A composite preservative for fecal microorganisms and metabolites thereof and a simultaneous multi-omics integrated detection technology

Abstract

The application provides a composite preservative for fecal microorganisms and metabolites thereof and a same-tube multi-omics joint detection integrated technology. The composite preservative contains main components of cetyltrimethylammonium bromide (CTAB), ammonium chloride (NH4Cl) and phosphate buffered saline (PBS), can keep the stability of microorganisms and metabolites thereof in a fecal sample during storage and transportation at room temperature, and guarantees the extraction efficiency of deoxyribonucleic acid (DNA) of the microorganisms in the fecal sample. The application also eliminates the influence of the preservative components on metabolite detection by adding a treatment reagent, significantly improves the response and ionization efficiency of the fecal sample metabolites in mass spectrometry, and thus improves the sensitivity and accuracy of the detection results. The application not only solves the timeliness problem of fecal sampling, but also provides a technical basis and idea for the feasibility study of the same-tube multi-omics of the fecal sample, and has a broad research and clinical application prospect.

Description

Technical Field

[0001] This invention relates to the fields of chemical analysis and medical testing, specifically to a composite preservative for fecal microorganisms and their metabolites and an integrated technology for multi-omics testing in the same tube. Background Technology

[0002] Fecal samples are a crucial source of direct information on gut microbiota and are considered to contain the richest amount of metabolites. Therefore, fecal samples play a vital role in the joint analysis of gut microbiota and their metabolites. The non-invasive collection method of feces has made them a focus of attention in early cancer risk assessment and screening, especially in the last two years, with the National Medical Products Administration approving several innovative fecal-based diagnostic products, demonstrating the feasibility and effectiveness of fecal samples in medical innovation. Fecal samples provide clinicians with more convenient and practical means of disease diagnosis and prevention. However, fecal samples are characterized by time sensitivity and heterogeneity, posing numerous challenges to sample collection, storage, transportation, multi-omics research, and clinical testing. Storing collected fecal samples at room temperature or 4°C for more than 12 hours can lead to significant changes in fecal microbiota and their metabolites. Therefore, immediate freezing at -80°C and dry ice transportation are widely recommended for fecal sample collection and storage. However, these stringent storage and transportation conditions make it difficult to implement home sampling for patients, large-scale community screening, cross-regional transportation, and even sampling of hospitalized patients.

[0003] To address the aforementioned issues, the use of various preservative solutions is an ideal approach. Currently, various DNA preservation solutions effectively maintain the room-temperature stability of fecal DNA, preserve the integrity of microbial information within the sample, and avoid the stringent requirements for sample freezing and transportation. Common DNA preservation solutions include ethanol, RNAlater, EDTA salts, and commercially available reagent kits such as OMNIgene GUT200 and Stool DNA Stabilizer, which are used in many clinical studies. While existing DNA-specific preservation agents offer advantages such as room-temperature storage and ease of transport, they also have limitations. The biggest drawback is that fecal samples with added DNA preservation agents cannot be qualitatively and quantitatively analyzed for metabolites using liquid chromatography-mass spectrometry (LC-MS / MS). This is because the high-salt components, chelating agents, or surfactants contained in DNA preservation agents can significantly inhibit or disrupt column separation and mass spectrometry ionization.

[0004] In summary, while the widespread use of DNA preservatives to protect fecal samples in clinical research cohorts has solved the challenges of fecal sample storage and transportation, it has limited the detection of metabolomics based on mass spectrometry. Ultimately, this means that multi-omics association analysis of clinical samples can only use samples with different preservation methods, which greatly weakens the correlation between information between multiple omics.

[0005] Therefore, this invention urgently needs to propose a composite preservative for fecal samples containing microorganisms and their metabolites, to facilitate the collection, storage, and transportation of clinical samples. Furthermore, this invention, by properly handling the mass spectrometry interference components of the preservative, enables the simultaneous detection of microorganisms and metabolites in the same solution system (simultaneous tube detection). Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a composite preservative for fecal microorganisms and their metabolites, an integrated multi-omics detection technology, and a method for pretreatment and quantitative detection of metabolites in fecal samples containing the preservative. The composite preservative comprises CTAB, NH4Cl, and PBS components, which can effectively maintain the stability of fecal sample DNA during room temperature storage and transportation. This invention proposes a preservative treatment and metabolite detection method that effectively eliminates the influence of the preservative on metabolite detection, significantly improves the response and ionization efficiency of fecal sample metabolites in mass spectrometry, and ensures the sensitivity and accuracy of detection.

[0007] On one hand, the present invention provides a fecal compound preservative, the components of which include: CTAB, NH4Cl and PBS.

[0008] In some embodiments, the present invention has compared and screened the components of the fecal composite preservative. The experimental results show that the DNA extraction efficiency of the fecal sample is the highest only when the components of the fecal composite preservative include CTAB, NH4Cl and PBS. The DNA content of the fecal sample stored at room temperature for 3 days remains stable, and the microbial composition of the fecal sample stored at room temperature for 5 days remains stable.

[0009] In some embodiments, based on the removal efficiency of CTAB in fecal samples, the composite preservative system was further screened in this invention. Experimental results showed that only when the optimal ratio of the composite preservative (i.e., including CTAB, NH4Cl, and PBS) was used, and the optimal amount of SDS (sodium dodecyl sulfate) was added, could CTAB be completely precipitated, achieving a removal efficiency of 100%. At this point, the analysis results of the bile acid metabolites in the fecal samples showed that their chromatographic retention times were normal. However, when any component in the composite preservative was changed, the removal efficiency of CTAB decreased significantly. In these cases, the chromatographic peak shape of the bile acid metabolites deteriorated significantly, and the retention times also shifted noticeably.

[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%, the content of NH4Cl is 20%, and the content of PBS is 10%.

[0012] In some embodiments, the content of different components in the composite preservative was screened. The experimental results showed that when the CTAB content was 2.0%, the NH4Cl content was 20%, and the PBS content was 10%, the DNA detection efficiency of the composite preservative prepared at this time reached the highest level, and the DNA in the fecal sample remained stable after 3 days at room temperature.

[0013] On the other hand, the present invention provides a method for preserving fecal samples, comprising the following steps:

[0014] (1) Preparation of fecal compound preservative: Prepare a compound preservative with active ingredients including CTAB, NH4Cl and PBS;

[0015] (2) Collection and storage of fecal samples: Homogenize the fecal samples, take 10-30mg of sample, add preservative, and shake well.

[0016] In another aspect, the present invention provides a pretreatment method for detecting metabolites in fecal samples containing a composite preservative, characterized by comprising the following steps:

[0017] (1) Take 5-50 μL of fecal sample containing the compound preservative and freeze-dry it at low temperature;

[0018] (2) Add the mixed organic solvent and magnetic beads to the freeze-dried sample in sequence, and homogenize;

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

[0020] (4) Prepare a sodium dodecyl sulfate (SDS) solution in the appropriate proportion according to the CTAB content;

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

[0022] In some embodiments, the inventors found that when using the optimal composite preservative, the results of metabolite detection in fecal samples were inaccurate, and all three components of the preservative affected the metabolite detection results. Therefore, the inventors screened methods for removing CTAB, NH4Cl, and PBS from mixed fecal samples and found that a mixture of methanol and acetonitrile (methanol:acetonitrile = 80:20, volume ratio) could completely remove NH4Cl and PBS, but not CTAB. The inventors utilized the principle of co-precipitation of anionic and cationic surfactants, adding SDS to form a precipitate with CTAB, thus removing CTAB. However, known conditions are not suitable for the effective removal of the CTAB component in the preservative system of this invention. Therefore, after a large number of screening experiments, the coprecipitation reaction conditions of SDS and CTAB were further optimized. The experimental results showed that only when the following three conditions are met simultaneously: (1) SDS and CTAB are coprecipitated in a CTAB+NH4Cl+PBS system; (2) the molar ratio of SDS to CTAB is 1:1; (3) the coprecipitation efficiency of CTAB and SDS reaches 100% when incubated in an ice bath at 10°C for 15 min.

