Artificial excrement and application of artificial excrement as reference product of excrement sample

Abstract

The invention discloses artificial excrement and application of the artificial excrement as a reference product of an excrement sample. The artificial excrement is prepared from sodium cholate, bilirubin, calcium chloride, magnesium chloride, amino acid, triglyceride, microcrystalline cellulose, small intestine epithelial cells, escherichia coli and water. The problems that a real excrement sample is difficult to collect, easy to be influenced by heterogeneity of the sample, large in environmental pollution and easy to deteriorate and degrade in the storage process are solved, and the artificial excrement with excellent uniformity, reproducibility and stability is provided; the scene of nucleic acid degradation and gene expression quantity detection of a real excrement sample at normal temperature can be objectively simulated, and long-term storage under a low-temperature condition can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to artificial feces and application thereof as a reference for feces samples. Background Art

[0002] In the past decade, the incidence and mortality of malignant tumors have been on a continuous rise, and malignant tumors have become the main cause of harm to residents' health. In recent years, gene mutation detection has developed rapidly and has important clinical significance in early tumor screening, precise targeted drug use and disease monitoring.

[0003] Non-invasive genetic testing, such as genetic testing based on stool samples, has attracted widespread attention due to its convenience and low invasiveness. Feces is an important biological testing sample. The nucleic acid components carried in feces come from the exfoliated epithelial cells of the digestive tract. When there is a digestive tract tumor, the tumor will shed cells into the feces. Therefore, stool samples play an important role in the detection of digestive tract tumors. For example, in colorectal cancer screening, compared with traditional invasive examinations (colonoscopy) and fecal occult blood tests, genetic testing technology for stool samples has shown great potential. The entire operation is completed in vitro, and tumor-related genetic variations can be detected to improve the detection rate of early cancer, with higher sensitivity and specificity.

[0004] However, stool samples are not easy to preserve, and the nucleic acids in the stool are easily degraded during storage and transportation. The nucleic acids finally extracted often have quality problems and cannot meet the subsequent testing requirements. It is necessary to develop preservation reagents and nucleic acid extraction reagents suitable for stool samples. In the process of developing these reagents, it is necessary to prepare stool reference products that can objectively reflect the efficiency of stool sample preservation and extraction in reality, which are used to verify the extraction yield, precision and other properties of the corresponding test kits. At present, the main source of stool reference products is stool samples collected from healthy people in the clinic. However, the number of samples is limited, and it is difficult to ensure the uniformity, consistency and stability of the reference products, and it cannot play the role of calibration of standardized reference products. Therefore, it is necessary to develop artificial stool samples as stool reference products that avoid individual differences, have more stable performance, and are convenient for calibration and quality control. Although some artificial feces samples have been disclosed in the prior art, such as the feces simulant in CN110857905A, its composition is relatively simple and mainly simulates the physical state of feces (simulating the crystalline particles in feces), it is still insufficient in simulating the complexity and stability of real samples. For example, it is impossible to simulate the degradation of nucleic acids contained in real feces samples at room temperature. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides an artificial feces and its application as a reference for feces samples.

[0006] The first object of the present invention is to provide an artificial feces.

[0007] The second object of the present invention is to provide the use of the artificial feces as a reference for fecal samples.

[0008] The third object of the present invention is to provide the use of the artificial feces in preparing nucleic acid extraction reagents for fecal samples.

[0009] A fourth object of the present invention is to provide a use of the artificial feces in detecting and / or evaluating the performance of nucleic acid extraction reagents of fecal samples.

[0010] In order to achieve the above object, the present invention is implemented by the following scheme:

[0011] An artificial feces composed of sodium cholate, bilirubin, calcium chloride, magnesium chloride, amino acids, triglycerides, microcrystalline cellulose, small intestinal epithelial cells, Escherichia coli and water.

