Composite preservative for fecal microorganisms and metabolites thereof and same-tube multi-omics joint inspection integrated technology

Abstract

The invention provides a composite preservative for fecal microorganisms and metabolites thereof and a same-tube multi-omics joint inspection integrated technology. The composite preservative comprises the following main components: cetyltrimethylammonium bromide (CTAB), ammonium chloride (NH4Cl) and phosphate buffer salt (PBS), the stability of microorganisms and metabolites thereof in the excrement sample in the room-temperature storage and transportation process can be kept, and the extraction efficiency of deoxyribonucleic acid (DNA) of the microorganisms in the excrement sample is guaranteed. In addition, by adding a treatment reagent, the influence of the preservative component on metabolite detection is eliminated, and the response and ionization efficiency of the excrement sample metabolite in the mass spectrum are remarkably improved, so that the sensitivity and accuracy of a detection result are improved. The method not only solves the problem of timeliness of excrement sampling, but also provides a technical basis and thought for feasibility research of the same-tube multi-omics of excrement samples, and has wide scientific research and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis and medical testing, and in particular to a composite preservative for fecal microorganisms and metabolites thereof and a same-tube multi-omics joint testing integrated technology. Background Art

[0002] Fecal samples are an important source of direct information about intestinal microorganisms and are considered to contain the most abundant metabolites. Therefore, fecal samples play a vital role in the joint analysis of intestinal microorganisms and their metabolites. The non-invasive collection of feces has made fecal samples a focus of attention in early cancer risk assessment and screening. In particular, in the past two years, the National Medical Products Administration has approved a number of innovative fecal-based diagnostic products, proving the feasibility and effectiveness of fecal samples in the field of medical innovation. Fecal samples provide more convenient and practical disease diagnosis and prevention methods for clinicians. However, fecal samples have the characteristics of timeliness and heterogeneity, which brings many challenges to sample collection, storage, transportation, multi-omics research and clinical testing. Storing the collected fecal samples at room temperature or 4°C for more than 12 hours will cause significant changes in fecal microorganisms and their metabolites. Therefore, the collection and storage of fecal samples are widely recommended to be frozen at -80°C and transported on dry ice. However, such harsh storage and transportation conditions make it difficult to implement home sampling, large-scale community screening, cross-regional transportation, and even sampling of hospitalized patients.

[0003] In order to solve the above problems, the use of various preservatives is an ideal solution. At present, various DNA preservatives can effectively maintain the room temperature stability of DNA in stool samples, maintain the integrity of microbial information in samples, and avoid the stringent requirements for sample freezing and transportation. Common DNA preservatives include ethanol, RNAlater, EDTA salts, and commercial kit products OMNIgene GUT200, Stool DNA Stabilizer, etc. DNA preservatives are used in many clinical studies. Although the existing DNA-specific preservatives have technical advantages such as room temperature storage and easy transportation, they also have limitations. The biggest drawback is that stool samples with DNA preservatives cannot be qualitatively and quantitatively detected by liquid chromatography-mass spectrometry. This is because the high salt components, chelating agents or surfactants contained in the DNA preservatives will significantly inhibit or destroy chromatographic column separation and mass spectrometry ionization.

[0004] In summary, clinical research cohorts widely use DNA preservatives to protect stool samples. Although this measure solves the challenges of stool sample storage and transportation, it limits the detection of metabolomics based on mass spectrometry. Ultimately, multi-omics association analysis of clinical samples can only use samples preserved in different ways, which greatly weakens the information correlation between multi-omics.

[0005] Therefore, the present invention urgently needs to propose a composite preservative for fecal sample microorganisms and their metabolites to provide convenience for clinical sample collection, storage and transportation. In addition, the present invention realizes simultaneous detection of microorganisms and metabolites in the same solution system (joint detection in the same tube) by properly disposing of the mass spectrometry detection interfering components of the preservative. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a composite preservative for fecal microorganisms and their metabolites and a same-tube multi-omics joint detection integrated technology, as well as a method for pre-treating a fecal sample containing a preservative and quantitatively detecting metabolites, wherein the composite preservative includes CTAB, NH 4 Cl and PBS components can effectively maintain the stability of stool sample DNA during storage and transportation at room temperature. The present invention proposes a preservative treatment and metabolite detection method, which can effectively eliminate the influence of the preservative on the detection of metabolites, significantly improve the response and ionization efficiency of stool sample metabolites in mass spectrometry, and ensure the sensitivity and accuracy of detection.

[0007] In one aspect, the present invention provides a feces composite preservative, the components of which include: CTAB, NH 4 Cl and PBS.

[0008] In some embodiments, the present invention compares and screens the components of the stool composite preservative, and the experimental results show that only when the components of the stool composite preservative include CTAB, NH 4 Cl and PBS, the extraction efficiency of DNA from stool samples was the highest, the DNA content of stool samples stored at room temperature for 3 days remained stable, and the microbial composition of stool samples stored at room temperature for 5 days remained stable.

[0009] In some embodiments, the present invention further screens the composite preservative system according to the removal efficiency of CTAB in fecal samples. The experimental results show that only when the composite preservative (i.e., including CTAB, NH 4 Cl and PBS), only after adding the optimal amount of SDS (sodium dodecyl sulfate) can CTAB be completely precipitated and the removal efficiency reaches 100%. At this time, the analysis results of metabolite bile acids in fecal samples show that their chromatographic retention time is normal; when any component in the composite preservative is changed, the removal efficiency of CTAB will decrease significantly. In these cases, the chromatographic peak shape of metabolite bile acids is significantly deteriorated, and the retention time is also significantly shifted.

[0010] Furthermore, the content of CTAB is 1% to 2.5%, the content of NH4Cl is 10% to 20%, and the content of PBS is 5% to 10%.

[0011] Preferably, the content of CTAB is 2.0%, NH 4 The content of Cl is 20%, and the content of PBS is 10%.

[0012] In some embodiments, the contents of different components in the composite preservative are screened. The experimental results show that when the CTAB content is 2.0%, NH 4 When the Cl content is 20% and the PBS content is 10%, the DNA detection efficiency of the composite preservative prepared at this time reaches the highest, and the DNA in the fecal sample remains stable after 3 days at room temperature.

[0013] In another aspect, the present invention provides a method for preserving a stool sample, comprising the following steps:

[0014] (1) Preparation of feces composite preservative: The active ingredients include CTAB, NH 4 A composite preservative of Cl and PBS;

[0015] (2) Fecal sample collection and storage: Fecal samples were homogenized, 10-30 mg of sample was added to the preservative and shaken well.

[0016] In another aspect, the present invention provides a pretreatment method for detecting metabolites in a stool sample containing a composite preservative, characterized in that it comprises the following steps:

[0017] (1) Take 5-50 μL of stool sample containing composite preservative and freeze-dry it;

[0018] (2) adding a mixed organic solvent and magnetic beads to the freeze-dried sample in sequence and homogenizing;

[0019] (3) centrifuging the homogenized sample at low temperature and high speed, taking 40-80 μL of the supernatant, and determining the CTAB content in the sample;

[0020] (4) According to the content of CTAB, accurately prepare the corresponding proportion of sodium dodecyl sulfate (SDS) solution;

[0021] (5) Co-precipitation elimination: Take an appropriate amount of the supernatant obtained in step (3), add 40-80 μL of the SDS solution prepared in step (4), incubate for a certain period of time, centrifuge at low temperature and high speed again, and take the final supernatant for metabolite detection.

[0022] In some embodiments, the inventors found that when the optimal composite preservative was used, the metabolite detection results of the stool samples were inaccurate, and the three components in the preservative all had an impact on the metabolite detection results. 4 The methods of Cl and PBS were screened and it was found that the mixed organic solvent methanol / acetonitrile (methanol: acetonitrile = 80:20, volume ratio) could completely remove NH 4 Cl and PBS, but cannot remove CTAB. The inventors used the co-precipitation principle of anionic and cationic surfactants to remove CTAB by adding SDS to form a precipitate with CTAB. However, the known conditions are not suitable for effectively removing the CTAB component in the preservative system of the present invention. Therefore, the present invention has further optimized the co-precipitation reaction conditions of SDS and CTAB after a large number of screening experiments. The experimental results show that only when the following three conditions are met at the same time: (1) in the CTAB+NH 4 SDS and CTAB were co-precipitated in a Cl+PBS system; (2) the molar ratio of SDS:CTAB was 1:1; (3) the co-precipitation efficiency of CTAB and SDS reached 100% after incubation at 10°C on ice for 15 min.

[0023] Furthermore, the fecal metabolites include fecal sample metabolites and composite preservative components; the fecal sample metabolites include GCA, TCA, GUDCA, TUDCA, TDCA, GDCA, TCDCA, GCDCA, TLCA, LCA, bCA, CA, UDCA, DCA and CDCA; the mixed organic solvent in step (2) is a methanol / acetonitrile mixture, and the volume ratio of methanol:acetonitrile is 80:20.

[0024] In some embodiments, the inventors have found that NH 4 Cl and PBS salts will precipitate in the liquid phase system and mass spectrometry ion source, which will not only seriously shorten the service life of the chromatographic column and equipment, but also significantly affect the metabolite detection results. Therefore, it was found through screening that the organic mixed solution of methanol / acetonitrile (methanol: acetonitrile = 80:20, volume ratio) can precipitate NH 4 Cl and PBS were effectively eliminated.

