Method and kit for extracting microbial dna from marine sediments

Abstract

This invention provides a method and kit for extracting microbial DNA from marine sediments. The extraction method includes: a) washing the marine sediments with a washing buffer to obtain a washed sample; wherein the washing buffer includes spermidine and sodium pyrophosphate; b) lysing the microbial cells in the washed sample to obtain a lysed sample; c) recovering, screening, and purifying the DNA from the lysed sample to obtain microbial DNA. This method solves the problem of poor DNA extraction efficiency in existing technologies and is applicable to the field of DNA extraction.

Description

Technical Field

[0001] This invention relates to the field of DNA extraction, and more specifically, to a method and kit for extracting microbial DNA from marine sediments. Background Technology

[0002] The ocean is the largest ecosystem on Earth, containing a vast number of bacterial and archaic cells, approximately 10^29, which play a central role in marine biogeochemical cycles and ecological processes. The broad taxonomic range and metabolic diversity of these microorganisms, along with their rapid adaptability to the environment, enable them to adapt to diverse conditions and play a crucial role in ecological functions. Metagenomic studies of marine sediments are of paramount importance for a deeper understanding of the operational mechanisms of marine ecosystems and for the protection of marine ecological environments.

[0003] In recent years, single-molecule sequencing technology based on long-read sequencing has received widespread attention due to its significant role in addressing the fragmentation problem of metagenomic MAGs (Metagenome Assembled Genomes). Compared with the short-read of NGS (Next Generation Sequencing) sequencing, single-molecule long-read sequences can cover longer DNA fragments, providing more accurate and complete genome assembly. This is crucial for distinguishing genomically similar species or strains, resolving splicing problems of repetitive sequence regions, and improving the accuracy and consistency of MAGs. At the same time, while providing superior sequencing results, single-molecule sequencing places higher demands on the DNA used; the DNA used for single-molecule sequencing needs to be at least 30kb in length and of high purity.

[0004] However, existing long-fragment DNA extraction technologies mainly focus on plant and animal samples. For marine sediment samples, a universal and efficient DNA extraction method is lacking. Marine sediment samples, due to their unique complexity (including but not limited to low biomass, tight cell-particle binding, and the presence of inhibitors such as humic acid and heavy metal ions), present significant challenges in extracting diverse and high-quality total microbial DNA. In particular, the abundance of minerals and heavy metal ions in deep-sea sediments creates additional barriers to downstream molecular reactions, significantly raising the technical threshold for DNA extraction.

[0005] Current DNA extraction procedures, such as lysis followed by precipitation or recovery of lysed DNA using silica purification columns, are primarily designed for plant and animal tissues. These tissues are characterized by relatively simple cellular composition, large sample volumes, and impurities that are mostly polysaccharides and polyphenols. These procedures are often unsuitable for DNA extraction from marine sediments because they fail to effectively address the issues of DNA length, purity, and inhibitors in marine sediment samples. Therefore, existing technologies have significant limitations in achieving efficient, high-purity, and long-fragment DNA extraction from marine sediments. Given these challenges and limitations, developing a highly efficient technology specifically for DNA extraction from marine sediments is particularly urgent. Summary of the Invention

[0006] The main objective of this invention is to provide a method and kit for extracting microbial DNA from marine sediments, thereby solving the problem of poor extraction efficiency of DNA from marine sediments in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for extracting microbial DNA from marine sediments is provided, the extraction method comprising: a) washing the marine sediments with a washing buffer to obtain a washed sample; wherein the washing buffer comprises spermidine and sodium pyrophosphate; b) lysing the microbial cells in the washed sample to obtain a lysed sample; and c) recovering, screening and purifying the DNA from the lysed sample to obtain microbial DNA.

[0008] Further, the concentration of spermidine in the washing buffer is 0.5 mM-2 mM; preferably, the concentration of sodium pyrophosphate in the washing buffer is 30-60 mM; preferably, the washing buffer includes 10-15 mM Tris-HCl and 10-35 mM... The washing buffer contains EDTA, 70-120 mM potassium chloride, 0.5-2 mM spermine, 0.5-2 mM spermidine, 0.2-0.8% (v / v) Triton X-100, 1-3 wt% polyvinylpyrrolidone 40, and 30-60 mM sodium pyrophosphate. Preferably, the pH of the washing buffer is 9.0-9.5. Preferably, a) includes washing the marine sediment at least twice with the washing buffer to obtain a washed sample. Preferably, the washing in a) includes mixing the marine sediment with the washing buffer, centrifuging, and removing the supernatant. Preferably, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 8-15 min. Preferably, the washing buffer is pre-cooled at 0-4°C before washing.

[0009] Further, wherein b) includes: b1) mixing the washed sample with a first lysis buffer and lysozyme, incubating, performing a first lysis, and obtaining a first lysis system; b2) mixing the first lysis system with a second lysis buffer, incubating, performing a second lysis, and obtaining a lysed sample; wherein the first lysis buffer includes triton, the second lysis buffer includes a protein denaturant, and the first lysis buffer and / or the second lysis buffer also contain a protease.

[0010] Further, the first lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1M phosphate ions, which also contains 20-50mM Tris-HCl, 0.5-3mM spermine, 0.5-3mM spermidine, 10-40mM EDTA, 0.5-1.5wt% PEG8000, 5-15mM NaCl, and 0.25%-1% (v / v) Triton X-100; preferably, the protease is proteinase K; preferably, the final concentration of lysozyme in the first lysis is 1 mg / mL; preferably, the final concentration of proteinase K in the second lysis is 0.16 mg / mL; preferably, the phosphate ions are derived from sodium dihydrogen phosphate and disodium hydrogen phosphate; preferably, the concentration of sodium dihydrogen phosphate in the phosphate buffer is 5.3mM-93.2mM, and correspondingly, the concentration of disodium hydrogen phosphate is 94.7mM-6.8mM.