[0023] Further, 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 organic solvent mixed in step (2) is a methanol / acetonitrile mixture, and the volume ratio of methanol to acetonitrile is 80:20.

[0024] In some embodiments, the inventors discovered that the two salts NH4Cl and PBS in the preservative precipitate in the liquid chromatography system and mass spectrometry ion source, which not only severely shortens the service life of the chromatographic column and equipment, but also significantly affects the metabolite detection results. Therefore, after screening, it was found that an organic mixture of methanol / acetonitrile (methanol:acetonitrile = 80:20, volume ratio) can effectively eliminate NH4Cl and PBS.

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

[0026] In another aspect, the present invention provides the use of sodium dodecyl sulfate in the preparation of formulations that enhance the response of fecal metabolites in mass spectrometry.

[0027] In some implementations, after successfully identifying 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 from fecal samples, thereby significantly improving the response of metabolites in mass spectrometry.

[0028] In another aspect, the present invention provides the use of sodium dodecyl sulfate in the preparation of formulations that improve the accuracy of retention time of fecal metabolites in mass spectrometry.

[0029] In some implementations, after successfully identifying the optimal ratio of SDS to CTAB, the optimal incubation conditions, and the optimal reaction system, the addition of SDS can effectively remove CTAB from fecal samples, thereby significantly improving the accuracy of metabolite detection.

[0030] In another aspect, the present invention provides the use of sodium dodecyl sulfate in the preparation of formulations that improve the ionization efficiency of fecal metabolites in mass spectrometry.

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

[0032] The beneficial effects achieved by this invention are as follows:

[0033] 1. This invention provides an effective method for preserving fecal samples, namely, using a composite preservative comprising 2.0% CTAB and 20%...

[0034] NH4Cl and 10% PBS can stably store and effectively extract DNA and metabolites from fecal samples. This innovation solves the problems caused by the timeliness and heterogeneity of fecal samples during collection, storage and transportation, greatly facilitating the sample processing flow, reducing dependence on harsh storage and transportation conditions, and making large-scale sample collection and research possible.

[0035] 2. This invention provides a method for the quantitative detection of CTAB, the active ingredient in compound preservatives. Using a methyl orange indicator and a full-wavelength scanner, it achieves precise quantitative detection of CTAB, a key component in compound preservatives. This method not only provides accurate data support for evaluating the efficacy of preservatives but also lays an important foundation for subsequent optimization of metabolite detection, helping to ensure the accuracy and reliability of experimental results.

[0036] 3. This invention optimizes the pretreatment method for metabolite detection: A method has been successfully developed to effectively remove interfering components (CTAB, NH4Cl, and PBS) from fecal samples containing a composite preservative. In the composite preservative CTAB+NH4Cl+PBS system, through the synergistic effect of a specific ratio of organic solvent methanol / acetonitrile mixture (methanol:acetonitrile = 80:20, volume ratio) and an equimolar amount of SDS with CTAB, under specific reaction conditions (incubation at 10°C on ice for 20 min), the mass spectrometry response and detection accuracy of fecal sample metabolites are significantly improved. This effectively removes the composite preservative components that affect metabolite detection, thus solving the problem of inhibition and interference of previous preservatives on metabolite detection.

[0037] 4. This 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 room temperature stability of fecal DNA and metabolites, providing technical basis 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 the metabolite mass spectrometry detection is eliminated, significantly improving the sensitivity and accuracy of metabolites. This provides a key technical method for the study of fecal sample metabolomics, and ultimately realizes the innovative idea of ​​multi-omics detection (simultaneous tube detection) of the same fecal sample, which has good scientific research and clinical application value.

[0038] 5. This invention achieves integrated multi-omics detection of fecal samples: The composite preservative and related detection methods provided by this invention enable the simultaneous detection of microorganisms and metabolites in the same fecal sample (simultaneous tube testing). This innovative approach overcomes the limitation of weakened correlation of multi-omics information caused by different preservation methods in traditional methods, providing strong technical support for a more comprehensive and in-depth understanding of the relationship between gut microbiota and metabolites. This has important guiding significance for the early diagnosis of intestinal diseases, research on pathogenesis, and the formulation of personalized treatment plans, and has broad application prospects and significant scientific value. Attached Figure Description

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

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

[0041] Figure 3 The image shows the qualitative detection results of fecal sample metabolites (bile acids) under the conditions of scheme 3 in Example 4 (with deionized water added).

[0042] Figure 4This is a graph showing the qualitative detection results of fecal sample metabolites (bile acids) under the conditions of scheme 2 in Example 4 (preservative was added but no treatment was performed).

[0043] Figure 5 The image shows the qualitative detection results of fecal sample metabolites (bile acids) under the conditions of Scheme 1 in Example 4 (preservative was added while removing influencing substances).

[0044] Figure 6 The figure shows the specific results of quantitative detection of bile acid metabolites under different conditions in Example 4.

[0045] Figure 7 This refers to the effect of the CTAB / SDS ratio on the micelle size of the mixed system mentioned in Example 5.

[0046] Figure 8 The image shows the peak shape detection results of each metabolite in the mixed fecal sample of Comparative Example 1 in Example 8 (conventional processing + organic solvent extraction).

[0047] Figure 9 The image shows the peak shape detection results of each metabolite in the mixed fecal sample of Comparative Example 2 in Example 8 (lyophilization + organic solvent extraction).

[0048] Figure 10 The image shows the peak shape detection results of each metabolite in the mixed fecal sample of Comparative Example 3 in Example 8 (lyophilization + organic solvent extraction + purification column pretreatment).

[0049] Figure 11 The image shows the peak shape detection results of each metabolite in the mixed fecal sample of Comparative Example 4 in Example 8 (lyophilization + organic solvent extraction + SDS elimination of CTAB).

[0050] Figure 12 The peak shape detection results of each metabolite in the fecal mixed sample of Example 1 in Example 8 are shown in Example 8 (freeze-drying + organic solvent extraction + determination of CTAB content, followed by the addition of SDS at a ratio of 1:1 to eliminate CTAB according to the determination results). Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased through legitimate channels.

[0052] In this invention, metabolites are first separated by chromatography, then progressively reach a mass spectrometry ion source. In the ion source, they are fragmented by different voltages, with different substances producing ion fragments with different charges—this is the ionization process. These ions are then collected by a mass spectrometer detector; 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, thus determining the total amount of substance. Therefore, the inventors determine the substance type based on retention time and fragment ions, and determine the substance concentration by the collected charge. However, when the retention time of the metabolite's chromatographic peak shifts significantly, the metabolite type cannot be clearly identified, making it impossible to compare relative ionization efficiencies. But when the retention time shift is small, the substance type can be clearly identified, allowing for the calculation of relative ionization efficiency.