[0012] Among them, sodium bile acid, bilirubin and triglycerides are the main DNA extraction interfering substances in stool samples; calcium chloride, magnesium chloride and amino acids are the main salt components in stool samples, which constitute interfering substances and maintain the stability of osmotic potential, and serve as basic substances to maintain the stability of cells and strains. Intestinal epithelial cells are cells that normally exist in stool and serve as a relatively stable source of human genomic DNA in stool samples. Escherichia coli is a Gram-negative bacterium that is commonly present in stool samples. It serves as a source of non-human genomic DNA in the samples and maintains a similar degradation rate between this protocol and real stool samples at room temperature. Microcrystalline cellulose and water constitute the remaining mass components of stool samples.

[0013] Preferably, the mass ratio of the sodium cholate, the bilirubin, the calcium chloride, the magnesium chloride and the microcrystalline cellulose is (0.05-0.1): (0.05-0.1): (0.02-0.1): (0.02-0.1): (1.5-2).

[0014] More preferably, the mass ratio of the sodium cholate, the bilirubin, the calcium chloride, the magnesium chloride and the microcrystalline cellulose is 0.05:0.05:0.02:0.02:1.5.

[0015] More preferably, the mass ratio of the sodium cholate, the bilirubin, the calcium chloride, the magnesium chloride and the microcrystalline cellulose is 0.1:0.1:0.1:0.1:2.

[0016] Preferably, in the artificial feces, measured by mass to volume ratio, the content of sodium cholate is (0.005-0.01) g / mL, the content of bilirubin is (0.005-0.01) g / mL, the content of calcium chloride is (0.002-0.01) g / mL, the content of magnesium chloride is (0.002-0.01) g / ml, and the content of microcrystalline cellulose is (0.5-0.2) g / mL.

[0017] More preferably, in the artificial feces, measured by mass to volume ratio, the content of sodium cholate is 0.005 g / mL, the content of bilirubin is 0.005 g / mL, the content of calcium chloride is 0.002 g / mL, the content of magnesium chloride is 0.002 g / ml, and the content of microcrystalline cellulose is 0.5 g / mL.

[0018] More preferably, in the artificial feces, measured by mass to volume ratio, the content of sodium cholate is 0.01 g / mL, the content of bilirubin is 0.01 g / mL, the content of calcium chloride is 0.01 g / mL, the content of magnesium chloride is 0.01 g / ml, and the content of microcrystalline cellulose is 0.2 g / mL.

[0019] Preferably, the content of small intestinal epithelial cells is 5×10 3 cells / mL~8×10 3 cells / mL.

[0020] More preferably, the content of small intestinal epithelial cells is 5×10 3 cells / mL.

[0021] Preferably, the human small intestinal epithelial cells are human small intestinal mucosal epithelial cells.

[0022] Preferably, the content of Escherichia coli is 10 8 cfu / mL~1.2×10 8 cfu / mL.

[0023] More preferably, the content of Escherichia coli is 10 8 cfu / mL.

[0024] Preferably, the Escherichia coli is a Rosetta 2 strain.

[0025] Preferably, the content of the amino acid is 0.3 μmol / mL to 0.6 μmol / mL.

[0026] More preferably, the concentration of the amino acid is 0.3 μmol / mL.

[0027] Preferably, the amino acids include L-aspartic acid, L-threonine, L-serine, L-glutamic acid, L-proline, glycine, L-alanine, L-cystine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-lysine, L-histidine, ammonium chloride and L-arginine.

[0028] Preferably, the concentration of triglyceride is 0.01 g / mL to 0.02 g / mL.

[0029] More preferably, the concentration of triglycerides is 0.01 g / mL.

[0030] Preferably, the triglycerides include triacetin (C2:0), tributyrin (C4:0), tricaproin (C6:0), tricaprylin (C8:0) and tricaprin (C10:0).

[0031] In some specific embodiments, each 10 mL of the artificial feces comprises 0.05 g to 0.1 g of sodium cholate, 0.05 g to 0.1 g of bilirubin, 0.02 g to 0.1 g of calcium chloride, 0.02 g to 0.1 g of magnesium chloride, 0.3 μmol / mL to 0.6 μmol / mL of amino acids, 0.01 g / mL to 0.02 g / mL of triglycerides, 1.5 g to 2 g of microcrystalline cellulose, 5×10 3 cells / mL~8×10 3 cells / mL small intestinal epithelial cells, 10 8 cfu / mL~1.2×10 8 cfu / mL Escherichia coli and water.