[0011] Further, the protein denaturing agent includes guanidine hydrochloride; preferably, the second lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1M phosphate ions, which also contains 20-50mM Tris-HCl, 0.5-3mM spermine, 0.5-3mM spermidine, 10-40mM EDTA, 0.5-1.5wt% PEG8000, 5-15mM NaCl, 0.25%-1% (v / v) Triton X-100, and 3-5M guanidine hydrochloride; preferably, the phosphate ions are derived from sodium dihydrogen phosphate and disodium hydrogen phosphate; preferably, the concentration of sodium dihydrogen phosphate in the phosphate buffer is 5.3mM-93.2mM, and correspondingly, the concentration of disodium hydrogen phosphate is 94.7mM-6.8mM.

[0012] Further, the temperature of the first lysis is 35-38℃, and the time of the first lysis is 30-40 min; preferably, b2) includes: mixing the first lysis system with the second lysis solution, wherein the volume ratio of the first lysis system to the second lysis solution is 3-5:1, and performing the second lysis to obtain the lysed sample; preferably, the temperature of the second lysis is 55-75℃, and the time of the second lysis is 40-80 min.

[0013] Further, c) includes: c1) mixing the lysed sample with an equal volume of phenol-chloroform-isoamyl alcohol solution, centrifuging and collecting the supernatant containing DNA; c2) adding isopropanol and sodium acetate solution to the supernatant containing DNA, allowing it to stand and then centrifuging to obtain DNA precipitate; c3) washing the DNA precipitate and resolving it to obtain DNA solution; c4) mixing the DNA solution, screening buffer 1 and screening buffer 2, centrifuging to obtain microbial DNA.

[0014] Further, in the phenol-chloroform-isoamyl alcohol mixture, the volume ratio of phenol, chloroform, and isoamyl alcohol is 25:24:1; preferably, the volume ratio of the DNA-containing supernatant to isopropanol is 1:0.7; preferably, the sodium acetate concentration in the sodium acetate solution is 3M, and the volume ratio of the DNA-containing supernatant to the sodium acetate solution is 1:0.1; preferably, the settling in c2) includes settling at 20-35°C for 30 min; preferably, the ionization in c2) includes centrifugation at 8000 rpm for 10 min; preferably, in c3), after washing the DNA precipitate twice with 70% (v / v) ethanol solution, the DNA precipitate is dissolved in TE buffer to obtain a DNA solution; preferably, the volume ratio of the DNA solution, screening buffer 1, and screening buffer 2 is 1:0.6:0.6; preferably, screening buffer 1 includes: 20 mM Tris-HCl; 2 mM EDTA; 10 wt% PEG8000; 1.6 M NaCl, the pH of screening buffer 1 is 8; preferably, screening buffer 2 comprises: 2% (w / v) PVP360000, 1.2M NaCl, 20mM Tris-HCl, and the pH of screening buffer 2 is 8.

[0015] To achieve the above objectives, according to a second aspect of the present invention, a kit for extracting microbial DNA from marine sediments is provided, the kit comprising a washing buffer comprising spermidine and sodium pyrophosphate.

[0016] Further, the concentration of spermidine in the washing buffer is 0.5 mM-2 mM; preferably, the concentration of sodium pyrophosphate in the washing buffer is 30-60 mM; preferably, the washing buffer comprises 10-15 mM Tris-HCl, 10-35 mM EDTA, 70-120 mM potassium chloride, 0.5-2 mM spermidine, 0.5-2 mM spermidine, 0.2-0.8% (v / v) Triton X-100, 1-3 wt% polyvinylpyrrolidone 40, and 30-60 mM sodium pyrophosphate; preferably, the pH of the washing buffer is 9.0-9.5; preferably, the kit also includes a lysis buffer for lysing microbial cells, the lysis buffer comprising a first lysis buffer and a second lysis buffer; the first lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1 M phosphate ions, and further containing 20-50 mM Tris-HCl, 0.5-3 mM spermine, 0.5-3 mM spermidine, 10-40 mM EDTA, 0.5-1.5 wt% PEG8000, 5-15 mM NaCl, 0.25%-1% (v / v) Triton X-100; the second lysis buffer is a phosphate buffer at pH 7.5-8.5 containing 0.1 M phosphate ions, which also contains 20-50 mM Tris-HCl, 0.5-3 mM spermine, 0.5-3 mM spermidine, 10-40 mM EDTA, 0.5-1.5 wt% PEG8000, 5-15 mM NaCl, 0.25%-1% (v / v) Triton X-100, 3-5M guanidine hydrochloride; preferably, the first lysis buffer also contains lysozyme; preferably, the first lysis buffer and / or the second lysis buffer also contain a protease; more preferably, the protease is proteinase K; preferably, the kit also includes screening buffer 1 and screening buffer 2 for screening and purifying DNA, screening buffer 1 comprising: 20mM Tris-HCl; 2mM EDTA; 10wt% PEG8000; 1.6M NaCl, the pH of screening buffer 1 is 8; screening buffer 2 comprises: 2% (w / v) PVP360000, 1.2M NaCl, 20mM Tris-HCl, the pH of screening buffer 2 is 8.

[0017] By applying the technical solution of this invention and utilizing the above-mentioned extraction method, by washing the marine sediment sample with a washing buffer containing spermidine and sodium pyrophosphate before lysis (preferably at least twice), it is possible to reduce interfering substances in the marine sediment and obtain DNA with high purity from the marine sediment, thus meeting the needs of subsequent sequencing of this DNA. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 An electrophoresis result diagram according to Embodiment 1 of the present invention is shown.

[0020] Figure 2 An electrophoresis result diagram according to Embodiment 2 of the present invention is shown. Detailed Implementation

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0022] Terminology Explanation:

[0023] Marine sediments: refers to the general term for seabed sediments formed by various marine sedimentation processes. Sediments are classified according to depth into nearshore sediments (0-20 meters), shallow sea sediments (20-200 meters), semi-deep sea sediments (200-2000 meters), and deep sea sediments (greater than 2000 meters).

[0024] As mentioned in the background section, existing DNA extraction methods are not suitable for extracting DNA from marine sediments and cannot meet the DNA sequencing sample requirements of technologies such as single-molecule sequencing. Therefore, in this application, the inventors attempted to develop a method for extracting microbial DNA from marine sediments, and based on this, proposed a series of protection schemes for this application.

