A broad-spectrum DNA extraction kit and method for removing polysaccharide and polyphenol interference

Abstract

The present application provides a broad-spectrum DNA extraction kit and extraction method for removing polysaccharide and polyphenol interference, and belongs to the technical field of nucleic acid extraction. The kit comprises BT reagent, Buffer A reagent, Buffer HH reagent, WB1 reagent and WB2 reagent. The BT reagent contains sucrose, KCl, PVP-40, Tris-HCl, EDTA, Triton X-100, NaOH, KOH, EGTA, LiCl, BSA, spermine, spermidine and Tween-20. The present application can effectively remove interfering substances such as polysaccharides and polyphenols through specific reagent combination and optimized operation process, and high-purity and high-integrity DNA can be obtained. Moreover, the kit and the matching method are not only suitable for polysaccharide and polyphenol plants, but also suitable for marine animals, mollusks, fungi and other difficult-to-extract samples rich in different types of polysaccharides, and have broad-spectrum applicability.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction technology, specifically to a broad-spectrum DNA extraction kit and extraction method capable of removing interfering substances such as polysaccharides and polyphenols. Background Technology

[0002] DNA extraction is a technique for isolating and purifying DNA from biological samples. Its core involves using physical, chemical, or biological methods to break down cells and remove impurities such as proteins, providing the basic material for downstream molecular biology applications such as PCR and sequencing. Currently, while DNA extraction technology is quite mature, it still has some specific limitations in certain biological samples: Existing conventional kits for extracting DNA from polysaccharide and polyphenolic plants (such as commercial kits based on CTAB or silica membrane column purification) often face the following problems: (1) Incomplete removal of polysaccharides and polyphenols leads to low purity of extracted DNA, with serious pigment and polysaccharide contamination, which seriously affects subsequent molecular experiments such as enzyme digestion, PCR, and sequencing; (2) Low and unstable DNA yield. For difficult-to-process samples, the total extraction amount is often less than 1 μg, which cannot meet the needs of library construction or multiple experiments; (3) Complex operation procedures or use of toxic reagents, such as phenol-chlorine extraction, which has poor safety and cumbersome steps; (4) Low throughput and difficulty in automation. Most methods rely on manual centrifugation, pipetting, and other steps, which are difficult to be compatible with automated liquid processing workstations and are not suitable for large-scale sample processing. Moreover, plants rich in secondary metabolites (such as rubber trees, Mongolian oaks, apricots, cacti, etc.) contain interfering substances such as resins, tannins, and alkaloids in addition to polysaccharides and polyphenols, which further increases the difficulty of DNA extraction.

[0003] Furthermore, existing technologies are less effective at extracting samples rich in polysaccharides or mucopolysaccharides, such as marine animals, mollusks, and fungi. Marine animals and mollusks (such as sea urchins, seahorses, clams, squid, and octopuses) contain a large amount of interfering substances such as mucopolysaccharides, glycogen, and glycoproteins. These substances co-precipitate with DNA during conventional extraction, forming a viscous gel-like substance that severely affects the solubility and purity of DNA and is difficult to remove effectively through conventional phenol-chloroform extraction or silica gel membrane column purification.

[0004] Fungal samples (such as shiitake mushrooms) have thick cell walls containing a large amount of polysaccharides such as chitin and glucan, which are difficult to lyse. Furthermore, the released polysaccharides severely interfere with DNA binding and elution, resulting in extremely low yields. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a DNA extraction kit and extraction method that can efficiently remove various polysaccharide interferences, is applicable to various sample types, and can be automated and operated in a high-throughput manner, aiming to solve the technical problem of poor DNA extraction effect for special samples.

[0006] To achieve the above objectives, the present invention specifically adopts the following solution: In a first aspect, the present invention provides a broad-spectrum DNA extraction kit for removing polysaccharide and polyphenol interference, which comprises at least the following reagents: BT reagent contains sucrose, KCl, PVP-40, Tris-HCl, EDTA, Triton X-100, NaOH, KOH, EGTA, LiCl, BSA, spermine, spermidine, Tween-20, and sterile water; Buffer A reagent contains Buffer GDB, SDS, RNase, Proteinase K and sterile water, wherein Buffer GDB reagent contains Tris-HCl, EDTA, NaCl and sterile water; Buffer HH reagent contains guanidine hydrochloride, Tris-HCl and sterile water; WB1 reagent contains guanidine hydrochloride, Tris-HCl, ethanol and sterile water; WB2 reagent contains sodium acetate, EDTA, ethanol, and sterile water.