[0032] In some specific embodiments, each 10 mL of the artificial feces comprises 0.05 g sodium cholate, 0.05 g bilirubin, 0.02 g calcium chloride, 0.02 g magnesium chloride, 0.025 μmol / mL amino acids, 0.01 g / mL triglycerides, 1.5 g microcrystalline cellulose, 5×10 3 cells / mL cells / mL small intestinal epithelial cells, 10 8 cfu / mL Escherichia coli and water.

[0033] In some specific embodiments, each 10 mL of the artificial feces comprises 0.1 g sodium cholate, 0.1 g bilirubin, 0.1 g calcium chloride, 0.1 g magnesium chloride, 0.05 μmol / mL amino acids, 0.02 g / mL triglycerides, 2 g microcrystalline cellulose, 8×10 3 cells / mL small intestinal epithelial cells, 1.2×10 8 cfu / mL Escherichia coli and water.

[0034] The method for preparing the artificial feces comprises the steps of uniformly mixing the sodium cholate, the bilirubin, the calcium chloride, the magnesium chloride, the small intestinal epithelial cells, the Escherichia coli and the water to obtain the artificial feces.

[0035] The present invention makes artificial feces with specific components so that the interfering substance content, shelf life, nucleic acid content and other properties are within the range of healthy human feces, and has very good uniformity, meets the requirements of being a reference product, simulates feces samples, can be used instead of real feces samples, is suitable for head-to-head comparison tests in performance testing of products such as nucleic acid extraction reagents or nucleic acid extraction kits for feces samples, and preservation reagents for feces samples, and evaluates the extraction and preservation effects of feces samples.

[0036] Therefore, the present invention also requests protection for the following contents:

[0037] The artificial feces is used as a reference for feces samples.

[0038] The artificial feces is used in preparing nucleic acid extraction reagents for feces samples.

[0039] The artificial feces is used in detecting and / or evaluating the performance of nucleic acid extraction reagents of fecal samples.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention overcomes the problems of real stool samples, such as difficulty in collecting, susceptibility to sample heterogeneity, high environmental pollution, and easy deterioration and degradation during storage, and provides artificial feces with excellent uniformity, reproducibility and stability. It can not only objectively simulate the scenarios of nucleic acid degradation and gene expression detection of real stool samples at room temperature, but also can be preserved for a long time under low temperature conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a trend chart of the amount of DNA extracted (ng) from the test samples extracted by commercial kit A and stored for different periods of time.

[0043] Figure 2 This is a trend chart of the amount of DNA extracted (ng) from the test samples extracted by commercial kit B and stored for different periods of time.

[0044] Figure 3 This is a trend chart of the amount of DNA extracted (ng) from the test samples extracted by commercial kit C and stored for different periods of time.

[0045] Figure 4 This is a trend chart of the amount of DNA extracted (ng) from the test samples extracted by commercial kit D and placed for different periods of time. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified. The main reagents involved in the following examples include: sodium cholate (CAS No.: 361-09-1), bilirubin (CAS No.: 635-65-4), calcium chloride (CAS No.: 10043-52-4), magnesium chloride (CAS No.: 7786-30-3), mixed amino acid solution (containing 12 kinds of amino acids (L-aspartic acid, L-threonine, L-serine, L-glutamic acid, L-proline, glycine, L-alanine, L-cystine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-lysine, L-histidine, ammonium chloride and L-arginine), each amino acid concentration is 2.50 μmol l / ml, Catalog No.: 013-08391, Manufacturer: Wako Pure Chemical Industries, Ltd.), triglyceride mixture (lipid standards (triacetin (C2:0), tributyrin (C4:0), tricaproin (C6:0), tricaprylin (C8:0) and tricaprin (C10:0)), Catalog No.: 17810, Manufacturer: Merck Supelco), microcrystalline cellulose powder (Catalog No. 1.02331, Manufacturer: millipore), human small intestinal epithelial cells (Catalog No.: CP-H039, Manufacturer: Wuhan Punosai Biotechnology Co., Ltd.) and Escherichia coli Rosetta 2 strain (Catalog No. B0067, Manufacturer: Baosai Biotechnology).