[0025] In a first typical embodiment of this application, a method for extracting microbial DNA from marine sediments is provided. The extraction method includes: a) washing the marine sediments with a washing buffer to obtain a washed sample; wherein the washing buffer includes spermidine and sodium pyrophosphate; b) lysing the microbial cells in the washed sample to obtain a lysed sample; and c) recovering, screening, and purifying the DNA from the lysed sample to obtain microbial DNA.

[0026] Using the above extraction method, washing marine sediments with a washing buffer can remove impurities that affect DNA extraction, such as humic substances and salts. The extraction method provided by this invention is low-cost and simple to operate, and can be used for both routine marine environmental samples, such as nearshore sediments (e.g., nearshore and shallow-sea sediments), and samples from deep-sea environments (e.g., semi-deep-sea and deep-sea sediments), such as those from cold seeps and seamounts. The nucleic acid extracted using this method yields a large quantity, high purity, and a length sufficient for single-molecule sequencing.

[0027] In the above extraction method, the inventors unexpectedly discovered that spermidine and sodium pyrophosphate can improve the extraction efficiency of microbial DNA from marine sediments. The hypothesized principle is as follows: spermidine can extend the DNA morphology and prevent DNA cleavage damage; sodium pyrophosphate can competitively bind to charged metal or mineral surfaces with nucleic acids, thereby increasing the amount of DNA finally extracted.

[0028] In a preferred embodiment, the concentration of spermidine in the washing buffer is 0.5 mM-2 mM, including but not limited to 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mM; preferably, the concentration of sodium pyrophosphate in the washing buffer is 30-60 mM, including but not limited to 30, 35, 40, 45, 50, 55 or 60 mM.

[0029] Using a washing buffer containing spermidine and sodium pyrophosphate at the aforementioned concentrations allows for the extraction of a larger quantity of DNA. Furthermore, the extracted DNA exhibits higher purity, with fewer residual proteins, RNA, and other impurities. The DNA purity characterization includes, but is not limited to, indicators such as OD260 / 280 and OD260 / 230. In other words, by using the aforementioned washing buffer to wash marine sediments in the extraction method, DNA with a purity sufficient for subsequent sequencing can be obtained.

[0030] In a preferred embodiment, the washing buffer comprises 10-15 mM Tris-HCl, 10-35 mM EDTA, 70-120 mM potassium chloride, 0.5-2 mM spermine, 0.5-2 mM spermidine, 0.2-0.8% (v / v) Triton X-100, 1-3 wt% polyvinylpyrrolidone 40 (PVP40), and 30-60 mM sodium pyrophosphate; preferably, the pH of the washing buffer is 9.0-9.5; preferably, a) comprises: washing the marine sediment at least twice with the washing buffer to obtain a washed sample; preferably, the washing in a) comprises: mixing the marine sediment with the washing buffer, centrifuging, and removing the supernatant; preferably, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 8-15 min; preferably, the washing buffer is pre-cooled at 0-4°C before washing.

[0031] In step a) above, it is preferable to wash the marine sediment sample at least twice using the aforementioned washing buffer to better remove interfering components from the sample. Multiple washing steps include, but are not limited to: mixing the marine sediment with the washing buffer, centrifuging, removing the supernatant, and then mixing the precipitate with fresh washing buffer for a second washing.

[0032] In a preferred embodiment, b) includes: b1) mixing the washed sample with a first lysis buffer and lysozyme, incubating, and performing a first lysis to obtain a first lysis system; b2) mixing the first lysis system with a second lysis buffer, incubating, and performing a second lysis to obtain a lysed sample; wherein the first lysis buffer includes Triton, and the second lysis buffer includes a protein denaturant; the first lysis buffer and / or the second lysis buffer also contain a protease.

[0033] In the above extraction method, a two-step lysis approach is preferably used to release DNA from the sample. The first lysis primarily aims to dissolve the cell walls of microorganisms in marine sediments under the combined action of lysozyme and Triton, resulting in more complete cell rupture and the release of proteins. The second lysis utilizes protein denaturants and proteases to further dissolve proteins at higher temperatures, leading to the disruption of cell structure and the secondary structure of nucleoproteins, thus detaching proteins from nucleic acids (DNA). This two-step lysis approach, by more fully releasing DNA and reducing the binding of DNA to impurities such as proteins, further improves the yield and purity of the subsequently obtained DNA. In this two-step lysis process, proteases can be introduced into the lysis system either in the first lysis or in the second lysis.

[0034] The protease (proteinase K) and lysozyme used in this application are commercially available products. The above-mentioned enzymes in the prior art can achieve the same technical effect in the extraction method of this application.

[0035] In a preferred embodiment, the first lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1 M phosphate ions, further containing 20-50 mM Tris-HCl, 0.5-3 mM spermine, 0.5-3 mM spermidine, 10-40 mM EDTA, 0.5-1.5 wt% PEG8000, 5-15 mM NaCl, and 0.25%-1% (v / v) Triton X-100; preferably, the protease is proteinase K; preferably, the final concentration of lysozyme is 1 mg / mL; preferably, in the second lysis, the final concentration of proteinase K is 0.16 mg / mL; preferably, the phosphate ions are derived from sodium dihydrogen phosphate and disodium hydrogen phosphate; preferably, the concentration of sodium dihydrogen phosphate in the phosphate buffer is 5.3 mM-93.2 mM, and correspondingly, the concentration of disodium hydrogen phosphate is 94.7 mM-6.8 mM.

[0036] In a preferred embodiment, the protein denaturing agent comprises guanidine hydrochloride; preferably, the second lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1M phosphate ions, further comprising 20-50mM Tris-HCl, 0.5-3mM spermine, 0.5-3mM spermidine, 10-40mM EDTA, 0.5-1.5wt% PEG8000, 5-15mM NaCl, 0.25%-1% (v / v) Triton X-100, and 3-5M guanidine hydrochloride, including but not limited to 3, 4, or 5M; preferably, the phosphate ions are derived from sodium dihydrogen phosphate and disodium hydrogen phosphate; preferably, the concentration of sodium dihydrogen phosphate in the phosphate buffer is 5.3mM-93.2mM, and correspondingly, the concentration of disodium hydrogen phosphate is 94.7mM-6.8mM.