[0053] Example 1: Optimal preparation method of compound preservative and co-detection method of fecal compound preservative samples

[0054] Currently, commonly used DNA preservatives can effectively protect the microbial DNA of fecal samples at room temperature. After adding a DNA preservative, fecal samples can be stored or transported at room temperature without altering the microbial composition. However, fecal samples containing DNA preservatives cannot be used for qualitative and quantitative mass spectrometry detection of metabolites. Therefore, this embodiment provides a fecal sample composite preservative with optimal performance. It can stably maintain DNA preservation and detection efficiency, and by adding sodium dodecyl sulfate (SDS) and organic solvents, it can effectively remove components that affect metabolite detection, enabling the detection of microorganisms and their metabolites in the same preservative sample, greatly improving the correlation between multiple omics studies.

[0055] I. Optimal Preservation Method for Fecal Samples

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

[0057] Step 1: Preparation of compound preservative: Prepare a compound preservative with active ingredients including CTAB, NH4Cl and PBS, wherein the content of CTAB is 2.0%, the content of NH4Cl is 20% and the content of PBS is 10%.

[0058] Step 2: Fecal sample collection and storage. Homogenize the fecal sample, weigh 10-30mg (20mg) of sample and add 160μL of preservative, then shake well.

[0059] Step 3: Add compound preservative and magnetic beads to the fecal sample, mix well, and then divide into different volumes of sample tubes for storage according to different testing purposes.

[0060] This optimal composite preservative has the same effect as common DNA preservatives, maintaining the stability of DNA in fecal samples at room temperature.

[0061] II. Detection Method for DNA in Fecal Samples with Added Compound Preservatives

[0062] This embodiment also provides a method for detecting DNA in fecal samples with added composite preservatives, including the following steps:

[0063] Step 1: Extraction of DNA from fecal samples

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

[0065] Step 2: PCR amplification

[0066] ① Template: Diluted genomic DNA;

[0067] ② Primers: Specific primers with barcodes are used according to the selected sequencing region; the primer for the 16S V4 region is 515F-806R;

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

[0069] ITS2-2043R, with ITS2 primers being ITS3-2024F and ITS4-2409R.

[0070] ③ Enzymes and buffers: High-Fidelity PCR MasterMix with GC from New England Biolabs was used.

[0071] Buffer. PCR is performed using highly efficient and high-fidelity enzymes to ensure amplification efficiency and accuracy.

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

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

[0074] Step 3: Mixing and purifying PCR products

[0075] Based on the PCR product concentration, samples were mixed at equal concentrations and thoroughly combined before purification using 2% agarose gel electrophoresis with a 1×TAE concentration.

[0076] For PCR products, sequences with a major band size between 400-450 bp were selected, and the target band was recovered by gel extraction. The GeneJET gel extraction kit from Thermo Scientific was used for product purification.

[0077] Step 4: Library construction and sequencing

[0078] Library construction was performed using the Illumina TruSeq DNA PCR-Free Library Preparation Kit.

[0079] After the constructed library was quantified using Qubit and tested, and passed the tests, it was sequenced using NovaSeq6000.

[0080] Step 5: Bioinformatics Analysis

[0081] ① Sequencing result preprocessing: The raw data obtained from sequencing contains a certain proportion of interference data (Dirty Data). In order to make the information analysis results more accurate and reliable, the raw data is first spliced ​​and filtered to obtain effective data (Clean Data).

[0082] ② Species Analysis and Annotation: Using DIAMOND software, Unigenes were compared with those from the NCBI NR database (Version).

[0083] On January 2, 2018, sequences of bacteria, fungi, archaea, and viruses extracted from https: / / www.ncbi.nlm.nih.gov / were compared.

[0084] ③ Data visualization processing: Using R data packages and custom programming, calculate 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 samples. Multiple sequence alignment was performed on the species and phylogenetic trees were constructed to further obtain the differences in community structure among different samples and groups, which were displayed using dimensionality reduction diagrams such as PCoA, PCA, and NMDS, as well as sample clustering trees.

[0086] Experimental results showed that when fecal samples were preserved using the above-mentioned preservative formulation with the best preservation effect, the detection results of the final fecal sample metabolites were inaccurate. All three components in the preservative affected the detection results. The inventors found that the reasons for this phenomenon were: (1) NH4Cl and PBS, two salts, precipitate in the liquid phase system and mass spectrometry ion source, which seriously affects the service life of the chromatographic column and equipment, and also significantly affects the detection results of bile acids; (2) CTAB carries a positive charge and will bind to some bile acids, which will not only affect the colorimetric results but also the detection results of bile acids. The spectroscopic behavior also leads to a significant shift in the retention time of amino acid-bound metabolites and significantly inhibits the ionization efficiency of non-amino acid-bound metabolites, resulting in a significant decrease in metabolite response. Therefore, metabolite detection requires effective removal of the three components of the composite preservative in fecal samples (① Effective removal in this invention is immediate detection and immediate elimination, removing the three components of the preservative before detection is required; ② Although the detection of metabolites will be affected after the addition of the composite preservative to the fecal sample, the addition of organic solvents and SDS can effectively eliminate the influence of the composite preservative on the detection of metabolites).

[0087] III. Methods for quantitative detection of CTAB, the active ingredient in compound preservatives, and methods to eliminate the influence of compound preservatives on metabolite detection.

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

[0089] (1) The quantitative detection method for the active ingredient CTAB in the compound preservative includes the following steps:

[0090] Step 1: Fecal sample pretreatment. The fecal sample is pretreated to obtain a pretreated fecal sample.

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

[0092] Step 3: Subtract the series of standard absorbance values ​​from the blank absorbance value to obtain the series of standard values, and perform the corresponding linear regression equation; subtract the absorbance value to be measured 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 solution.

[0093] (2) The method for eliminating CTAB, NH4Cl and PBS includes the following steps:

[0094] Step 1: After adding MeOH solution to the mixed fecal sample, CTAB quantification was performed in the mixed sample;

[0095] Step 2: Prepare SDS by measuring the CTAB content to prepare the appropriate proportion of SDS;

[0096] Step 3: Co-precipitation elimination. For mixed samples, add the same volume of SDS as CTAB: 60 μL, mix well, centrifuge to remove the precipitate and retain the supernatant.

[0097] In summary, this embodiment provides an optimal composite preservative containing 2.0% CTAB, 20% NH4Cl, and 10% PBS, which effectively extracts DNA from fecal samples, significantly improving DNA detection efficiency and maintaining the stability of microorganisms and metabolites at room temperature or during transportation. Simultaneously, to ensure that metabolite quantification is not affected and to improve detection accuracy, this embodiment, while maintaining DNA stability, eliminates NH4Cl and PBS by adding an organic solvent methanol / acetonitrile mixture (methanol:acetonitrile = 80:20), and eliminates CTAB by adding SDS and co-precipitating with CTAB. The specific steps are as follows:

[0098] ① Sample pretreatment after adding compound preservative to fecal samples:

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

[0100] 2) The sample was freeze-dried overnight;

[0101] 3) Add 10 mg of magnetic beads to the EP tube and redissolve them in 200 μL of methanol / acetonitrile solution containing internal standard;

[0102] 4) Grind for 2 minutes;

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

[0104] 6) Centrifuge at 4℃, 18000g for 25 minutes;

[0105] 7) Dispense the supernatant into two 1.5 mL EP tubes, labeling them i and ii;