[0047] Example 1 An artificial feces sample

[0048] 0.05 g sodium cholate, 0.05 g bilirubin, 0.02 g calcium chloride, 0.02 g magnesium chloride, 0.1 ml mixed amino acid solution, 0.1 g triglyceride mixture, 1.5 g microcrystalline cellulose, 0.3 g human small intestinal epithelial cells cultured routinely in the laboratory (containing 5 × 10 4 cells) and 1 mL of routine laboratory culture of Escherichia coli Rosetta 2 (containing 10 9 cfu / ml) was added into a clean sterile centrifuge tube, filled with water to 10 mL, and placed on a shaker to mix thoroughly for 10 min to prepare an artificial feces sample.

[0049] Example 2 An artificial feces sample

[0050] 0.1 g sodium cholate, 0.1 g bilirubin, 0.1 g calcium chloride, 0.1 g magnesium chloride, 0.2 ml mixed amino acid solution, 0.2 g triglyceride mixture, 2 g microcrystalline powdered cellulose, 0.5 g human small intestinal epithelial cells cultured routinely in the laboratory (containing 8 × 10 4 cells) and 1.2 mL of routine laboratory culture of Escherichia coli Rosetta 2 (containing 10 9 cfu / ml) was added into a clean sterile centrifuge tube, filled with water to 10 mL, and placed on a shaker to mix thoroughly for 10 min to prepare an artificial feces sample.

[0051] Comparative Example 1: An artificial feces sample

[0052] The artificial feces sample was prepared by reacting barium chloride with sulfuric acid to obtain a 0.05 mol / L barium sulfate solution, which was then diluted 10 times with water.

[0053] Comparative Example 2: An artificial feces sample

[0054] The artificial feces sample was prepared by reacting ammonium oxalate monohydrate and calcium chloride to obtain a 0.1 mol / L calcium oxalate solution, which was then diluted 10 times with water.

[0055] Comparative Example 3: An artificial feces sample

[0056] Barium sulfate solution is prepared by reacting barium chloride with sulfuric acid.

[0057] Barium sulfate solution (the final concentration of barium sulfate is 0.05 mol / L), 0.05 g sodium cholate, 0.05 g bilirubin, 0.1 ml mixed amino acid solution, 0.1 g triglyceride mixture, 1.5 g microcrystalline cellulose, 0.3 g human small intestinal epithelial cells cultured routinely in the laboratory (containing 5 × 10 4 cells) and 1 mL of routine laboratory culture of Escherichia coli Rosetta 2 (containing 10 9 cfu / ml) was added into a clean sterile centrifuge tube, filled with water to 10 mL, and placed on a shaker to mix thoroughly for 10 min to prepare an artificial feces sample.

[0058] Comparative Example 4: an artificial feces sample

[0059] The calcium oxalate solution is prepared by reacting ammonium oxalate monohydrate and calcium chloride.

[0060] Calcium oxalate solution (the final concentration of calcium oxalate is 0.01 mol / L), 0.05 g sodium cholate, 0.05 g bilirubin, 0.1 ml mixed amino acid solution, 0.1 g triglyceride mixture, 1.5 g microcrystalline cellulose, 0.3 g human small intestinal epithelial cells cultured routinely in the laboratory (containing 5 × 104 cells) and 1 mL of routine laboratory culture of Escherichia coli Rosetta 2 (containing 10 9 cfu / ml) was added into a clean sterile centrifuge tube, filled with water to 10 mL, and placed on a shaker to mix thoroughly for 10 min to prepare an artificial feces sample.