[0007] Preferably, in the above kit, BT reagent contains 3.0 M sucrose, 1.0 M KCl, 0.6 M PVP-40, 0.3 M Tris-HCl, 0.4 M EDTA, 3% Triton X-100, 0.1 M NaOH, 0.1 M KOH, 0.08 M EGTA, 0.1 M LiCl, 5% BSA, 3 M spermine, 3 M spermidine, 2% Tween-20, and the balance being sterile water; Buffer A reagent consists of 90% Buffer GDB, 5% SDS, 2% RNase (10 mg / ml), 2% Proteinase K (20 mg / ml), and the balance being sterile water, and Buffer GDB reagent consists of 1.0 M Tris-HCl, 1 M EDTA, 2 M NaCl, and the balance being sterile water; Buffer HH reagent consists of 3 M guanidine hydrochloride, 0.8 M Tris-HCl, and the balance being sterile water; WB1 reagent consists of 3 M guanidine hydrochloride, 1.0 M... The reagent consists of Tris-HCl, 30% anhydrous ethanol, and the remainder sterile water; the WB2 reagent consists of 2 M sodium acetate, 1 M EDTA, 70% anhydrous ethanol, and the remainder sterile water.

[0008] Preferably, the above kit also includes magnetic beads for purifying DNA, as well as other auxiliary reagents or consumables (such as 80% ethanol, isopropanol, enzyme-free water, deep well plates, steel beads, centrifuge tubes, etc.). Secondly, the present invention provides a DNA extraction method for removing interference from polysaccharides and polyphenols. This method is implemented based on the broad-spectrum DNA extraction kit provided by the present invention. Specifically, the method includes the following steps: S1. After freezing the biological sample, pulverize it and add BT reagent to the resulting powder for washing. S2. Add Buffer A reagent to the obtained precipitate for lysis, centrifuge, and take the supernatant into a new tube; S3. Add Buffer HH reagent, isopropanol and magnetic beads, mix well and let stand, then discard the supernatant. S4. Wash the magnetic beads sequentially with WB1 reagent, WB2 reagent and 80% ethanol, dry the magnetic beads and then add enzyme-free water to dissolve them to obtain the nucleic acid product.

[0009] Preferably, in step S1 of the above method, the BT reagent is used to wash twice, and the washing method is as follows: after adding the BT reagent, shake to mix, then centrifuge at 2000-5000 g and discard the supernatant.

[0010] Preferably, in step S2 of the above method, the pyrolysis conditions are pyrolysis at 37-65℃ for 20-70 minutes, and the centrifugation speed is 9000-12500 rpm.

[0011] Thirdly, the present invention provides the application of the broad-spectrum DNA extraction kit and extraction method provided by the present invention in the extraction of nucleic acids from biological samples, wherein the biological samples are rich in polysaccharides and / or polyphenols, and such biological samples include, but are not limited to, samples derived from papaya, baobab, strawberry, kiwi, grape, cactus, apricot, moss, shiitake mushroom, rubber tree, Mongolian oak, sea urchin, seahorse, clam or squid.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Cross-species applicability: A single kit is applicable to DNA extraction from three major biological groups: the plant kingdom (angiosperms, gymnosperms, bryophytes), the animal kingdom (marine animals, mollusks), and the fungi kingdom. This has not been reported in the existing technology.

[0013] (2) Removal capacity across polysaccharide types: It can remove multiple polysaccharide interfering substances with different structures, such as plant polysaccharides (starch, pectin), animal mucopolysaccharides (glycosaminoglycans, glycogen), and fungal polysaccharides (chitin, glucan), indicating that the composite formulation of BT reagent has produced a synergistic enhancement effect, rather than a simple summation of the components.

