Method for extracting DNA (deoxyribonucleic acid) of microorganisms on plastic surface of culture sea area

Abstract

The invention discloses a method for extracting DNA (deoxyribonucleic acid) of microorganisms on a plastic surface of an aquaculture sea area, which comprises the following steps of: performing sterile treatment on a plastic sample, cutting into pieces, putting into a conical flask, adding 2.5% NaCl solution, performing gradient oscillation elution, and performing elution-transfer circulation treatment for multiple times until 500 ml of NaCl solution is used up, so as to ensure that the microorganisms are effectively separated from the plastic surface; physically crushing the microbe-enriched filter membrane, placing the crushed filter membrane in a lysis tube, and adding a lysis solution for cell lysis; a DNA product obtained after purification treatment is subjected to agarose gel electrophoresis detection to display strip integrity, and an ultramicro spectrophotometer is used for detecting DNA concentration and purity. The obtained DNA meets the experimental requirements of metagenomics research of microorganisms on the plastic surface, and can be directly used for subsequent DNA fragmentation, database establishment and high-throughput sequencing. According to the method, through an optimized elution-enrichment-extraction process, the technical problems of low microorganism recovery rate, insufficient DNA purity and the like in the existing method are effectively solved, and the method is particularly suitable for DNA extraction of microorganisms on the surfaces of various environmental plastic samples.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a method for extracting microbial DNA from the surface of plastics in aquaculture areas. Background Technology

[0002] Plastics are widely used in various industrial sectors due to their chemical stability, lightweight, and plasticity. In the natural environment, microplastics / plastic fragments formed through ultraviolet radiation, mechanical wear, and biodegradation adsorb dissolved organic matter, proteins, and other nutrients from the environmental medium, providing a unique "plastic zone" ecological niche for microbial colonization and reproduction, thus forming complex microbial communities on the plastic surface. This microenvironment not only improves the survival rate of microorganisms by providing nutrients and physical support but also promotes the enrichment of specific functional microorganisms, potentially making them carriers of pollutants and pathogens, which can then spread to aquatic organisms or humans through various pathways. Furthermore, plastics have a strong adsorption capacity for organic pollutants, heavy metals, and antibiotics, further exacerbating their ecological risks. Therefore, microorganisms on plastic surfaces are closely related to various environments and even humans; plastics are both carriers of microorganisms and various pollutants and media for their transmission. A thorough understanding of the diversity, community composition, and functions of microorganisms on plastic surfaces is beneficial not only for in-depth analysis of the molecular mechanisms of plastic biodegradation but also for accurately assessing their ecological risks as carriers of pathogenic microorganisms.

[0003] In microbial molecular biology research, obtaining high-quality DNA is a crucial prerequisite for achieving high-throughput sequencing and metagenomic analysis. However, extracting microbial DNA from plastic surfaces faces significant technical challenges: First, the abundance of microorganisms on plastic surfaces is far lower than that in media such as water, soil, and sediments in the natural environment; second, the various substances adsorbed on plastic surfaces are complex, leading to a significant reduction in DNA purity. These problems severely restrict the accuracy and reliability of microbial research on plastic surfaces.

[0004] Currently, there are two main methods for extracting microbial DNA from plastic surfaces: The first is the traditional CTAB manual extraction method: the plastic is directly cut into small pieces and placed into centrifuge tubes, then cells are lysed with cetyltrimethylammonium bromide, followed by DNA extraction and purification using a phenol-chloroform organic solvent. This method suffers from drawbacks such as cumbersome procedures, long processing times per cycle, and the use of highly toxic chloroform. The second method involves directly cutting the plastic into small pieces and placing them into centrifuge tubes, then using a commercial kit for DNA extraction. While this method solves the problems of the first method, the sample volume per cycle is limited, and coupled with the properties of the plastic itself, the obtained DNA concentration and purity are highly unlikely to meet the requirements of metagenomic sequencing. In summary, these technical limitations make it difficult to obtain DNA samples that meet the requirements of metagenomic sequencing using existing methods, severely hindering in-depth research on microorganisms on plastic surfaces. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting microbial DNA from the surface of plastics in aquaculture areas, which solves the problems of complex operation, long time consumption, low efficiency and poor extraction quality in current methods for extracting microbial DNA from plastic surfaces.

