Identification method for comparing cross-habitat living microorganisms based on environment DNA technology

Abstract

The invention discloses an identification method for comparing cross-habitat living microorganisms based on an environmental DNA technology, and relates to the technical field of environmental microorganisms and molecular biology. According to the method, through an effective pretreatment method, microorganisms are collected through a filter membrane with the pore size of 0.22 mu m and then subjected to PMA dyeing treatment, the comparability of living microorganisms in the water-soil-sediment habitat is enhanced, and the quality of the extracted DNA meets the requirements of high-throughput sequencing and metagenome sequencing; the research on diversity and ecological functions of living microbiome in the environment, especially cross-habitat propagation and ecological health risk assessment of living pathogenic microorganisms, is assisted. The identification method disclosed by the invention has the advantages of unique features, high efficiency, strong operability, simple and conventional required equipment and great potential in the aspects of large-scale popularization and application, and is expected to become an optimal scheme widely applied to related fields.

Description

Technical Field

[0001] The present invention relates to the technical fields of environmental microorganisms and molecular biology, and specifically to an identification method for comparing living microorganisms across habitats based on environmental DNA technology. Background Art

[0002] Environmental DNA extracted from natural ecosystems generally includes DNA of viable cells with intact cell membranes and active metabolism and DNA of dead cells with damaged cell membranes. DNA of dead microorganisms is ubiquitous in natural environments and has a relatively high abundance, even higher than that of viable microorganisms. It is reported that DNA of dead microorganisms is widely distributed in the ocean, fresh water, soil, sediment, air, and sometimes can reach 80% of the total DNA content. If there is interference from dead microorganisms in the sample, then conventional sampling and sequencing methods based on DNA may overestimate the abundance, diversity, and functions of viable microorganisms in the overall community. That is to say, based on the analysis of overall pathogenic microorganisms without distinguishing between live and dead microorganisms, it does not always provide more accurate and clear information for the study of ecosystem processes, has limited support for the accurate assessment of ecosystem health and safety, and may greatly overestimate the spread and ecological risks of harmful microorganisms (such as pathogenic microorganisms).

[0003] Currently, there have been studies using Propidium monoazide (PMA) staining to identify dead and viable microorganisms in environmental samples. For example, the evaluation of antibacterial effects in sewage treatment plants, the detection of viable pathogenic bacteria in food, the monitoring of viable pathogenic bacteria in public places, and even the evaluation of viable and dead microorganisms in rivers and reservoirs. However, due to the high complexity of different habitats such as soil and sediment habitats, existing methods often only target single habitats such as water bodies or soil alone, and the treatment and identification methods for different habitats vary. Currently, there is no reported effective method for simultaneously comparing viable microbiomes in different habitats. In order to make the comparability between microorganisms in different habitats higher, and considering that there is currently no systematic and effective sampling and identification method for comparing living microbiomes across habitats, therefore, there is an urgent need to develop a rapid, accurate, and efficient technical method to compare, evaluate, and study the living microbiomes across habitats (water body - soil - sediment). So we propose an identification method for comparing living microorganisms across habitats based on environmental DNA technology to solve the problems raised above.

[0004] The above information disclosed in this background art is only used to increase the understanding of the background art of the present invention. Therefore, it may include prior art that is not known to those of ordinary skill in the art. Summary of the Invention

[0005] The object of the present invention is to provide a method for identifying living microorganisms across habitats based on environmental DNA technology, which can minimize the differences in sample processing methods for water-soil-sediment cross-habitat samples, achieve comparability between living microorganisms in different habitats, reduce the error of human factors, and finally obtain high-quality DNA of the living microbiome to meet the requirements of amplicon and metagenomic library construction sequencing.

[0006] To achieve the above object, the present invention provides the following technical solution: A method for identifying living microorganisms across habitats based on environmental DNA technology, comprising the following steps:

[0007] Step (1): Weigh 3-5 g of each fresh soil / sediment sample and place it in a 150 mL conical flask.

[0008] Step (2): Add 100 mL of phosphate buffer saline (PBS) to the conical flask containing the sample in step (1), cover it with tin foil, and prepare a soil / sediment solution with a concentration of 4%.

[0009] Step (3): Place the conical flask in step (2) into a constant temperature shaking incubator at a temperature of 25 °C and a rotation speed of 220 rpm, and shake for 10 min to fully release the soil / sediment particle microorganisms into the PBS solution.

[0010] Step (4): Filter using a nylon sieve with a pore size of 200 μm to remove soil / sediment particles and macro-metazoans, and obtain the filtrate.

[0011] Step (5): Filter the filtrate obtained in step (4) through a polycarbonate membrane with a pore size of 0.22 μm under a vacuum filtration pressure of 0.02 MPa for no less than 30 min.

