Pretreatment reagent and method for extracting nucleic acid of excrement sample

Abstract

The invention provides a pretreatment reagent and a method for nucleic acid extraction of an excrement sample. The pretreatment reagent is a solution comprising TritonX-100, PEG6000, SDS, guanidine isothiocyanate, KAl (SO4) 2 and ammonium acetate. According to the reagent provided by the invention, the scheme of the excrement pretreatment reagent capable of realizing one-step pyrolysis and inhibitor sedimentation can be realized, the operation process and time of manual uncovering and sample transfer are greatly shortened, pyrolysis, flocculation and flocculation termination are all completed in the same tube, only one centrifugation step is needed, and the clinical operability is greatly improved. Meanwhile, the scheme has more excellent performance than a commercial excrement pretreatment reagent.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a pretreatment reagent and method for nucleic acid extraction from fecal samples. Background Art

[0002] As one of the commonly used clinical samples, feces contain key nucleic acid markers such as gastrointestinal pathogens, intestinal tumor markers, and intestinal flora, which are crucial for the diagnosis of infectious diseases, tumor screening, and flora analysis. Nucleic acid detection based on feces is also widely used in rapid pathogen detection, drug resistance gene screening, flora research, and early tumor screening due to its high sensitivity and specificity. However, there are still the following difficulties in extracting and detecting nucleic acids from fecal samples: 1. The feces have a high viscosity, making it difficult to sample; 2. The fecal samples contain a large amount of soluble PCR inhibitors, and traditional nucleic acid extraction methods are difficult to remove this part of the components; 3. The feces have a strong odor, and operators generally resist direct contact with the samples.

[0003] In the prior art, when extracting nucleic acids from fecal samples, the sample lysis solution and the sample flocculant are used separately one after another, which requires multiple sample tube replacements, multiple lid-opening operations, and multiple centrifugation operations, making it difficult to achieve automated operation. The presence of the sample flocculant will cause the nucleic acid to fail during the binding process with magnetic beads, so it is necessary to terminate the flocculant. Conventional termination solutions use phosphates or carbonates to precipitate aluminum salts, but this requires multiple centrifugation steps, with cumbersome operations and long time consumption. Finally, a binding solution is added to the pretreated supernatant for solid-phase extraction of nucleic acids. The entire process has cumbersome operations and involves multiple steps of lid-opening and manual transfer of fecal fluid. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a pretreatment reagent for nucleic acid extraction from fecal samples, and the pretreatment reagent is a solution comprising TritonX-100, PEG6000, SDS, guanidine isothiocyanate, KAl(SO4)2, and ammonium acetate.

[0005] In one embodiment, the concentration of the ammonium acetate is 1.5M - 6M.

[0006] In one embodiment, the concentration of the KAl(SO4)2 is 0.15M - 0.2M.

[0007] In one embodiment, the pretreatment reagent further comprises an EDTA termination solution.

[0008] In one embodiment, the EDTA termination solution is an aqueous solution containing ethanol, and the concentration of EDTA is 0.09M - 0.2M.

[0009] In one embodiment, the present invention provides a pretreatment method for nucleic acid extraction from fecal samples, and the method comprises the following steps: Step 1: Add a pretreatment reagent for nucleic acid extraction from fecal samples into a sample tube containing fecal samples, and the pretreatment reagent is a solution comprising Triton X-100, PEG6000, SDS, guanidine isothiocyanate, KAl(SO4)2 and ammonium acetate; vortex and mix well to make the fecal samples and the above pretreatment reagent in a homogenized state; Step 2: After homogenization, heat-treat and centrifuge the sample tube; Step 3: After centrifugation, take the supernatant, and add an EDTA termination solution to the supernatant; and Step 4: After adding the EDTA termination solution, directly use it for nucleic acid extraction from fecal samples without centrifugation.

[0010] In one embodiment, nucleic acid extraction from fecal samples is carried out by the magnetic bead method.