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

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

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

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

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

[0111] 13) Centrifuge at 4℃, 18000g for 25 minutes;

[0112] 14) Take 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 thoroughly;

[0115] 17) 4℃, 1450rpm, shake for 15 minutes;

[0116] 18) Centrifuge at 4℃, 18000g for 25 minutes;

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

[0118] ② Liquid chromatography and mass spectrometry conditions:

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

[0120] 2) The operating conditions for the mass spectrometer are as follows: ion source temperature 220℃; injection volume 0.5-5 μL; splitless injection; ion source voltage 70 eV; mass spectrometry scan range 38-650 m / z; acquisition rate 20-25 spectra / s. The column temperature and sample tray temperature are 30℃ and 10℃, respectively. Mobile phase A is 0.01% formic acid solution (pH=4), mobile phase B is acetonitrile:methanol = 80:20, and the initial mobile phase ratio A:B = 95:5. The flow rate is 0.4 mL / min, with gradient conditions: 0-1 min (5% B); 1-3 min (5-30% B); 3-15 min (30-100% B); 15-16 min (100-5% B); 16-17 min (5% B). The ion source temperature and dissolution temperature are 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 these conditions, each metabolite in the fecal sample will have a good signal, ensuring the accuracy of quantitative analysis.

[0121] ③ Qualitative and quantitative analysis:

[0122] Qualitative and quantitative analysis, based on a database of derivatives of commercially procured standard substances, achieves accurate qualitative and quantitative analysis of the analyte information by comparing the retention indices and mass spectrometry fragmentation information of different analytes. Specifically:

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

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

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

[0126] Example 2: Screening of the components and concentrations of the compound preservative

[0127] To obtain the best-performing composite preservative as shown in Example 1, the components and concentrations 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; however, CTAB alone does not have DNA preservation properties. Therefore, in this example, the inventors added NH4Cl and PBS to CTAB and compared the ratios of the three components. The specific experimental scheme and results are shown in Table 2.

[0128] Table 1. Effects of different components and ratios of CTAB, NH4Cl, and PBS on DNA concentration in fecal samples.

[0129]

[0130] Note: 1) In this embodiment, the theoretical DNA concentration in fecal samples of the same mass should be comparable. Therefore, the DNA concentration results in this embodiment reflect the DNA extraction efficiency and detection status. Higher DNA concentrations in the table indicate better performance under these conditions. 2) In this embodiment, the optimal pH range for fecal DNA preservation is 6.4–6.6. Within this pH range, fecal DNA remains stable. When the pH exceeds this range, metabolite mass spectrometry detection will be affected. 3) Fecal samples are highly heterogeneous, and DNA concentrations at different sampling locations may vary, but are generally within the same order of magnitude. Furthermore, DNA concentrations may fluctuate with increasing preservation time. 4) The PSP Stool DNA in the table... The imported manufacturer and batch number of Stabilizer is Invitek, 1038111100. It is a commercially available reagent kit with good DNA preservation effect. However, its disadvantages are that the import cycle of the reagent is long, the supply is unstable and the cost is high. In addition, the ingredients in this reagent kit are unknown. The table shows the components and their concentration ranges as shown in the instructions. Therefore, it is not recommended as a preservation sample for mass spectrometry detection of fecal sample metabolites. 5) The DNA concentration in the table may also vary due to the amount of sample weighed, but this difference is considered to be a normal error.

[0131] Table 1 shows that: 1) The commercially available DNA preservation kit (PSP Stool DNA Stabilizer) has a pH of 6.50, high DNA detection efficiency, and the DNA concentration in fecal samples stored at room temperature remains stable after 3 days. However, imported reagents have long lead times, unstable supply, high cost, unclear component information, and wide content ranges in the instructions, significantly reducing the accuracy of metabolite detection results. Therefore, it is not suitable for detecting metabolites in fecal samples; 2) DNA preservation solutions containing only CTAB or NH4Cl have unstable pH values ​​and are therefore excluded; 3) DNA preservation solutions containing only PBS have a pH of approximately 7.25–7.35, which is not within the optimal pH range. This system has high DNA extraction efficiency, but the DNA concentration in fecal samples decreases significantly after 3 days at room temperature. 4) The pH value of the CTAB+NH4Cl+PBS composite preservative system of the present invention is very stable and is at the pH value required for optimal detection of metabolites. At this time, the DNA concentration is also at its maximum. The DNA concentration in fecal samples at room temperature for 3 days remains basically stable. Compared with commercially available PSP kits, the DNA concentration in fecal samples after zero days and 3 days of storage at room temperature is close. At the same time, the components of the composite preservative of the present invention are clear and the concentration is fixed. Through appropriate pretreatment, the defect of PSP kits in not being able to detect metabolites in samples is solved. 5) Any change in any component in the CTAB+NH4Cl+PBS composite preservative system will affect the pH value and the ion system of the preservative system.

[0132] Therefore, the optimal formulation of the composite preservative proposed in this invention is a CTAB + NH4Cl + PBS system. In this embodiment, different components and their contents were screened, and the optimal formulation was determined to be 2.0% CTAB, 20% NH4Cl, and 10% PBS. The DNA concentration of the composite preservative of this invention remains basically stable when storing fecal samples at room temperature.

[0133] Example 3: Performance of the fecal preservative sample from Example 1

[0134] This embodiment provides a composite preservative for fecal samples, used to store microorganisms and their metabolites in feces at room temperature. Specifically, it comprises: 1-10% CTAB, 10-20% NH4Cl, 10-20% PBS, and deionized water. In this embodiment, the inventors tested the effect of the preservative on microorganisms and their metabolites under optimal preparation conditions. The specific experimental scheme and results are shown below:

[0135] The method for preparing the composite preservative for fecal samples includes the following steps:

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

[0137] 2) Depending on the purpose of the test, fecal samples containing preservatives were packaged separately and stored in individual tubes.

[0138] 3) After placing the aliquoted samples at room temperature for 0, 3 and 5 days, the samples were collected for microbial sequencing and quantitative detection of metabolites.

[0139] The microorganisms and metabolites of the fecal sample used as a preservative were obtained using the method mentioned in Example 1.

[0140] from Figure 1 As can be seen from the above, 1) when no preservative is used, the microbial composition of fecal samples changes after 3 and 5 days of storage. Compared with the 0-day sample, the changes in fecal microorganisms are more significant after 5 days of storage at room temperature. Therefore, fecal samples stored at room temperature are not suitable for microbial sequencing; 2) when the fecal sample is stored with the commercial OM200 preservative, the microbial composition is not significantly changed after 3 and 5 days of storage at room temperature, and the fecal biological information remains stable; 3) when the fecal sample is stored with the optimal proportion of the preservative of this invention, the microbial composition is also unaffected after 3 and 5 days of storage at room temperature, and the fecal microorganisms remain stable; in addition, the commercial kit is recognized as having excellent DNA preservation effect, and the preservative of this invention can achieve a preservation effect close to that of fecal microorganisms at room temperature.