[0014] (3) The unity of automation and broad applicability: While achieving broad applicability, it maintains perfect compatibility with high-throughput automation platforms, solving the technical contradiction that "broad-spectrum reagent kits are difficult to automate, and automated reagent kits have limited sample types". Attached Figure Description

[0015] Figure 1 This is a DNA gel electrophoresis image of each papaya leaf sample from Example 4; Figure 2 This is a gel electrophoresis image of DNA from various sea urchin gonad samples in Example 5. Figure 3 The image shows DNA gel electrophoresis images of various baobab tree samples from Example 5. Figure 4 The images show DNA gel electrophoresis results of the squid, strawberry, kiwi, and grape samples from Example 5. Figure 5 DNA gel electrophoresis images of cactus, apricot, moss, and seahorse samples from Example 5. Figure 6 DNA gel electrophoresis images of samples from shiitake mushrooms, rubber trees, Mongolian oaks, and clam viscera / foot from Example 5; Figure 7 DNA gel electrophoresis images of various samples of Siberian apricot leaves in Comparative Example 1; Figure 8 The image shows DNA gel electrophoresis results from various baobab tree samples in Comparative Example 2. Figure 9 DNA gel electrophoresis images of strawberry, kiwi, grape, cactus, and apricot samples in Comparative Example 2; Figure 10 DNA gel electrophoresis images of various moss samples in Comparative Example 2; Figure 11 DNA gel electrophoresis images of various samples from shiitake mushrooms and rubber trees in Comparative Example 2; Figure 12 DNA gel electrophoresis images of various samples of Mongolian oak in Comparative Example 2; Figure 13 The images show DNA gel electrophoresis results of kiwifruit samples under different BT reagents in Comparative Example 3. Figure 14 The images show DNA gel electrophoresis results of various shiitake mushroom samples under different BT reagents in Comparative Example 3. Figure 15 DNA gel electrophoresis images of kiwifruit samples under different treatment methods in Comparative Example 4; Figure 16 DNA gel electrophoresis images of shiitake mushroom samples under different treatment methods in Comparative Example 4; Detailed Implementation Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0016] When the inventors used conventional DNA extraction kits and methods to extract DNA from Siberian apricot leaves and other biological samples rich in polysaccharides and / or polyphenols, the results were mostly extremely low total DNA content (mostly <1 μg) and poor purity (A260 / 280 <1.7), indicating the limitations of existing technologies in processing such special samples rich in polysaccharides and interfering substances. To address this technical problem, this invention provides a DNA extraction kit and its accompanying extraction method for removing polysaccharide and polyphenol interference. Through specific reagent combinations and optimized operating procedures, interfering substances such as polysaccharides and polyphenols can be effectively removed, yielding high-purity, high-integrity DNA, thus enabling subsequent molecular biology experiments.

[0017] The DNA extraction kit provided in this embodiment of the invention contains at least the following reagents: BT reagent contains sucrose, KCl, PVP-40, Tris-HCl, EDTA, Triton X-100, NaOH, KOH, EGTA, LiCl, BSA, spermine, spermidine, Tween-20, and sterile water; Buffer A reagent contains Buffer GDB, SDS, RNase, Proteinase K and sterile water, wherein Buffer GDB reagent contains Tris-HCl, EDTA, NaCl and sterile water; Buffer HH reagent contains guanidine hydrochloride, Tris-HCl and sterile water; WB1 reagent contains guanidine hydrochloride, Tris-HCl, ethanol and sterile water; WB2 reagent contains sodium acetate, EDTA, ethanol, and sterile water.

[0018] In this invention, the BT reagent creatively combines multiple polysaccharide removal components: EGTA (chelates calcium ions, disrupting the structure of mucopolysaccharides), LiCl (selectively precipitates polysaccharides and RNA at high concentrations), PVP-40 (binds polyphenols), BSA (blocks polyphenol oxidation and non-specific adsorption), and spermine / spermine (neutralizes the negative charge of nucleic acids, protects DNA integrity, and promotes the separation of polysaccharides from DNA). The synergistic effect of these components allows for the simultaneous removal of various interfering substances, including plant polysaccharides, animal mucopolysaccharides, and fungal polysaccharides—a technical effect that cannot be achieved by simply combining single or multiple components.

[0019] This invention provides a corresponding DNA extraction method, including the following operations: 1) Freeze and then pulverize the biological sample; 2) Add BT reagent to the obtained powder, mix well, centrifuge at 4000 g and discard the supernatant; 3) Add BT reagent to the obtained precipitate, mix well, and centrifuge again at 4000 g to discard the supernatant; 4) Add Buffer A reagent to the obtained precipitate for lysis, centrifuge at 12000 rpm and collect the supernatant into a new tube; 5) Add Buffer HH reagent, isopropanol and magnetic beads (such as Magbeads PN), mix well and let stand, then discard the supernatant; 6) Wash the magnetic beads sequentially with WB1 reagent, WB2 reagent and 80% ethanol, dry the magnetic beads and then add enzyme-free water to dissolve them to obtain the nucleic acid product.

