A method for extracting microbial DNA from the surface of distillers' grains or wheat samples

Abstract

This invention relates to the field of molecular biology, specifically to a method for extracting microbial DNA from the surface of distillers' grains or wheat samples. The method involves weighing an appropriate amount of frozen or fermented distillers' grains or wheat sample into a 15 mL sterile centrifuge tube, adding 3-5 glass beads and 1×PBS solution, and eluting by shaking. This is repeated through multiple elution-transfer cycles until 12 mL of 1×PBS solution is exhausted, ensuring effective detachment of surface microorganisms from the sample surface. The eluted 1×PBS solution is centrifuged to obtain a bottom precipitate, which is then resuspended in 1.5 mL of 1×PBS solution and transferred to a 2 mL sterile centrifuge tube. After high-speed centrifugation, the supernatant is discarded, and finally, 200 μL of 1×PBS solution is added to resuspend the bottom precipitate. The resuspended sample can be stored at -80°C or directly used for downstream extraction and purification. The purified DNA product shows clear bands on agarose gel electrophoresis and its concentration can be determined using the Qubit reagent. The obtained DNA is also suitable for downstream quantitative detection, and the resulting nucleic acid quality is excellent.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to a method for extracting microbial DNA from the surface of distillers' grains or wheat samples. Background Technology

[0002] Distillers' grains and wheat are two core raw material and by-product substrates in brewing, koji making, and solid-state fermentation industries. The surface of distillers' grains is rich in microorganisms such as yeasts, bacteria, and molds that participate in alcoholic fermentation and flavor formation; the surface of wheat is covered with natural microbial communities, including lactic acid bacteria, Bacillus, fungi, and some pathogenic microorganisms. These microorganisms profoundly influence fermentation efficiency and product quality through community succession and metabolic regulation during koji making, stacking fermentation, and saccharification. Therefore, systematically analyzing the diversity, community structure, and functional genes of microorganisms on the surfaces of distillers' grains and wheat has significant theoretical value and application prospects for optimizing fermentation processes, preventing grain mold growth, identifying functional strains, and ensuring food safety.

[0003] Obtaining high-quality, high-purity microbial genomic DNA is a prerequisite for high-throughput sequencing and metagenomic analysis. However, the extraction of microbial DNA from distillers' grains and wheat surfaces faces significant technical challenges, and the interfering matrices for each have distinct characteristics. Distillers' grains contain abundant residual starch, cellulose, hemicellulose, lignin, pectin, and phenolic compounds, which readily co-precipitate or adsorb with DNA. Furthermore, the abundant polysaccharides and proteins in distillers' grains form viscous colloids after lysis, severely inhibiting lysin activity and the throughput of DNA purification columns. As for wheat surfaces, the wheat grain epidermis is covered with a waxy layer, lipids, and cuticle, hindering sufficient contact between the lysis buffer and surface microorganisms. Additionally, polysaccharides from within the wheat grains and secondary metabolites such as phenolic acids and tannins released from the bran are continuously released during extraction, competing with DNA for adsorption sites and inhibiting downstream enzyme reactions. In addition, both share the challenge that: both distiller's grains and wheat are solid particles, with surface microorganisms embedded in dense biofilms or tightly bound to matrix pores, making it difficult to fully release cellular DNA using conventional lysis methods; at the same time, both contain high levels of PCR inhibitors (such as humic acid analogs, phenols, metal ions, etc.), which means that even if the extracted DNA reaches the required concentration, it cannot be used for library construction or amplification.