[0025] Furthermore, in the step (3), after centrifugation, the supernatant is taken for OD value measurement to determine the actual accurate content of CTAB in the system; in the step (5), SDS is added according to the molar ratio of SDS:CTAB of 1:1, the reaction temperature is 10°C, and the reaction time is 20 min.

[0026] In yet another aspect, the present invention provides a use of sodium dodecyl sulfate for preparing a preparation for improving the response of fecal metabolites in mass spectrometry.

[0027] In some embodiments, after successfully screening the optimal ratio of SDS to CTAB, the optimal conditions for the precipitation reaction, and the optimal reaction system, the addition of SDS can effectively remove CTAB in the stool sample, thereby significantly improving the response of the metabolite in the mass spectrometer.

[0028] In yet another aspect, the present invention provides a use of sodium dodecyl sulfate for preparing a preparation for improving the accuracy of retention time of fecal metabolites in mass spectrometry.

[0029] In some embodiments, after successfully screening out the optimal ratio of SDS to CTAB, the optimal conditions for the incubation, and the optimal reaction system, the addition of SDS can effectively remove CTAB in the stool sample, thereby significantly improving the accuracy of metabolite detection.

[0030] In yet another aspect, the present invention provides a use of sodium dodecyl sulfate for preparing a preparation for improving the ionization efficiency of fecal metabolites in mass spectrometry.

[0031] In another aspect, the present invention provides a feces composite preservative for use in preparing a preparation for improving the response and detection accuracy of feces metabolites in mass spectrometry, wherein the feces composite preservative is as described in any one of the above technical solutions.

[0032] Beneficial effects achieved by the present invention:

[0033] 1. The present invention provides an effective method for preserving stool samples, namely, using a composite preservative, including 2.0% CTAB, 20%

[0034] NH 4 Cl and 10% PBS can stably store and effectively extract DNA and metabolites from fecal samples. This innovation solves the problems caused by timeliness and heterogeneity in the collection, storage and transportation of fecal samples, greatly facilitates the sample processing process, reduces dependence on harsh storage and transportation conditions, and makes large-scale sample collection and research possible.

[0035] 2. The present invention provides a method for quantitative detection of CTAB, an effective ingredient of a composite preservative, which achieves accurate quantitative detection of CTAB, a key ingredient in the composite preservative, by using a methyl orange indicator and a full-wavelength scanner. This method not only provides accurate data support for evaluating the effect of the preservative, but also provides an important basis for subsequent optimization of metabolite detection, helping to ensure the accuracy and reliability of the experimental results.

[0036] 3. The present invention optimizes the pretreatment method for metabolite detection: a method that can effectively remove interfering components (CTAB, NH4Cl and PBS) in stool samples containing a composite preservative is successfully developed. 4 In the Cl+PBS system, through the synergistic effect of a specific ratio of organic solvent methanol / acetonitrile mixture (methanol:acetonitrile = 80:20, volume ratio) and SDS in an amount equimolar to CTAB, under specific reaction conditions (incubation at 10°C in an ice bath for 20 minutes), the mass spectrometric response and detection accuracy of metabolites in fecal samples were significantly improved, thereby removing the composite preservative components that affect metabolite detection, effectively solving the problem of inhibition and interference of preservatives on metabolite detection in the past.

[0037] 4. The present invention provides a composite preservative and a method for detecting microorganisms and their metabolites in the same preservative system. The composite preservative effectively ensures the stability of fecal DNA and metabolites at room temperature, providing technical basic data for the room temperature storage and transportation of fecal samples. At the same time, by adding SDS and organic mixed reagents, the influence of the composite preservative components on metabolite mass spectrometry detection is eliminated, and the sensitivity and accuracy of metabolites are significantly improved, which provides a key technical method for the study of fecal sample metabolomics, and finally realizes the innovative idea of ​​multi-omics detection of the same fecal sample (joint detection in the same tube), which has good scientific research and clinical application value.

[0038] 5. The present invention realizes the integration of multi-omics detection of fecal samples: the composite preservative and related detection methods provided by the present invention enable the simultaneous detection of microorganisms and metabolites in the same fecal sample (joint detection in the same tube). This innovative idea breaks the limitation of the traditional method that the correlation of multi-omics information is weakened due to different preservation methods, and provides strong technical support for a more comprehensive and in-depth understanding of the relationship between intestinal microorganisms and metabolites. This has important guiding significance for the early diagnosis of intestinal diseases, the study of pathogenesis, and the formulation of personalized treatment plans, and has broad application prospects and important scientific value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a graph showing the room temperature stability results of fecal sample microorganisms under different storage conditions in Example 3.

[0040] Figure 2 This is a graph showing the room temperature stability results of bile acids in fecal samples under different storage conditions in Example 3.

[0041] Figure 3 This is a qualitative test result diagram of fecal sample metabolites (bile acid) under the conditions of Scheme 3 in Example 4 (deionized water was added).

[0042] Figure 4This is a qualitative test result diagram of fecal sample metabolites (bile acid) under the conditions of Scheme 2 in Example 4 (preservative added but no treatment).

[0043] Figure 5 This is a diagram showing the qualitative detection results of fecal sample metabolites (bile acid) under the conditions of Scheme 1 in Example 4 (preservative was added while influencing substances were removed).

[0044] Figure 6 This is a specific result diagram of the quantitative detection of bile acid metabolites under different conditions in Example 4.

[0045] Figure 7 Example 5 mentions the effect of the CTAB / SDS ratio on the micelle particle size of the mixed system in the literature.

[0046] Figure 8 This is a graph showing the peak shape detection results of each metabolite in the mixed stool sample of Comparative Example 1 in Example 8 (conventional treatment + organic solvent extraction).

[0047] Fig. 9 This is a peak shape detection result diagram of each metabolite in the fecal mixed sample of Comparative Example 2 in Example 8 (lyophilization treatment + organic solvent extraction).

[0048] Fig.10 This is a graph showing the peak shape detection results of each metabolite in the mixed stool sample of Comparative Example 3 in Example 8 (lyophilization + organic solvent extraction + purification column pretreatment).

[0049] Fig.11 This is a peak shape detection result diagram of each metabolite in the fecal mixed sample of Comparative Example 4 in Example 8 (lyophilization + organic solvent extraction + SDS elimination of CTAB).

[0050] Fig.12 This is a graph showing the peak shape detection results of each metabolite in the fecal mixed sample of Example 1 in Example 8 (freeze drying + organic solvent extraction + determination of CTAB content followed by addition of SDS in a 1:1 ratio to eliminate CTAB based on the determination results). DETAILED DESCRIPTION

[0051] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be understood by people with general skills in the field to which the present invention belongs. The reagents or instruments used without indicating the manufacturer are conventional products that can be purchased through regular channels.

[0052] In the present invention, metabolites first enter the chromatogram for separation, and then gradually reach the mass spectrometry ion source, where they are broken into fragments by different voltages, and different substances produce different charged ion fragments, which is the ionization process. Afterwards, the ions are collected by the mass spectrometry detector, and different ions are collected by different detector channels. The total ion charge is calculated, and the total amount of fragments is quantified by the charge carried by the ions, and then the total amount of substances is quantified. Therefore, the inventors determine the type of substance based on the retention time and the fragment ions, and determine the concentration of the substance by the collected charge. When the retention time of the chromatographic peak of the metabolite is offset greatly, the metabolite cannot be clearly identified, so the relative ionization efficiency cannot be compared. However, when the retention time offset is less, the relative ionization efficiency can be calculated when the type of substance can be clearly identified.

[0053] Example 1: Optimal preparation method of composite preservative and co-detection method of composite preservative sample in stool

[0054] At present, commonly used DNA preservatives can effectively protect the microbial DNA of room temperature feces. After the fecal sample is added with the DNA preservative, it can be stored or transported at room temperature without changing the microbial composition. However, the fecal sample containing the DNA preservative cannot be used for qualitative and quantitative mass spectrometry detection of metabolites. Therefore, in this embodiment, a composite preservative for fecal samples with the best effect is provided, which can not only stably maintain the DNA preservation and detection efficiency, but also effectively remove the components that affect the detection of metabolites by adding sodium dodecyl sulfate (SDS) and organic solvents, so as to realize the detection of microorganisms and their metabolites in the same preservative sample, and greatly improve the correlation between multiple omics.

[0055] 1. Best way to store stool samples

[0056] This embodiment provides an optimal method for preserving stool samples, comprising the following steps:

[0057] Step 1: Prepare the composite preservative: prepare the active ingredients including CTAB, NH 4Cl and PBS composite preservative, the content of CTAB is 2.0%, NH 4 The content of Cl is 20% and the content of PBS is 10%;

[0058] Step 2: Fecal sample collection and storage: Fecal sample homogenization, weigh 10-30 mg (20 mg) of sample, add 160 μL of preservative, and shake well;

[0059] Step 3: Add the composite preservative and magnetic beads to the stool sample, mix well, and store in individual tubes of different volumes according to the purpose of the test.