[0061] Comparative Example 5: An artificial feces sample

[0062] 0.05 g sodium cholate, 0.05 g bilirubin, 0.1 ml mixed amino acid solution, 0.1 g triglyceride mixture, 0.3 g human small intestinal epithelial cells cultured in the laboratory (containing 5 × 10 4 cells) and 1 mL of routine laboratory culture of Escherichia coli Rosetta 2 (containing 10 9 cfu / ml) was added into a clean sterile centrifuge tube, filled with water to 10 mL, and placed on a shaker to mix thoroughly for 10 min to prepare an artificial feces sample.

[0063] Comparative Example 6: An artificial feces sample

[0064] 0.05 g sodium cholate, 0.05 g bilirubin, 0.02 g calcium chloride, 0.02 g magnesium chloride, 0.1 ml mixed amino acid solution, 0.1 g triglyceride mixture, 1.5 g microcrystalline powder cellulose and 1 mL of laboratory routine cultured Escherichia coli Rosetta 2 bacterial solution (containing 10 9 cfu / ml) was added into a clean sterile centrifuge tube, filled with water to 10 mL, and placed on a shaker to mix thoroughly for 10 min to prepare an artificial feces sample.

[0065] Comparative Example 7: An artificial feces sample

[0066] 0.05 g sodium cholate, 0.05 g bilirubin, 0.02 g calcium chloride, 0.02 g magnesium chloride, 0.1 ml mixed amino acid solution, 0.1 g triglyceride mixture, 1.5 g microcrystalline powdered cellulose and 0.3 g human small intestinal epithelial cells cultured routinely in the laboratory (containing 5 × 10 4 The artificial feces sample was prepared by adding 10 mL of 1% dapoxetine (100 cells) into a clean sterile centrifuge tube, adding water to 10 mL, and placing it on a shaker to mix thoroughly for 10 min.

[0067] Comparative Example 8: an artificial feces sample

[0068] 0.05 g sodium cholate, 0.05 g bilirubin, 0.05 g calcium chloride, 0.02 g magnesium chloride, 0.1 ml mixed amino acid solution, 0.1 g triglyceride mixture, 1.5 g microcrystalline powdered cellulose, 0.15 g human small intestinal epithelial cells cultured in the laboratory (containing 2.5 × 104 cells) and 0.5 mL of routine laboratory culture of Escherichia coli Rosetta 2 (containing 10 9 cfu / ml) was added into a clean sterile centrifuge tube, filled with water to 10 mL, and placed on a shaker to mix thoroughly for 10 min to prepare an artificial feces sample.

[0069] Application Example 1: Artificial feces samples simulate nucleic acid degradation in real feces samples

[0070] 1. Preparation of samples to be tested

[0071] The freshly prepared artificial stool samples of Examples 1 to 2 and Comparative Examples 1 to 8, as well as an equal amount of fresh stool samples of healthy subjects collected clinically (i.e., real stool samples), were respectively added with 100 ng of a new coronavirus-positive reference substance (article number: BDS-BW-117, manufacturer: Guangzhou Bondeson Biotechnology Co., Ltd.), mixed to obtain 12 groups of samples to be tested, and placed at room temperature for 1 hour.

[0072] 2. Nucleic acid extraction

[0073] At 0 h, 0.5 h, and 1 h, equal amounts of stool samples were taken from each sample to be tested and DNA was extracted using four different commercial kits (A: QIAamp Fast DNA Stool Mini Kit, B: QIAsymphony Power Fecal Pro DNA Kit, C: Tiangen Fecal Genomic DNA Extraction Kit, and D: Kangwei Century Magbead Stool DNA Midi Kit).

[0074] 3. Nucleic acid testing

[0075] For the extracted DNA, the concentration was measured using Invitrogen Qubit3.0, and the Agilent 4200 gene analyzer characterized the integrity of the genomic DNA (i.e., DIN value. In the construction of the second-generation sequencing library, a nucleic acid DIN value ≥ 2.5 is a qualified sample for library construction, and a DIN value < 2.5 is a risk in library construction and sequencing). Afterwards, a new coronavirus nucleic acid detection kit (Cat. No.: SJ-HX-804-2, Manufacturer: Shanghai Berger Medical Technology Co., Ltd.) was used for fluorescent quantitative PCR to detect the content of the virus ORF1ab gene.