[0011] In the prior art, lysis and inhibitor precipitation require two steps because both Al 3+ and Fe 3+ need to be stored in an acidic environment, and among them, Al 3+ is more commonly used. When the pH value is higher than 4.5, Al 3+ will react with OH - in the solution to form Al(OH)3 precipitate. Generally, the lysis buffer and the magnetic bead binding buffer are both alkaline, which is more conducive to protecting the stability of the nucleic acid template, and is conducive to cell membrane lysis and denaturation of various proteins. Secondly, the alkaline environment is conducive to the binding of the nucleic acid template to the surface of the magnetic beads. Therefore, in the general understanding of those skilled in the art, lysis and inhibitor precipitation need to be completed in two steps.

[0012] To overcome the above-mentioned prior art prejudice, an ammonium acetate solution is selected as the buffer solution in the present invention. When the concentration of ammonium acetate reaches a certain value, the high-concentration acetate ions in the solution environment form a weakly ionized state with the aluminum salt, thus competing with the aluminum salt and inhibiting the reaction of forming hydroxide precipitate. Thereby, the stable coexistence of the aluminum salt and the lysis buffer in the same solution can be achieved, and at the same time, the precipitation of the flocculant aluminum salt is avoided. In this way, the cell lysis buffer and the flocculant for inhibitor precipitation can coexist in the same reaction system, and thus lysis and inhibitor flocculation can be carried out in one tube reaction.

[0013] In the present invention, based on the special function of the ammonium acetate buffer system, the dissolution stability of the aluminum salt in a slightly neutral environment can be stabilized, and at the same time, a suitable pH value environment can be provided for the binding of the aluminum salt and the inhibitor.

[0014] Meanwhile, the presence of the flocculant will cause failure in the process of nucleic acid binding to magnetic beads. Therefore, it is necessary to terminate the flocculant. Conventional termination solutions use phosphates or carbonates to precipitate aluminum salts, but this requires multiple centrifugation steps, which are cumbersome and time-consuming. The present invention uses an EDTA termination solution containing ethanol. Based on the formulation of the EDTA-based flocculation termination binding solution, it simultaneously achieves aluminum salt termination and provides a suitable environment for nucleic acid binding to magnetic beads. It reduces manual centrifugation operations and is conducive to the realization of full-process automation. Based on the formulation of the pretreatment reagent for fecal sample nucleic acid extraction of the present invention, pretreatment is achieved in one step, reducing manual operation time and facilitating the automation of fecal treatment.

[0015] In summary, the solution of the fecal pretreatment reagent provided by the present invention that can achieve one-step lysis and inhibitor sedimentation greatly reduces the operation process and time of manual tube opening and sample transfer. Lysis, flocculation, and flocculation termination are all completed in the same tube, and only one centrifugation step is required, greatly increasing clinical operability. At the same time, this solution has better performance than commercial fecal pretreatment reagents.

[0016] In the prior art, it is still difficult to precipitate inhibitors in feces without affecting the nucleic acid recovery rate. Through the pretreatment reagent of the present invention, not only can inhibitors in fecal samples be precipitated without affecting the nucleic acid recovery rate, but also the subsequent PCR amplification operation after nucleic acid extraction can be efficiently carried out. Detailed implementation manners

[0017] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0018] Example 1 Selection of the buffer system of the pretreatment reagent for fecal sample nucleic acid extraction of the present invention

[0019] In order to explore the integration of the lysis solution and the flocculant components and make them coexist in a solution environment, the present invention uses different buffer systems.

[0020] I. Reagent formulation

[0021] The pretreatment reagents for nucleic acid extraction from fecal samples include 2.5% Triton X-100, 5% PEG6000, 0.05 M SDS, 2 M guanidine isothiocyanate (GuSCN), and 0.18 M KAl(SO4)2. Among them, different types and concentrations of buffer systems are designed according to different experiments. The existing termination binding solution includes 0.1 M K2HPO4 and 50% aqueous ethanol solution.