[0037] In a preferred embodiment, the temperature of the first lysis is 33-38°C and the time of the first lysis is 30-40 min; preferably, b2) includes: mixing the first lysis system with the second lysis solution, wherein the volume ratio of the first lysis system to the second lysis solution is 3-5:1, and performing the second lysis to obtain the lysed sample; preferably, the temperature of the second lysis is 55-75°C and the time of the second lysis is 40-80 min.

[0038] In a preferred embodiment, c) includes: c1) mixing the lysed sample with an equal volume of phenol-chloroform-isoamyl alcohol solution, centrifuging and collecting the supernatant containing DNA; c2) adding isopropanol and sodium acetate solution to the supernatant containing DNA, allowing it to stand and then centrifuging to obtain DNA precipitate; c3) washing the DNA precipitate and resolving it to obtain DNA solution; c4) mixing the DNA solution, screening buffer 1 and screening buffer 2, centrifuging to obtain microbial DNA.

[0039] In the above extraction method, DNA is separated and purified from the lysed sample using the method in step c). During this process, small DNA fragments that do not meet the requirements for single-molecule sequencing are removed, further ensuring that the OD260 / 280, OD260 / 230 and other parameters of the obtained DNA product meet the requirements for sequencing (such as single-molecule sequencing).

[0040] In a preferred embodiment, the volume ratio of phenol, chloroform, and isoamyl alcohol in the phenol-chloroform-isoamyl alcohol mixture is 25:24:1; preferably, the volume ratio of the DNA-containing supernatant to isopropanol is 1:0.7; preferably, the sodium acetate concentration in the sodium acetate solution is 3M, and the volume ratio of the DNA-containing supernatant to the sodium acetate solution is 1:0.1; preferably, the settling in c2) includes settling at 20-35°C for 30 min; preferably, the ion setting in c2) includes centrifugation at 8000 rpm for 10 min; preferably, in c3), after washing the DNA precipitate twice with 70% (v / v) ethanol solution, the DNA precipitate is dissolved in TE buffer to obtain a DNA solution; preferably, the volume ratio of the DNA solution, screening buffer 1, and screening buffer 2 is 1:0.6:0.6; preferably, screening buffer 1 includes: 20 mM Tris-HCl; 2 mM EDTA; 10 wt% PEG8000; 1.6 M NaCl, the pH of screening buffer 1 is 8; preferably, screening buffer 2 comprises: 2% (w / v) PVP360000, 1.2M NaCl, 20mM Tris-HCl, and the pH of screening buffer 2 is 8.

[0041] Using the above extraction method, DNA is gradually purified through extraction (extraction) in c1), precipitation in c2), and washing in c3), ensuring high purity and low impurities in the subsequent DNA product. Further, in step c4), the DNA is mixed with screening buffer 1 and screening buffer 2 to remove impurities and small DNA fragments smaller than 10kb, increasing the proportion of longer DNA fragments in the DNA product (i.e., microbial DNA), thus making it more suitable for technologies and platforms with high requirements for extraction length, such as single-molecule sequencing technology.

[0042] It can remove shorter fragments from DNA to obtain marine environmental sample DNA with better integrity and purity, meeting the requirements of Cyclone Seq platform, as well as other single-molecule sequencing technologies and platforms such as Oxford Nanopore Technology and Pacbio that have high requirements for extraction length, thereby obtaining more complete metagenomic MAGs and other data.

[0043] In a second typical embodiment of this application, a kit for extracting microbial DNA from marine sediments is provided. The kit includes a washing buffer comprising spermidine and sodium pyrophosphate.

[0044] In a preferred embodiment, the concentration of spermidine in the washing buffer is 0.5 mM-2 mM; preferably, the concentration of sodium pyrophosphate in the washing buffer is 30-60 mM; preferably, the washing buffer comprises 10-15 mM Tris-HCl and 10-35 mM... EDTA, 70-120 mM potassium chloride, 0.5-2 mM spermine, 0.5-2 mM spermidine, 0.2-0.8% (v / v) Triton X-100, 1-3 wt% (polyvinylpyrrolidone 40, 30-60 mM sodium pyrophosphate); preferably, the washing buffer pH is 9.0-9.5; preferably, the kit also includes a lysis buffer for lysing microbial cells, the lysis buffer comprising a first lysis buffer and a second lysis buffer; the first lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1 M phosphate ions, further containing 20-50 mM Tris-HCl, 0.5-3 mM spermine, 0.5-3 mM spermidine, 10-40 mM The first lysis buffer contains EDTA, 0.5-1.5 wt% PEG8000, 5-15 mM NaCl, and 0.25%-1% (v / v) Triton X-100; the second lysis buffer is a phosphate buffer at pH 7.5-8.5 containing 0.1 M phosphate ions, further containing 20-50 mM Tris-HCl, 0.5-3 mM spermine, 0.5-3 mM spermidine, 10-40 mM EDTA, 0.5-1.5 wt% PEG8000, 5-15 mM NaCl, 0.25%-1% (v / v) Triton X-100, and 3-5 M guanidine hydrochloride; preferably, the first lysis buffer also contains lysozyme; preferably, the first and / or second lysis buffers also contain a protease; more preferably, the protease is proteinase K; preferably, the kit also includes screening buffer 1 and screening buffer 2 for screening and purifying DNA, screening buffer 1 comprising: 20 mM Tris-HCl; 2 mM EDTA; 10 wt% PEG8000; 1.6 M NaCl, the pH of screening buffer 1 is 8; screening buffer 2 consists of: 2% (w / v) PVP360000, 1.2 M NaCl, 20 mM Tris-HCl, the pH of screening buffer 2 is 8.

[0045] The above-mentioned kit includes, but is not limited to, the reagents used in the above extraction methods. These reagents enable the efficient extraction of microbial DNA from marine sediments, yielding DNA products with high yield and purity. Preferably, the washing buffer and lysis buffer work together to improve DNA extraction efficiency. Furthermore, the two screening buffers described above can remove short fragments from the DNA, obtaining marine environmental sample DNA with good integrity and purity, meeting the requirements of the Cyclone Seq platform, as well as other single-molecule sequencing technologies and platforms such as Oxford Nanopore Technology and PacBio that have high requirements for extraction length, thereby obtaining more complete metagenomic MAGs and other data.