[0020] Existing patents for DNA extraction from polysaccharide and polyphenol plants (such as the modified CTAB method and centrifuge column method) mostly rely on manual operation, which is cumbersome and difficult to automate. In contrast, the method of this invention is based on the principle of magnetic bead purification, and its reagent system and operating procedures are specifically optimized for automated platforms. It can seamlessly adapt to 32-channel and 96-channel automated extraction instruments, solving the long-standing throughput bottleneck in this field and achieving a leap from manual laboratory operation to a high-throughput automated platform. This automation adaptability is a prominent technical feature not reported in currently published patents for polysaccharide and polyphenol plant extraction.

[0021] The embodiments of this invention demonstrate the successful application of the reagent kit and its supporting methods to various biological samples, including plants, animals, and fungi. This overcomes the limitations of traditional reagent kits, such as their limited sample type and ineffectiveness on specific samples like marine animals. Specifically: (1) Positive effects on marine animals and mollusks.

[0022] Marine animal tissues (such as sea urchins, seahorses, clams, and squid) contain abundant mucopolysaccharides, glycogen, and glycoproteins. These substances differ significantly in structure from plant polysaccharides, and conventional plant DNA extraction kits are completely ineffective for such samples—mucopolysaccharides co-precipitate with DNA, forming a gel-like substance that renders the DNA insoluble or of extremely low purity. This invention utilizes the synergistic effect of EGTA and LiCl in the BT reagent: EGTA chelates calcium ions, disrupting the calcium bridge structure of mucopolysaccharides and causing them to dissociate from the DNA; LiCl selectively precipitates mucopolysaccharides at high concentrations, effectively removing them before the magnetic bead binding step; spermine / spermidine protects the DNA from the effects of mucopolysaccharide co-precipitation. As shown in some examples, this invention yields high-quality DNA from various marine animal samples, including sea urchin gonads, seahorse tissue, clam viscera, and squid: the A260 / 280 ratio is concentrated in the ideal range of 1.8-2.0, with a maximum total amount of up to 12.7 μg (clams), significantly higher than conventional methods (conventional methods typically cannot obtain detectable DNA from such samples).

[0023] (2) Positive effects on fungal samples.

[0024] Fungi (such as shiitake mushrooms) have thick cell walls containing large amounts of chitin and glucan, making lysis difficult and subject to significant polysaccharide interference. The high concentration of LiCl and alkaline components (NaOH, KOH) in the BT reagent of this invention effectively promote cell wall lysis, while PVP-40 and BSA synergistically remove released polysaccharides and polyphenols. As shown in some examples, this invention extracts a total DNA amount of 7.6 μg from shiitake mushroom samples, with an A260 / 280 ratio between 2.03 and 2.15, indicating thorough removal of polysaccharides and proteins, allowing for direct use in downstream molecular experiments.

[0025] (3) Excellent results on highly difficult plant samples.

[0026] The present invention also performs excellently for woody plants rich in resins, tannins, and alkaloids (such as rubber trees, Mongolian oaks, and apricots), as well as fruits rich in polysaccharides and polyphenols (strawberries, kiwifruit, and grapes). In particular, for Mongolian oaks (woody plants with extremely high tannin content), which are generally considered extremely difficult to extract from, the present invention can still yield a total of 0.6-4.2 μg of DNA, while conventional methods often yield very low results for such samples.

[0027] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0028] Example 1 This example provides a broad-spectrum DNA extraction kit capable of removing interference from polysaccharides and polyphenols, comprising the following components: BT reagent: Composed of 3.0 M sucrose, 1.0 M KCl, 0.6 M PVP-40, 0.3 M Tris-HCl, 0.4 M EDTA, 3% Triton X-100, 0.1 M NaOH, 0.1 M KOH, 0.08 M EGTA, 0.1 M LiCl, 5% BSA, 3 M spermine, 3 M spermidine, 2% Tween-20, and the balance being sterile water.

[0029] Buffer A reagent: consists of 90% Buffer GDB, 5% SDS, 2% RNase (10 mg / ml), 2% Proteinase K (20 mg / ml) and the remainder sterile water.