[0022] II. Test steps

[0023] 1. Use a blade to take about 200 mg of fecal sample into a 2 mL centrifuge tube;

[0024] 2. Add 800 μL of lysis precipitation agent to the above centrifuge tube, vortex thoroughly to mix evenly, so that the feces and liquid reagent are in a homogenized state;

[0025] 3. Place the above centrifuge tube on a metal bath and heat at 70 °C for 5 min;

[0026] 4. Centrifuge the above centrifuge tube at 12000 rpm for 1 min;

[0027] 5. Take 600 μL of the supernatant and add it to a new centrifuge tube, and add 1200 μL of the termination binding solution, vortex to mix evenly;

[0028] 6. Centrifuge at 12000 rpm for 1 min

[0029] 7. Take all the supernatant and bind it to magnetic beads, and perform subsequent magnetic bead-based nucleic acid extraction and nucleic acid PCR amplification.

[0030] III. Test scheme design

[0031] Scheme 1: Set the concentration of KAl(SO4)2 to 0.18 M, prepare the pretreatment reagents for nucleic acid extraction from fecal samples with a pH of 6.0 under different buffer systems, and add 10 μl of plasmid with a concentration of 10 3 copies / μL to the fecal samples of healthy people as simulated samples, and perform plasmid-specific qPCR detection on the elution products after extraction to verify the plasmid recovery rate.

[0032] Scheme 2: Set the gradient NH4OAc concentrations to 6 M, 3 M, 1.5 M, 0.75 M, and 0.375 M respectively, keep the concentrations and pH of the remaining components unchanged, and repeat the above plasmid recovery experiment.

[0033] IV. Experimental results

[0034] Table 1. Amplification results (Ct values) of extraction products with different types of buffer systems

[0035]

[0036] First, due to different solubilities, the concentrations of MOPS and MES buffers were set at 0.5 M and 0.2 M respectively, while the concentrations of NH4OAc and Tris-HCl were set at 2 M. From the above results, it can be seen that compared with the other three buffers, ammonium acetate has the best effect, the lowest Ct value compared with other buffer systems, and the lowest data dispersion of Ct values. This indicates that when the pretreatment reagent of the present invention using ammonium acetate buffer is used, the DNA concentration extracted from the sample is high and the amplification efficiency is high; the low data dispersion of Ct values indicates that the results of multiple repeated experiments (technical replicates or biological replicates) are highly consistent, indicating that the results are stable and have good consistency when the pretreatment reagent of the present invention using ammonium acetate buffer is used.

[0037] At the same time, during the storage of the solution, white precipitates appeared in both Tris-HCl and MOPS, while although no precipitate appeared in MES, positive results could not be successfully obtained in the subsequent nucleic acid extraction. Therefore, ammonium acetate was selected as the buffer used in the system.

[0038] Table 2. Amplification results (Ct values) of extraction products at different NH4OAc concentrations

[0039]

[0040] From the above results, it can be seen that complete nucleic acid extraction cannot be achieved without the addition of NH4OAc or with too low a concentration of NH4OAc added, and the amplification of the obtained products is all negative. When the concentration of NH4OAc is higher than 0.375 M, positive results begin to appear. When the concentration of NH4OAc reaches 1.5 M and above, the Ct value reaches the lowest and approaches stability at this time, proving that the extraction effect is optimal at this time. The acetate ion concentration and aluminum ion concentration are approximately 10:1 at this time. Continuing to increase the concentration of ammonium acetate fails to further increase the Ct value, but in an environment with too high ammonium acetate, the reagents of the pretreatment reagent for nucleic acid extraction of some fecal samples begin to reach the solubility saturation state. The inventor believes that high-concentration acetate ions form a weakly ionized state with aluminum salts in the solution environment, thus competing with aluminum salts and inhibiting the reaction of forming hydroxide precipitates.

[0041] Based on the above results, in this example, the concentration of NH4OAc is preferably between 1.5 M and 6 M, among which the nucleic acid recovery rate is the best at a concentration of 3 M and the dissolution of each reagent is good, which is suitable for application in the present invention.

[0042] Example 2 Experiment on the concentration of flocculant KAl(SO4)2

[0043] To optimize the concentration of the flocculant during nucleic acid extraction, this example studied the effects of different concentrations and the absence of the aluminum salt flocculant KAl(SO4)2 on nucleic acid recovery.