[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 plastic in aquaculture areas includes the following steps: S1. Sample preparation: Cut the plastic sample into small pieces with sterile scissors and put them into a 250 ml sterilized conical flask; S2. Elution of microorganisms on plastic surface: Add 2.5% NaCl solution to the conical flask and elute by shaking to allow microorganisms to detach from the plastic surface and aggregate in the solution; S3. Collection of eluent and secondary elution: Collect the supernatant of the eluent, add 2.5% NaCl solution to the conical flask again, elute again, and repeat step S2 until 500 ml of 2.5% NaCl solution is used up.

[0007] S4. Enrichment of microorganisms on plastic surfaces: Filter all collected supernatants onto a 0.22 μm aqueous filter membrane; S5. Extraction of microbial DNA from plastic surfaces: After cutting the filter membrane into pieces, transfer it to a lysis tube and add lysis buffer. After bead milling and lysis, heat for further lysis. Then, elute and collect the DNA. Finally, detect the DNA by agarose gel electrophoresis and detect the DNA concentration and purity by ultra-micro spectrophotometer. S6, DNA quality inspection; S7, Metagenomic sequencing of microbial DNA on plastic surfaces.

[0008] More preferably, in S1, the sample size is determined by assessing the microbial content on the object surface: 8g of rope samples are cut into shorter pieces; 4g of mesh samples are cut into smaller pieces; and 70cm of foam samples are scraped from the surface using a sterile knife. 2 about.

[0009] More preferably, the elution method in S2 is to add a pre-cooled and sterile 2.5% NaCl solution at 0-4℃ to an Erlenmeyer flask, the amount of which should cover the plastic sample, and then shake it in a shaker at 4℃ and 200rpm for 30 minutes.

[0010] More preferably, in step S3: the supernatant of the eluent is collected into a sterilized 500 ml beaker and stored at 4°C. Then, 2.5% NaCl solution is added again to the conical flask, and the mixture is shaken in a shaker at 4°C and 200 rpm for 30 min to perform a second elution.

[0011] More preferably, in step S4: a water-based mixed fiber filter membrane with a pore size of 0.22 μm and a diameter of 47-50 mm is used to perform negative pressure filtration on all the collected supernatant, and the beaker containing the supernatant is rinsed with a 2.5% NaCl solution, and the rinsing solution is also filtered onto the filter membrane.

[0012] More preferably, in step S5: the filter membrane is cut into 0.1 cm pieces using sterile scissors in a UV-sterilized clean bench. 2 The fragments were transferred to a 2 ml Bead Tube lysis tube provided by the HiPure Soil DNA Kit. After adding 0.8 ml Buffer SOL Plus lysis buffer, the sample was lysed by vortexing for 30 min and then incubated at 70 °C for 10 min for further lysis. The DNA extraction was then performed according to the DNA extraction steps in the kit. Finally, the extracted DNA was detected by agarose gel electrophoresis.

[0013] More preferably, in step S6: the concentration of microbial DNA on the plastic surface is determined using a Micro Drop ultra-micro spectrophotometer, and electrophoresis is performed on a 1% agarose gel at a voltage of 5 V / cm for 20 min. After electrophoresis, the gel is observed and photographed using a gel imaging system.

[0014] More preferably, in step S7: DNA is fragmented, and fragments of approximately 350 bp are selected for constructing a PE library, and finally metagenomic sequencing is performed.

