ProClin 300 as an antibacterial agent. It has a broad spectrum of antibacterial properties, a fast action rate, can quickly penetrate the cell membrane, and inhibit specific enzymes that are essential for cell respiration; at the same time, a small dosage of 6-20ppm ProClin300 can achieve the purpose of antibacterial. In addition, ProClin's safety toxicity is much lower than similar products, it can be mixed with water in any ratio, and has good compatibility with enzymes and diagnostic factors, and will not affect the subsequent reactions with enzymes or antibodies.

[0046] In addition, the preservation solution also includes one or more inorganic salts. Exemplary inorganic salts include sodium chloride, potassium chloride, sodium fluoride, lithium chloride, sodium iodide, etc. Sodium chloride is preferred in the present invention.

[0047] In another preferred embodiment, the content of the chaotropic agent is 0.01-1M, preferably 0.05-0.5M, and more preferably 0.1-0.5M, based on the total amount of the stool sample nucleic acid preservation solution.

[0048] In another preferred embodiment, the content of the chelating agent is 0.01-2M, preferably 0.01-0.05M, based on the total amount of the stool sample nucleic acid preservation solution.

[0049] In another preferred embodiment, the content of the buffer is 0.01-2M, preferably 0.02-0.05M, based on the total amount of the stool sample nucleic acid preservation solution.

[0050] In another preferred embodiment, the content of the antibacterial agent is 1-100 ppm, preferably 8-15 ppm, based on the total amount of the stool sample nucleic acid preservation solution.

[0051] In another preferred embodiment, the content of the inorganic salt is 0.1-5M, preferably 0.2-0.5M, based on the total amount of the stool sample nucleic acid preservation solution.

[0052] In another embodiment of the present invention, a use of the above-mentioned stool sample nucleic acid preservation solution is provided, characterized in that it is used to (i) maintain the stability of host exfoliated cells and microbial DNA in stool samples; and / or (ii) release the DNA of host exfoliated cells and Gram-negative bacteria in stool samples.

[0053] In another preferred embodiment, the feces are derived from feces of humans or other mammals other than Homo sapiens.

[0054] In another preferred embodiment, according to the present invention, the mass volume ratio of the stool sample to the preservation reagent is 1:20-1:4.

[0055] In another preferred embodiment, the DNA includes intestinal microbial DNA or host exfoliated cell DNA.

[0056] In another embodiment of the present invention, a method for preparing the above-mentioned stool sample DNA preservation solution is provided, comprising the steps of:

[0057] (1) Mix the required components according to the set ratio of chaotropic agent, chelating agent, buffer, antibacterial agent and inorganic salt, and make up to volume with deionized water;

[0058] (2) Filter to obtain the storage solution.

[0059] Stool Sample Nucleic Acid Processing Kit

[0060] The present invention also provides a stool sample nucleic acid processing kit for (i) maintaining the stability of host exfoliated cells and microbial DNA in stool samples; and / or (ii) releasing host exfoliated cells and Gram-negative bacteria DNA in stool samples.

[0061] In the present invention, the kit of the present invention contains a stool sample nucleic acid preservation solution of the following components: a chaotropic agent, a chelating agent, a buffer, an inorganic salt and an antibacterial agent, and the pH of the preservation solution is 7.0-9.0.

[0062] In the present invention, the ratio between the components in the kit of the present invention and the content of the components may not be particularly limited.

[0063] The components in the kit of the present invention can be purchased or prepared by conventional methods.

[0064] In the present invention, the various components of the preservation solution may be located in different containers or in the same container.

[0065] The kit of the present invention can (i) maintain the stability of host exfoliated cells and microbial DNA in stool samples; and / or (ii) release host exfoliated cells and Gram-negative bacteria DNA in stool samples.

[0066] The main advantages of the present invention include:

[0067] (1) Room temperature transportation and storage: The stool preservation solution provided by the present invention can effectively preserve the host intestinal exfoliated cell DNA and intestinal microbial DNA under room temperature conditions, and effectively prevent nucleic acid degradation during sample storage and transportation at room temperature. The samples can be stored at 15-37°C for at least 30 days, reducing the possibility of nucleic acid degradation due to improper transportation or storage, solving the problem that the old stool preservation requires low-temperature storage, reducing the dependence on liquid nitrogen dry ice and freezing equipment, and can effectively reduce the cost of sample transportation and storage.

[0068] (2) Effectively lyse the host's intestinal exfoliated cells and Gram-negative bacterial DNA: The stool preservation solution provided by the present invention can effectively lyse and release the host's intestinal exfoliated cell DNA and Gram-negative bacterial DNA, with less lysis of Gram-positive bacterial DNA, so that it can be used for screening various Gram-negative pathogens, and can also be used for the initial screening of host diseases such as colorectal cancer, while avoiding the interference of Gram-positive bacterial nucleic acids in detection.