[0012] Step (6): After filtration, divide the filter membrane into two parts: one part is used for PMA staining treatment, and the other part is not treated with PMA staining; for PMA treatment, place the filter membrane in a petri dish with a diameter of 5 cm, add 500 μL of PMA dye to soak the filter membrane, and keep it in the dark for 20 min, then irradiate it with a 200 W LED lamp at a distance of 20 cm from the petri dish for 15 min.

[0013] Step (7): For the filter membrane not treated with PMA staining, place it in a petri dish with a diameter of 5 cm and incubate it together with the filter membrane treated with PMA; after light treatment, place the filter membrane in a sterilized 2 mL centrifuge tube for storage and freeze it in an -80 °C refrigerator until DNA extraction.

[0014] Step (8): The filter membrane treated with PMA is used to detect viable microorganisms, and the filter membrane not treated with PMA is used for the analysis of the total microbiome;

[0015] Step (9): For water habitat samples, take 300 - 1000 mL of water samples and pre-filter them through a nylon mesh sieve with a pore size of 200 μm to remove macrozoa and particles. Then, sequentially pass them through a polycarbonate membrane with a pore size of 0.22 μm under a vacuum filtration pressure of 0.02 MPa, and the filtration time is at least 30 min;

[0016] Step (10): The subsequent treatment steps for the water habitat are the same as those described in steps (6), (7), and (8) of the soil / sediment habitat;

[0017] Step (11): Use a DNA extraction kit to obtain the DNA of the viable microbiome and the total microbiome.

[0018] Preferably, the soil types of the soil / sediment samples include, but are not limited to, urban green belt soil, forest soil, farmland soil, lake sediment, reservoir sediment, and river sediment. The water types of the water samples include, but are not limited to, lakes, reservoirs, and rivers. The samples are fresh samples within 4 - 12 hours after sampling.

[0019] Preferably, for the 150 mL conical flask used in step (1), first wash it thoroughly with ultrapure water, seal it with tin foil, sterilize it in an autoclave at 121 °C for 30 min, and dry it in an oven at a temperature of 60 °C.

[0020] Preferably, in step (2), the preparation method of the PBS buffer solution is: purchase ready-to-use dry powder with a specification of 2 L / pH 7.2 - 7.4, that is, dissolve each pack of dry powder in 2 L of ultrapure water and stir until dissolved to obtain the PBS buffer solution (pH = 7.2 - 7.4).

[0021] Preferably, in step (4), when filtering the solution, after pouring out the solution from the conical flask, wash the conical flask 3 times, each time using 5 - 10 mL of PBS solution and filtering it to prevent microorganisms from remaining in the conical flask and causing errors.

[0022] Preferably, in step (6), the final concentration of the PMA dye is 50 μg / mL.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] (1) Through an effective pretreatment method, the present invention finally collects microorganisms through a 0.22 μm pore size filter membrane and then performs PMA staining treatment, enhancing the comparability of viable microorganisms among water-soil-sediment habitats. The quality of the DNA extracted by the present invention meets the requirements of high-throughput sequencing and metagenomic sequencing, and can be used for the study of the diversity and ecological functions of viable microbiomes in the environment, especially the cross-habitat transmission and ecological health risk assessment of viable pathogenic microorganisms.

[0025] (2) The identification method of the present invention is unique, with high efficiency, strong operability, and simple and conventional required equipment. It has great potential in large-scale promotion and application and is expected to become an optimal solution widely used in related fields.

[0026] The above summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present invention will be readily apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic flow chart of the present invention;

[0028] Figure 2 It is the DNA quality detection result of 54 cross-habitat samples in Example 1 of the present invention;

[0029] Figure 3 It is the DNA electrophoresis detection chart of the overall microbiome and viable microbiome in the water-soil-sediment cross-habitat in Example 1 of the present invention;

[0030] Figure 4 It is a schematic diagram of the differences between the bacterial community and eukaryotic microbial community in the water-soil-sediment cross-habitat in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0032] Example 1

[0033] In this Example 1, the water-soil-sediment cross-habitat samples from 3 urban parks were used as the research objects. For each park, 3 sampling sites were set for each habitat, and the total microbiome and viable microbiome DNA were collected and extracted to identify the viable microbiome. A total of 54 samples were set, including 27 total microbiomes and 27 viable microbiomes: 9 surface water samples, 9 soil samples, and 9 sediment samples. The treatment and extraction steps for each sample group are as follows:

[0034] Step (1): Weigh 3 - 5 g of each fresh soil / sediment sample and place it in a 150 mL conical flask. Among them, the 150 mL conical flasks used were first washed clean with ultrapure water, sealed with tin foil, sterilized in an autoclave at 121 °C for 30 min, and dried in an oven at a temperature of 60 °C.