[0060] This optimal composite preservative has the same effect as common DNA preservatives and can keep the DNA in stool samples stable at room temperature.

[0061] 2. DNA detection method of stool samples with composite preservatives

[0062] This embodiment also provides a method for detecting DNA in a stool sample to which a composite preservative has been added, comprising the following steps:

[0063] Step 1: DNA extraction from stool samples

[0064] The DNA in the sample was extracted using the CTAB or SDS method, and the purity and concentration of the DNA were detected by agarose gel electrophoresis. 10 to 20 μL (10 μL) was taken in a centrifuge tube and the sample was diluted to 1 ng / μl using sterile water.

[0065] Step 2: PCR amplification

[0066] ① Template: diluted genomic DNA;

[0067] ② Primers: Use specific primers with barcodes according to the selection of sequencing regions; the primers for the 16S V4 region are 515F-806R;

[0068] The primers for 18S V4 region were 528F-706R; the primers for 18S V9 region were 380F-1510R; the primers for ITS1 region were ITS5-1737F,

[0069] ITS2-2043R, primers for ITS2 region are ITS3-2024F and ITS4-2409R

[0070] ③ Enzyme and buffer: High-Fidelity PCR MasterMix with GC from New England Biolabs

[0071] Buffer. Use high-efficiency and high-fidelity enzymes for PCR to ensure amplification efficiency and accuracy.

[0072] ④PCR instrument: Bio-rad T100 gradient PCR instrument

[0073] ⑤PCR products were detected by electrophoresis using 2% agarose gel;

[0074] Step 3: Mixing and purification of PCR products

[0075] Mix samples of equal concentration according to the concentration of PCR products, and purify them by electrophoresis using 1×TAE 2% agarose gel

[0076] For PCR products, the sequence with the main band size between 400-450 bp was selected, and the target band was recovered by gel cutting. The product purification kit used was the GeneJET gel recovery kit from Thermo Scientific.

[0077] Step 4: Library construction and sequencing

[0078] The library was constructed using the Illumina TruSeq DNA PCR-Free Library Preparation Kit.

[0079] The constructed library was subjected to Qubit quantification and library detection. Once qualified, it was sequenced using NovaSeq6000.

[0080] Step 5: Bioinformatics analysis

[0081] ① Preprocessing of sequencing results: The raw data obtained by sequencing contains a certain proportion of dirty data. In order to make the results of information analysis more accurate and reliable, the raw data is first spliced ​​and filtered to obtain valid data.

[0082] ② Species analysis annotation: Unigenes were annotated with the NR database (Version 2.0) from NCBI using DIAMOND software.

[0083] 2018-01-02,https: / / www.ncbi.nlm.nih.gov / ) were used to compare the sequences of bacteria, fungi, archaea and viruses extracted from the ncbi.nlm.nih.gov / .

[0084] ③Data visualization: Using R data package and custom programming, calculate the abundance and Alpha diversity of species data.

[0085] Venn diagrams and petal diagrams were used to obtain information on species richness and evenness within the sample. Multiple sequence alignments were performed on species and phylogenetic trees were constructed to further obtain differences in community structure among different samples and groups, which were displayed through dimensionality reduction diagrams such as PCoA, PCA, and NMDS, as well as sample clustering trees.

[0086] The experimental results show that when the stool samples were preserved using the above-mentioned preservative formula with the best preservation effect, the test results in the final stool sample metabolite test were inaccurate. The three components in the preservative all had an impact on the test results. The inventors found that the reasons for this phenomenon were: (1) NH 4 Cl and PBS salts will precipitate in the liquid phase system and mass spectrometry ion source, seriously affecting the service life of the chromatographic column and equipment, and will also significantly affect the detection results of metabolite bile acids; (2) CTAB carries a positive charge and will bind to some metabolites of bile acids, which will not only affect the chromatographic behavior, but also cause a significant shift in the retention time of amino acid-bound metabolites, while significantly inhibiting the ionization efficiency of non-amino acid-bound metabolites in mass spectrometry, resulting in a significant decrease in metabolite response. Therefore, metabolite detection requires effective removal of the three components of the composite preservative in the fecal sample (① The effective removal in the present invention is real-time detection and real-time elimination, and the three components of the preservative are removed before detection is required; ② After the composite preservative is added to the fecal sample, the detection of metabolites will be affected, but the addition of organic solvents and SDS can effectively eliminate the effect of the composite preservative on metabolite detection).

[0087] 3. Methods for quantitative detection of CTAB, the active ingredient of composite preservatives, and methods for removing the influence of composite preservatives on metabolite detection

[0088] Therefore, based on the above results, this embodiment also provides a method for quantitative detection of CTAB, an effective ingredient of a composite preservative, and a method for removing the influence of a composite preservative on metabolite detection. First, the CTAB in a fecal sample is quantitatively detected by a methyl orange indicator and a full wavelength scanner to obtain accurate CTAB concentration information; then, an SDS solution with an equimolar amount to CTAB is added to eliminate CTAB in the fecal sample, so as to perform normal detection of metabolites in the fecal mixed sample.

[0089] (1) The quantitative detection method of CTAB, an active ingredient of the composite preservative, comprises the following steps:

[0090] Step 1: pre-treatment of the stool sample, pre-treating the stool sample to obtain a pre-treated stool sample;

[0091] Step 2: absorbance detection of CTAB standard solution, blank solution and test solution;

[0092] Step 3: Subtract the series standard absorbance value from the blank absorbance value to obtain the series standard value, and make a corresponding linear regression equation; subtract the absorbance value to be tested from the blank absorbance value to obtain the test value, and then substitute the test value into the linear regression equation to obtain the CTAB content of the test liquid.

[0093] (2) The elimination of CTAB, NH 4 The method of Cl and PBS comprises the following steps:

[0094] Step 1: Add MeOH solution to the fecal mixed sample and quantify the CTAB in the mixed sample;

[0095] Step 2: Prepare SDS, and prepare SDS of corresponding proportion by measuring the CTAB content;

[0096] Step 3: Eliminate co-precipitation. Add 60 μL of SDS in the same volume as CTAB to the mixed sample, mix well, centrifuge to remove the precipitate and retain the supernatant.

[0097] In summary, in this embodiment, an optimal composite preservative is provided, comprising 2.0% CTAB, 20% NH 4 Cl and 10% PBS can effectively extract DNA from stool samples, significantly improve the detection efficiency of DNA, and keep microorganisms and metabolites stable at room temperature or during transportation. At the same time, in order to ensure that the metabolite quantification is not affected and the accuracy of the detection is improved, in this embodiment, under the premise of ensuring the stability of DNA, an organic solvent methanol / acetonitrile mixture (methanol: acetonitrile = 80:20) is added to eliminate NH 4 Cl and PBS, add SDS and CTAB to co-precipitate to eliminate CTAB. The specific steps are as follows:

[0098] ① After adding the composite preservative to the stool sample, sample pretreatment:

[0099] 1) Accurately transfer 40 μL of stool sample into a 1.5 mL EP tube;

[0100] 2) The samples were freeze-dried overnight;

[0101] 3) Add 10 mg of magnetic beads to the EP tube and reconstitute with 200 μL of methanol / acetonitrile mixture containing internal standard;

[0102] 4) Grind for 2 minutes;

[0103] 5) Freeze at -20°C for 20 minutes;

[0104] 6) Centrifuge at 18,000 g for 25 min at 4°C;

[0105] 7) Divide the supernatant into two 1.5 mL EP tubes, numbered i and ii;

[0106] 8) Take tube i and dilute it 50 times with deionized water;

[0107] 9) Take 400 μL of the dilution in tube i and mix it with an equal volume of methyl orange indicator;

[0108] 10) Shake at room temperature for 20 minutes;

[0109] 11) Determine the OD value of the solution and calculate the CTAB content in the sample;

[0110] 12) Add an equal volume of SDS to the supernatant and the same concentration as CTAB to tube ii, and vortex to mix;

[0111] 13) Centrifuge at 18,000 g for 25 min at 4°C;

[0112] 14) taking the supernatant;

[0113] 15) Add 60 μL of mobile phase A:mobile phase B (1:1) to the supernatant;

[0114] 16) Vortex the mixed solution for 2 minutes to mix well;

[0115] 17) 4°C, 1450 rpm, shake for 15 min;

[0116] 18) Centrifuge at 18,000 g for 25 min at 4°C;

[0117] 19) Take the supernatant into the sample injection plate;

[0118] ②Liquid chromatography and mass spectrometry conditions:

[0119] 1) The operating conditions of the liquid chromatograph are as follows: the chromatographic columns are ACQUITY UPLC Cortecs C18 1.6μM VanGuard pre-column (2.1×5mm) and ACQUITY UPLC Cortecs C18 1.6μM analytical column (2.1×100mm). The operating temperature range is 60°C-350°C; the injection port temperature of the liquid chromatograph is 270°C, and the transfer tube temperature is 270°C.