[0069] (3) Simple and user-friendly operation: The stool preservation solution provided by the present invention can lyse and release nucleic acids. The operator only needs to take the supernatant from the stool preservation solution containing the sample after centrifugation to perform the nucleic acid extraction step without the need for a series of complicated pre-treatment operations, which not only saves time and material costs, but also reduces the discomfort of the operator.

[0070] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0071] Unless otherwise specified, the materials and cell lines used in the present invention are commercially available products.

[0072] Example 1. Feces preservation solution formulation

[0073] The stool sample preservation solution of the present invention mainly comprises the following components: 0.1-0.5M sodium carbonate, 0.01-0.05M EDTA-2Na, 0.02-0.05M Tris, 8-15ppm ProClin300, 0.2-0.5M sodium chloride, and the pH value of the preservation solution is 7.0-9.0, preferably 7.5-8.5.

[0074] Table 1. Stool preservation solution formula

[0075]

[0076] Example 2. Method for storing reagent configuration

[0077] Preparation method of No. 1 fecal preservation solution: weigh 10.6g sodium carbonate, 16.8g EDTA-2Na, 3.6g Tris, and 17.53g sodium chloride, use a pipette to absorb 8μL ProClin300, add purified water to 980mL, add appropriate amount of sodium hydroxide to adjust the pH to 8.0, and then add purified water to make up to 1L.

[0078] Preparation method of No. 2 stool preservation solution: weigh 26.5g sodium carbonate, 3.36g EDTA-2Na, 2.42g Tris, and 29.22g sodium chloride, use a pipette to absorb 13μL ProClin300, add purified water to 980mL, add appropriate amount of sodium hydroxide to adjust the pH to 8.0, and then add purified water to make up to 1L.

[0079] Preparation method of No. 3 fecal preservation solution: weigh 53g sodium carbonate, 6.72g EDTA-2Na, 6.05g Tris, and 11.7g sodium chloride, use a pipette to absorb 15μL ProClin300, add purified water to 980mL, add appropriate amount of sodium hydroxide to adjust the pH to 8.0, and then add purified water to make up to 1L.

[0080] Example 3. Study on the nucleic acid preservation ability of feces preservation fluid

[0081] Experimental materials: feces preservation solutions No. 1, 2, and 3 were prepared according to the methods described in Examples 1 and 2, and fresh feces of volunteers.

[0082] Experimental method: Add equal amounts (about 2 grams) of fresh feces to 20 mL of prepared feces preservation solution No. 1, 2, and 3, respectively, and mix them by inversion; each preservation solution is divided into four equal parts and stored at room temperature for 0 days, 15 days, 30 days, and 45 days, respectively. Take 300 μL of preservation solution sample from each part and use the fecal genomic DNA extraction kit (Kangwei Century, product number CW2556) for DNA extraction. For specific operating steps, please refer to the product manual. The sample is eluted in 70 μL elution solution, and 5 μL of each sample is taken as a template for fluorescent quantitative qPCR detection.

[0083] The extracted DNA nucleic acid samples were tested by fluorescence quantitative PCR using Kangwei Century qPCR detection reagent (Cat. No. CW0957). The human gene detection primers used were human internal reference gene β-actin primers, and the primer sequence information was β-actin-F: 5'-GCGCCGTTCCGAAAGTT-3' (SEQ ID No. 1), β-actin-R: 5'-CGGCGGATCGGCAAA-3' (SEQ ID No. 2); the bacterial detection primers used were 16S rRNA genes, and the primer sequence information was 16S-F: 5'-CCTACGGGAGGCAGCAG-3' (SEQ ID No. 3), 16S-R: 5'-ATTACCGCGGCTGCTGG-3' (SEQ ID No. 4). For specific operations, refer to the instructions. The reaction system is as shown in the following table.

[0084] Table 2 PCR reaction system

[0085]

[0086]

[0087] Amplification was performed on an ABI 7500 fluorescent quantitative PCR instrument. The PCR reaction conditions are shown in Table 3:

[0088] Table 3 PCR reaction conditions

[0089]

[0090] Table 4 qPCR detection of the preservation effect of fecal preservation solution on human nucleic acid

[0091]

[0092] Table 5 qPCR detection of the preservation effect of feces preservation solution on bacterial nucleic acid

[0093]

[0094] Experimental results: The results are shown in Tables 4 and 5. Compared with the Ct of the 0-day control group, the Ct differences of samples preserved in preservation solutions 1, 2, and 3 after 15 and 30 days at room temperature were basically within 1, and after 45 days, the Ct differences were basically greater than 1, indicating that the three preservation solutions can effectively maintain the stability of DNA in fecal samples at room temperature and can ensure effective detection of nucleic acids even after 30 days of storage.