[0076] 4. Test results

[0077] Table 1 The extraction amount (ng) and integrity results of the DNA samples placed for 0 h

[0078]

[0079] Table 2 Results of DNA extraction (ng) and integrity of samples placed for 0.5 h

[0080]

[0081] Table 3 Results of DNA extraction amount (ng) and integrity of samples placed for 1 h

[0082]

[0083]

[0084] As shown in Tables 1 to 3, when the DNA extracted by the same kit was placed for different periods of time, the amount of DNA extracted in Examples 1 to 2 was equivalent to or even greater than that in the real stool samples, and the DIN values ​​all reached the qualified standard (DIN value ≥ 2.5), while the amount of DNA extracted in Comparative Examples 1 to 8 was significantly lower than that in the real stool samples, and the DIN values ​​of Comparative Examples 1 to 2, Comparative Examples 5 and Comparative Examples 7 were unqualified (DIN value < 2.5), indicating that the components contained in the artificial stool samples of Comparative Examples 1 to 2, Comparative Examples 5 and Comparative Examples 7 directly caused nucleic acid degradation during preparation, and continued to cause nucleic acid degradation during subsequent storage.

[0085] In addition, if Figures 1 to 4 As shown, with the extension of the storage time, the change trend of the DNA extraction amount of Examples 1 to 2 is basically consistent with that of the real stool samples, while the change trend of the DNA extraction amount of Comparative Examples 1 to 8 is obviously inconsistent with the real stool samples, indicating that the artificial stool samples of Comparative Examples 1 to 8 cannot objectively simulate the nucleic acid degradation of real stool when placed at room temperature.

[0086] Table 4 Ct value determination results of viral ORF1ab gene in the samples placed for different time (h)

[0087]

[0088] As shown in Table 4, in the DNA extracted by the same kit, as the placement time increases, the Ct value change trend of the ORF1ab gene in Examples 1 to 2 is basically the same as that of the real stool sample, and the Ct value is less than 38, while the Ct value change trend of the ORF1ab gene in Comparative Examples 1 to 3 and 5 to 8 is inconsistent with the real stool sample, and most of the Ct values ​​are greater than 40 (false negative results) when stored for 1 hour. Although the Ct value of the ORF1ab gene in Comparative Example 4 has a change trend close to that of the real stool sample, the Ct value is mostly close to 39 when placed for 1 hour, which is easy to cause a misjudgment of a negative result in the actual quantitative PCR test. This shows that compared with Comparative Examples 1 to 8, the artificial stool samples of Examples 1 to 2 can more objectively simulate the detection of gene expression after nucleic acid extraction in real stool.

[0089] Application Example 2 Reproducibility of artificial feces samples

[0090] 1. Preparation of samples to be tested

[0091] Ten batches of artificial feces samples of Examples 1 to 2 were prepared in parallel, 2 mL was taken from each sample, 100 ng of a positive reference substance for the new coronavirus (article number: BDS-BW-117, manufacturer: Guangzhou Bondeson Biotechnology Co., Ltd.) was added, and mixed to obtain 30 groups of samples to be tested.

[0092] 2. Nucleic acid extraction

[0093] DNA was extracted from freshly prepared samples using the QIAamp Fast DNA Stool Mini Kit.

[0094] 3. Nucleic acid testing

[0095] For the extracted DNA, the concentration was measured using Invitrogen Qubit3.0, and the genomic DNA integrity (DIN value) was characterized by Agilent 4200 gene analyzer. Afterwards, the novel coronavirus nucleic acid detection kit (Cat. No.: SJ-HX-804-2, Manufacturer: Shanghai Berger Medical Technology Co., Ltd.) was used to perform fluorescence quantitative PCR to detect the content of the viral ORF1ab gene.