[0030] Buffer GDB reagent: consists of 1.0 M Tris-HCl, 1 M EDTA, 2 M NaCl and the remainder sterile water.

[0031] Buffer HH reagent: consists of 3 M guanidine hydrochloride, 0.8 M Tris-HCl and the remainder sterile water.

[0032] WB1 reagent consists of 3 M guanidine hydrochloride, 1.0 M Tris-HCl, 30% anhydrous ethanol, and the remainder sterile water.

[0033] WB2 reagent: consists of 2 M sodium acetate, 1 M EDTA, 70% anhydrous ethanol and the remainder sterile water.

[0034] In addition to the reagents mentioned above, the kit may also contain magnetic beads (such as Magbeads PN) for DNA purification, as well as auxiliary reagents such as 80% ethanol, isopropanol, and enzyme-free water.

[0035] Example 2 Based on the kit in Example 1, this example provides a method for manually extracting DNA, including the following steps: ① Take about 0.2 g of sample, freeze it with liquid nitrogen, add it to a centrifuge tube containing steel balls, and grind it into powder using a grinder; ② Add 1 mL of BT reagent, shake to mix, centrifuge at 4℃ and 4000 g for 10 minutes, and discard the supernatant; ③ Add 1 mL of BT reagent to the precipitate, mix well, centrifuge at 4℃ and 4000 g for 5 minutes, and discard the supernatant; ④ Add Buffer A reagent to the precipitate, mix well by pipetting, lyse at 56℃ for 30 minutes, centrifuge at 12000 rpm for 1 minute, and transfer the supernatant to a new tube; ⑤ Add 280 μL Buffer HH reagent, 320 μL isopropanol, and 20 μL Magbeads PN. Vortex to mix and let stand at room temperature for 10 minutes. ⑥ Place on a magnetic rack and let stand for 30 seconds, then discard the supernatant; ⑦ Add 500 μL of WB1 reagent to the centrifuge tube, keep the centrifuge tube on the magnetic rack, rotate the centrifuge tube 360 ​​degrees, so that the magnetic bead shuttles back and forth in the reagent, repeat several times and then remove it. ⑧ Add 500 μL of WB2 reagent to the centrifuge tube, keep the centrifuge tube on the magnetic rack, rotate the centrifuge tube 360 ​​degrees, so that the magnetic bead shuttles back and forth in the reagent, repeat several times and then remove it. ⑨ Add 500 μL of 80% alcohol to the centrifuge tube, keep the centrifuge tube on the magnetic rack, rotate the centrifuge tube 360 ​​degrees, so that the magnetic beads shuttle back and forth in the alcohol, repeat several times and then remove the tube; ⑩ Dry the magnetic beads until they are matte, add 100 μL of enzyme-free water and re-dissolve them at 37℃ for 15-30 minutes, then remove them.

[0036] DNA purity and concentration were detected using Nanodrop and Qubit (quality testing), and DNA length was determined using gel electrophoresis (integrity testing).

[0037] Example 3 Based on the kit in Example 1, this example provides a method for automated DNA extraction, including the following operations: ① Add two 5 mm steel balls to a 2 mL centrifuge tube, pour liquid nitrogen into a bowl, crush 0.2 g of sample, place the sample in a 2 mL centrifuge tube and grind it into powder using a grinder; ② Add 1 mL of BT reagent to the sample tube, shake well to mix, centrifuge at 4℃ and 4000 g for 10 minutes, and discard the supernatant; ③ After centrifugation, add 1 mL of BT reagent to the precipitate, mix well, centrifuge at 4000 g for 5 minutes at 4℃, and discard the supernatant; ④ After centrifugation, add the precipitate to the prepared Buffer A and mix by suction and stirring. Then, lyse and digest at 56°C for 30 minutes, shaking once in the middle. ⑤ After lysis, the sample was centrifuged at 12,000 rpm for 1 minute. The supernatant was then transferred to a dispensed deep-well plate. ⑥ Instrument setup: Place the deep well plate with reagents into the instrument and run the program. The instrument used can be a 32-channel or 96-channel automated extractor. You can refer to the table for sample loading layout.

[0038] Table 1

[0039] After the procedure is complete, transfer the nucleic acid product to a 1.5 mL centrifuge tube. Use Nanodrop and Qubit to detect DNA purity and concentration, and use gel electrophoresis to determine DNA length.