[0095] Example 4. Study on the ability of stool preservation fluid to release nucleic acids from human and Gram-negative bacteria in samples

[0096] Experimental group preservation solution: No. 1 feces preservation solution was prepared according to the method described in Examples 1 and 2;

[0097] Comparative group preservation solution: Referring to patent CN 111197042 B, a stool sample preservation solution (including 0.5% v / v TritonX-100, 0.1% w / v SDS, 50mM EDTA, 50mM Tris, 150mM sodium chloride, 2% w / v mannitol, 0.1% w / v lauryl methacrylate, 5% v / v ethanol, 0.1% w / v peppermint essential oil, 0.25% w / v jasmine essential oil, the balance is water, pH 8.0) was prepared as a comparative example.

[0098] Fecal sample collection and DNA extraction: Fecal samples were collected from 4 volunteers. An equal amount of feces (4 g) was taken from each sample and put into two kinds of fecal preservation solutions. The mixture was kept at room temperature for 7 days. Then, 300 μL of the preservation solution was taken from each sample and DNA was extracted using a fecal genomic DNA extraction kit (Kangwei Century, catalog number CW2556). For specific operating steps, please refer to the product manual. The samples were eluted in 70 μL of elution solution, and 5 μL of each sample was taken as a template for fluorescence quantitative qPCR detection.

[0099] The extracted DNA nucleic acid samples were tested by fluorescent quantitative PCR using Kangwei Century qPCR detection reagent (Cat. No. CW0957). The human gene detection primers used were human internal reference gene β-actin primers, and the primer sequence information was β-actin-F: 5'-GCGCCGTTCCGAAAGTT-3' (SEQ ID No. 1), β-actin-R: 5'-CGGCGGATCGGCAAA-3' (SEQ ID No. 2); the Gram-negative bacteria used were Helicobacter pylori (HP) specific primers, and the primer sequence information was HP-F: 5'-AGGGCTTTGGCTCTTATGG-3' (SEQ ID No. 5), HP-R: 5'-TCAAACTACCCACCAAGCATT-3' (SEQ ID No. 6). For specific operations, refer to the instructions. The reaction system and PCR reaction conditions are as in Example 3.

[0100] Table 6 The release capacity of different stool preservation solutions for nucleic acid of human origin and Gram-negative bacteria in samples

[0101]

[0102]

[0103] Experimental results: The results are shown in Table 6. Compared with the control group, the preservation solution of the experimental group had no difference in amplifying the human reference gene β-actin (Ct<1), and the Ct value was significantly reduced when amplifying the Helicobacter pylori specific primer. This shows that the preservation solution of the present invention has no difference from the preservation solution of the control group in preserving and lysing the nucleic acid of human fecal intestinal exfoliated cells, avoiding the inhibitory effect of microbial nucleic acid on human nucleic acid, and can effectively preserve and lyse the nucleic acid of Gram-negative bacteria.

[0104] Example 5. Study on the ability of stool preservation fluid to release human and bacterial nucleic acids in samples

[0105] Experimental group preservation solution: No. 1 feces preservation solution was prepared according to the method described in Examples 1 and 2;

[0106] Comparative group preservation solution: Referring to patent CN107937391A, a stool sample preservation solution (including 5M guanidine isothiocyanate, 5% Triton-100, 3% NP-40, 10mM EDTA, 100mM Tris-HCl, the balance is water, pH 8.0) was prepared as a comparative example.

[0107] Fecal sample collection and DNA extraction: Fecal samples were collected from 4 volunteers. An equal amount of feces (4 g) was taken from each sample and put into two kinds of fecal preservation solutions. The mixture was kept at room temperature for 7 days. Then, 300 μL of the preservation solution was taken from each sample and DNA was extracted using a fecal genomic DNA extraction kit (Kangwei Century, catalog number CW2556). For specific operating steps, please refer to the product manual. The samples were eluted in 70 μL of elution solution, and 5 μL of each sample was taken as a template for fluorescence quantitative qPCR detection.