[0120] 2) The conditions for using the mass spectrometer are: ion source temperature of 220℃; injection volume of 0.5-5μL; splitless injection; ion source voltage of 70eV; mass spectrometry scanning range of 38-650m / z; acquisition rate of 20-25spectra / s. The column temperature and sample tray temperature were 30℃ and 10℃, respectively. Mobile phase A was 0.01% formic acid water (pH=4), mobile phase B was acetonitrile: methanol = 80:20, and the initial mobile phase ratio A: B = 95:5. The flow rate was 0.4mL / min, and the gradient conditions were: 0-1 minute (5% B); 1-3 minutes (5-30% B); 3-15 minutes (30-100% B); 15-16 minutes (100-5% B); 16-17 minutes (5% B). The ion source temperature and dissolution temperature were 150℃ and 550℃, respectively. Specifically, a UPLC-MS / MS system (ACQUITY UPLC-XevoTQ-S, Waters Corp., Milford, MA, USA) can be used for detection. Under this condition, each metabolite in the fecal sample can have a good signal, ensuring the accuracy of quantitative analysis.

[0121] ③Qualitative and quantitative analysis:

[0122] Qualitative and quantitative analysis, based on a database established by derivatives of commercially purchased standard substances, by comparing the retention index and mass spectrum fragment information of different substances to be analyzed, accurate qualitative and quantitative analysis of the information to be analyzed is achieved, specifically:

[0123] Data processing: MassLynx's TargetLynx software was used to extract data, integrate, establish standard quantitative curves, conduct quality control review, and output final report concentrations for the raw data files generated by UPLC-MS / MS. Different information to be analyzed corresponding to different metabolites was obtained, including mass spectrometry parameters and retention index (RI).

[0124] Quantitative analysis: given in combination with the standard curve; the final data will be exported in CSV format, and its information includes sample name, metabolite, Kovats-RI, quantitative ion, peak area and concentration; the final quantitative results are obtained by establishing a standard curve.

[0125] It can be seen that the preservative provided in this embodiment has the best preservation effect of fecal sample microbial DNA. At the same time, the method for detecting metabolites in fecal samples can ensure the stability of fecal DNA while eliminating the influence of the preservative on metabolites, thereby improving the accuracy of fecal sample metabolite detection.

[0126] Example 2: Screening of the composition and concentration of the composite preservative

[0127] In order to obtain the composite preservative with the best effect in Example 1, the composition and concentration of the composite preservative were screened in this example. CTAB is widely used for DNA extraction from plant tissues, animal tissues or other biological samples, but CTAB alone does not have the function of preserving DNA. Therefore, in this example, the inventors added NH 4 Cl and PBS, and the ratio of the three ingredients was compared and tested. The specific experimental scheme and results are shown in Table 2:

[0128] Table 1. CTAB, NH 4 Effects of different components and ratios of Cl and PBS on DNA concentration in fecal samples

[0129]

[0130] Note: 1) In this example, the same mass of fecal samples were weighed, and the theoretical concentration of DNA in the feces should be equivalent. Therefore, the DNA concentration results of this example reflect the DNA extraction efficiency and detection status, that is, the higher the DNA concentration in the table, the better the condition; 2) In this example, the optimal pH range for fecal DNA preservation is 6.4-6.6. Within this pH range, fecal DNA can remain stable, and when the pH exceeds this range, the metabolite mass spectrometry detection will be affected; 3) Fecal samples are samples with greater heterogeneity. The DNA concentrations at different sampling locations may vary, but are generally at the same order of magnitude. In addition, the DNA concentration will fluctuate with the increase of storage time; 4) PSP Stool DNA in the table The foreign import manufacturer and batch number of Stabilizer are: Invitek, 1038111100. It is a commercially recognized kit with good DNA preservation effect, but its disadvantages are that the imported reagent cycle is long, the supply is unstable and the cost is high. In addition, the ingredients in the kit are unknown, and the table shows the components and their content ranges shown in the instructions. Therefore, it is not recommended as a preservative sample for mass spectrometry detection of fecal sample metabolites; 5) The DNA concentration in the table will also be affected by the difference in the weighed sample amount, but this difference is considered to be a normal error.

[0131] As shown in Table 1, 1) the pH value of the commercially available DNA preservative kit (PSP Stool DNA Stabilizer) is 6.50, which has high DNA detection efficiency. At the same time, the DNA concentration in the stool sample stored at room temperature for 3 days remains stable. However, the imported reagent has a long cycle, unstable supply and high cost, and the components and content range shown in the instructions are unclear. The accuracy of the metabolite detection results extracted by this preservative is significantly reduced, so it is not suitable for the detection of metabolites in stool samples; 2) The reagent only contains CTAB or NH 4The pH value of the DNA preservative solution containing Cl is unstable, so it is directly excluded; 3) The pH value of the DNA preservative containing only PBS is about 7.25-7.35, which is not within the optimal pH range; the DNA concentration extraction efficiency in this system is high, but the DNA concentration in the fecal sample decreases significantly after 3 days at room temperature; 4) The CTAB+NH 4 The pH value of the Cl+PBS composite preservative system is very stable and at the pH value required for optimal detection of metabolites, the DNA concentration is also the maximum; the DNA concentration in the stool sample at room temperature for 3 days remains basically stable; compared with the commercially available PSP kit, the DNA concentration in the stool sample after zero day and 3 days of storage at room temperature of the composite preservative of the present invention is close, and the composite preservative of the present invention has clear components and fixed concentrations. Through appropriate pretreatment, the defect that the PSP kit cannot detect metabolites in the sample is solved; 5) Composite preservative CTAB+NH 4 Any change in any component in the Cl+PBS system will affect the pH value of the preservative system and the change in the solution ion system.

[0132] It can be seen that the optimal formula of the composite preservative proposed in the present invention is CTAB+NH 4 Cl+PBS system. In this embodiment, different compositions and contents were screened and the optimal formula was determined to be 2.0% CTAB, NH 4 The Cl content is 20%, and the PBS content is 10%. The composite preservative of the present invention stores stool samples at room temperature, and the DNA concentration thereof is also substantially stable.

[0133] Example 3: Performance of the stool preservative sample in Example 1

[0134] This embodiment provides a stool sample composite preservative for storing microorganisms and their metabolites in stool at room temperature, specifically comprising: 1-10% CTAB, 10%-20% NH4Cl, 10%-20% PBS and deionized water. In this embodiment, the inventor tested the effect of the composite preservative on microorganisms and their metabolites under the optimal preparation conditions of the composite preservative. The specific experimental scheme and results are as follows:

[0135] The method for preparing the composite preservative for stool samples comprises the following steps:

[0136] 1) Fecal sample collection and storage: Weigh 20 mg of fresh fecal sample, add 160 μL of composite preservative, and shake well;

[0137] 2) Depending on the purpose of the test, the stool samples containing the preservatives are packaged separately and stored in separate tubes.

[0138] 3) Place the subpackaged samples at room temperature for 0, 3, and 5 days, then collect the samples for microbial sequencing and metabolite quantitative detection.

[0139] The microorganisms and metabolites of the fecal sample in the preservative are prepared using the method mentioned in Example 1.

[0140] from Figure 1 It can be seen that 1) when no preservative is used, the microbial composition of the stool sample changes after being placed for 3 and 5 days. Compared with the 0-day sample, the fecal microbial changes are more obvious after being placed at room temperature for 5 days. Therefore, the fecal sample is not suitable for microbial sequencing after being placed at room temperature; 2) when the feces is added with the OM200 commercial preservative, the microbial composition is not significantly changed after being placed at room temperature for 3 and 5 days, and the fecal biological information remains stable; 3) when the fecal sample is added with the optimal preservative ratio of the present invention, the microbial composition is also not affected compared with the 0-day sample after being placed at room temperature for 3 and 5 days, and the fecal microorganisms remain stable; in addition, the commercial kit is recognized as having excellent DNA preservation effect, and the preservative of the present invention can approach the preservation effect of its fecal microorganisms under room temperature conditions.

[0141] from Figure 2 It can be seen that 1) when no preservative is used, the bile acid composition of the stool sample changes after being placed for 3 and 5 days. The proportion of primary bile acid decreases, and the proportion of secondary bile acid increases. Therefore, the stool sample is not suitable for metabolite detection after being placed at room temperature; 2) the stool is stored at -80°C, and after 3 and 5 days, the bile acid composition does not change, and the stool sample remains stable; 3) when the stool sample is added with the optimal preservative ratio of the present invention, the bile acid composition is also not affected after being placed at room temperature for 3 and 5 days, and the fecal bile acid composition remains stable; in addition, the -80°C storage is recognized as the "gold standard" for the storage of fecal metabolites, and the preservative of the present invention can approach the preservation effect of fecal bile acid at room temperature.

[0142] It can be seen that the preservative provided by the present invention can ensure that the microorganisms and metabolites in the stool sample remain stable and basically unchanged after being placed at room temperature for 3 days and 5 days.