[0040] As can be seen, simply loading the samples and reagents into the deep well plate and running the preset program can complete the parallel extraction of 32 / 96 samples at once. The processing time for a single batch is only 1-2 hours, which greatly improves experimental throughput and efficiency and reduces human error.

[0041] Example 4 Following the method described in Example 3, DNA extraction was performed on papaya leaves as the sample to be tested. A total of 6 samples were collected, and the detection results of the DNA from different samples are shown in Table 2 and... Figure 1 As shown.

[0042] Table 2

[0043] Example 5 Referring to the method in Example 3, DNA extraction was performed on various molluscs, various plant samples rich in polysaccharides and polyphenols, and fungi as samples to be tested.

[0044] The results of DNA testing for each sample are shown in Table 3. Figure 2 This is a gel electrophoresis image of DNA from a gonad sample of *Sargassum fusiforme*. Figure 3 This is a gel electrophoresis image of DNA from a baobab tree sample. Figure 4 DNA gel electrophoresis images of squid (lanes 1-10), strawberry, kiwi, and grape (lanes 11-40) samples. Figure 5 DNA gel electrophoresis images of cactus, apricot, moss, and seahorse samples. Figure 6 DNA gel electrophoresis images of samples from shiitake mushrooms, rubber trees, Mongolian oaks, and clam viscera.

[0045] Table 3

[0046] Comparative Example 1 In this example, a commercially available standard DNA extraction kit was used to extract DNA from multiple Siberian apricot leaves. The specific extraction procedure is as follows: Lysis: Transfer the sample to a 1.5 mL centrifuge tube, add 20 μL proteinase K + 200 μL lysis buffer (containing SDS, Tris buffer, EDTA, and NaCl), and incubate at 56°C for 10-20 minutes. Binding: Add 300 μL isopropanol + 15-25 μL magnetic beads, vortex for 1 minute, incubate at room temperature for 5 minutes, place on a magnetic rack for 30 seconds, and discard the supernatant. Rinse (repeat twice). Remove the magnetic rack, add 500 μL washing buffer (Tris buffer + EDTA), and vortex to resuspend. Place on a magnetic rack for 30 seconds and discard the supernatant. Drying: Open the cap and air dry for 2-5 minutes. Elution: Add 50-100 μL elution buffer and resuspend by pipetting. Place on a magnetic rack for 2 minutes and transfer the supernatant to a new tube.

[0047] The extracted samples underwent quality testing, and the results are shown in Table 4 and... Figure 7 As shown.

[0048] Table 4

[0049] Comparative Example 2 In this example, a commercially available standard DNA extraction kit (same as Comparative Example 1) was used to extract DNA from the same sample as in Example 5. The results are shown in Table 5 and... Figure 8-12 As shown, where Figure 8 This is a gel electrophoresis image of baobab DNA. Figure 9 DNA gel electrophoresis images of strawberries, kiwis, grapes, cacti, and apricots. Figure 10 This is a gel electrophoresis image of moss DNA. Figure 11 These are gel electrophoresis images of DNA from shiitake mushrooms and rubber trees. Figure 12 Image of Mongolian oak DNA gel electrophoresis.

[0050] Table 5

[0051] Comparing the data from Examples 4-5 and Comparative Examples 1-2, it is evident that the method of the present invention differs significantly from the prior art in DNA yield (see Table 6) and DNA purity, as detailed below: Table 6

[0052] As shown in Table 6, the total DNA extraction yield of this invention is significantly higher than that of conventional kits for various types of samples, with most samples showing an increase of more than 10 times. Conventional methods are completely ineffective for difficult-to-extract samples such as marine animals, while this invention can obtain high-quality DNA.

[0053] Regarding DNA purity, the A260 / 280 ratio of the method of this invention is concentrated in the ideal range of 1.8-2.1, indicating that impurities such as proteins and polysaccharides are thoroughly removed and can be directly used for downstream molecular experiments; while in Comparative Examples 1-2, the A260 / 280 ratio is mostly between 1.1-1.7, and some samples are even below 1.3, indicating serious polysaccharide and protein contamination, which cannot meet the requirements of downstream experiments.

[0054] Meanwhile, the method of this invention can successfully extract samples from more than 10 types of samples, including marine animals, mollusks, fungi, woody plants, fruits, and mosses, achieving broad applicability across species. In contrast, Comparative Examples 1-2 performed poorly on all types of samples tested, especially for high-difficulty samples such as marine animals and fungi, with extremely low yields or complete ineffectiveness.