[0035] Step (2): Add 100 mL of phosphate buffer saline (PBS) to the conical flask containing the sample in Step (1), cover it with tin foil, and configure it into a 4% soil / sediment solution. The preparation method of the PBS buffer solution is as follows: Purchase ready-to-use dry powder with a specification of 2 L / pH 7.2 - 7.4, that is, dissolve each pack of dry powder in 2 L of ultrapure water and stir until dissolved to obtain the PBS buffer solution (pH = 7.2 - 7.4).

[0036] Step (3): Place the conical flask in Step (2) into a constant temperature shaking incubator at a temperature of 25 °C and a rotation speed of 220 rpm, and shake for 10 min to fully release the soil / sediment particle microorganisms into the PBS solution.

[0037] Step (4): Filter using a nylon sieve with a pore size of 200 μm to remove soil / sediment particles and large metazoans to obtain the filtrate. When filtering the solution, after pouring out the solution from the conical flask, the conical flask needs to be washed 3 times, and each time 5 - 10 mL of PBS solution is used for washing and filtering to prevent microorganisms from remaining in the conical flask and causing errors.

[0038] Step (5): Filter the filtrate obtained in Step (4) through a polycarbonate membrane with a pore size of 0.22 μm under a vacuum filtration pressure of 0.02 MPa, and the filtration time is not less than 30 min.

[0039] Step (6): After filtration, divide the filter membrane into two parts: one part is used for PMA staining treatment, and the other part is not treated with PMA staining. For the PMA treatment, place the filter membrane in a petri dish with a diameter of 5 cm, soak the filter membrane in 500 μL of PMA dye (final concentration of 50 μg / mL), and keep it in the dark for 20 min, then irradiate it with a 200 W LED lamp at a distance of 20 cm from the petri dish for 15 min.

[0040] Step (7): For the filter membranes not treated with PMA staining, place them in a petri dish with a diameter of 5 cm and incubate them together with the PMA-treated filter membranes. After light treatment, place the filter membranes in a sterilized 2 mL centrifuge tube for storage and freeze them at -80 °C in a refrigerator until DNA extraction;

[0041] Step (8): Among them, the filter membranes treated with PMA are used to detect viable microorganisms, and the filter membranes not treated with PMA are used for the analysis of the total microbiome;

[0042] Step (9): For water habitat samples, take 300 - 1000 mL of water samples and pre-filter them through a nylon mesh sieve with a pore size of 200 μm to remove macrozoa and particles. Then, sequentially filter them through a polycarbonate membrane with a pore size of 0.22 μm under a vacuum filtration pressure of 0.02 MPa, and the filtration time is at least 30 min;

[0043] Step (10): The subsequent treatment steps for the water habitat are the same as those described in steps (6), (7), and (8) of the soil / sediment habitat;

[0044] Step (11): Use a DNA extraction kit to obtain the DNA of the viable microbiome and the total microbiome.

[0045] It should be noted that the specific dosages of the reagents in Example 1 are only examples. During the actual operation process, the reagents can be increased or decreased in the total configuration amount as needed, as long as the final concentrations of each component remain unchanged.

[0046] To further prove the effect of Example 1, quality inspection is carried out on the experimental results of Example 1. The quality inspection conclusion meets the requirements of second-generation high-throughput sequencing and metagenomic sequencing. The specific results are shown in Figure 2 and Figure 3 .

[0047] Figure 2 are the DNA quality inspection results of 54 cross-habitat samples in Example 1. The DNA concentration is detected by a Qubit 4.0 instrument. The quality grades are divided into 5 grades: Class A represents that the sample quality is qualified and the total amount meets the requirements for library construction 2 or more times; Class B represents that the sample quality is qualified and the total amount meets the requirements for 1 time but is less than the library construction requirements of Class A. For such samples, subsequent experiments can be carried out. If the library construction is not ideal, samples need to be prepared again; Class C represents that the sample does not fully meet the library construction requirements and risk library construction can be tried; Class D represents that the sample does not meet the library construction requirements and is not recommended for use. The quality inspection results of Example 1 show that the quality of all samples is qualified, meeting and meeting the requirements for further library construction and sequencing. Among them, 90% of the samples are qualified for quality inspection as Class A, and 10% of the samples are qualified for quality inspection as Class B.

[0048] Figure 3It is the DNA electrophoresis detection diagram of the overall microbiome and viable microbiome of 54 cross-habitat samples in Example 1. Among them, the Marker is DL15000, and the loading amount of the Marker is 5 μL for all; the DNA loading amount is 20 - 60 ng. The electrophoresis conditions are: 1.2% agarose gel, the voltage is 120 V, the current is 80 mA, and the electrophoresis time is 20 min.