[0015] In summary, the present invention has the following beneficial effects: The extraction method of this invention, through an optimized elution-enrichment-extraction process, has the advantages of high microbial recovery rate and high DNA purity. It can efficiently obtain high-quality and high-concentration DNA, meet the requirements of metagenomic sequencing, and promote in-depth research on microorganisms on plastic surfaces. Attached Figure Description

[0016] Figure 1 This is a flowchart of the operation of the present invention; Figure 2 This is an agarose gel electrophoresis result of the DNA of microorganisms extracted from the plastic surface in Example 1. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings.

[0018] Example: A method for extracting microbial DNA from the surface of plastic in aquaculture areas, such as... Figure 1 As shown, this example uses samples of netting, nylon rope, and foam collected from the aquaculture area of ​​Zhanjiang Bay as samples to extract microbial DNA from the surfaces of these three types of plastics. The specific operation steps are as follows: S1. Sample Preparation: To assess the microbial content on the object surface and determine the sample size, approximately 4 g of mesh sample is shredded with sterile scissors, approximately 9 g of nylon rope is shortened with sterile scissors, and 70 cm of foam is scraped from the surface with a sterile knife. 2 Place the two flasks into sterilized 250 ml Erlenmeyer flasks using sterile forceps.

[0019] S2. Elution of microorganisms from plastic surface: Add pre-cooled and sterile 2.5% NaCl solution (0-4℃) to each conical flask, ensuring the solution completely covers the plastic sample. Then, shake the conical flask in a shaker at 4℃ and 200 rpm for 30 minutes. This shaking elution process causes microorganisms to detach from the plastic surface and aggregate in the solution.

[0020] S3. Collection of eluent and secondary elution: Slowly pour the supernatant of the eluent into a sterilized 500 ml beaker and store at 4°C. Then, add 2.5% NaCl solution to the conical flask again and shake in a shaker at 4°C and 200 rpm for 30 min for secondary elution. Repeat the above steps until all 500 ml of 2.5% NaCl solution is used.

[0021] S4. Enrichment of microorganisms on plastic surfaces: All collected supernatants were filtered under negative pressure using an aqueous mixed fiber filter membrane with a pore size of 0.22 μm and a diameter of 47-50 mm. The beaker containing the supernatant was rinsed with a 2.5% NaCl solution, and the rinsing solution was also filtered onto the filter membrane.

[0022] S5. Extraction of microbial DNA from plastic surfaces: In a UV-sterilized clean bench, use sterile scissors to cut the filter membrane into 0.1 cm pieces. 2 The fragments were collected and transferred to 2 ml Bead Tubes lysis tubes provided with the HiPure Soil DNA Kit. 0.8 ml of Buffer SOL Plus lysis buffer was added, and the samples were lysed using a vortex mixer for 30 min, followed by incubation at 70°C for 10 min for further lysis. Subsequent DNA extraction was performed following the DNA extraction steps in the kit. Finally, the extracted DNA was analyzed by agarose gel electrophoresis.

[0023] S6. DNA quality inspection: The concentration of microbial DNA on the plastic surface was determined using a Micro Drop ultra-micro spectrophotometer. Electrophoresis was performed on a 1% agarose gel at 5 V / cm for 20 min. After electrophoresis, the gel was observed and photographed using a gel imaging system.

[0024] S7. Metagenomic sequencing of microbial DNA on plastic surfaces: DNA fragmentation is performed, and fragments of about 350 bp are selected for constructing PE libraries, followed by metagenomic sequencing.

[0025] Table 1. Results of DNA concentration of microorganisms extracted from plastic surfaces as detected by Micro Drop ultra-micro spectrophotometer. The results are as follows Figure 2 As shown, the obtained DNA product was detected by agarose gel electrophoresis and the bands were intact. The OD260 / 280 ratio was stable between 1.8 and 2.0 by ultra-micro spectrophotometer. The concentration met the requirements of ≥20 ng / μl for metagenomic sequencing and ≥0.5 ng / μl for Illumina PE library.