[0143] Example 4: Comparative test and result verification of the fecal sample metabolite detection method in Example 1

[0144] In order to verify that the method for detecting metabolites in stool samples in Example 1 is the best detection method, different detection methods were compared and tested in this example, specifically including the following three schemes (the remaining conditions are the best conditions in Example 1):

[0145] 1. Solution 1:

[0146] The optimal detection method for fecal sample metabolites in Example 1 (adding a preservative to eliminate CTAB and NH in the fecal mixed sample) 4 Cl and PBS);

[0147] 2. Solution 2:

[0148] Specifically, the following steps are included (only adding the preservative, without adding CTAB, NH 4 Cl and PBS elimination):

[0149] ① Sample pretreatment;

[0150] 1) Weigh 10-30 mg (20 mg) of feces and add it to the preservative, mix thoroughly, and accurately transfer 40 μL of the mixed sample into a 1.5 mL EP tube;

[0151] 2) The samples were freeze-dried overnight;

[0152] 3) Add 10 mg of magnetic beads to the EP tube and reconstitute with 200 μL of methanol / acetonitrile mixture containing internal standard;

[0153] 4) Grind for 2 minutes;

[0154] 5) Freeze at -20°C for 20 minutes;

[0155] 6) Centrifuge at 18,000 g for 25 min at 4°C;

[0156] 7) Take 60 μL of supernatant into a new 1.5 ml EP tube;

[0157] 8) Add 60 μL of mobile phase A:mobile phase B (1:1) to the supernatant;

[0158] 9) Vortex the mixed solution for 2 minutes to mix well;

[0159] 10) 4°C, 1450 rpm, shake for 15 min;

[0160] 11) Centrifuge at 18,000 g for 25 min at 4°C;

[0161] 12) Take the supernatant into the sample injection plate;

[0162] ②Liquid chromatography and mass spectrometry conditions

[0163] The same as the method for detecting metabolites in stool samples in Example 1;

[0164] ③Qualitative and quantitative analysis

[0165] The same as the method for detecting metabolites in stool samples in Example 1;

[0166] 3. Scheme 3 (Comparative Example 1):

[0167] No preservative was added, and deionized water was used instead of feces preservative;

[0168] The sample metabolite detection method has the same steps as those in Example 1.

[0169] The specific results of qualitative detection of metabolites under different conditions are as follows Figure 3 to Figure 5 As shown, Figure 3 This is the result of qualitative detection of metabolites in fecal samples under the conditions of Scheme 3 (deionized water). Figure 4 This is the result of qualitative detection of metabolites in fecal samples under the conditions of Scheme 2 (with preservative added). Figure 5 The results of qualitative detection of metabolites in fecal samples under the conditions of Scheme 1 (preservatives were added but their effective components were removed). Figure 3 to Figure 5 The changes in metabolite detection results using metabolites TUDCA (RT = 6.43 min), TCA (RT = 7.45 min), and GUDCA (RT = 7.98 min) as examples describe the effects of different schemes on metabolite detection results. The specific results are as follows:

[0170] Table 2. Effects of different protocols on the detection results of fecal metabolites TUDCA, TCA, and GUDCA

[0171]

[0172]

[0173] Note: It is known that the metabolites detectable in the stool samples of the present invention and their retention times are: GCA (8.45min), TUDCA (6.43min), GUDCA (7.98min), TCA (7.45min), TDCA (9.67min), GDCA (10.21min), TCDCA (9.45min), GCDCA (10.02min), TLCA (11.12min), LCA (14.70min), bCA (9.34min), CA (10.900min), UDCA (10.77min), DCA (12.97min) and CDCA (12.72min).

[0174] The calculation formula of relative ionization efficiency is: relative ionization efficiency = (ion response of experimental group / ion response of standard solution) * 100%.

[0175] Combination Figure 3 to Figure 5As shown in the results in Table 2, after using Scheme 2 (adding a preservative but not eliminating it), in the detection results of fecal sample metabolites: 1) two small peaks should appear at 5.39min and 5.58min, but after adding the preservative, the two peaks overlapped and no peak appeared at 5.58min; while in Scheme 1 and Scheme 3, after using the best detection method in Example 1 and adding deionized water instead of the preservative, two small peaks still appeared, and the separation met the requirements and no overlap occurred; 2) bile acid TCA peaked at around 7.45min, but after adding the DNA preservative, the peak at 7.45min disappeared; while in Scheme 1 and Scheme 3, after using the best detection method in Example 1, the peak at 7.45min disappeared. After adding deionized water to replace the preservative, TCA peaked normally at 7.45min; 3) The retention time of bile acid GUDCA was about 7.98min, but after adding the preservative, its retention time shifted significantly to about 8.02min, and the peak height decreased significantly, indicating that the response of the metabolite in the mass spectrometry was significantly reduced. After adding the preservative in Scheme 1 to eliminate its effective components, the retention time of bile acid returned to 7.97min, which showed that the influence of the preservative on the metabolite detection results was eliminated, and the accuracy of the detection results was improved; 4) At the same time, compared with Scheme 3 (deionized water group) and Scheme 1 (elimination of CTAB, NH 4 Cl and PBS), the relative ionization efficiency of metabolites in Scheme 2 was significantly reduced, which was because CTAB, NH 4 The elimination of the three components of Cl and PBS significantly reduced the response of each metabolite, resulting in a significant decrease in the final relative ionization efficiency.

[0176] The specific results of metabolite quantitative detection under different conditions are as follows Figure 6 As shown in the figure, A is the deionized water group of scheme 3, B is the preservative group of scheme 2, and C is the preservative group of scheme 1 but without CTAB and NH 4 Cl and PBS groups. Figure 6 It can be seen that the total bile acid content in the control group A is about 13000ng / mg, and after adding the preservative using scheme 2 (B), the total bile acid content detected is significantly reduced; and when the detection method of scheme 1 (C) is used, the total bile acid content is close to 13000ng / mg, and there is no significant decrease, which is not significantly different from the results of the control group. Therefore, using scheme 1 to eliminate the influence of the preservative on the metabolite detection results can significantly improve the accuracy of the metabolite detection results.

[0177] It can be seen that compared with the control group (deionized water group), the qualitative and quantitative metabolites were affected after the preservative was added to the feces in Scheme 2, the metabolite identification was interfered, and the quantitative results were inaccurate. In Scheme 1, compared with the control group, the metabolite identification was accurate and the quantitative results were not affected. Therefore, after the optimal metabolite detection method in Scheme 1 is optimized, the fecal sample can be accurately qualitatively and quantitatively treated after the preservative is added, which is beneficial to the multi-omics association analysis of fecal samples. Therefore, it is verified in this embodiment that Scheme 1 (metabolite detection method in Example 1) is the optimal detection method for fecal sample metabolites in the present invention.

[0178] Example 5: Screening of NH4Cl and PBS elimination methods in mixed stool samples

[0179] In order to obtain the best detection method for fecal sample metabolites in Example 1 and improve the accuracy of sample metabolite detection, this example eliminates CTAB and NH 4 Cl and PBS were screened, and this example finally found that NH 4 Cl and PBS, therefore, in this embodiment, NH 4 Cl and PBS methods were screened and explored to completely remove NH 4 The effects of Cl and PBS on the metabolite detection results of fecal samples specifically include the following methods (the remaining conditions are the optimal conditions in Example 1, and CTAB in this example has been completely removed using the optimal method in Example 1):

[0180] 1. Use deionized water;

[0181] 2. The organic solvent used is: methanol (MeOH);

[0182] 3. The organic solvent used is: acetonitrile (ACN);

[0183] 4. Use mixed organic solvent: methanol: acetonitrile = 1:1 (volume ratio);

[0184] 5. Use mixed organic solvent: methanol: acetonitrile = 80:20 (volume ratio);

[0185] The specific results are shown in Table 3:

[0186] Table 3. Effects of different organic solvents on NH 4 The removal effect of Cl and PBS and its influence on the detection results of metabolites in fecal samples

[0187]

[0188] Note: 1) It is known that the metabolites detectable in the stool samples of the present invention are bile acids, and the retention times of different bile acids are: GCA (8.45min), TUDCA (6.43min), GUDCA (7.97min), TCA (7.45min), TDCA (9.67min), GDCA (10.21min), TCDCA (9.45min), GCDCA (10.02min), TLCA (11.12min), LCA (14.70min), bC A (9.34min), CA (10.900min), UDCA (10.77min), DCA (12.97min) and CDCA (12.72min); 2) Relative ionization efficiency calculation formula: relative ionization efficiency = (experimental group ion response / standard solution ion response) * 100%; In the relative ionization efficiency result, "--" means: due to excessive retention time deviation, the peak shape cannot be collected normally, resulting in inaccurate qualitative analysis of the substance, so the relative ionization efficiency cannot be compared and no specific calculation is performed.

[0189] As shown in Table 3, 1) when only deionized water or a single organic solvent such as acetonitrile or methanol was used, NH 4 Cl and PBS, thus affecting the final detection results of fecal sample metabolites, making some metabolites undetectable, retention times significantly shifted, and responses of some metabolites significantly reduced; 2) When the organic solvent was a mixed solution of methanol and acetonitrile, the NH 4 The removal effect of Cl and PBS was significantly improved, and when methanol:acetonitrile = 80:20, the removal of NH 4 The removal efficiency of Cl and PBS can reach 100%, thus eliminating NH 4 The effects of Cl and PBS on the metabolite detection results of fecal samples. All metabolites showed normal peaks and good responses.

[0190] It can be seen that in this embodiment, the preferred organic solvent is methanol: acetonitrile = 80:20 (volume ratio) to remove NH 4 Elimination of Cl and PBS.