[0004] Currently, there are two main types of methods for extracting microbial DNA from solid matrices such as distiller's grains and wheat surfaces: The first type is the traditional CTAB manual extraction method, which typically involves grinding or mincing the sample, lysing cells using CTAB, and then purifying the DNA using a phenol-chloroform organic solvent. This method is cumbersome, time-consuming, and requires the use of highly toxic chloroform, posing a high safety risk. Furthermore, polysaccharides in distiller's grains and phenolic acids in wheat are difficult to completely remove through organic extraction, resulting in significant residues of inhibitors in the DNA. The second type involves directly using commercial soil or grain DNA extraction kits, adding a small amount of sample to a lysis tube, followed by mechanical disruption and centrifugation for purification. However, these kits are designed for high-biomass homogeneous samples and are poorly suited for heterogeneous samples such as distiller's grains and wheat, which contain high levels of starch, polysaccharides, and lipids and have low surface microbial abundance. The sample volume processed per batch is typically less than 0.5 g (equivalent to only a few grains of wheat or less than 0.3 g of wet weight in distiller's grains), resulting in low total DNA concentration and numerous impurities, which completely fails to meet the quality standards required for metagenomic sequencing. In summary, existing technologies cannot simultaneously solve the problems of severe interference from the surface matrix of distillers' grains and wheat, difficulty in cell release, and insufficient DNA purity and yield, making it difficult to obtain qualified DNA samples and severely restricting the in-depth research on the structure and function of the microbial community in the aforementioned matrix. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention proposes a method for extracting microbial DNA from the surface of distiller's grains or wheat samples. This method solves the problems of complex operation, toxicity, low extraction efficiency, and poor extraction quality in the existing methods for extracting microorganisms from the surface of distiller's grains or wheat.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A method for extracting microbial DNA from the surface of distillers' grains or wheat samples includes the following steps: S1. Sample weighing and reagent / consumable preparation: Weigh an appropriate amount of sample and add it to a 15mL sterile centrifuge tube, along with freshly prepared 1×PBS solution and sterilized glass beads. S2, Elution of microorganisms on the sample surface: Add the sample, 1×PBS solution and sterilized glass beads to the solid-liquid mixture, shake to mix well, so that the surface microorganisms detach from the surface of the distillers' grains, wheat seeds, etc. and accumulate in the solution; S3. Collection and multiple elution of the eluent: Centrifuge the solid-liquid mixture obtained in step S2 after shaking and mixing, and collect the supernatant. Keep the solid precipitate, and then add fresh 1×PBS solution to the solid precipitate for elution. Shake and mix, centrifuge and collect the supernatant. Repeat this elution step multiple times, and combine all the supernatants from centrifugation to obtain the eluent. S4. Enrichment of surface microorganisms: By centrifuging the eluent obtained in step S3, the surface microorganisms are enriched to the bottom of a 15 mL centrifuge tube. The supernatant is removed, and the bottom precipitate is resuspended with 1×PBS solution and transferred to a 2 mL centrifuge tube. The supernatant is removed by high-speed centrifugation, and the bottom precipitate is resuspended with 1×PBS solution. After resuspending, the sample can be stored in a -80℃ freezer or directly extracted and purified downstream. S5. DNA quality control: The extracted and purified nucleic acids were subjected to agarose gel electrophoresis, Qubit concentration determination, and quantitative PCR amplification.

[0007] More preferably, the sample weight in S1 is usually between 3-5g; 1×PBS solution is usually added to 900 mL of sterile water with 100 mL of 10×PBS solution (CW0040S), mixed thoroughly, and used immediately; glass beads are sterilized by autoclaving and dried after sterilization.

[0008] More preferably, the elution method in S2 refers to adding 5g of sample, 4mL of 1×PBS solution and 3 sterilized glass beads (Sangon Biotech: A500478) at the same time, and then shaking the sample at 2800rpm for 5min using a shaker.

[0009] More preferably, the collection and multiple elution of the eluent in S3 specifically involves centrifuging the solid-liquid mixture obtained in step S2 at 1500 r / min for 5 min, collecting the supernatant and transferring it to a new sterile 15 mL centrifuge tube, retaining the solid precipitate, and adding 4 mL of new 1×PBS to the solid precipitate for elution, oscillating at 2800 rpm for 5 min to mix, and then centrifuging again under the same conditions to collect the supernatant. This elution step is repeated once until 12 mL of 1×PBS solution is consumed (one 4 mL 1×PBS elution in S2, two 4 mL 1×PBS elutions in S3, for a total of 12 mL 1×PBS). All the supernatants from the three centrifugations are combined to obtain the eluent.