[0046] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0047] Example 1: Extraction of deep-sea cold seep sediments

[0048] Extraction was performed using the method of this invention and a commercially available reagent kit, and the results were compared.

[0049] The commercially available kit used was the MGIEAsy fecal genomic DNA (meta) extraction kit, and extraction was performed according to the instructions.

[0050] The method of the present invention is carried out according to the following steps:

[0051] 1. Sediment washing: Weigh 5g of cold seep sediment, place the weighed sediment into a centrifuge tube, add 25ml of washing buffer, mix well, and centrifuge at 8000rpm for 10min. The washing buffer needs to be pre-cooled at 4℃. Wash twice.

[0052] The washing buffer consists of: 10 mM Tris-HCl, 10 mM EDTA, 80 mM potassium chloride, 1 mM spermine, 1 mM spermidine, 0.5% Triton X-100 (volume), 2% PVP40 (polyvinylpyrrolidone 40), and 50 mM sodium pyrophosphate.

[0053] It should be noted that the pH of the cleaning solution needs to be adjusted to 9, and sodium pyrophosphate and Triton should be added immediately before use.

[0054] 2. Pre-lysis of bacterial cells in sediment: Resuspend the washed sediment in 20 mL of extraction buffer, add 200 μL of 20 mg / mL proteinase K (product number GPK003001, manufacturer Gproan) and 200 μL of 100 mg / mL lysozyme (product number A610308-0005, manufacturer BBI), and lyse at 37 °C for 30 minutes.

[0055] The components of the extraction buffer are: Tris-HCl 30mM; spermine 1mM; spermidine 1mM; EDTA 20mM; PEG 8000 1%; NaCl 10mM; Triton 0.5%; sodium dihydrogen phosphate 5.3mM; disodium hydrogen phosphate 94.7mM; pH 8.0.

[0056] 3. Sediment lysis: Add 5 mL of lysis buffer to the above extraction centrifuge tube, invert to mix, transfer to 65℃ and continue lysis for 1 hour.

[0057] The components of the lysis buffer are: Tris-HCl 30mM, spermine 1mM, spermidine 1mM, EDTA 20mM, PEG 8000 1%, NaCl 10mM, Triton 0.5%, sodium dihydrogen phosphate 5.3mM, disodium hydrogen phosphate 94.7mM, and guanidine hydrochloride 4M.

[0058] 4. DNA Recovery: Add an equal volume of phenol, chloroform, and isoamyl alcohol (25:24:1), centrifuge at 8000 rpm at room temperature to obtain the supernatant, transfer the supernatant to a new centrifuge tube, add 0.7 volumes of isopropanol and 1 / 10 volume of 3M sodium acetate solution, let stand at room temperature for 30 minutes, then centrifuge at 8000 rpm for 10 minutes at room temperature to precipitate the DNA. Resuspend the precipitate with a wide-mouth pipette tip and wash the precipitate twice with 5 mL of 70% anhydrous ethanol, then dissolve it in 200 μL of TE buffer.

[0059] 5. DNA screening and purification: Add 0.6 volumes of screening buffer 1 and screening buffer 2, and centrifuge at 10,000 rpm at room temperature for 25 minutes to separate impurities and small DNA molecules.

[0060] The components of the screening buffer 1 are: Tris-HCl 20mM, EDTA 2mM, PEG8000 10%, NaCl 1.6M, pH=8;

[0061] The components of the screening buffer 2 are: 2% w / v PVP 360000, 1.2M NaCl, 20mM Tris-HCl, pH=8.

[0062] 6. DNA Detection and Sequencing: DNA fragment lengths are determined using pulsed-field electrophoresis, and the results are as follows: Figure 1 As shown in Table 1, the concentration was detected using qubit4.0 and the purity was detected using nanodrop.

[0063] The results in Table 1 show that for deep-sea cold seep sediment samples, the total amount and purity of DNA extracted using the present invention are superior to those of commercial kits. The DNA extracted by the method of the present invention meets the sequencing requirements for DNA purity of third-generation sequencing PacBio sequencing: OD260 / 280 1.8-2.0 and OD260 / 230 1.8-2.5.

[0064] After passing the tests, the DNA extracted using this method was further sequenced using the PacBio platform. The data quality is shown in Table 2.

[0065] Example 2: Extraction of sediments from the East China Sea continental shelf

[0066] Extraction was performed using the method of this invention and a commercially available reagent kit, and the results were compared.

[0067] The commercially available kit used was the MGIEAsy fecal genomic DNA (meta) extraction kit, and extraction was performed according to the instructions.

[0068] The method of the present invention is carried out according to the following steps:

[0069] 1. Sediment washing: Weigh 7g of sediment from the East China Sea continental shelf, place the weighed sediment into a centrifuge tube, add 25mL of washing buffer, mix well, and centrifuge at 8000rpm for 10min. The washing buffer needs to be pre-cooled at 4℃. Wash twice.

[0070] The washing buffer consists of: 10 mM Tris-HCl, 10 mM EDTA, 80 mM potassium chloride, 1 mM spermine, 1 mM spermidine, 0.5% Triton X-100 (volume), 2% PVP40 (polyvinylpyrrolidone), and 50 mM sodium pyrophosphate.

[0071] It should be noted that the pH of the cleaning solution needs to be adjusted to 9, and sodium pyrophosphate and triton should be added immediately before use.

[0072] 2. Pre-lysis of bacterial cells in sediment: Resuspend the washed sediment in 20 mL of extraction buffer, add 200 μL of 20 mg / mL proteinase K and 200 μL of 100 mg / mL lysozyme, and lyse at 37 °C for 30 minutes.

[0073] The components of the extraction buffer are: Tris-HCl 30mM; spermine 1mM; spermidine 1mM; EDTA 20mM; PEG 8000 1%; NaCl 10mM; 0.5% Triton X100 (volume), sodium dihydrogen phosphate 5.3mM, disodium hydrogen phosphate 94.7mM, pH 8.0.