[0108] The extracted DNA nucleic acid samples were subjected to fluorescent quantitative PCR detection using Kangwei Century qPCR detection reagent (Cat. No. CW0957). The human gene detection primers used were human internal reference gene β-actin primers, and the primer sequence information was β-actin-F: 5'-GCGCCGTTCCGAAAGTT-3' (SEQ ID No. 1), β-actin-R: 5'-CGGCGGATCGGCAAA-3' (SEQ ID No. 2); the Gram-negative bacteria used were Helicobacter pylori (HP) specific primers, and the primer sequence information was HP-F: 5'-AGGGCTTTGGCTCTTATGG-3' (SEQ ID No. 5), HP-R: 5'-TCAAACTACCCACCAAGCATT-3' (SEQ ID No.6); the Gram-positive bacteria used are Bifidobacterium (BIF) specific primers, and the primer sequence information is BIF-F: 5'-TTACCTGGGCTTGACATGTTCCC-3' (SEQ ID No.7), BIF-R: 5'-AACACGGGGCGAGGGTT-3' (SEQ ID No.8). For specific operations, refer to the instructions. The reaction system and PCR reaction conditions are as in Example 3.

[0109] Table 7 The release capacity of different stool preservation solutions on human and bacterial nucleic acids in samples

[0110]

[0111] Experimental results: The results are shown in Table 7. Compared with the control group, the preservation solution of the experimental group has a significantly lower Ct value in amplifying the human reference gene β-actin, no difference in the Ct value when amplifying Helicobacter pylori HP-specific primers, and a significantly increased Ct value when amplifying Bifidobacterium. This shows that the preservation solution of the present invention is no different from the preservation solution of the control group in terms of preservation and lysis of Gram-negative bacteria, and the ability to lyse and release Gram-positive bacteria is significantly less than that of the preservation solution of the control group. Because the lysis ability of bacteria is weak, the huge microbial nucleic acid is prevented from interfering with the amplification of human nucleic acid, so the amplification of human reference genes is better than the preservation solution of the control group. Therefore, the preservation solution of the present invention not only takes into account the preservation of human nucleic acids, but also can be used for early screening of diseases such as colorectal cancer, and is also suitable for screening of Gram-negative pathogens.

[0112] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A stool sample nucleic acid storage solution, characterized in that: The preservation solution contains a chaotropic agent, a chelating agent, a buffer, an inorganic salt and an antibacterial agent.

2. The stool sample nucleic acid storage solution according to claim 1, characterized in that: The chaotropic agent is selected from one or more of the following groups: sodium carbonate, lithium perchlorate, lithium thiocyanate, potassium thiocyanate, ammonium thiocyanate, and guanidine sulfate; preferably, the chaotropic agent is sodium carbonate.

3. The stool sample nucleic acid storage solution according to claim 1, characterized in that: The chelating agent is selected from one or more of: disodium ethylenediaminetetraacetate (EDTA-2Na), citric acid, ethylene glycol diethyl ether diaminetetraacetic acid (EGTA) and trisodium nitrilotriacetic acid monohydrate (NTA); preferably, the chelating agent is EDTA-2Na.

4. The stool sample nucleic acid storage solution according to claim 1, characterized in that: The buffer is selected from Tris, ethanolamine, triethanolamine, glycine, alanine, mannose and trisodium citrate, preferably Tris buffer.

5. The stool sample nucleic acid storage solution according to claim 1, wherein: The antibacterial agent is selected from the group consisting of Proclin 300, β-propiolactone (BPL), or a combination thereof, preferably Proclin 300.

6. The stool sample nucleic acid storage solution according to claim 1, characterized in that: The inorganic salt is selected from the group consisting of one or more of sodium chloride, potassium chloride, sodium fluoride, lithium chloride, and sodium iodide; preferably, the inorganic salt is sodium chloride.

7. Use of the stool sample nucleic acid preservation solution according to claim 1, for (i) maintaining the stability of host exfoliated cells and microbial DNA in stool samples; and / or (ii) releasing DNA of host exfoliated cells and Gram-negative bacteria in stool samples.

8. A stool sample nucleic acid processing kit, characterized in that: The kit contains the stool sample nucleic acid preservation solution according to claim 1; and A label or instructions indicating that the kit is used to (i) maintain the stability of host exfoliated cells and microbial DNA in a stool sample; and / or (ii) release DNA of host exfoliated cells and Gram-negative bacteria in a stool sample.

9. A method for releasing DNA from host exfoliated cells and Gram-negative bacteria in a stool sample, comprising the steps of: The stool sample is processed in the presence of the stool sample nucleic acid storage solution of claim 1.

10. A method for preparing the stool preservation solution according to claim 1, characterized in that: Includes steps: (1) Mix the required components according to the set ratio of chaotropic agent, chelating agent, buffer, antibacterial agent and inorganic salt, and make up to volume with deionized water; (2) Filter to obtain the storage solution.

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