[0026] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for extracting microbial DNA from the surface of plastic in aquaculture areas, characterized in that, Includes the following steps: S1. Sample preparation: Cut the plastic sample into small pieces with sterile scissors and put them into a 250 ml sterilized conical flask; S2. Elution of microorganisms on plastic surface: Add 2.5% NaCl solution to the conical flask and elute by shaking to allow microorganisms to detach from the plastic surface and aggregate in the solution; S3. Collection of eluent and secondary elution: Collect the supernatant of the eluent, add 2.5% NaCl solution to the conical flask again, elute again, and repeat step S2 until 500 ml of 2.5% NaCl solution is used up. S4. Enrichment of microorganisms on plastic surfaces: Filter all collected supernatants onto a 0.22 μm aqueous filter membrane; S5. Extraction of microbial DNA from plastic surfaces: After cutting the filter membrane into pieces, transfer it to a lysis tube and add lysis buffer. After bead milling and lysis, heat for further lysis. Then, elute and collect the DNA. Finally, detect the DNA by agarose gel electrophoresis and detect the DNA concentration and purity by ultra-micro spectrophotometer. S6, DNA quality inspection; S7, Metagenomic sequencing of microbial DNA on plastic surfaces.

2. The method for extracting microbial DNA from the surface of plastic in aquaculture areas according to claim 1, characterized in that: For the plastics in S1, the sample size is determined by assessing the microbial content on the object surface. Rope samples are cut into short pieces (9g), mesh samples are cut into small pieces (4g), and foam samples are scraped from the surface using a sterile knife (70 cm). 2 about.

3. The method for extracting microbial DNA from the surface of plastic in aquaculture areas according to claim 2, characterized in that: The elution method in S2 is to add a pre-cooled and sterile 2.5% NaCl solution at 0-4℃ to an Erlenmeyer flask, the amount of which should cover the plastic sample, and then shake it in a shaker at 4℃ and 200rpm for 30 minutes.

4. The method for extracting microbial DNA from the surface of plastic in aquaculture areas according to claim 3, characterized in that, In step S3: the supernatant of the eluent is collected into a sterilized 500 ml beaker and stored at 4°C. Then, 2.5% NaCl solution is added again to the conical flask, and the mixture is shaken for 30 min at 4°C and 200 rpm for a second elution.

5. The method for extracting microbial DNA from the surface of plastic in aquaculture areas according to claim 4, characterized in that, In step S4: a water-based mixed fiber filter membrane with a pore size of 0.22 μm and a diameter of 47-50 mm is used to perform negative pressure filtration on all the collected supernatant. The beaker containing the supernatant is rinsed with a 2.5% NaCl solution, and the rinsing solution is also filtered onto the filter membrane.

6. The method for extracting microbial DNA from the surface of plastic in aquaculture areas according to claim 5, characterized in that, In step S5: the filter membrane is cut into 0.1 cm pieces using sterile scissors inside a UV-sterilized clean bench. 2 The fragments were transferred to a 2 ml Bead Tube lysis tube provided by the HiPure Soil DNA Kit. After adding 0.8 ml Buffer SOL Plus lysis buffer, the sample was lysed by vortexing for 30 min and then incubated at 70 °C for 10 min for further lysis. The DNA extraction was then performed according to the DNA extraction steps in the kit. Finally, the extracted DNA was detected by agarose gel electrophoresis.

7. The method for extracting microbial DNA from the surface of plastic in aquaculture areas according to claim 6, characterized in that, In step S6: the concentration of microbial DNA on the plastic surface was determined using a Micro Drop ultra-micro spectrophotometer, and electrophoresis was performed on a 1% agarose gel at a voltage of 5 V / cm for 20 min. After electrophoresis, the gel was observed and photographed using a gel imaging system.

8. The method for extracting microbial DNA from the surface of plastic in aquaculture areas according to claim 7, characterized in that, In S7: DNA fragmentation is performed, and fragments of approximately 350 bp are selected for constructing a PE library, followed by metagenomic sequencing.

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