[0074] 3. Sediment lysis: Add 5 mL of lysis buffer to the above extraction centrifuge tube, invert to mix, transfer to 65℃ and continue lysis for 1 hour.

[0075] The components of the lysis buffer are: Tris-HCl 30mM; spermine 1mM; spermidine 1mM; EDTA 20mM; PEG 8000 1%; NaCl 10mM; 0.5% Triton (volume), sodium dihydrogen phosphate 5.3mM, disodium hydrogen phosphate 94.7mM, and guanidine hydrochloride 4M.

[0076] 4. DNA Recovery: Add an equal volume of phenol, chloroform, and isoamyl alcohol (25:24:1), centrifuge at 8000 rpm at room temperature to obtain the supernatant, transfer the supernatant to a new centrifuge tube, add 0.7 volumes of isopropanol and 1 / 10 volume of 3M sodium acetate solution, let stand at room temperature for 30 minutes, then centrifuge at 8000 rpm for 10 minutes at room temperature to precipitate the DNA. Resuspend the precipitate with a wide-mouth pipette tip and wash the precipitate twice with 5 mL of 70% anhydrous ethanol, then dissolve it in 200 μL of TE buffer.

[0077] 5. DNA screening and purification: Add 0.6 volumes of screening buffer 1 and screening buffer 2, and centrifuge at 10,000 rpm at room temperature for 25 minutes to separate impurities and small DNA molecules.

[0078] The components of screening buffer 1 are: Tris-HCl 20mM; EDTA 2mM; PEG8000 10%; NaCl 1.6M pH=8; the components of screening buffer 2 are: 2% w / v PVP 360000, 1.2M NaCl, 20mM Tris-HCl pH=8.

[0079] 6. DNA Detection and Sequencing: DNA fragment lengths are determined using pulsed-field electrophoresis with parameters ranging from 2k to 300k. Results are as follows: Figure 1 As shown in Table 1, the concentration was detected using qubit4.0 and the purity was detected using nanodrop.

[0080] Figure 1 The samples are as follows: Lane 1, deep-sea cold seep sediments + commercial reagent kit method; Lane 2, East China Sea continental shelf sediments + commercial reagent kit method; Lane 3, deep-sea cold seep sediments + method of the present invention; Lane 4, East China Sea continental shelf sediments + method of the present invention.

[0081] from Figure 1 As can be seen, commercially available kits yield DNA with a main band length of around 20kb, which cannot meet the DNA length requirements of third-generation sequencing. The method of this invention can obtain DNA with a main band length of 48.5kb or more.

[0082] The results in Table 1 show that for sediment samples from the East China Sea continental shelf, the total amount and purity of DNA extracted using this invention are superior to those of commercial kits. The DNA extracted by the method of this invention meets the sequencing requirements for DNA purity of third-generation sequencing PacBio sequencing: OD260 / 280 1.8-2.0 and OD260 / 230 1.8-2.5.

[0083] After the DNA extracted using the method of this embodiment of the invention passed the test, it was further sequenced using the PacBio platform. The data quality is shown in Table 2.

[0084] Table 1

[0085]

[0086]

[0087] Table 2

[0088]

[0089] Note: In Table 2, "N50" refers to the sequence length calculated by accumulating the sequence lengths from the beginning after sorting the sequences by size until the sum reaches half of the total sequence length. The sequence length at that position is the N50 value. For sediments, an N50 exceeding 10k (10000) indicates that the quality meets the requirements.

[0090] Example 3: Effect of the amount of sodium pyrophosphate, the main component of reagent cleaning, on extraction results

[0091] 1. Sediment Washing: Weigh 7g of sediment from the East China Sea continental shelf, totaling 6 samples, numbered 1-6. Place the weighed sediments into centrifuge tubes. Wash samples 1-2 with washing buffer 1, samples 3-4 with washing buffer 2, and samples 5-6 with washing buffer 3. After weighing, add 25mL of washing buffer, mix well, and centrifuge at 8000rpm for 10min. The washing buffer needs to be pre-cooled at 4℃. Wash twice.

[0092] The components of the cleaning buffer are:

[0093] Washing buffer 1: 10 mM Tris-HCl, 10 mM EDTA, 80 mM potassium chloride, 1 mM spermine, 0.2 mM spermidine, 0.5% volume Triton X-100, 2% PVP40 (polyvinylpyrrolidone), 100 mM sodium pyrophosphate.

[0094] Washing buffer 2: 10 mM Tris-HCl, 10 mM EDTA, 80 mM potassium chloride, 1 mM spermine, 10 mM spermidine, 0.5% volume Triton X-100, 2% PVP40 (polyvinylpyrrolidone), 10 mM sodium pyrophosphate.

[0095] Washing buffer 3: 10 mM Tris-HCl, 10 mM EDTA, 80 mM potassium chloride, 1 mM spermine, 1 mM spermidine, 0.5% volume Triton X-100, 2% PVP40 (polyvinylpyrrolidone), 50 mM sodium pyrophosphate.

[0096] It should be noted that the pH of the cleaning solution needs to be adjusted to 9, and sodium pyrophosphate and Triton should be added immediately before use.

[0097] 2. Pre-lysis of bacterial cells in sediment: Resuspend the washed sediment in 20 mL of extraction buffer, add 200 μL of 20 mg / mL proteinase K and 200 μL of 100 mg / mL lysozyme, and lyse at 37 °C for 30 minutes.

[0098] The components of the extraction buffer are: Tris-HCl 30mM, spermine 1mM, spermidine 1mM, EDTA 20mM, PEG 8000 1%, NaCl 10mM, Triton 0.5%, sodium dihydrogen phosphate 5.3mM, disodium hydrogen phosphate 94.7mM, pH 8.0.

[0099] 3. Sediment lysis: Add 5 mL of lysis buffer to the above extraction centrifuge tube, invert to mix, transfer to 65℃ and continue lysis for 1 hour.

[0100] The components of the lysis buffer are: Tris-HCl 30mM, spermine 1mM, spermidine 1mM, EDTA 20mM, PEG 8000 1%, NaCl 10mM, Triton 0.5%, sodium dihydrogen phosphate 5.3mM, disodium hydrogen phosphate 94.7mM, and guanidine hydrochloride 4M.