[0055] Comparative Example 3 This example modifies the BT reagent in the kit to verify the synergistic effect between the components of the BT reagent. The specific experimental and detection results are as follows: Prepare different BT reagents as shown in Table 7.

[0056] Table 7

[0057] Other reagents were the same as in Example 1. Taking shiitake mushrooms and kiwifruit as examples, the DNA extraction effects of different kits were tested according to the method in Example 3, and the results are shown in Table 8. Figure 13-14 As shown (lanes 1-8 correspond to formula groups 1-8 in sequence).

[0058] Table 8

[0059] From the above results, we can conclude that: The complete formulation group 1 achieved ideal results in both samples: kiwi fruit yield 4.97 μg, A260 / 280=1.93, shiitake mushroom yield 13.23 μg, A260 / 280=2.07, with clear and complete bands.

[0060] The absence of a single component led to a precipitous drop in yield: the yields of groups 2-6 (lacking any one component) decreased by 79%-99.8% compared to group 1, with varying degrees of degradation in the bands and significant deviations in purity from the ideal range. Specifically, the absence of EGTA or LiCl resulted in a 79%-94% decrease in yield and band degradation; the absence of BSA, spermine, or spermidine resulted in yields exceeding 99%, with severe band degradation or extremely low yields. This demonstrates that each of the five components plays a crucial role and none can be omitted.

[0061] Some component combinations were completely ineffective: the yields of Group 7 (EGTA + LiCl only) and Group 8 (without EGTA / LiCl) were only 0-4% of the complete formulation, with extremely low or severely degraded band yields, far lower than Groups 2-6 which lacked a single component. This proves that all five components must be present simultaneously to achieve a synergistic effect.

[0062] Sample variability in component contributions: BSA, spermine, and spermidine have more prominent effects in fungal samples (yield decreases by more than 99.5% when absent), while EGTA and LiCl have more significant effects in plant samples (yield decreases by 93%-94% when absent). The five components complement and synergistically achieve broad applicability to a wide range of samples.

[0063] The above results indicate that the simultaneous presence of all five components (EGTA, LiCl, BSA, spermine, and spermidine) in the BT reagent produces a significant synergistic effect, and its extraction efficiency is far superior to that of formulations lacking any one component or containing only some of the components.

[0064] Comparative Example 4 Unlike Example 2, this example modifies the extraction method, as detailed in Table 9: Table 9

[0065] Taking shiitake mushrooms and kiwifruit as examples, DNA was extracted and nucleic acid products were detected using the different processing methods described above. The results are shown in Table 10. Figure 15-16 As shown (lanes 1-5 correspond to treatment groups 1-5 in sequence).

[0066] Table 10

[0067] From the above results, we can conclude that: The method of the present invention (i.e., treatment group 1) performed excellently in both samples: kiwi fruit yield 14.28 μg, A260 / 280=1.98, shiitake mushroom yield 5.96 μg, A260 / 280=1.85, with clear and complete bands.

[0068] The BT washing step is indispensable: After omitting BT washing in treatment group 2, the yield of kiwi fruit decreased by 99.3%, DNA in shiitake mushrooms was completely undetectable, the purity was extremely poor (A260 / 280≤0.52), and bands could not be produced or the yield was extremely low.

[0069] The order of steps cannot be changed: In treatment group 3, BT washing was placed after lysis, and DNA could not be detected in either sample (yield was zero), the purity was extremely poor, and no bands could be generated.

[0070] Washing solution must be removed: In treatment group 4, the washing solution was not discarded, resulting in a 91%-99.6% decrease in yield, severe deterioration in purity (A260 / 280≤0.25), and band purity exceeding the acceptable range.

[0071] The purification method is irreplaceable: In treatment group 5, the magnetic bead purification was replaced with isopropanol precipitation, and the yield decreased by 90.8%-95.7%, the purity was severely deteriorated (A260 / 280≤0.98), the bands were degraded or the purity was poor, indicating that there is a specific coordination relationship between magnetic bead purification and the previous steps.

[0072] The above results indicate that the steps in the method of the present invention have a close synergistic relationship. The omission of any step, the change of order, or the replacement of the purification method all lead to a decrease in extraction efficiency of more than 90%, indicating that the overall technical solution of the present invention is indivisible.