[0044] I. Reagent Formulation

[0045] The pretreatment reagents for nucleic acid extraction from fecal samples include 2.5% Triton X-100, 5% PEG6000, 0.05 M SDS, 2 M GuSCN, and 3 M NH4OAc. Among them, a gradient concentration of potassium alum [KAl(SO4)2] is set, and 0.1 M, 0.15 M, 0.18 M, 0.2 M, 0.25 M, and higher concentrations of [KAl(SO4)2] are used as flocculation components respectively. The termination binding solution includes 0.1 M K2HPO4 and 50% aqueous ethanol solution.

[0046] II. Experimental procedures are the same as in Example 1

[0047] III. Experimental Design

[0048] Add 10 μl of plasmid with a concentration of 10 3 copies / μL to a healthy human fecal sample as a simulated sample, and perform plasmid-specific qPCR detection on the elution product after extraction to verify the plasmid recovery rate. And determine the nucleic acid recovery rate.

[0049] IV. Experimental Results

[0050] Table 3. Nucleic acid recovery rate (%) at different flocculant concentrations

[0051]

[0052] As can be seen from the above results, in the absence of a flocculant, the pure magnetic bead-based nucleic acid extraction reagent alone cannot achieve complete fecal nucleic acid detection, because a large number of inhibitors result in false-negative test results.

[0053] As the flocculant concentration gradually increases to 0.18 M, the amplification positivity gradually increases.

[0054] When the flocculant concentration is lower than 0.15 M, the nucleic acid recovery rate is relatively low (for example, the recovery rate range at 0.1 M concentration is 6.8% - 14.8%). This is because the insufficient flocculant concentration leads to incomplete precipitation of nucleic acid inhibitors (such as proteins and polysaccharides) in the sample, thus affecting the purity and efficiency of nucleic acid extraction.

[0055] When the flocculant concentration increases to the range of 0.15 M - 0.2 M, the nucleic acid recovery rate increases significantly (for example, the recovery rate range at 0.18 M concentration is 49.9% - 61.3%). This is because this concentration range can effectively precipitate impurities in the sample while causing less interference to nucleic acid molecules, achieving the best balance.

[0056] When the concentration of the flocculant exceeds 0.2 M (for example, 0.25 M and above), the nucleic acid recovery rate begins to decline sharply (for example, the recovery rate at a concentration of 0.25 M ranges from 3.1% to 6.2%). Analysis shows that too high a concentration of the flocculant may remain in the nucleic acid solution and bind non-specifically to nucleic acid molecules, resulting in the loss of nucleic acids.

[0057] Based on the above results, in this example, the preferred concentration of the flocculant [KAl(SO4)2] is between 0.15 M and 0.2 M, and the nucleic acid recovery rate is the best at a concentration of 0.18 M, which is suitable for application in subsequent nucleic acid extraction processes.

[0058] Example 3. Experiment on the termination solution

[0059] To verify the necessity and optimal concentration of EDTA in terminating residual aluminum salts, this example studied the effects of different concentrations and the absence of EDTA on the nucleic acid recovery rate (Ct value).

[0060] I. Reagent formulation

[0061] The pretreatment reagents for nucleic acid extraction from fecal samples include 2.5% TritonX-100, 5% PEG6000, 0.05 M SDS, 2 M GuSCN, 0.18 M KAl(SO4)2, and 3 M NH4OAc. The termination binding solution includes 66.7% aqueous ethanol solution and one termination solution component, and the remaining termination solution components are set with different types and conditions according to the following experimental conditions.