[0101] 4. DNA Recovery: Add an equal volume of phenol, chloroform, and isoamyl alcohol (25:24:1), centrifuge at 8000 rpm at room temperature to obtain the supernatant, transfer the supernatant to a new centrifuge tube, add 0.7 volumes of isopropanol and 1 / 10 volume of 3M sodium acetate solution, let stand at room temperature for 30 minutes, then centrifuge at 8000 rpm for 10 minutes at room temperature to precipitate the DNA. Resuspend the precipitate with a wide-mouth pipette tip and wash the precipitate twice with 5 mL of 70% anhydrous ethanol, then dissolve it in 200 μL of TE buffer.

[0102] 5. DNA screening and purification: Add 0.6 volumes of screening buffer 1 and screening buffer 2, and centrifuge at 10,000 rpm at room temperature for 25 minutes to separate impurities and small DNA molecules.

[0103] The components of the screening buffer 1 are: Tris-HCl 20mM, EDTA 2mM, PEG8000 10%, NaCl 1.6M, pH=8;

[0104] The components of the screening buffer 2 are: 2% w / v PVP 360000, 1.2M NaCl, 20mM Tris-HCl, pH=8.

[0105] 6. DNA Detection and Sequencing: DNA fragment lengths are determined using pulsed-field electrophoresis. Figure 2 The concentration was detected using qubit4.0 and the purity was detected using nanodrop. The results are shown in Table 3.

[0106] Figure 2 The samples are as follows: Lanes 1 and 2: Extraction results of washing buffer 1; Lanes 3 and 4: Extraction results of washing buffer 2; Lanes 5 and 6: Extraction results of washing buffer 3.

[0107] from Figure 2 As can be seen, the main band length of DNA obtained from washing buffers 1 and 2 (lanes 1-4) is around 20kb, which cannot meet the DNA length requirements of third-generation sequencing. Washing buffer 3 (lanes 5-6) can obtain DNA with a main band length of over 48.5kb.

[0108] Table 3

[0109] Sample Name method Concentration (ng / μL) Volume (μL) OD260 / 280 OD260 / 230 Deep-sea cold seep sediments Washing buffer 1 21 200 1.66 0.98 East China Sea continental shelf sediments Washing buffer 1 18 200 1.57 1.10 Deep-sea cold seep sediments Washing buffer 2 49 200 1.43 1.33 East China Sea continental shelf sediments Washing buffer 2 33 200 1.51 1.27 Deep-sea cold seep sediments Washing buffer 3 66 200 1.87 1.89 East China Sea continental shelf sediments Washing buffer 3 56 200 1.80 2.24

[0110] Among them, using washing buffer 3, which is the preferred concentration range of sodium pyrophosphate and spermidine in this invention, the total amount of DNA extracted is higher than that of washing buffer 1 and washing buffer 2. At the same time, the DNA extracted by washing buffer 3 can meet the sequencing requirements of PacBio third-generation sequencing for DNA purity: OD260 / 280 1.8-2.0, OD260 / 230 1.8-2.5.

[0111] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0112] The washing buffer developed in this invention can effectively remove impurities and improve DNA purity. Furthermore, the extraction method utilizes a two-step lysis process to fully lyse microorganisms in marine sediments. This effectively lyses various microorganisms such as archaea, bacteria, and fungi in marine sediments. In addition, this invention discloses two screening buffers that can improve purity and perform fragment selection. Using screening buffer 1 and screening buffer 2 to screen and purify DNA can remove DNA fragments smaller than 10kb, reducing the impact of small DNA fragments on DNA purity and subsequent sequencing.

[0113] This invention develops a method for extracting microbial DNA from marine sediments, mainly involving steps such as washing, pre-lysis, lysis, DNA recovery, fragment screening, and purification. Effective steps to improve length and purity are added upstream and downstream of the classic DNA extraction process, and various microbial components in marine sediments are fully lysed through a two-step lysis process. This extraction method can handle a wider range of sample types, from deep-sea cold seep sediments to nearshore sediments, allowing for the acquisition of long fragments with high purity.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting microbial DNA from marine sediments, characterized by, The extraction method includes: a) The marine sediments were cleaned using a cleaning buffer to obtain a cleaned sample; The washing buffer solution includes spermidine and sodium pyrophosphate; b) Lyse the microbial cells in the cleaned sample to obtain a lysed sample; c) The lysed sample is subjected to DNA recovery, screening and purification to obtain the microbial DNA.

2. The extraction method according to claim 1, characterized in that, The concentration of spermidine in the washing buffer is 0.5 mM-2 mM; Preferably, the concentration of sodium pyrophosphate in the washing buffer is 30-60 mM; Preferably, the washing buffer comprises 10-15 mM Tris-HCl, 10-35 mM EDTA, 70-120 mM potassium chloride, 0.5-2 mM spermine, 0.5-2 mM spermidine, 0.2-0.8% (v / v) Triton X-100, 1-3 wt% polyvinylpyrrolidone 40, and 30-60 mM sodium pyrophosphate; Preferably, the pH of the washing buffer solution is 9.0-9.5; Preferably, step a) includes: washing the marine sediment at least twice using the washing buffer to obtain the washed sample; Preferably, the washing in step a) includes: mixing the marine sediment with the washing buffer, centrifuging, and removing the supernatant; Preferably, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 8-15 min; Preferably, the cleaning buffer solution is pre-cooled at 0-4°C before the cleaning is performed.

3. The extraction method according to claim 1, wherein b) comprises: b1) The washed sample is mixed with the first lysis buffer and lysozyme, incubated, and subjected to the first lysis to obtain the first lysis system; b2) Mix the first lysis system with the second lysis solution, incubate, and perform a second lysis to obtain the lysed sample; The first lysis buffer contains triton, the second lysis buffer contains a protein denaturant, and the first lysis buffer and / or the second lysis buffer also contain a protease.