[0191] Example 6: Screening of CTAB elimination methods in mixed stool samples

[0192] On the basis of Example 4, in order to obtain the best detection method for fecal sample metabolites in Example 1 and improve the accuracy of sample metabolite detection, the method of eliminating CTAB in fecal mixed samples was further screened in this example, and the influence of complete removal of CTAB on the detection results of fecal sample metabolites was explored. The inventors found that in a master's thesis in materials science "Shen Teng. Properties of CTAB / SDS mixed aqueous solutions and their template effect on the synthesis of mesoporous materials [D]. Shandong: Shandong University of Science and Technology, 2016.", the application of CTAB and SDS was mentioned. The purpose of the document was to study the formation of ordered combinations of positive and negative ion surfactants. It was mentioned that different mixing ratios of CTAB and SDS would affect the micelle particle size of the system. When the CTAB: (SDS+CTAB) pairing ratio is between 0.4 and 0.6, a precipitate will be formed. Specifically, Figure 7 As shown, Figure 7 The effect of the ratio of CTAB / SDS on the micelle size of the mixed system. Therefore, the present invention draws on the principle of anion and cation coprecipitation mentioned in the literature, and removes CTAB by coprecipitation of SDS and CTAB. However, the research focus in the literature is the preparation of liquid crystal materials. In order to produce a crystal structure, its reaction system (temperature, time, ratio) is completely unsuitable for the method of the present invention. Therefore, in this embodiment, on the one hand, substances that can be coprecipitated with CTAB are screened, and on the other hand, the reaction ratio, reaction temperature and reaction time of CTAB and SDS are improved and further optimized, and the effects of coprecipitation of different substances with CTAB and the reaction system in the literature are compared with the reaction conditions in this embodiment, specifically including the following methods (the remaining conditions are the optimal conditions in Example 1, and NH in this embodiment 4 Cl and PBS have been completely removed using the best method in Example 1):

[0193] 1. Use deionized water without adding other substances;

[0194] 2. Use the reaction conditions in the literature to co-precipitate SDS and CTAB: (1) SDS: CTAB = 0.43-1.5: 1; (2) Reaction temperature is 35°C in a water bath; (3) Reaction time is 24h;

[0195] 3. The optimal reaction conditions optimized in Example 1 of the present invention were used to carry out co-precipitation of SDS and CTAB: (1) SDS: CTAB = 1: 1; (2) the incubation temperature was an ice bath at 10° C.; (3) the incubation time was 15 min;

[0196] 4. Use sodium tetrapropylene benzene sulfonate and CTAB for co-precipitation;

[0197] 5. Use sodium oleoyloxyethanesulfonate and CTAB for co-precipitation;

[0198] The specific results are shown in Table 4:

[0199] Table 4. Effects of different methods on CTAB removal and metabolite detection results in fecal samples

[0200]

[0201] Note: 1) It is known that the metabolites detectable in the stool samples of the present invention are bile acids, and the retention times of different bile acids are: GCA (8.45min), TUDCA (6.43min), GUDCA (7.97min), TCA (7.45min), TDCA (9.67min), GDCA (10.21min), TCDCA (9.45min), GCDCA (10.02min), TLCA (11.12min), LCA (14.70min). in), bCA (9.34min), CA (10.900min), UDCA (10.77min), DCA (12.97min) and CDCA (12.72min); 2) the conjugated bile acids include TCA, GCA, TCDCA, GCDCA, TUDCA, GUDCA, TLCA, TDCA, GDCA; 2) relative ionization efficiency calculation formula: relative ionization efficiency = (experimental group ion response / standard solution ion response) * 100%.

[0202] As can be seen from Table 4, 1) when the reaction conditions in the literature are used, ① at the ratio of SDS:CTAB = 0.43 to 1.5:1, although precipitation will occur, the purpose of the literature is to study the crystal structure in a non-precipitated state, hoping to find the conditions under which SDS and CTAB react without precipitation, so the problems solved are different; ② the reaction temperature in the literature is 35°C in a water bath for the purpose of preparing a liquid crystal reaction system, and increasing the temperature thereby increases the reaction efficiency; ③ the reaction time in the literature is 24h, and the yield is increased by extending the reaction time; therefore, the purpose reported in the literature is completely different from the co-precipitation of SDS and CTAB expected in the present invention. At the same time, under the reaction conditions reported in the literature, when SDS and CTAB are co-precipitated, the removal effect of CTAB is only 52%, so this result is bound to affect the final detection results of fecal sample metabolites. , making it basically impossible to detect metabolites, and at the same time, its relative ionization efficiency is extremely low; 2) when deionized water or sodium tetrapropylene benzene sulfonate or sodium oleoyloxyethane sulfonate is used to remove CTAB, CTAB in the mixed stool sample cannot be completely removed, thereby affecting the final detection results of metabolites in the stool sample, making some metabolites unable to be detected, retention times significantly shifted, and responses of some metabolites significantly reduced, and the relative ionization efficiency is significantly reduced; 3) when the optimized reaction conditions in Example 1 of the present invention are used for co-precipitation of SDS and CTAB, the removal effect of CTAB in the mixed stool sample can reach 100%, thereby eliminating the influence of CTAB on the detection results of metabolites in the stool sample, so that all metabolites can peak normally and respond well, and the relative ionization efficiency can reach 90% to 98%.

[0203] At the same time, in order to further improve the removal effect of CTAB and the accuracy of metabolite detection results, the reaction conditions were further optimized in this example. The specific scheme and results are shown in Tables 5 and 6: (1) SDS: CTAB = 0.8-1.2: 1; (2) the reaction temperature was an ice bath at 10°C; (3) the reaction time was 5-20 min;

[0204] Table 5. Effects of different ratios of SDS to CTAB on the removal of CTAB and the detection results of metabolites (bile acids) in fecal samples

[0205]

[0206] Note: 1) It is known that the metabolites detectable in the stool samples of the present invention are bile acids, and the retention times of different bile acids are: GCA (8.45min), TUDCA (6.43min), GUDCA (7.97min), TCA (7.45min), TDCA (9.67min), GDCA (10.21min), TCDCA (9.45min), GCDCA (10.02min), TLCA (11.12min), LCA (14.70min), bCA (9.34min), CA (10.90min), UDCA (10.77min), DCA (12 .97min) and CDCA (12.72min); 2) the conjugated bile acids include TCA, GCA, TCDCA, GCDCA, TUDCA, GUDCA, TLCA, TDCA, GDCA; 3) only the ratio of SDS:CTAB was changed here, and the other reaction conditions were the optimal reaction conditions; 4) relative ionization efficiency calculation formula: relative ionization efficiency = (experimental group ion response / standard solution ion response) * 100%; in the relative ionization efficiency result, "--" means: due to excessive retention time deviation, the peak shape could not be collected normally, resulting in inaccurate qualitative analysis of the substance, so the relative ionization efficiency cannot be compared and no specific calculation is performed.

[0207] As can be seen from Table 5, 1) when SDS: CTAB = 0.8:1, the removal effect of CTAB is only 71%, and CTAB in the fecal mixed sample cannot be completely removed, thus affecting the final detection results of fecal sample metabolites, making some metabolites unable to be detected, the retention time of conjugated bile acids significantly shifted backwards, and the response of all bile acids significantly decreased, and the combined retention time of conjugated bile acids shifted and reached 3.15±1min. This may be because the amount of SDS added is insufficient and CTAB cannot be completely removed, so the remaining CTAB still affects the detection results of bile acids, causing their retention time to shift backwards; 2) when SDS: CTAB = 1:1, CTAB in the fecal mixed sample can be completely removed at this time, thereby eliminating the influence of CTAB on the detection results of fecal sample metabolites, so that all metabolites can peak normally and respond well, and the retention time is basically not shifted. This may be because when the ratio of the two is 1:1, CTAB in the fecal sample is almost completely eliminated and no longer affects the detection of bile acids. 3) When SDS:CTAB=1.2:1, although CTAB in the fecal mixed sample can be completely removed at this time, the retention time of conjugated bile acids still shifts forward significantly and the response of some bile acids is significantly reduced. The retention time of conjugated bile acids shifts and reaches 1.8±0.2min. This may be because SDS is excessive at this time. Although CTAB is completely removed, the remaining SDS may also affect the detection results of bile acids, eventually shifting its retention time forward and interfering with the detection results of metabolites.

[0208] It can be seen that the optimal reaction ratio of SDS to CTAB in this embodiment is preferably 1:1.

[0209] Table 6. Effects of different reaction times on the removal of CTAB and the detection results of fecal sample metabolites (bile acid)

[0210]

[0211] Note: 1) It is known that the metabolites detectable in the stool samples of the present invention are bile acids, and the retention times of different bile acids are: GCA (8.45min), TUDCA (6.43min), GUDCA (7.97min), TCA (7.45min), TDCA (9.67min), GDCA (10.21min), TCDCA (9.45min), GCDCA (10.02min), TLCA (11.12min), LCA (14.70min), bCA (9.34min), CA (10.900min), UDCA (10.77min), DCA (12.97min) and CDCA (12.72min); 2) Only the reaction time was changed here, and the other reaction conditions were all optimal reaction conditions; 3) Relative ionization efficiency calculation formula: relative ionization efficiency = (experimental group ion response / standard solution ion response) * 100%; In the relative ionization efficiency result, "--" means: due to excessive retention time deviation, the peak shape could not be collected normally, resulting in inaccurate qualitative analysis of the substance, so the relative ionization efficiency cannot be compared and no specific calculation is performed.