[0141] from Figure 2 As can be seen from the above, 1) when no preservative is used, the bile acid composition of fecal samples changes after 3 and 5 days of storage. The proportion of primary bile acids decreases, while the proportion of secondary bile acids increases. Therefore, fecal samples stored at room temperature are not suitable for metabolite detection; 2) when fecal samples are stored at -80°C, the bile acid composition does not change after 3 and 5 days, and the fecal samples remain stable; 3) when the optimal proportion of the preservative of this invention is added to the fecal samples, the bile acid composition is also unaffected after 3 and 5 days of storage at room temperature, and the fecal bile acid composition remains stable; furthermore, the -80°C storage is recognized as the "gold standard" for fecal metabolite storage, and the preservative of this invention can achieve a preservation effect close to that of fecal bile acids at room temperature.

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

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

[0144] To verify that the detection method for fecal sample metabolites in Example 1 is the optimal method, this example compares and tests different detection methods, specifically including the following three schemes (all other conditions are the optimal conditions in Example 1):

[0145] 1. Option 1:

[0146] The optimal detection method for fecal sample metabolites in Example 1 (eliminating CTAB, NH4Cl and PBS in mixed fecal samples after adding a preservative);

[0147] 2. Option 2:

[0148] Specifically, this includes the following steps (only the preservative is added; CTAB, NH4Cl, and PBS are not eliminated from the mixed fecal sample):

[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 sample was freeze-dried overnight;

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

[0153] 4) Grind for 2 minutes;

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

[0155] 6) Centrifuge at 4℃, 18000g for 25 minutes;

[0156] 7) Transfer 60 μL of the supernatant to 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 thoroughly;

[0159] 10) 4℃, 1450rpm, shake for 15 minutes;

[0160] 11) Centrifuge at 4℃, 18000g for 25 minutes;

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

[0162] ② Liquid Chromatography and Mass Spectrometry Conditions

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

[0164] ③ Qualitative and quantitative analysis

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

[0166] 3. Option 3 (Compare with Example 1):

[0167] No preservatives are added; deionized water is used instead of fecal preservatives.

[0168] The sample metabolite detection method described herein follows the same steps as in Example 1.

[0169] Specific results of qualitative detection of metabolites under different conditions are as follows: Figures 3-5 As shown, where, Figure 3 The image shows the qualitative detection results of fecal metabolites (deionized water) under Scheme 3 conditions. Figure 4 This is a graph showing the qualitative detection results of fecal metabolites under Scheme 2 conditions (with preservative added). Figure 5 The image shows the qualitative detection results of fecal metabolites under Scheme 1 conditions (preservative added but its active ingredients removed). Table 2 summarizes the results. Figures 3-5 The changes in metabolite detection results, taking TUDCA (RT = 6.43 min), TCA (RT = 7.45 min), and GUDCA (RT = 7.98 min) as examples, are described, illustrating the impact of different protocols on metabolite detection results. Specific results are shown below:

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

[0171]

[0172]

[0173] Note: The known metabolites and their retention times in fecal samples in this invention are as follows: GCA (8.45 min), TUDCA (6.43 min), GUDCA (7.98 min), TCA (7.45 min), TDCA (9.67 min), GDCA (10.21 min), TCDCA (9.45 min), GCDCA (10.02 min), TLCA (11.12 min), LCA (14.70 min), bCA (9.34 min), CA (10.900 min), UDCA (10.77 min), DCA (12.97 min), and CDCA (12.72 min).

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

[0175] Combination Figures 3-5 As shown in Table 2, after using Scheme 2 (adding a preservative but not eliminating it), the results of the detection of metabolites in the fecal samples were as follows: 1) Two small peaks should have appeared at 5.39 min and 5.58 min, but after adding the preservative, the two peaks overlapped, and no peak appeared at 5.58 min; while in Schemes 1 and 3, after using the optimal detection method in Example 1 and adding deionized water instead of the preservative, two small peaks still appeared, and the separation met the requirements, without overlap; 2) Bile acid TCA showed a peak at around 7.45 min, but the peak at 7.45 min disappeared after adding the DNA preservative; while in Schemes 1 and 3, after using the optimal detection method in Example 1 and adding deionized water instead of the preservative, TCA showed a normal peak at 7.45 min; 3) The retention time of bile acid GUDCA was approximately The retention time was 7.98 min, but after adding the preservative, the retention time shifted significantly to about 8.02 min, and the peak height decreased significantly, indicating that the response of the metabolite in the mass spectrometer was significantly reduced. In Scheme 1, after adding the preservative and eliminating its effective components, the retention time of bile acids recovered to 7.97 min, which shows 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, NH4Cl and PBS), the relative ionization efficiency of the metabolite in Scheme 2 was significantly reduced. This is because the three components CTAB, NH4Cl and PBS were not eliminated in Scheme 2, which significantly reduced the response of each metabolite, resulting in a significant decrease in the final relative ionization efficiency.

[0176] Specific results of quantitative detection of metabolites under different conditions are as follows: Figure 6 As shown, A represents Scheme 3 (deionized water group), B represents Scheme 2 (group with preservative added), and C represents Scheme 1 (group with preservative added but CTAB, NH4Cl, and PBS removed). From... Figure 6 As can be seen, the total bile acid content in control group A was approximately 13000 ng / mg. However, after adding the preservative using scheme 2 (B), the final detected total bile acid content decreased significantly. When using scheme 1 (C), the total bile acid content was close to 13000 ng / mg, showing no significant decrease and no significant difference from the control group. Therefore, using scheme 1 to eliminate the influence of the preservative on metabolite detection results can significantly improve the accuracy of metabolite detection results.

[0177] Therefore, compared with the control group (deionized water group), the qualitative and quantitative analysis of metabolites was affected after adding the preservative to the feces in Scheme 2, resulting in interference with metabolite identification and inaccurate quantitative results. In Scheme 1, compared with the control group, metabolite identification was accurate, and the quantitative results were not affected. Therefore, after optimization of the optimal metabolite detection method in Scheme 1, the addition of the preservative to fecal samples allows for accurate qualitative and quantitative analysis of metabolites, which is beneficial for multi-omics correlation analysis of fecal samples. Therefore, this embodiment verifies that Scheme 1 (the metabolite detection method in Example 1) is the optimal detection method for fecal sample metabolites in this invention.

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

[0179] To obtain the optimal detection method for fecal sample metabolites in Example 1 and improve the accuracy of sample metabolite detection, this example screened methods for removing CTAB, NH4Cl, and PBS from mixed fecal samples. This example ultimately found that NH4Cl and PBS could be removed by organic solvent extraction. Therefore, in this example, methods for removing NH4Cl and PBS from mixed fecal samples were first screened, and the impact of complete removal of NH4Cl and PBS on the detection results of fecal sample metabolites was explored. Specifically, this included the following methods (all other conditions were the optimal conditions in Example 1, and CTAB was completely removed in this example 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 a mixed organic solvent: methanol: acetonitrile = 1:1 (volume ratio);

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

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

[0186] Table 3. Removal effects of different organic solvents on NH4Cl and PBS and their influence on the detection results of metabolites in fecal samples.

[0187]

[0188] Note: 1) It is known that bile acids are detectable metabolites in fecal samples of this invention. The retention times of different bile acids are as follows: GCA (8.45 min), TUDCA (6.43 min), GUDCA (7.97 min), TCA (7.45 min), TDCA (9.67 min), GDCA (10.21 min), TCDCA (9.45 min), GCDCA (10.02 min), TLCA (11.12 min), LCA (14.70 min), bC A (9.34 min), CA (10.900 min), UDCA (10.77 min), DCA (12.97 min), and CDCA (12.72 min); 2) Formula for calculating relative ionization efficiency: Relative ionization efficiency = (Ion response of experimental group / Ion response of standard solution) * 100%; In the relative ionization efficiency results, "--" indicates that: due to excessive retention time shift, the peak shape could not be collected normally, resulting in inaccurate qualitative analysis of substances. Therefore, the relative ionization efficiency cannot be compared and is not specifically calculated.