4. The extraction method according to claim 3, characterized in that, The first lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1M phosphate ions, and also contains 20-50mM Tris-HCl, 0.5-3mM spermine, 0.5-3mM spermidine, 10-40mM EDTA, 0.5-1.5wt% PEG8000, 5-15mM NaCl, and 0.25%-1% (v / v) Triton X-100; Preferably, the protease is proteinase K; Preferably, in the first lysis, the final concentration of the lysozyme is 1 mg / mL; Preferably, in the second lysis, the final concentration of proteinase K is 0.16 mg / mL; Preferably, the phosphate ions are derived from sodium dihydrogen phosphate and disodium hydrogen phosphate; Preferably, the concentration of sodium dihydrogen phosphate in the phosphate buffer solution is 5.3 mM-93.2 mM, and correspondingly, the concentration of disodium hydrogen phosphate is 94.7 mM-6.8 mM.

5. The extraction method according to claim 3, characterized in that, The protein denaturing agent includes guanidine hydrochloride; Preferably, the second lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1M phosphate ions, and further contains 20-50mM Tris-HCl, 0.5-3mM spermine, 0.5-3mM spermidine, 10-40mM EDTA, 0.5-1.5wt% PEG8000, 5-15mM NaCl, 0.25%-1% (v / v) Triton X-100, and 3-5M guanidine hydrochloride; Preferably, the phosphate ions are derived from sodium dihydrogen phosphate and disodium hydrogen phosphate; Preferably, the concentration of sodium dihydrogen phosphate in the phosphate buffer solution is 5.3 mM-93.2 mM, and correspondingly, the concentration of disodium hydrogen phosphate is 94.7 mM-6.8 mM.

6. The extraction method according to any one of claims 3-5, characterized in that, The temperature of the first pyrolysis is 35-38℃, and the time of the first pyrolysis is 30-40 min; Preferably, b2) includes: The first lysis system is mixed with the second lysis solution, wherein the volume ratio of the first lysis system to the second lysis solution is 3-5:1, and the second lysis is performed to obtain the lysed sample; Preferably, the temperature of the second pyrolysis is 55-75°C, and the time of the second pyrolysis is 40-80 min.

7. The extraction method of claim 1, wherein, c) includes: c1) Mix the lysed sample with an equal volume of phenol-chloroform-isoamyl alcohol solution, centrifuge, and collect the supernatant containing DNA; c2) Add isopropanol and sodium acetate solution to the supernatant containing DNA, let stand, and then centrifuge to obtain DNA precipitate; c3) After washing the DNA precipitate, redissolve it to obtain a DNA solution; c4) Mix the DNA solution, screening buffer 1 and screening buffer 2, centrifuge, and obtain the microbial DNA.

8. The extraction method according to claim 7, characterized in that, In the phenol-chloroform-isoamyl alcohol mixture, the volume ratio of phenol, chloroform, and isoamyl alcohol is 25:24:

1. Preferably, the volume ratio of the DNA-containing supernatant to isopropanol is 1:0.7; Preferably, the concentration of sodium acetate in the sodium acetate solution is 3M, and the volume ratio of the DNA-containing supernatant to the sodium acetate solution is 1:0.

1. Preferably, the standing time in c2) includes: standing at 20-35°C for 30 minutes; Preferably, the ions in c2) include those obtained by centrifugation at 8000 rpm for 10 min; Preferably, in step c3), after washing the DNA precipitate twice with 70% (v / v) ethanol solution, the DNA precipitate is dissolved in TE buffer to obtain the DNA solution; Preferably, the volume ratio of the DNA solution, the screening buffer 1, and the screening buffer 2 is 1:0.6:0.6; Preferably, the screening buffer 1 comprises: 20 mM Tris-HCl; 2 mM EDTA; 10 wt% PEG8000; 1.6M NaCl, the pH of the screening buffer 1 is 8; Preferably, the screening buffer 2 comprises: 2% (w / v) PVP360000, 1.2M NaCl, 20mM Tris-HCl, and the pH of the screening buffer 2 is 8.

9. A kit for extracting microbial DNA from marine sediments, characterized in that, The kit includes a washing buffer comprising spermidine and sodium pyrophosphate.

10. The kit of claim 9, wherein The concentration of spermidine in the washing buffer is 0.5 mM-2 mM; Preferably, the concentration of sodium pyrophosphate in the washing buffer is 30-60 mM; Preferably, the washing buffer comprises 10-15 mM Tris-HCl, 10-35 mM EDTA, 70-120 mM potassium chloride, 0.5-2 mM spermine, 0.5-2 mM spermidine, 0.2-0.8% (v / v) Triton X-100, 1-3 wt% polyvinylpyrrolidone 40, and 30-60 mM sodium pyrophosphate; Preferably, the pH of the washing buffer solution is 9.0-9.5; Preferably, the kit further includes a lysis buffer for lysing the cells of the microorganism, the lysis buffer comprising a first lysis buffer and a second lysis buffer; The first lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1M phosphate ions, and also contains 20-50mM Tris-HCl, 0.5-3mM spermine, 0.5-3mM spermidine, 10-40mM EDTA, 0.5-1.5wt% PEG8000, 5-15mM NaCl, and 0.25%-1% (v / v) Triton X-100; The second lysis buffer is a phosphate buffer with pH 7.5-8.5 containing 0.1M phosphate ions, and also contains 20-50mM Tris-HCl, 0.5-3mM spermine, 0.5-3mM spermidine, 10-40mM EDTA, 0.5-1.5wt% PEG8000, 5-15mM NaCl, 0.25%-1% (v / v) Triton X-100, and 3-5M guanidine hydrochloride; Preferably, the first lysis buffer further contains lysozyme; Preferably, the first lysis buffer and / or the second lysis buffer further contain a protease; More preferably, the protease is proteinase K; Preferably, the kit further includes screening buffer 1 and screening buffer 2 for screening and purifying DNA, wherein screening buffer 1 comprises: 20 mM Tris-HCl; 2 mM EDTA; 10 wt% PEG8000; 1.6 M NaCl, and the pH of screening buffer 1 is 8; The screening buffer 2 comprises: 2% (w / v) PVP360000, 1.2M NaCl, 20mM Tris-HCl, and the pH of the screening buffer 2 is 8.

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