[0212] As can be seen from Table 6, when the reaction time is too short (5 min), CTAB cannot be completely removed, thereby affecting the final detection results of fecal sample metabolites, making some bile acids undetectable, retention times significantly shifted, and all bile acid responses significantly reduced; when the reaction time is 15 min or 20 min, CTAB can be completely removed, and at this time the retention times of different metabolites are close to the normal retention time, with almost no shift. Therefore, considering the comprehensive experimental results and time cost, in this embodiment, the optimal reaction time is preferably 15 min.

[0213] In this embodiment, the optimal reaction temperature was also screened, and the experimental results showed that the optimal reaction temperature was an ice bath of 10°C. This is because the metabolites of biological samples are unstable, and the metabolic enzyme activity in fecal samples can be significantly inhibited under low temperature conditions, reducing the physiological and biochemical reactions and maintaining the original state of metabolites in the sample as much as possible. Therefore, the preferred optimal reaction temperature is an ice bath of 10°C.

[0214] Example 7: Test on the effect of the optimal DNA preservative system in Example 1 on the co-precipitation efficiency of SDS and CTAB

[0215] The best stool DNA preservative system has been screened out in Example 3. In order to further explore the effect of the DNA preservative system on the co-precipitation efficiency of SDS and CTAB, the best stool preservative system in Example 1 and the DNA preservative systems of other components were compared and tested in this example, specifically including the following methods (the rest of the conditions are the best conditions in Example 1):

[0216] 1. DNA preservative system: including CTAB, NH 4 Cl and PBS;

[0217] 2. DNA preservative system: including CTAB, NaCl and PBS;

[0218] 3. DNA preservative system: including CTAB, NH 4 Cl and HBSS;

[0219] The specific results are shown in Table 7:

[0220] Table 7. Effects of different DNA preservative systems on CTAB precipitation efficiency and metabolite detection results in fecal samples

[0221]

[0222] Note: 1) It is known that the metabolites detectable in the stool samples of the present invention are bile acids, and the retention times of different bile acids are: GCA (8.45min), TUDCA (6.43min), GUDCA (7.97min), TCA (7.45min), TDCA (9.67min), GDCA (10.21min), TCDCA (9.45min), GCDCA (10.02min), TLCA (11.12min), LCA (14.70min), bC A (9.34min), CA (10.900min), UDCA (10.77min), DCA (12.97min) and CDCA (12.72min); 2) Relative ionization efficiency calculation formula: relative ionization efficiency = (experimental group ion response / standard solution ion response) * 100%; In the relative ionization efficiency result, "--" means: due to excessive retention time deviation, the peak shape cannot be collected normally, resulting in inaccurate qualitative analysis of the substance, so the relative ionization efficiency cannot be compared and no specific calculation is performed.

[0223] As can be seen from Table 7, only when the optimal DNA preservative system in Example 1 is used, after adding 60 μL of SDS, can CTAB be completely precipitated, and the precipitation efficiency reaches 100%. At this time, all metabolites can be normally eluted and respond well, and the retention time is basically not shifted. At the same time, the ionization efficiency returns to the normal level, which is 100%. When any component in the system is replaced, the precipitation efficiency of CTAB will be significantly reduced, and some metabolites cannot be detected, the retention time is significantly shifted, and the sample DNA cannot remain stable. This may be because a certain proportion of NH 4 Cl can make the system in a high-proportion salt solution state. At this time, the physiological and biochemical reactions in the sample are inhibited, DNA can remain stable, and NH 4Cl is also helpful for the acid-base balance of the solution system; at the same time, PBS is crucial for the pH adjustment of the system. After the sample is added to the DNA preservative system, a more appropriate pH value can be maintained to ensure the stability of metabolites and DNA; and when NH 4 When any one of the components of Cl and PBS is replaced, the precipitation efficiency of CTAB will be significantly reduced, and the detection results of bile acids in the sample will be affected, making them undetectable, the retention time shifted, or the response significantly reduced.

[0224] It can be seen that this example proves that the stool DNA preservative system in Example 1 can significantly improve the co-precipitation efficiency of SDS and CTAB, and further proves that the stool DNA preservative system in Example 1 is the best system, including CTAB, NH 4 Cl and PBS, which had the best CTAB precipitation efficiency, also significantly improved the accuracy of metabolite detection results in fecal samples.

[0225] Example 8: Screening of fecal sample pretreatment methods

[0226] In order to obtain the best method for detecting metabolites in stool samples in Example 1, the pretreatment methods of stool samples containing composite preservatives were further compared and screened in this example, specifically including the following methods (the remaining conditions are the best conditions in Example 1). The stool samples here are mixed stool samples from different sources to ensure that the test dosage is sufficient, and the same mixed stool sample can eliminate individual errors and ensure the parallel comparability of the following results:

[0227] 1. Option 1: Conventional treatment - feces preservative sample (Comparative Example 1)

[0228] ① Take 5-50 μL of stool sample suspension and add 100 μL-1 mL of internal standard methanol / acetonitrile mixture (methanol:acetonitrile = 80:20, volume ratio) to the stool sample;

[0229] ② Add 10-15 mg of magnetic beads to the mixed sample;

[0230] ③ Add the mixed sample to the tissue homogenizer and grind;

[0231] ④After grinding, the mixed sample is subjected to low-temperature ultra-high-speed centrifugation, and the supernatant is transferred to the sample injection plate for detection;

[0232] 2. Option 2: Freeze-drying + organic solvent extraction - feces preservative sample (Comparative Example 2)

[0233] ① Take 5-50 μL of stool sample suspension and freeze-dry the sample in a low-temperature freeze vacuum dryer;

[0234] ② Add 100 μL-1 mL of methanol / acetonitrile mixture containing internal standard (methanol:acetonitrile = 80:20, volume ratio) to the freeze-dried sample;

[0235] ③ Add 10-15 mg of magnetic beads to the mixed sample;

[0236] ④ Add the mixed sample to the tissue homogenizer and grind it;

[0237] ⑤After grinding, the mixed sample was subjected to low-temperature ultra-high-speed centrifugation, and 90 μL of supernatant was taken to the sample injection plate for detection;

[0238] 3. Scheme 3: Freeze-drying + organic solvent extraction + purification column pretreatment - feces preservative sample (Comparative Example 3)

[0239] ① Take 5-50 μL of stool sample suspension and freeze-dry the sample in a low-temperature freeze vacuum dryer;

[0240] ② Add 100 μL to 1 mL of internal standard methanol / acetonitrile mixture (methanol:acetonitrile = 80:20, volume ratio) to the freeze-dried stool sample;

[0241] ③ Add 10-15 mg of magnetic beads to the mixed sample;

[0242] ④ Add the mixed sample to the tissue homogenizer and grind it;

[0243] ⑤After grinding, the mixed sample is subjected to low-temperature ultra-high-speed centrifugation;

[0244] ⑥After centrifugation, take 1-50 μL of supernatant into a 1.5 mL centrifuge tube;

[0245] ⑦ Add 10-1000 μL ultrapure water to the supernatant and mix well;

[0246] ⑧Take out the solid phase extraction column from the low temperature refrigerator and return it to room temperature;

[0247] ⑨ Use 10 to 20 column volumes of methanol to activate the solid phase extraction column;

[0248] ⑩ Use a certain volume of methanol / acetonitrile mixture (80 / 20, volume ratio) to balance the solid phase extraction column;

[0249] The mixed supernatant is loaded into a solid phase extraction column;

[0250] Elute three times with 1-10 mL of ultrapure water and discard the eluent;

[0251] Elute with 1-10 mL of methanol three times, collect and combine the eluates, and vortex to mix;

[0252] Take 1-5 mL of the eluate into an EP tube and freeze-dry;

[0253] 90-120 μL of initial mobile phase was reconstituted and tested on the instrument;

[0254] 4. Scheme 4: Elimination of CTAB by SDS (Comparative Example 4)

[0255] ① Take 5-50 μL of stool sample suspension and freeze-dry the sample in a low-temperature freeze vacuum dryer;

[0256] ② Add 100 μL-1 mL of methanol / acetonitrile mixture containing internal standard (methanol:acetonitrile = 80:20, volume ratio) to the freeze-dried sample;

[0257] ③ Add 10-15 mg of magnetic beads to the mixed sample;

[0258] ④ Add the mixed sample to the tissue homogenizer and grind it;

[0259] ⑤After grinding, the mixed sample was subjected to low-temperature ultra-high-speed centrifugation, and 40-80 μL (60 μL) of the supernatant was taken into a 1.5 mL EP tube;

[0260] ⑥ Add an equal volume of 1% to 1.5% SDS to the EP tube and oscillate to mix;

[0261] ⑦ Centrifuge the mixed sample and take the supernatant to the sample injection plate for detection;

[0262] 5. Scheme 5: Elimination of CTAB by SDS (best detection method in Example 1)