[0189] As shown in Table 3, 1) when only deionized water or a single organic solvent such as acetonitrile or methanol is used, it is impossible to completely remove NH4Cl and PBS from the fecal mixed sample, thus affecting the final detection results of fecal metabolites, resulting in some metabolites being undetectable, retention times shifting significantly, and responses of some metabolites decreasing significantly; 2) when a mixed solution of methanol and acetonitrile is used, the removal effect of NH4Cl and PBS in the fecal mixed sample is significantly improved, and when methanol:acetonitrile = 80:20, the removal effect of NH4Cl and PBS in the fecal mixed sample can reach 100%, thus eliminating the influence of NH4Cl and PBS on the detection results of fecal metabolites, and all metabolites can be detected normally with good response.

[0190] Therefore, in this embodiment, the preferred organic solvent is methanol:acetonitrile = 80:20 (volume ratio) to eliminate NH4Cl and PBS from the fecal mixed sample.

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

[0192] Based on Example 4, in order to obtain the optimal detection method for fecal sample metabolites in Example 1 and improve the accuracy of sample metabolite detection, this example further screened the method for eliminating CTAB in mixed fecal samples and explored the impact of completely removing CTAB on the detection results of fecal sample metabolites. The inventors discovered that a materials science master's thesis, "Shen Teng. Properties of CTAB / SDS mixed aqueous solution and its template effect on the synthesis of mesoporous materials [D]. Shandong: Shandong University of Science and Technology, 2016," mentions the application of CTAB and SDS. The purpose of this thesis is to study the formation of ordered assemblages by positive and negative ionic surfactants. It mentions that different mixing ratios of CTAB and SDS affect the micelle size of the system. When the CTAB:(SDS+CTAB) pairing ratio is between 0.4 and 0.6, precipitation will form, specifically as follows... Figure 7 As shown, Figure 7 This study investigates the effect of the CTAB / SDS ratio on the micelle size of the mixed system. Therefore, this invention draws upon the principle of cation-anion coprecipitation mentioned in the literature, removing CTAB through the coprecipitation of SDS and CTAB. However, the research in the literature focuses on liquid crystal material preparation, aiming to form a crystal structure; therefore, its reaction system (temperature, time, ratio) is completely unsuitable for the method of this invention. Therefore, in this embodiment, on the one hand, substances that can coprecipitate with CTAB were screened; on the other hand, the reaction ratio of CTAB to SDS, reaction temperature, and reaction time were improved and further optimized. The effects of coprecipitating CTAB with different substances and the reaction conditions in this embodiment were compared with those in the literature, specifically including the following methods (all other conditions are the optimal conditions in Example 1, and NH4Cl and PBS in this embodiment have been completely removed using the optimal method in Example 1):

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

[0194] 2. Co-precipitation of SDS and CTAB was carried out using the reaction conditions described in the literature: (1) SDS:CTAB = 0.43-1.5:1; (2) Reaction temperature was 35℃ in a water bath; (3) Reaction time was 24h;

[0195] 3. Co-precipitation of SDS and CTAB was carried out using the optimal reaction conditions optimized in Example 1 of the present invention: (1) SDS:CTAB = 1:1; (2) Incubation temperature was 10°C in an ice bath; (3) Incubation time was 15 min;

[0196] 4. Use sodium tetrapolybenzenesulfonate to co-precipitate with CTAB;

[0197] 5. Use sodium oleoyloxyethanesulfonate to co-precipitate with CTAB;

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

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

[0200]

[0201] Note: 1) It is known that bile acids are detectable metabolites in fecal samples of this invention. The retention times of different bile acids are as follows: GCA (8.45 min), TUDCA (6.43 min), GUDCA (7.97 min), TCA (7.45 min), TDCA (9.67 min), GDCA (10.21 min), TCDCA (9.45 min), GCDCA (10.02 min), TLCA (11.12 min), LCA (14.70 min). The ionization efficiency was calculated as follows: 1) in), bCA (9.34 min), CA (10.900 min), UDCA (10.77 min), DCA (12.97 min), and CDCA (12.72 min); 2) The bound bile acids included TCA, GCA, TCDCA, GCDCA, TUDCA, TLCA, TDCA, and GDCA; 2) The formula for calculating the relative ionization efficiency was: Relative ionization efficiency = (Ion response of experimental group / Ion response of standard solution) * 100%.

[0202] As shown in Table 4, 1) when using the reaction conditions in the literature, ① although precipitation occurs at an SDS:CTAB ratio of 0.43–1.5:1, the purpose of this 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, thus addressing different problems; ② the purpose of the reaction temperature in the literature, at 35°C in a water bath, is to prepare the liquid crystal reaction system, increasing the temperature to increase reaction efficiency; ③ the reaction time in the literature is 24 hours, extending the reaction time to improve the yield. Therefore, the purpose reported in the literature is completely different from the expected co-precipitation of SDS and CTAB in this invention. Furthermore, under the reaction conditions reported in the literature, the removal efficiency of CTAB during SDS and CTAB co-precipitation is only 52%, which will inevitably affect the final detection results of metabolites in the fecal sample. 1) This makes the metabolites virtually undetectable, and the relative ionization efficiency is extremely low; 2) When using deionized water or sodium tetrapolybenzenesulfonate or sodium oleoyloxyethanesulfonate to remove CTAB, it is also impossible to completely remove CTAB from the fecal mixed sample, thus affecting the final detection results of fecal sample metabolites, making some metabolites undetectable, causing a significant shift in retention time, and significantly reducing the response of some metabolites, while the relative ionization efficiency is significantly reduced; 3) However, when using the optimized reaction conditions in Example 1 of this invention for the co-precipitation of SDS and CTAB, the removal effect of CTAB in the fecal mixed sample can reach 100%, thereby eliminating the influence of CTAB on the detection results of fecal sample metabolites, allowing all metabolites to elute normally and respond well, while the relative ionization efficiency can reach 90% to 98%.

[0203] Meanwhile, 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 embodiment. The specific scheme and results are shown in Tables 5 and 6: (1) SDS:CTAB = 0.8~1.2:1; (2) The reaction temperature is 10℃ ice bath; (3) The reaction time is 5~20min.

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

[0205]

[0206] Note: 1) It is known that bile acids are detectable metabolites in fecal samples of this invention. The retention times of different bile acids are as follows: GCA (8.45 min), TUDCA (6.43 min), GUDCA (7.97 min), TCA (7.45 min), TDCA (9.67 min), GDCA (10.21 min), TCDCA (9.45 min), GCDCA (10.02 min), TLCA (11.12 min), LCA (14.70 min), bCA (9.34 min), CA (10.900 min), UDCA (10.77 min), DCA (12 min). 1) 0.97 min) and CDCA (12.72 min); 2) The bound bile acids include TCA, GCA, TCDCA, GCDCA, TUDCA, GUDCA, TLCA, TDCA, and GDCA; 3) Here, only the ratio of SDS to CTAB is changed, and other reaction conditions are the optimal reaction conditions; 4) The formula for calculating the relative ionization efficiency is: Relative ionization efficiency = (Ion response of experimental group / Ion response of standard solution) * 100%; In the relative ionization efficiency result, "--" indicates that: due to excessive retention time shift, 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 is not specifically calculated.