[0010] Further preferably, the specific method for enriching surface microorganisms in step S4 is as follows: The elution buffer obtained in step S3 is centrifuged at 4500 r / min for 5 min to enrich the surface microorganisms to the bottom of a 15 mL centrifuge tube. The supernatant is removed, and the bottom precipitate is resuspended in 1.5 mL of 1×PBS solution. The resuspended precipitate is then transferred to a 2 mL centrifuge tube and centrifuged at 12000 g for 3 min to remove the supernatant. Finally, the bottom precipitate is resuspended in 200 μL of 1×PBS solution. After resuspending, the sample can be stored at -80℃ or directly subjected to downstream extraction and purification. Nucleic acid extraction and purification are performed using the Kangwei Century-Magnetic Bead Method Soil and Fecal DNA Extraction Kit (CW2556M). The extracted DNA undergoes downstream DNA quality control.

[0011] Further preferably, in S5: the extracted and purified DNA is electrophoresed on a 1% agarose gel at 121V / cm for 20 minutes and then observed by gel imaging; the Qubit concentration determination reagent is the Kangwei Century-1×dsDNA High Sensitivity Detection Kit CW3102M, 197μL of 1×dsDNA HS Working Solution and 3μL of the sample to be tested are thoroughly mixed and measured using a Thermo Fisher Invitrogen Qubit 4 fluorometer.

[0012] More preferably, the primers used for quantitative PCR amplification in S5 include primer pairs targeting the V3 and V4 variable regions of the bacterial 16S rRNA gene (referred to as 16Sv3-v4 primer pairs) and primer pairs targeting the fungal ITS gene (referred to as ITS primer pairs). The upstream and downstream primer sequences of the 16Sv3-v4 primer pair are as follows: Upstream primer: 5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO:1) Downstream primer: 5'-GACTACHVGGGTATCTAATCC-3' (SEQ ID NO:2) The upstream and downstream primer sequences of the ITS primer pair are as follows: Upstream primer: 5'-CTTGGTCATTTAGAGGAAGTAA-3' (SEQ ID NO:3) Downstream primer: 5'-GCTGCGTTCTTCATCGATGC-3' (SEQ ID NO:4).

[0013] In the above sequences SEQ ID NO:1-4, the degenerate bases are defined as follows: N is A, T, C or G; W is A or T; H is A, T or C; V is A, C or G.

[0014] More preferably, in S5: the system used for quantitative PCR amplification is: ddH2O 7.6μL, forward and reverse primers 0.4μL each, DNA template 2.0μL, 2×SuperPro qPCR Mix 10μL.

[0015] More preferably, in S5, the amplification conditions are: 95℃ pre-denaturation for 30s; 95℃ denaturation for 6s, 55℃ annealing extension for 30s, 72℃ annealing extension for 30s, fluorescence collection, for 40 cycles.

[0016] The present invention has the following beneficial effects: 1) This invention proposes a method for enriching microorganisms on the surface of distiller's grains or wheat. Through multiple elutions, sufficient microbial DNA can be extracted for downstream experiments, solving the problem of difficult or impossible extraction of surface microorganisms.

[0017] 2) The extraction method of the present invention, through an optimized elution-enrichment-extraction process, has high microbial recovery rate and high DNA purity, and can efficiently obtain high-quality and high-concentration DNA, meeting the requirements of quantitative PCR and metagenomic sequencing, and promoting in-depth research on surface microorganisms such as distillers' grains or wheat. Attached Figure Description

[0018] Figure 1 This is an agarose gel electrophoresis result of the DNA of microorganisms extracted from the surface of distiller's grains in Example 1; Figure 2 This is a diagram showing the results of quantitative PCR amplification of DNA from microorganisms extracted from the surface of distiller's grains in Example 1; Figure 3 This is an agarose gel electrophoresis result of the DNA extracted from wheat surface microorganisms in Example 2; Figure 4 This is a diagram showing the quantitative PCR amplification results of DNA extracted from wheat surface microorganisms in Example 2; Figure 5 This is a comparison of the agarose gel electrophoresis results of DNA extracted from microorganisms on the surface of distiller's grains using two methods: enrichment and non-enrichment, in Comparative Example 1. Figure 6 This is a graph showing the results of quantitative PCR amplification of DNA from microorganisms on the surface of distiller's grains extracted using two methods, enrichment and non-enrichment, in Comparative Example 1. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0020] The experimental materials used in the embodiments of this invention are from Shanghai Haoweitai Biotechnology Co., Ltd.