[0062] II. Operating procedures for different termination solutions

[0063] Experimental method for EDTA termination solution:

[0064] 1. Use a blade to take about 200 mg of fecal sample into a 2 mL centrifuge tube;

[0065] 2. Add 800 μL of lysis and precipitation agent to the above centrifuge tube, vortex thoroughly to make the feces and liquid reagent in a homogenized state;

[0066] 3. Place the above centrifuge tube on a metal bath and heat at 70 °C for 5 min;

[0067] 4. Centrifuge the above centrifuge tube at 12000 rpm for 1 min;

[0068] 5. Take 800 μL of the supernatant and add it to a new centrifuge tube, and add 800 μL of the termination binding solution;

[0069] 6. Complete the pretreatment steps, and then it can be directly combined with magnetic beads and the subsequent magnetic bead method for nucleic acid extraction can be implemented.

[0070] Experimental method for K2HPO4 termination solution:

[0071] 1. Take about 200 mg of fecal sample with a blade and put it into a 2 mL centrifuge tube;

[0072] 2. Add 800 μL of lysis precipitation agent to the above centrifuge tube, vortex thoroughly to mix evenly, so that the feces and the liquid reagent are in a homogenized state;

[0073] 3. Place the above centrifuge tube on a metal bath and heat it at 70 °C for 5 min;

[0074] 4. Centrifuge the above centrifuge tube at 12000 rpm for 1 min;

[0075] 5. Take 600 μL of the supernatant and add it to a new centrifuge tube, and add 600 μL of the termination solution, then vortex to mix evenly;

[0076] 6. Centrifuge at 12000 rpm for 1 min;

[0077] 7. Take all the supernatant, add 600 μL of absolute ethanol, and vortex to mix evenly;

[0078] 8. Perform subsequent nucleic acid extraction by the magnetic bead method.

[0079] III. Different experimental designs

[0080] Experimental design 1: To verify the use of EDTA instead of phosphate for terminating the reaction with aluminum salt, 10 μl of plasmid with a concentration of 10 3 copies / μL was added to a healthy human fecal sample as a simulated sample, and plasmid-specific qPCR detection was performed on the elution product after extraction to verify the plasmid recovery rate. Set the composition of the termination solution to Na2CO3, K2HPO4, and EDTA-2Na with a concentration of 0.2 M each.

[0081] Experimental design 2: To test the dosage effect of EDTA at different concentrations, the experimental conditions were basically the same as those in Experiment 1. The only difference was that the concentrations of other reagent components were kept unchanged. EDTA at concentrations of 0 M, 0.017 M, 0.045 M, 0.09 M, 0.18 M, and 0.2 M was used as the aluminum salt terminator respectively, and the nucleic acid amplification was measured.

[0082] IV. Experimental results

[0083] Table 4. Effects of different termination solutions in the experiment at a concentration of 0.2 M (Ct values)

[0084]

[0085] As can be seen from the above results, first of all, EDTA can achieve a similar termination effect as phosphate, while carbonate cannot achieve a similar effect. Further, using EDTA for termination can eliminate the operation process of secondary centrifugation.

[0086] Table 5. Amplification results (Ct values) of extraction products at different EDTA concentrations

[0087]

[0088] As can be seen from the above results, in the absence of EDTA and when too low a concentration of EDTA is added, complete nucleic acid extraction cannot be achieved, and the amplification of the obtained products is all negative. When the EDTA concentration is higher than 0.09 M, positive results start to appear. At this time, the molar ratio of EDTA to the aluminum salt in the pretreatment reagent for fecal sample nucleic acid extraction is 1:2; when the EDTA concentration reaches 0.18 M and above, the molar ratio of EDTA to the substances in the pretreatment reagent for fecal sample nucleic acid extraction is 1:1, and its Ct value reaches the lowest, proving that the extraction effect is optimal at this time. When the EDTA reaches 0.2 M and above, due to the high ethanol content in the solvent, the EDTA reaches solubility saturation, white precipitates appear in the solution, and the Ct value does not increase further.

[0089] Based on the above results, in this example, the concentration of EDTA is preferably between 0.09 M and 0.2 M, and the nucleic acid recovery rate is the best at a concentration of 0.18 M, which is suitable for application in subsequent nucleic acid extraction processes.