[0073] In summary, this invention solves the technical problems of low purity, poor yield, and difficulty in high-throughput automation of DNA extraction from various polysaccharide-rich samples such as plants, marine animals, mollusks, and fungi by using BT reagents with specific synergistic combinations of components and an optimized extraction process. It also achieves the technical effect of unifying the removal of polysaccharide interference and broad applicability.

[0074] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A broad-spectrum DNA extraction kit for removing interference from polysaccharides and polyphenols, characterized in that, It contains at least the following reagents: BT reagent contains sucrose, KCl, PVP-40, Tris-HCl, EDTA, Triton X-100, NaOH, KOH, EGTA, LiCl, BSA, spermine, spermidine, Tween-20, and sterile water; Buffer A reagent contains Buffer GDB, SDS, RNase, Proteinase K and sterile water, wherein Buffer GDB reagent contains Tris-HCl, EDTA, NaCl and sterile water; Buffer HH reagent contains guanidine hydrochloride, Tris-HCl and sterile water; WB1 reagent contains guanidine hydrochloride, Tris-HCl, ethanol and sterile water; WB2 reagent contains sodium acetate, EDTA, ethanol, and sterile water.

2. The broad-spectrum DNA extraction kit according to claim 1, characterized in that, The BT reagent contains 3.0 M sucrose, 1.0 M KCl, 0.6 M PVP-40, 0.3 M Tris-HCl, 0.4 M EDTA, 3% Triton X-100, 0.1 M NaOH, 0.1 M KOH, 0.08 M EGTA, 0.1 M LiCl, 5% BSA, 3 M spermine, 3 M spermidine, 2% Tween-20, and the balance being sterile water; the Buffer A reagent consists of 90% Buffer GDB, 5% SDS, 2% 10 mg / ml RNase, 2% 20 mg / ml Proteinase K, and the balance being sterile water, and the Buffer GDB reagent consists of 1.0 M Tris-HCl, 1 M EDTA, 2 M NaCl, and the balance being sterile water.

3. The broad-spectrum DNA extraction kit according to claim 2, characterized in that, The Buffer HH reagent consists of 3M guanidine hydrochloride, 0.8 M Tris-HCl, and the remainder sterile water; the WB1 reagent consists of 3 M guanidine hydrochloride, 1.0 M Tris-HCl, 30% anhydrous ethanol, and the remainder sterile water; and the WB2 reagent consists of 2 M sodium acetate, 1 M EDTA, 70% anhydrous ethanol, and the remainder sterile water.

4. The broad-spectrum DNA extraction kit according to claim 1, characterized in that, It also contains magnetic beads for purifying DNA.

5. The application of the broad-spectrum DNA extraction kit as described in any one of claims 1-4 in the extraction of nucleic acids from biological samples.

6. The application according to claim 5, characterized in that, The biological sample is rich in polysaccharides and / or polyphenols, and the biological sample includes tissue samples derived from papaya, baobab, strawberry, kiwi, grape, cactus, apricot, moss, shiitake mushroom, rubber tree, Mongolian oak, sea urchin, seahorse, clam or squid.

7. A method for DNA extraction that removes interference from polysaccharides and polyphenols, characterized in that, Extraction was performed using the broad-spectrum DNA extraction kit according to any one of claims 1-4.

8. The method according to claim 7, characterized in that, Includes the following steps: S1. After freezing the biological sample, pulverize it and add BT reagent to the resulting powder for washing. S2. Add Buffer A reagent to the obtained precipitate for lysis, centrifuge, and take the supernatant into a new tube; S3. Add Buffer HH reagent, isopropanol and magnetic beads, mix well and let stand, then discard the supernatant. S4. Wash the magnetic beads sequentially with WB1 reagent, WB2 reagent and 80% ethanol, dry the magnetic beads and then add enzyme-free water to dissolve them to obtain the nucleic acid product.

9. The method according to claim 8, characterized in that, In step S1, the BT reagent is used to wash twice, and the washing method is as follows: after adding the BT reagent, the mixture is shaken and mixed, and then the supernatant is discarded after centrifugation at 2000-5000 g.

10. The method according to claim 8, characterized in that, In step S2, the pyrolysis conditions are pyrolysis at 37-65℃ for 20-70 minutes, and the centrifugation speed is 9000-12500 rpm.

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