[0021] Example 1

[0022] A method for enriching and extracting microbial DNA from the surface of distiller's grains samples involves elution, enrichment, extraction, purification, and quality control of microorganisms on the surface of distiller's grains samples. The specific operational steps are as follows: S1. Sample weighing and preparation of reagents and consumables: Prepare fresh 1× PBS solution: The specific preparation method is to add 100 mL of 10× PBS solution to 900 mL of sterile water and mix thoroughly; put glass beads into a 50 mL centrifuge tube, sterilize in an autoclave, dry in an oven, and then place at room temperature for later use; weigh 5 g of the distiller's grains sample and put it into a 15 mL sterile centrifuge tube.

[0023] S2. Elution of microorganisms on the sample surface: Add 4 mL of 1×PBS solution and 3 sterile glass beads to a 15 mL sterile centrifuge tube containing 5 g of distiller's grains sample to form a solid-liquid mixture. Shake the mixture at 2800 rpm for 5 min to allow the surface microorganisms to detach from the surface of the distiller's grains and accumulate in the solution. S3. Collection and repeated elution of the eluent: Centrifuge the solid-liquid mixture obtained in step S2 at 1500 r / min for 5 min, collect the supernatant and transfer it to a new sterile 15 mL centrifuge tube. Retain the solid precipitate and add 4 mL of fresh 1×PBS to the solid precipitate for elution. Mix by shaking at 2800 rpm for 5 min, and then centrifuge under the same conditions to collect the supernatant. Repeat this elution step once until 12 mL of 1×PBS solution is consumed. Combine all the supernatants from the three centrifugations to obtain the eluent.

[0024] S4. Enrichment of surface microorganisms: Centrifuge the eluent obtained in step S3 at 4500 r / min for 5 min to enrich the surface microorganisms to the bottom of a 15 mL centrifuge tube. Discard the supernatant, resuspend the bottom precipitate in 1.5 mL of 1× PBS solution, transfer it to a 2 mL centrifuge tube, and centrifuge at 12000 g for 3 min. Discard the supernatant and resuspend the bottom precipitate in 200 μL of 1× PBS solution. After resuspending, the sample can be stored at -80℃ or directly used for downstream extraction and purification. Nucleic acid extraction and purification were performed using the Kangwei Century-Magnetic Bead Method Soil and Fecal DNA Extraction Kit (CW2556M). The extracted DNA can be used for downstream DNA quality control. S5. DNA Quality Inspection: Gel electrophoresis detection: The extracted and purified DNA was electrophoresed on a 1% agarose gel at 121V / cm for 20 min and then observed by gel imaging. Qubit concentration was determined using the Kangwei Century-1×dsDNA High Sensitivity Detection Kit CW3102M. 197 μL of 1×dsDNA HS Working Solution and 3 μL of the sample to be tested were thoroughly mixed and measured using a Thermo Fisher Invitrogen Qubit 4 fluorometer. The primers used for quantitative PCR amplification in S5 include primer pairs targeting the V3 and V4 variable regions of the bacterial 16S rRNA gene (referred to as 16Sv3-v4 primer pairs) and primer pairs targeting the fungal ITS gene (referred to as ITS primer pairs). The upstream and downstream primer sequences of the 16Sv3-v4 primer pair are as follows: Upstream primer: 5'-CCTACGGGAGGCAGCAG-3'; (SEQ ID NO:5) Downstream primer: 5'-GACTACAGGGGTATCTAATCC-3'; (SEQ ID NO:6) The upstream and downstream primer sequences of the ITS primer pair are as follows: Upstream primer: 5'-CTTGGTCATTTAGAGGAAGTAA-3'; (SEQ ID NO:3) Downstream primer: 5'-GCTGCGTTCTTCATCGATGC-3'; (SEQ ID NO:4).

[0025] In S5: the system used for quantitative PCR amplification is: ddH2O 7.6μL, forward and reverse primers 0.4μL each, DNA template 2.0μL, 2×SuperPro qPCR Mix 10μL.