[0090] Example 4 Comparative experiment between the pretreatment reagent for fecal sample nucleic acid extraction of the present invention and existing commercial reagents

[0091] To verify the performance difference between the pretreatment reagent for fecal sample nucleic acid extraction of the present invention and existing reagents, the following tests were carried out.

[0092] I. Reagent formulation

[0093] The pretreatment reagent for fecal sample nucleic acid extraction includes 2.5% TritonX-100, 5% PEG6000, 0.05 M SDS, 2 M GuSCN, 0.18 M KAl(SO4)2, and 3 M NH4OAc. Among them, the termination binding solution includes 0.18 M EDTA-2Na and 66.7% ethanol aqueous solution. Among them, EDTA-2Na is mainly used to chelate aluminum ions, and the ethanol solution is used to promote the binding of nucleic acid to magnetic beads.

[0094] II. Specific experimental steps

[0095] 1. Use a blade to take about 200 mg of fecal sample into a 2 mL centrifuge tube;

[0096] 2. Add 800 μL of lysis precipitant to the above centrifuge tube, vortex thoroughly to mix evenly, so that the feces and the liquid reagent are in a homogenized state;

[0097] 3. Place the above centrifuge tube on a metal bath and heat at 70 °C for 5 min;

[0098] 4. Centrifuge the above centrifuge tube at 12,000 rpm for 1 min;

[0099] 5. Take 800 μL of the supernatant and add it to a new centrifuge tube, and then add 800 μL of binding termination solution;

[0100] 6. Complete the pretreatment step, and then it can be directly combined with magnetic beads to perform subsequent magnetic bead-based nucleic acid extraction.

[0101] III. Experimental Scheme Design

[0102] Conduct a comparative experiment with the fecal inhibitor removal reagents in the commercially available BEAVER fecal nucleic acid extraction kit, QIAGEN FastDNA Stool Mini Kit, and CWBIOMagbead Pathogenic Microbiome DNA / RNA Kit (hereinafter referred to as "traditional kits").

[0103] Scheme 1: In the absorbance test experiment, two real fecal samples are used, and nucleic acid extraction is carried out using four different methods respectively. Each extraction product is measured for absorbance three times, and the average value is taken. The experimental determination includes nucleic acid yield (ng / μL), A260 / A280 ratio (characterization of protein impurity residue), and A260 / A230 ratio (characterization of small molecule organic compound residue).

[0104] Scheme 2: Further verify the effect of the pretreatment reagent for nucleic acid extraction from fecal samples based on external reference standards. Add external reference standards (including pure plasmid solution and Escherichia coli transformed with the corresponding plasmid) to fecal samples to simulate real samples for recovery rate testing. In the experiment, the same tube of feces is used, and an equal amount of external reference standards is added to prepare simulated positive samples, and then nucleic acid extraction is carried out.

[0105] IV. Experimental Results

[0106] The experimental results of Scheme 1 are shown in Table 6 below.

[0107] Table 6. Absorbance test of products extracted from simulated samples by different methods

[0108]

[0109]

[0110] The experimental results of Scheme 1 show that the pretreatment reagent system for nucleic acid extraction from fecal samples can achieve higher fecal nucleic acid extraction efficiency than other commercial kits, including nucleic acid yield and nucleic acid purity. The traditional kit has the highest nucleic acid concentration, but the ratios of A260 / A280 and A260 / A230 are relatively low, indicating that there are more residues of proteins and small molecule inhibitors in its extraction products, which may lead to a false increase in nucleic acid concentration. Further, the nucleic acid extraction products of the traditional kit show obvious color, while the extraction products of the pretreatment reagent system for nucleic acid extraction from fecal samples are more transparent, indicating that it has a better effect on removing fecal inhibitors.

[0111] The experimental results of Scheme 2 are shown in Tables 7 and 8 below.

[0112] Table 7. Verification of extraction of bacterial external reference simulation samples

[0113]

[0114] Table 8. Verification of extraction of plasmid external reference simulation samples

[0115]

[0116] The results of Scheme 2 show that compared with two commercial reagents, the pretreatment reagent for nucleic acid extraction from fecal samples of the present invention can effectively remove PCR inhibitors in feces while ensuring a nucleic acid recovery rate not lower than that of the reference reagent. Since the traditional kit is not optimized for fecal samples and cannot remove inhibitors, it shows false negative results during the amplification of undiluted extraction products, indicating that its extraction products contain a large amount of PCR inhibitors.