[0049] As Figure 4 shown, compared with the overall microbiome, there are significant differences in the composition of the bacterial community and eukaryotic microbial community obtained from the PMA-treated samples, and there are significant differences in the bacterial community and eukaryotic microbial community among the 3 sampling habitats. There are also significant differences between different habitats, as well as between the overall bacterial / eukaryotic microbial community and the viable bacterial / eukaryotic microbial community.

[0050] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0051] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An identification method for comparing living microorganisms across habitats based on environmental DNA technology, characterized in that, It includes the following steps: Step (1): Weigh 3 - 5 g of each fresh soil / sediment sample and place it in a 150 mL conical flask; Step (2): Add 100 mL of Phosphate Buffer Saline (PBS) to the conical flask containing the sample in Step (1), cover it with tin foil, and prepare a soil / sediment solution with a concentration of 4%; Step (3): Place the conical flask in Step (2) into a constant temperature shaking incubator at a temperature of 25°C and a rotation speed of 220 rpm, and shake for 10 min to fully release the soil / sediment particle microorganisms into the PBS solution; Step (4): Filter using a nylon sieve with a pore size of 200 μm to remove soil / sediment particles and macrozoa, and obtain the filtrate; Step (5): Filter the filtrate obtained in Step (4) through a polycarbonate membrane with a pore size of 0.22 μm under a vacuum filtration pressure of 0.02 MPa, and the filtration time is not less than 30 min; Step (6): After filtration, divide the filter membrane into two parts: one part is used for PMA staining treatment, and the other part is not treated with PMA; for the PMA treatment, place the filter membrane in a petri dish with a diameter of 5 cm, add 500 μL of PMA dye to soak the filter membrane, and keep it in the dark for 20 min, then irradiate it with a 200 W LED lamp at a distance of 20 cm from the petri dish for 15 min; Step (7): For the filter membrane not treated with PMA staining, place it in a petri dish with a diameter of 5 cm and incubate it together with the filter membrane treated with PMA; after light treatment, place the filter membrane in a sterilized 2 mL centrifuge tube for storage and freeze it in a -80°C refrigerator until DNA extraction; Step (8): Among them, the filter membrane treated with PMA is used to detect viable microorganisms, and the filter membrane not treated with PMA is used for the analysis of the total microbiome; Step (9): For water habitat samples, take 300 - 1000 mL of water samples and pre-filter them through a nylon mesh sieve with a pore size of 200 μm to remove macrozoa and particles, and then sequentially filter them through a polycarbonate membrane with a pore size of 0.22 μm under a vacuum filtration pressure of 0.02 MPa, and the filtration time is at least 30 min; Step (10): The subsequent treatment steps for the water habitat are the same as those described in Steps (6)(7)(8) of the soil / sediment habitat; Step (11): Use a DNA extraction kit to obtain the DNA of the viable microbiome and the total microbiome.

2. The identification method for comparing living microorganisms across habitats based on environmental DNA technology according to claim 1, characterized in that: The soil types of the soil / sediment samples include, but are not limited to, urban green belt soil, forest soil, farmland soil, lake reservoir sediment, reservoir sediment, river sediment. The water types of the water samples include, but are not limited to, lakes, reservoirs, rivers. The samples are fresh samples within 4 - 12 hours after sampling.

3. The identification method for comparing living microorganisms across habitats based on environmental DNA technology according to claim 1, characterized in that: The 150 mL conical flask used in Step (1) is first washed clean with ultrapure water, sealed with tin foil, sterilized in an autoclave at 121°C for 30 min, and dried in an oven at a temperature of 60°C.

4. The identification method for comparing living microorganisms across habitats based on the environmental DNA technology according to claim 1, wherein: In the step (2), the preparation method of the PBS buffer solution is as follows: purchase ready-to-use dry powder with a specification of 2L / pH 7.2 - 7.4, that is, dissolve each pack of dry powder in 2L of ultrapure water by stirring to obtain the PBS buffer solution (pH = 7.2 - 7.4).

5. The identification method for comparing live microorganisms across habitats based on environmental DNA technology according to claim 1, characterized in that: In the step (4), when filtering the solution, after pouring out the solution from the conical flask, the conical flask needs to be washed 3 times, and each washing uses 5 - 10 mL of PBS solution and is filtered to prevent microorganisms from remaining in the conical flask and causing errors.

6. The identification method for comparing live microorganisms across habitats based on environmental DNA technology according to claim 1, wherein: In the step (6), the final concentration of the PMA dye is 50 μg / mL.

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