[0026] In S5, the quantitative PCR amplification conditions are: 95℃ pre-denaturation for 30s; 95℃ denaturation for 6s, 55℃ annealing extension for 30s, 72℃ annealing extension for 30s, fluorescence collection, 40 cycles.

[0027] The results are as follows Figure 1-2 As shown in Table 1.

[0028] Table 1: Results of Qubit assay for the concentration of microbial DNA extracted from the surface of distiller's grains samples Sample 1 14.645 Sample 2 19.492 Sample 3 5.883 Sample 4 22.747 Sample 5 15.916 .

[0029] As can be seen from Table 1, the concentration of microbial nucleic acids extracted from the surface of distiller's grains using the method of the present invention is high, which meets the requirements of subsequent experiments.

[0030] Figure 1This is the agarose gel electrophoresis result of the DNA extracted from the surface microorganisms of the lees in Example 1. The result shows that the band brightness corresponds to that in Table 1, indicating that the DNA extracted from the surface microorganisms of the lees by the method provided by the present invention can be easily detected by gel electrophoresis to determine the quality of nucleic acid extraction, which is the basis for downstream experimental extraction. Figure 2 This image shows the quantitative PCR amplification results of the DNA extracted from the surface of distiller's grains in Example 1. It indicates that the extracted DNA from the microorganisms on the surface of the distiller's grains contains a relatively large number of fungi. It also demonstrates that the DNA extracted from the microorganisms on the surface of the distiller's grains using the method provided in this invention can be successfully subjected to downstream quantitative PCR experiments without significant inhibition, indicating excellent nucleic acid quality. This method is fully applicable to the extraction of microbial DNA from the surface of distiller's grains.

[0031] Example 2 A method for enriching and extracting DNA from microorganisms on the surface of wheat samples involves elution, enrichment, extraction, purification, and quality control of microorganisms on the surface of wheat samples. The specific steps are as follows: S1. Sample weighing and preparation of reagents and consumables: Prepare fresh 1× PBS solution: The specific preparation method is to add 100 mL of 10× PBS solution to 900 mL of sterile water and mix thoroughly; put glass beads into a 50 mL centrifuge tube, sterilize in an autoclave, dry in an oven, and then place at room temperature for later use; weigh 5 g of wheat sample and put it into a 15 mL sterile centrifuge tube.

[0032] S2. Elution of microorganisms on the sample surface: Add 4 mL of 1×PBS solution and 3 sterile glass beads to a 15 mL sterile centrifuge tube containing 5 g of wheat sample to form a solid-liquid mixture. Shake the mixture at 2800 rpm for 5 min to allow the surface microorganisms to detach from the wheat surface and aggregate in the solution. S3. Collection and repeated elution of the eluent: Centrifuge the solid-liquid mixture obtained in step S2 at 1500 r / min for 5 min, collect the supernatant and transfer it to a new sterile 15 mL centrifuge tube. Retain the solid precipitate and add 4 mL of fresh 1×PBS to the solid precipitate for elution. Mix by shaking at 2800 rpm for 5 min, and then centrifuge under the same conditions to collect the supernatant. Repeat this elution step once until 12 mL of 1×PBS solution is consumed. Combine all the supernatants from the three centrifugations to obtain the eluent.