[0096] 4. Test results

[0097] Table 5 The extraction amount and integrity of different batches of sample DNA and the determination results of the Ct value of the viral ORF1ab gene

[0098]

[0099]

[0100] As shown in Table 5, the coefficients of variation (CV less than 10%) of DNA extraction from 10 batches of artificial feces samples of Examples 1 to 2 were 0.058, 0.075 and 0.095, respectively, and the DIN values ​​all met the qualified requirements (DIN value ≥ 2.5), and the coefficients of variation of the Ct values ​​of the ORF1ab gene were 0.087, 0.095 and 0.087, respectively. This indicates that there is no significant difference in the DNA extraction effects of different batches of artificial feces samples of Examples 1 to 2, and the reproducibility is good.

[0101] Application Example 3: Stability of artificial feces samples

[0102] 1. Preparation of samples to be tested

[0103] The artificial stool samples prepared in Examples 1 to 2 and fresh stool samples of healthy subjects collected from the clinic (ie, real stool samples) from the same batch were stored at 4° C. for 12 months.

[0104] 2. Nucleic acid extraction and detection

[0105] At 0, 1, 3, 5, 7, 9 and 12 months of storage, equal amounts of artificial feces samples were taken and DNA was extracted using the QIAampFast DNAStool Mini Kit. The concentration was then measured using the Invitrogen Qubit 3.0 and the genomic DNA integrity (DIN value) was characterized using the Agilent 4200 Gene Analyzer.

[0106] 3. Protein detection

[0107] Equal amounts of artificial feces samples were taken at 0, 1, 3, 5, 7, 9 and 12 months of storage, and the content of human tubulin (TUB protein) was detected by ELISA.

[0108] 4. Test results

[0109] Table 6 The extraction amount and integrity of DNA and the determination results of TUB protein content in the samples stored at 4°C for different time periods (months)

[0110]

[0111]

[0112] As shown in Table 6, the DNA extraction amount and integrity of the real feces samples decreased significantly after being stored at 4°C for 1 month, and no DNA could be extracted after being stored for 7 months; in contrast, the DNA content and protein content of the bacteria and cells contained in the artificial feces samples of Examples 1-2 only slightly decreased during the 12-month storage at 4°C, and the integrity was still qualified. This shows that the artificial feces samples of Examples 1-2 have good stability and there is no obvious deterioration and degradation of the components after being stored at 4°C for more than one year.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An artificial feces, characterized in that: It is composed of sodium cholate, bilirubin, calcium chloride, magnesium chloride, amino acids, triglycerides, microcrystalline cellulose, small intestinal epithelial cells, Escherichia coli and water.

2. The artificial feces according to claim 1, characterized in that The mass ratio of the sodium cholate, the bilirubin, the calcium chloride, the magnesium chloride and the microcrystalline cellulose is (0.05-0.1): (0.05-0.1): (0.02-0.1): (0.02-0.1): (1.5-2).

3. The artificial feces according to claim 1, characterized in that: Calculated by mass to volume ratio, in the artificial feces, the content of sodium cholate is (0.005-0.01) g / mL, the content of bilirubin is (0.005-0.01) g / mL, the content of calcium chloride is (0.002-0.01) g / mL, the content of magnesium chloride is (0.002-0.01) g / ml, and the content of microcrystalline cellulose is (0.15-0.2) g / mL.

4. The artificial feces according to claim 1, characterized in that: The content of small intestinal epithelial cells is 5×10 3 cells / mL~8×10 3 cells / mL.

5. The artificial feces according to claim 1, characterized in that: The content of Escherichia coli is 10 8 cfu / mL~1.2×10 8 cfu / mL.

6. The artificial feces according to claim 1, characterized in that: The content of the amino acid is 0.3 μmol / mL to 0.6 μmol / mL.

7. The artificial feces according to claim 1, characterized in that: The content of triglyceride is 0.01 g / mL to 0.02 g / mL.

8. Use of the artificial feces according to any one of claims 1 to 7 as a reference for fecal samples.

9. Use of the artificial feces according to any one of claims 1 to 7 in preparing a nucleic acid extraction reagent for a fecal sample.

10. Use of the artificial feces according to any one of claims 1 to 7 in detecting and / or evaluating the performance of nucleic acid extraction reagents of fecal samples.

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