[0207] Table 5 shows that: 1) When SDS:CTAB = 0.8:1, the removal efficiency of CTAB is only 71%, which cannot completely remove CTAB from the mixed fecal sample. This affects the final detection results of fecal metabolites, resulting in some metabolites being undetectable, the retention time of conjugated bile acids shifting significantly, and the response of all bile acids decreasing significantly. The retention time shift of conjugated bile acids reaches 3.15 ± 1 min. This may be because the amount of SDS added is insufficient to completely remove CTAB. Therefore, the remaining CTAB still affects the detection results of bile acids, causing their retention time to shift. 2) When SDS:CTAB = 1:1, CTAB in the mixed fecal sample can be completely removed, thus eliminating the influence of CTAB on the detection results of fecal metabolites. All metabolites can show normal peaks and good responses, and the retention time does not shift significantly. This may be because when the ratio is 1:1, CTAB in the fecal sample is almost completely eliminated and no longer affects the detection of bile acids. The retention time of SDS and CTAB is adjusted back to the normal level. 3) When the ratio of SDS to CTAB is 1.2:1, although CTAB in the fecal mixed sample can be completely removed, the retention time of bound bile acids is significantly shifted forward and the response of some bile acids is significantly reduced. The retention time of bound bile acids is shifted to 1.8 ± 0.2 min. This may be because there is an excess of SDS at this time. Although CTAB is completely removed, the remaining SDS may also affect the detection results of bile acids, ultimately causing the retention time to shift forward and interfering with the detection results of metabolites.

[0208] Therefore, in this embodiment, the optimal reaction ratio of SDS to CTAB is 1:1.

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

[0210]

[0211] Note: 1) It is known that bile acids are detectable metabolites in fecal samples of this invention. The retention times of different bile acids are as follows: GCA (8.45 min), TUDCA (6.43 min), GUDCA (7.97 min), TCA (7.45 min), TDCA (9.67 min), GDCA (10.21 min), TCDCA (9.45 min), GCDCA (10.02 min), TLCA (11.12 min), LCA (14.70 min), bCA (9.34 min), CA (10.900 min), UDCA (10.77 min), DCA (12.97 min), and CDCA (12.72 min); 2) Only the reaction time was changed here, and the other reaction conditions were the optimal reaction conditions; 3) The formula for calculating the relative ionization efficiency is: Relative ionization efficiency = (Ion response of experimental group / Ion response of standard solution) * 100%; In the relative ionization efficiency results, "--" indicates that: due to excessive retention time shift, the peak shape could not be collected normally, resulting in inaccurate qualitative analysis of the substance. Therefore, the relative ionization efficiency cannot be compared and is not specifically calculated.

[0212] As shown in Table 6, when the reaction time is too short (5 min), CTAB cannot be completely removed, thus affecting the final detection results of metabolites in the fecal sample. This results in some bile acids being undetectable, a significant shift in retention time, and a significant decrease in the response of all bile acids. When the reaction time is 15 min or 20 min, CTAB can be completely removed, and the retention times of different metabolites are similar to the normal retention times, with almost no shift. Therefore, considering the experimental results and time cost, the optimal reaction time in this embodiment is preferably 15 min.

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

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

[0215] In Example 3, the optimal fecal DNA preservative system was screened. To further investigate the effect of the DNA preservative system on the co-precipitation efficiency of SDS and CTAB, this example compares the optimal fecal preservative system from Example 1 with other DNA preservative systems, specifically including the following methods (all other conditions are the optimal conditions in Example 1):

[0216] 1. DNA preservation system: including CTAB, NH4Cl and PBS;

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

[0218] 3. DNA preservation system: including CTAB, NH4Cl and HBSS;

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

[0220] Table 7. Effects of different DNA preservation agent systems on CTAB precipitation efficiency and fecal sample metabolite detection results

[0221]

[0222] Note: 1) It is known that bile acids are detectable metabolites in fecal samples of this invention. The retention times of different bile acids are as follows: GCA (8.45 min), TUDCA (6.43 min), GUDCA (7.97 min), TCA (7.45 min), TDCA (9.67 min), GDCA (10.21 min), TCDCA (9.45 min), GCDCA (10.02 min), TLCA (11.12 min), LCA (14.70 min), bC A (9.34 min), CA (10.900 min), UDCA (10.77 min), DCA (12.97 min), and CDCA (12.72 min); 2) Formula for calculating relative ionization efficiency: Relative ionization efficiency = (Ion response of experimental group / Ion response of standard solution) * 100%; In the relative ionization efficiency results, "--" indicates that: due to excessive retention time shift, the peak shape could not be collected normally, resulting in inaccurate qualitative analysis of substances. Therefore, the relative ionization efficiency cannot be compared and is not specifically calculated.

[0223] As shown in Table 7, CTAB can only be completely precipitated when the optimal DNA preservation system in Example 1 is used and 60 μL of SDS is added, with a precipitation efficiency of 100%. At this time, all metabolites can be eluted normally and respond well, the retention time does not change significantly, and the ionization efficiency returns to the normal level of 100%. However, when any component in the system is replaced, the precipitation efficiency of CTAB will decrease significantly, some metabolites will not be detected, the retention time will change significantly, and the sample DNA will not remain stable. This may be because a certain proportion of NH4Cl can keep the system in a high-salt solution state, at which point physiological and biochemical reactions in the sample are inhibited, and DNA can remain stable. At the same time, NH4Cl also helps with the acid-base balance of the solution system. Meanwhile, PBS is crucial for the pH regulation of the system. After adding DNA preservation agent to the sample, it can maintain a relatively suitable pH value and ensure the stability of metabolites and DNA. When either NH4Cl or PBS is replaced, the precipitation efficiency of CTAB will be significantly reduced, and the detection results of bile acids in the sample will also be affected, making them undetectable, causing a shift in retention time, or significantly reducing the response.

[0224] Therefore, this embodiment demonstrates that the fecal DNA preservative system in Example 1 can significantly improve the co-precipitation efficiency of SDS and CTAB, further proving that the fecal DNA preservative system in Example 1 is the optimal system, including CTAB, NH4Cl and PBS, with the best CTAB precipitation efficiency, while significantly improving the accuracy of fecal sample metabolite detection results.

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

[0226] To obtain the optimal method for detecting fecal metabolites as described in Example 1, this example further compared and screened the pretreatment methods for fecal samples containing a compound preservative. Specifically, the methods included the following (all other conditions were the optimal conditions described in Example 1). Here, the fecal samples were mixed samples from different sources to ensure sufficient experimental quantity, and the use of the same mixed fecal sample could eliminate individual errors, ensuring the parallel comparability of the following results:

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

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

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

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

[0231] ④ After grinding, the mixed sample is centrifuged at low temperature and high speed, and the supernatant is transferred to the sample plate for detection;

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

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

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

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

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

[0237] ⑤ After grinding, the mixed sample is centrifuged at low temperature and high speed, and 90 μL of supernatant is transferred to the sample plate for detection;

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

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

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

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

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

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

[0244] ⑥ After centrifugation, transfer 1–50 μL of the supernatant to a 1.5 mL centrifuge tube;

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

[0246] ⑧ Remove the solid-phase extraction column from the low-temperature freezer and allow it to return to room temperature;

[0247] ⑨ Activate the solid-phase extraction column with 10–20 column volumes of methanol;

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

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

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

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

[0252] Transfer 1–5 mL of eluent into an EP tube and freeze-dry.