[0117] The pretreatment reagent for nucleic acid extraction from fecal samples is superior in terms of inhibitor removal and nucleic acid recovery rate, comparable to other commercial kits. However, the traditional kit cannot detect positive signals when undiluted samples are loaded, and the diluted templates still do not show a nucleic acid recovery rate higher than that of the pretreatment reagent for nucleic acid extraction from fecal samples. In addition, in the experiments of pure plasmid and bacterial plasmid extraction, the nucleic acid recovery rates of fecal matrix samples are lower than those of pure culture extraction methods without fecal interference, showing a nucleic acid loss of more than 2 times (△Ct≥1). Among them, in the plasmid-based simulation sample experiment of the pretreatment reagent system for nucleic acid extraction from fecal samples, the difference in the average Ct value compared with pure plasmid extraction is 1.37, and in the bacterial-based simulation sample experiment, this difference is 0.83, indicating that the pretreatment reagent for nucleic acid extraction from fecal samples can still achieve a high nucleic acid release efficiency in complex matrix samples, ensuring the reliability of PCR amplification detection.

[0118] In summary, this embodiment shows that the automated nucleic acid extraction system with a pretreatment reagent for nucleic acid extraction from fecal samples performs better than existing commercial kits in nucleic acid extraction from fecal samples, can effectively remove PCR inhibitors, improve nucleic acid purity, and ensure a nucleic acid recovery rate not lower than that of the reference reagent. Its extraction method is applicable to high-throughput nucleic acid detection scenarios and can be widely used in the field of nucleic acid detection of fecal samples.

[0119] It should be understood that the present invention disclosed herein is not limited to the specific methods, protocols, and materials described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0120] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also encompassed by the appended claims.

Claims

1. A pretreatment reagent for nucleic acid extraction from stool samples, characterized in that: The pretreatment reagent is a solution including TritonX-100, PEG6000, SDS, guanidine isothiocyanate, KAl(SO4)2 and ammonium acetate.

2. The pretreatment reagent according to claim 1, characterized in that The concentration of the ammonium acetate is 1.5M-6M.

3. The pretreatment reagent according to claim 1, characterized in that The concentration of KAl(SO4)2 is 0.15M-0.2M.

4. A pretreatment reagent for nucleic acid extraction from stool samples, characterized in that: The pre-treatment reagent also includes EDTA stop solution.

5. The pretreatment reagent according to claim 4, characterized in that The EDTA stop solution is an aqueous solution containing ethanol, and the concentration of EDTA is 0.09M-0.2M.

6. A pretreatment method for extracting nucleic acid from stool samples, characterized in that: The method comprises the following steps: Step 1: Add a pretreatment reagent for nucleic acid extraction from a stool sample into a sample tube containing a stool sample, wherein the pretreatment reagent is a solution comprising TritonX-100, PEG6000, SDS, guanidine isothiocyanate, KAl(SO4)2, and ammonium acetate; vortex mix thoroughly to make the stool sample and the pretreatment reagent in a homogenous state; Step 2: After homogenization, heat and centrifuge the sample tube; Step 3: After centrifugation, take the supernatant and add EDTA stop solution to the supernatant; Step 4: After adding EDTA stop solution, use it directly for nucleic acid extraction from stool samples without centrifugation.

7. The method according to claim 6, characterized in that The concentration of the ammonium acetate is 1.5M-6M.

8. The method according to claim 6, characterized in that The concentration of KAl(SO4)2 is 0.15M-0.2M.

9. The method according to claim 6, characterized in that The EDTA stop solution is an aqueous solution containing ethanol, and the concentration of EDTA is 0.09M-0.2M.

10. The method according to claim 6, characterized in that Nucleic acid extraction from stool samples was performed using the magnetic bead method.

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