[0033] S4. Enrichment of surface microorganisms: Centrifuge the eluent obtained in step S3 at 4500 r / min for 5 min to enrich the surface microorganisms to the bottom of a 15 mL centrifuge tube. Discard the supernatant, resuspend the bottom precipitate in 1.5 mL of 1×PBS solution, transfer it to a 2 mL centrifuge tube, and centrifuge at 12000 g for 3 min. Discard the supernatant and resuspend the bottom precipitate in 200 μL of 1×PBS solution. After resuspending, the sample can be stored at -80℃ or directly used for downstream extraction and purification. Nucleic acid extraction and purification were performed using the Kangwei Century-Magnetic Bead Method Soil and Fecal DNA Extraction Kit (CW2556M). The extracted DNA can be used for downstream DNA quality control. S5. DNA Quality Inspection: Gel electrophoresis detection: The extracted and purified DNA was electrophoresed on a 1% agarose gel at 121V / cm for 20 min and then observed by gel imaging. Qubit concentration was determined using the Kangwei Century-1×dsDNA High Sensitivity Detection Kit CW3102M. 197 μL of 1×dsDNA HS Working Solution and 3 μL of the sample to be tested were thoroughly mixed and measured using a Thermo Fisher Invitrogen Qubit 4 fluorometer. The primers used for quantitative PCR amplification in S5 include primer pairs targeting the V3 and V4 variable regions of the bacterial 16S rRNA gene (referred to as 16Sv3-v4 primer pairs) and primer pairs targeting the fungal ITS gene (referred to as ITS primer pairs). The upstream and downstream primer sequences of the 16Sv3-v4 primer pair are as follows: Upstream primer: 5'-CCTACGGGAGGCAGCAG-3'; (SEQ ID NO:5) Downstream primer: 5'-GACTACAGGGGTATCTAATCC-3'; (SEQ ID NO:6) The upstream and downstream primer sequences of the ITS primer pair are as follows: Upstream primer: 5'-CTTGGTCATTTAGAGGAAGTAA-3'; (SEQ ID NO:3) Downstream primer: 5'-GCTGCGTTCTTCATCGATGC-3'; (SEQ ID NO:4).

[0034] In S5: the system used for quantitative PCR amplification is: ddH2O 7.6μL, forward and reverse primers 0.4μL each, DNA template 2.0μL, 2X SuperPro qPCR Mix 10μL.

[0035] In S5, the quantitative PCR amplification conditions are: 95℃ pre-denaturation for 30s; 95℃ denaturation for 6s, 55℃ annealing extension for 30s, 72℃ annealing extension for 30s, fluorescence collection, 40 cycles.

[0036] The results are as follows Figure 3-4 As shown in Table 2.

[0037] Table 2: Results of Qubit assay for concentration of microbial DNA extracted from wheat sample surface Sample 1 26.4 Sample 2 13.4 Sample 3 21.8 Sample 4 43.7 Sample 5 25.1 .

[0038] As can be seen from Table 2, the DNA concentration of wheat surface microorganisms extracted by the method of the present invention is high, which can fully meet the requirements of subsequent experiments.

[0039] Figure 3 This is an agarose gel electrophoresis result of the DNA extracted from wheat surface microorganisms in Example 2; Figure 4 This is a graph showing the quantitative PCR amplification results of DNA extracted from wheat surface microorganisms in Example 2. From... Figure 3 and Figure 4 It can be seen that the method of the present invention is also applicable to the extraction of DNA from microorganisms on the surface of wheat, and the obtained nucleic acid concentration is high. The quality of nucleic acid can be detected by gel electrophoresis, and there is no inhibition on downstream quantitative PCR, with excellent purity.

[0040] Comparative Example 1 A comparison was made between the method of directly eluting and shaking with 12 mL of 1×PBS instead of multiple elution-enrichment and the multiple elution-enrichment method of the present invention: The specific steps of the non-multiple elution-enrichment method are as follows: S1. Sample weighing and preparation of reagents and consumables: Prepare fresh 1×PBS solution: The specific preparation method is to add 100 mL of 10×PBS solution to 900 mL of sterile water and mix thoroughly; put glass beads into a 50 mL centrifuge tube, sterilize in an autoclave, dry in an oven, and then place at room temperature for later use; weigh 5 g of the distiller's grains sample and put it into a 15 mL sterile centrifuge tube.

[0041] S2. Elution of microorganisms on the sample surface: Add 12 mL of 1×PBS solution and 3 sterile glass beads to a 15 mL sterile centrifuge tube containing 5 g of distiller's grains sample to form a solid-liquid mixture. Shake the mixture at 2800 rpm for 5 min to allow the surface microorganisms to detach from the surface of the distiller's grains and accumulate in the solution. S3. Enrichment of surface microorganisms: The supernatant obtained from step S2 was centrifuged at 4500 rpm for 5 min to enrich the surface microorganisms to the bottom of a 15 mL centrifuge tube. The supernatant was discarded, and the bottom precipitate was resuspended in 1.5 mL of 1×PBS solution. The resuspended precipitate was then transferred to a 2 mL centrifuge tube and centrifuged at 12000 g for 3 min. The supernatant was discarded, and the bottom precipitate was resuspended in 200 μL of 1×PBS solution. After resuspending, the sample can be stored at -80°C or directly used for downstream extraction and purification. Nucleic acid extraction and purification were performed using the Kangwei Century-Magnetic Bead Method Soil and Fecal DNA Extraction Kit (CW2556M). The extracted DNA can be used for downstream DNA quality control.