[0253] Redissolve 90–120 μL of the initial mobile phase and analyze the solution.

[0254] 4. Option 4: SDS to eliminate CTAB (Comparative Example 4)

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

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

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

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

[0259] ⑤ After grinding, the mixed sample is centrifuged at low temperature and high speed, and 40-80 μL (60 μL) of supernatant is transferred to a 1.5 mL EP tube;

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

[0261] ⑦ Centrifuge the mixed samples and collect the supernatant into the sample plate for testing;

[0262] 5. Option Five: SDS Elimination of CTAB (Optimal Detection Method in Example 1)

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

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

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

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

[0267] ⑤ After grinding, the mixed sample is centrifuged at low temperature and high speed, and 120 μL of supernatant is transferred to a 1.5 mL EP tube;

[0268] ⑥ Take 40-80 μL (60 μL) of supernatant to determine the OD value. Based on the determination results, add SDS at a molar ratio of 1:1, mix and shake well.

[0269] ⑦ Centrifuge the mixed samples and collect the supernatant into the sample plate for testing;

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

[0271] Table 8. Effects of different pretreatment methods on the detection results of metabolites in fecal samples containing compound preservatives.

[0272]

[0273] Note: 1) The differences between the different schemes are as follows: Comparative Example 1 is conventional treatment + organic solvent extraction; Comparative Example 2 is freeze-drying + 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 followed by addition of SDS at a 1:1 ratio to eliminate CTAB; 2) It is known that the metabolites detectable in the fecal samples of this invention are bile acids, and the retention times of different bile acids are: GCA (8.45 min), 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 Comparative Examples 1, 2, and 3, GCA, TCA, and TDCA bile acids in the fecal samples could not be detected, indicating that simply removing NH4Cl and PBS from the composite preservative still significantly affected the detection results of metabolites in the fecal samples; 2) when using the scheme in Comparative Example 4, adding an equal volume of SDS to the supernatant (equimolar ratio), compared with the retention time of the standard for qualitative analysis, the retention times of GCA, TCA, GUDCA, TUDCA, TDCA, GDCA, TCDCA, GCDCA, and TLCA bile acids in the fecal samples were significantly advanced, resulting in inaccurate metabolite detection results; 3) when using the optimal scheme in Example 1, the retention time of metabolites in the fecal samples was normal, and the quantitative results were accurate.

[0275] Therefore, in this embodiment, the optimal pretreatment method in Example 1 is preferred for detecting metabolites in fecal samples.

[0276] Under various conditions, the peak shapes of metabolites in mixed fecal samples are as follows: Figures 8-12 As shown, where, Figure 8 The peak shape detection results of each metabolite in the mixed fecal sample under the conditions of Comparative Example 1 (conventional processing + organic solvent extraction); Figure 9 The peak shape detection results of each metabolite in the mixed fecal sample under the conditions of Comparative Example 2 (lyophilization + organic solvent extraction); Figure 10 The peak shape detection results of each metabolite in the mixed fecal sample under the conditions of Comparative Example 3 (lyophilization + organic solvent extraction + purification column pretreatment); Figure 11 The peak shape detection results of each metabolite in the mixed fecal sample under the conditions of Comparative Example 4 (lyophilization + organic solvent extraction + SDS elimination of CTAB); Figure 12 The peak shape detection results of each metabolite in the fecal mixed sample under the conditions of Example 1 (freeze-drying + organic solvent extraction + determination of CTAB content, followed by the addition of SDS at a ratio of 1:1 to eliminate CTAB according to the determination results). Figures 8-12 The figure shows nine bile acids whose retention time, peak shape, or response were significantly affected; the remaining bile acids were not significantly affected and therefore are not shown in the figure. Table 9 summarizes... Figures 8-12 The changes in peak shape, response, and ionization efficiency of various metabolites in mixed fecal samples are shown in the following results:

[0277] Table 9. Changes in peak shape and response of various metabolites in mixed fecal samples under different treatments

[0278]

[0279] Note: 1) It is known that bile acids are detectable metabolites in fecal samples of this invention. The retention times of different bile acids are as follows: GCA (8.45 min), TUDCA (6.43 min), GUDCA (7.97 min), TCA (7.45 min), TDCA (9.67 min), GDCA (10.21 min), TCDCA (9.45 min), GCDCA (10.02 min), TLCA (11.12 min), LCA (14.70 min), bC A (9.34 min), CA (10.900 min), UDCA (10.77 min), DCA (12.97 min), and CDCA (12.72 min); 2) Formula for calculating relative ionization efficiency: Relative ionization efficiency = (Ion response of experimental group / Ion response of standard solution) * 100%; In the relative ionization efficiency results, "--" indicates that: due to excessive retention time shift, the peak shape could not be collected normally, resulting in inaccurate qualitative analysis of substances. Therefore, the relative ionization efficiency cannot be compared and is not specifically calculated.

[0280] Combination Figures 8-12 As shown in Table 9, 1) when using Comparative Example 1, Comparative Example 2, and Comparative Example 3, bile acids in the fecal samples could not be fully detected, and the peak shapes of the four detectable metabolites were poor, the separation between different metabolites did not meet the standard, and the responses of each metabolite were significantly lower; 2) when using the scheme in Comparative Example 4, adding an equal volume of SDS to the supernatant, bile acids in the fecal samples could be fully detected and the response was improved, but the responses of each metabolite were still significantly lower, such as the response of TCDCA reaching only 1.2E+6, and the ionization efficiency was significantly reduced; 3) when using the optimal scheme in Example 1, bile acids in the fecal samples could be fully detected, the responses of each metabolite were significantly improved and the separation met the standard, and the retention times were accurate with minimal shift.

[0281] Therefore, it is further confirmed that the optimal pretreatment method in Example 1 of this embodiment is preferred for the detection of metabolites in fecal samples.

[0282] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for detecting metabolites in fecal samples containing a compound preservative for non-disease diagnostic purposes, characterized in that, It consists of the following steps: (1) Take 5-50 μL of fecal sample containing the compound preservative and freeze-dry it at low temperature; (2) Add the mixed organic solvent and magnetic beads to the freeze-dried sample in sequence and homogenize; the mixed organic solvent is a methanol / acetonitrile mixture; (3) Centrifuge the homogenized sample at low temperature and high speed, take 40-80 μL of supernatant, and determine the accurate content of CTAB in the sample; (4) Prepare a sodium dodecyl sulfate (SDS) solution in the appropriate proportion according to the CTAB content; (5) Coprecipitation elimination: Take an appropriate amount of supernatant obtained in step (3), add 40-80 μL of SDS solution prepared in step (4), shake and incubate for a certain period of time, then centrifuge again at low temperature and high speed, and take the final supernatant for metabolite detection; In step (5), SDS is added at a molar ratio of 1:1 of SDS:CTAB, the reaction temperature is 10℃, and the reaction time is 20 min. The composite preservative consists of 2.0% CTAB, 20% NH4Cl and 10% PBS.

2. The detection method as described in claim 1, characterized in that, The metabolites in the fecal samples include GCA, TCA, GUDCA, TUDCA, TDCA, GDCA, TCDCA, GCDCA, TLCA, LCA, bCA, CA, UDCA, DCA, and CDCA.

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