[0263] ① Take 5-50 μL of stool sample suspension and freeze-dry the sample in a low-temperature freeze vacuum dryer;

[0264] ② Add 100 μL-1 mL of methanol / acetonitrile mixture containing internal standard (methanol:acetonitrile = 80:20, volume ratio) to the freeze-dried sample;

[0265] ③ Add 10-15 mg of magnetic beads to the mixed sample;

[0266] ④ Add the mixed sample to the tissue homogenizer and grind it;

[0267] ⑤After grinding, the mixed sample was subjected to low-temperature ultra-high-speed centrifugation, and 120 μL of supernatant was taken into a 1.5 mL EP tube;

[0268] ⑥ Take 40-80 μL (60 μL) of the supernatant to measure the OD value. According to the measurement results, add SDS at a molar ratio of 1:1 and mix and shake;

[0269] ⑦ Centrifuge the mixed sample and take the supernatant to the sample injection plate for detection;

[0270] The retention time detection results of different metabolites are shown in Table 8:

[0271] Table 8. Effects of different pretreatment schemes on metabolite detection results of stool samples containing composite preservatives

[0272]

[0273] Note: 1) The differences among the different schemes are as follows: Comparative Example 1 is conventional treatment + organic solvent extraction; Comparative Example 2 is freeze-drying treatment + organic solvent extraction; Comparative Example 3 is freeze-drying + organic solvent extraction + purification column pretreatment; Comparative Example 4 is freeze-drying + organic solvent extraction + SDS elimination of CTAB; Example 1 is freeze-drying + organic solvent extraction + determination of CTAB content and then adding SDS to eliminate CTAB in a 1:1 ratio according to the determination result; 2) It is known that the metabolites that can be detected in the stool sample of the present invention are bile acids, and the retention times of different bile acids are: GCA (8.45min), TUDCA ( 6.43min), GUDCA(7.97min), TCA(7.45min), TDCA(9.67min), GDCA(10.21min), TCDCA(9.45min), GCDCA(10.02min), T LCA (11.12min), LCA (14.70min), bCA (9.34min), CA (10.900min), UDCA (10.77min), DCA (12.97min) and CDCA (12.72min).

[0274] As shown in Table 8, 1) when using the schemes of Comparative Example 1, Comparative Example 2 and Comparative Example 3, GCA, TCA and TDCA bile acids in the fecal samples could not be detected, indicating that only the NH 4 Cl and PBS, the detection results of metabolites in stool samples will still be significantly affected; 2) When the scheme in Comparative Example 4 is used, when an equal volume of SDS to the supernatant is added (equal molar ratio), compared with the qualitative retention time of the standard, the retention times of GCA, TCA, GUDCA, TUDCA, TDCA, GDCA, TCDCA, GCDCA and TLCA bile acids in the stool samples are significantly shifted forward, resulting in inaccurate metabolite detection results; 3) When the best scheme in Example 1 is used, the retention time of metabolites in stool samples is normal and the quantitative results are accurate.

[0275] Therefore, it can be seen that in this embodiment, the best pretreatment method in Example 1 is preferably used to detect metabolites in stool samples.

[0276] The peak shape results of metabolites in fecal mixed samples under each condition are as follows: Figure 8 to Figure 12 As shown, Figure 8 This is a peak shape detection result diagram of each metabolite in the fecal mixed sample under the conditions of Comparative Example 1 (conventional treatment + organic solvent extraction); Fig. 9 This is a peak shape detection result diagram of each metabolite in the fecal mixed sample under the conditions of Comparative Example 2 (lyophilization + organic solvent extraction); Fig.10 This is a peak shape detection result diagram of each metabolite in the fecal mixed sample under the conditions of Comparative Example 3 (lyophilization + organic solvent extraction + purification column pretreatment); Fig.11 This is a peak shape detection result diagram of each metabolite in the fecal mixed sample under the conditions of Comparative Example 4 (lyophilization + organic solvent extraction + SDS elimination of CTAB); Fig.12 This is a peak shape detection result diagram of each metabolite in the fecal mixed sample under the conditions of Example 1 (freeze drying + organic solvent extraction + determination of CTAB content and then adding SDS in a 1:1 ratio to eliminate CTAB according to the determination results). Figure 8 to Figure 12 In the figure, nine bile acids whose retention time, peak shape or response were significantly affected are shown, while the remaining bile acids were not significantly affected and are therefore not shown. Figure 8 to Figure 12 The changes in the peak shape, response and ionization efficiency of each metabolite in the mixed feces sample are shown below:

[0277] Table 9. Changes in peak shape and response of metabolites in mixed fecal samples by different schemes

[0278]

[0279] Note: 1) It is known that the metabolites detectable in the stool samples of the present invention are bile acids, and the retention times of different bile acids are: GCA (8.45min), TUDCA (6.43min), GUDCA (7.97min), TCA (7.45min), TDCA (9.67min), GDCA (10.21min), TCDCA (9.45min), GCDCA (10.02min), TLCA (11.12min), LCA (14.70min), bC A (9.34min), CA (10.900min), UDCA (10.77min), DCA (12.97min) and CDCA (12.72min); 2) Relative ionization efficiency calculation formula: relative ionization efficiency = (experimental group ion response / standard solution ion response) * 100%; In the relative ionization efficiency result, "--" means: due to excessive retention time deviation, the peak shape cannot be collected normally, resulting in inaccurate qualitative analysis of the substance, so the relative ionization efficiency cannot be compared and no specific calculation is performed.

[0280] Combination Figure 8 to Figure 12As shown in the results in Table 9, 1) when the schemes of Comparative Examples 1, 2 and 3 are used, the bile acids in the stool samples cannot be detected completely, and the peak shapes of the four metabolites that can be detected are poor, the separation between different metabolites does not meet the standard, and the response of each metabolite is also significantly smaller; 2) when the scheme in Comparative Example 4 is used, when SDS with an equal volume to the supernatant is added, although all the bile acids in the stool samples can be detected and the response is improved, the metabolites are still significantly low, such as the response of TCDCA can only reach 1.2E+6, and the ionization efficiency is significantly reduced; 3) when the best scheme in Example 1 is used, all the bile acids in the stool samples can be detected, the response of each metabolite is significantly improved and the separation meets the standard, and the retention time is accurate and basically no deviation occurs.

[0281] Therefore, it is further confirmed that the best pretreatment method in Example 1 is preferred in this embodiment for detecting metabolites in stool samples.

[0282] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A composite feces preservative, characterized in that: The components of the preservative include: CTAB, NH4Cl and PBS.

2. The feces composite preservative according to claim 1, characterized in that: The content of CTAB is 1% to 2.5%, The content of NH4Cl is 10% to 20%, and the content of PBS is 5% to 10%.

3. The feces composite preservative according to claim 2, characterized in that: The content of CTAB is 2.0%, the content of NH4Cl is 20%, and the content of PBS is 10%.

4. A method for preserving a stool sample, characterized in that: The following steps are involved: (1) Preparing a compound feces preservative: preparing a compound preservative containing active ingredients including CTAB, NH4Cl and PBS; (2) Fecal sample collection and storage: Fecal samples were homogenized, 10-30 mg of sample was added to the preservative and shaken well.

5. A pretreatment method for detecting metabolites in a stool sample containing a composite preservative, characterized in that: The following steps are involved: (1) Take 5-50 μL of stool sample containing composite preservative and freeze-dry it; (2) adding a mixed organic solvent and magnetic beads to the freeze-dried sample in sequence and homogenizing; (3) Centrifuge the homogenized sample at low temperature and high speed, take 40-80 μL of the supernatant, and determine the exact content of CTAB in the sample; (4) According to the content of CTAB, accurately prepare the corresponding proportion of sodium dodecyl sulfate (SDS) solution; (5) Co-precipitation elimination: Take an appropriate amount of the supernatant obtained in step (3), add 40-80 μL of the SDS solution prepared in step (4), incubate for a certain period of time, centrifuge again at low temperature and high speed, and take the final supernatant for metabolite detection.

6. The pretreatment method according to claim 5, characterized in that The fecal metabolites include fecal sample metabolites and composite preservative components; the fecal sample metabolites include GCA, TCA, GUDCA, TUDCA, TDCA, GDCA, TCDCA, GCDCA, TLCA, LCA, bCA, CA, UDCA, DCA and CDCA; the mixed organic solvent in step (2) is a methanol / acetonitrile mixture, and the volume ratio of methanol:acetonitrile is 80:

20.

7. The pretreatment method according to claim 6, characterized in that: In the step (3), after centrifugation, the supernatant is taken for OD value measurement to determine the actual accurate content of CTAB in the system; in the step (5), SDS is added according to the molar ratio of SDS:CTAB of 1:1, the reaction temperature is 10°C, and the reaction time is 20 minutes.

8. Use of sodium dodecyl sulfate for preparing a preparation for improving the response of fecal metabolites in mass spectrometry.

9. Use of sodium dodecyl sulfate for preparing a preparation for improving the accuracy of retention time of fecal metabolites in mass spectrometry.

10. Use of a feces composite preservative for preparing a preparation for improving the response and detection accuracy of feces metabolites in mass spectrometry, characterized in that: The composite feces preservative is as described in any one of claims 1-3.

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