[0042] The method of multiple elution-enrichment-extraction in this embodiment is the same as that in Example 1, and the DNA quality control method is the same as that in Example 1.

[0043] The experimental results of the non-multiple elution-enrichment method and the multiple elution-enrichment method of the present invention are shown in Table 3. Figure 5-6 As shown.

[0044] Table 3: Results of microbial DNA concentration determination on the surface of distiller's grains samples extracted by the Qubit enrichment and non-enrichment methods Sample 1 18.1 9.267 Sample 2 32.01 26.8 Sample 3 35.588 26.5 Sample 4 30.863 20.7 Sample 5 45.723 29.8 .

[0045] As can be seen from Table 3, there is a significant difference in the nucleic acid concentration obtained by the enrichment and non-enrichment methods. The enrichment method provided by this invention increases the concentration by one time compared to the non-enrichment method.

[0046] Figure 5 This is a comparison of the agarose gel electrophoresis results of the DNA extracted from the surface microorganisms of distiller's grains using the enrichment method and the non-enrichment method in Comparative Example 1. It can be seen that the DNA from the surface microorganisms of distiller's grains extracted using the enrichment method of this invention has a significantly brighter band on the gel electrophoresis than the DNA from the surface microorganisms of distiller's grains extracted using the non-enrichment method.

[0047] Figure 6 This is a quantitative PCR amplification result of DNA extracted from microorganisms on the surface of distiller's grains using two methods: enrichment and non-enrichment, in Comparative Example 1. It can be seen that the peak of DNA extracted from microorganisms on the surface of distiller's grains using the enrichment method is significantly earlier than that extracted using the non-enrichment method.

[0048] Comparative experiments show that the nucleic acid concentration, gel image brightness, and quantitative values ​​obtained by the multiple elution-enrichment-extraction method of this invention are significantly better than those obtained by the non-enrichment extraction method. The nucleic acid concentration obtained by this invention is high, the gel image brightness is high, and no inhibition occurs when using downstream quantitative analysis.

[0049] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for extracting microbial DNA from the surface of distillers' grains or wheat samples, characterized in that, Includes the following steps: S1. Sample weighing and reagent / consumable preparation: Weigh an appropriate amount of sample and add it to a 15mL sterile centrifuge tube, along with freshly prepared 1×PBS solution and sterilized glass beads. S2, Elution of microorganisms on the sample surface: Add the sample, 1×PBS solution and sterilized glass beads to the solid-liquid mixture, shake to mix well, so that the surface microorganisms are detached from the surface of the distillers' grains or wheat sample and aggregate in the solution; S3. Collection and multiple elution of the eluent: Centrifuge the solid-liquid mixture obtained in step S2 after shaking and mixing, and collect the supernatant. Keep the solid precipitate, and then add fresh 1×PBS solution to the solid precipitate for elution. Shake and mix, centrifuge and collect the supernatant. Repeat this elution step multiple times, and combine all the supernatants from centrifugation to obtain the eluent. S4. Enrichment of surface microorganisms: By centrifuging the eluent obtained in step S3, the surface microorganisms are enriched to the bottom of a 15 mL centrifuge tube. The supernatant is removed, and the bottom precipitate is resuspended with 1×PBS solution and transferred to a 2 mL centrifuge tube. The supernatant is removed by high-speed centrifugation, and the bottom precipitate is resuspended with 1×PBS solution. After resuspending, the sample can be stored in a -80℃ freezer or directly extracted and purified downstream. S5. DNA quality control: The extracted and purified nucleic acids were subjected to agarose gel electrophoresis, Qubit concentration determination, and quantitative PCR amplification.

2. The method for extracting microbial DNA from the surface of distillers' grains or wheat samples according to claim 1, characterized in that, The sample in S1 is weighed to a mass of 3-5g; the 1×PBS solution is prepared by adding 100 mL of 10×PBS solution to 900 mL of sterile water and mixing thoroughly; the glass beads are sterilized by autoclaving and dried after sterilization.

3. The method for extracting microbial DNA from the surface of distillers' grains or wheat samples according to claim 1, characterized in that, The elution method in S2 refers to adding 5g of sample, 4mL of 1×PBS solution and 3 sterilized glass beads at the same time, and then shaking the sample at 2800rpm for 5min.

4. The method for extracting microbial DNA from the surface of distillers' grains or wheat samples according to claim 1, characterized in that, The collection and multiple elutions of the eluent in step S3 are as follows: the solid-liquid mixture obtained in step S2 is centrifuged at 1500 r / min for 5 min, the supernatant is collected and transferred to a new sterile 15 mL centrifuge tube, the solid precipitate is retained, and 4 mL of new 1×PBS is added to the solid precipitate for elution. The mixture is oscillated at 2800 rpm for 5 min to mix, and then centrifuged again under the same conditions to collect the supernatant. This elution step is repeated once until 12 mL of 1×PBS solution is consumed. All the supernatants from the three centrifugations are combined to obtain the eluent.

5. The method for extracting microbial DNA from the surface of distillers' grains or wheat samples according to claim 1, characterized in that, The specific method for enriching surface microorganisms in step S4 is as follows: centrifuge the eluent obtained in step S3 at 4500 r / min for 5 min to enrich the surface microorganisms to the bottom of a 15 mL centrifuge tube, remove the supernatant, resuspend the bottom precipitate with 1.5 mL of 1×PBS solution, transfer it to a 2 mL centrifuge tube, and centrifuge at 12000 g for 3 min to remove the supernatant. Finally, resuspend the bottom precipitate with 200 μL of 1×PBS solution. After resuspending, the sample can be stored in a -80℃ freezer or directly extracted and purified downstream.

6. The method for extracting microbial DNA from the surface of distillers' grains or wheat samples according to claim 1, characterized in that, In S5: the extracted and purified DNA was electrophoresed on a 1% agarose gel at 121V / cm for 20 min and then observed by gel imaging; the Qubit concentration was determined using the Kangwei Century-1×dsDNA High Sensitivity Detection Kit CW3102M, 197μL of 1×dsDNAHS Working Solution and 3μL of the sample to be tested were thoroughly mixed and measured using a Thermo Fisher Invitrogen Qubit 4 fluorometer.

7. The method for extracting microbial DNA from the surface of distiller's grains or wheat samples according to claim 1, characterized in that, The primers used for quantitative PCR amplification in S5 include primer pairs targeting the V3 and V4 genes of bacterial 16S rRNA (referred to as 16Sv3-v4 primer pairs) and primer pairs targeting the ITS gene of fungi (referred to as ITS primer pairs). The upstream and downstream primer sequences of the 16Sv3-v4 primer pair are as follows: Upstream primer: 5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO:1) Downstream primer: 5'-GACTACHVGGGTATCTAATCC-3' (SEQ ID NO:2) The upstream and downstream primer sequences of the ITS primer pair are as follows: Upstream primer: 5'-CTTGGTCATTTAGAGGAAGTAA-3' (SEQ ID NO:3) Downstream primer: 5'-GCTGCGTTCTTCATCGATGC-3' (SEQ ID NO:4).

8. The method for extracting microbial DNA from the surface of distillers' grains or wheat samples according to claim 1, characterized in that, In S5: the system used for quantitative PCR amplification is: ddH2O 7.6μL, forward and reverse primers 0.4μL each, DNA template 2.0μL, 2×SuperPro qPCR Mix 10μL.

9. The method for extracting microbial DNA from the surface of distillers' grains or wheat samples according to claim 1, characterized in that, In S5, the amplification conditions are: 95℃ pre-denaturation for 30s; 95℃ denaturation for 6s; 55℃ annealing extension for 30s; 72℃ annealing extension for 30s; fluorescence collection; 40 cycles.

Images