CN112111384B - Water body environment DNA sampling device and method - Google Patents
Water body environment DNA sampling device and method Download PDFInfo
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- CN112111384B CN112111384B CN202010930987.2A CN202010930987A CN112111384B CN 112111384 B CN112111384 B CN 112111384B CN 202010930987 A CN202010930987 A CN 202010930987A CN 112111384 B CN112111384 B CN 112111384B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/20—Controlling water pollution; Waste water treatment
Abstract
The invention discloses a water body environment DNA sampling device, which mainly comprises: the device comprises a first sampling column for sampling a larger volume of DNA, a plurality of second sampling columns capable of completing sampling of smaller DNA from the first sampling columns, first sampling plates arranged at the middle ends of the first sampling columns in a penetrating manner, second sampling plates arranged at the positions, close to the bottom ends, of the second sampling columns in a penetrating manner, and a driving mechanism for completing simultaneous sampling of the first sampling columns and the second sampling columns; the invention can collect different DNA samples in the water body in a mode of simultaneously sampling a plurality of sampling columns, has simple use method, can directly cause suspended particles in the water to be deposited on the filter membrane during sampling, saves the step of large deposition after the previous collection, has better sampling effect and higher sampling speed, and is suitable for wide popularization.
Description
Technical Field
The invention relates to the technical field of water body environment inspection, in particular to a water body environment DNA sampling device and method.
Background
The water sampling is the basis for detecting various research problems such as water quality, water heavy metal and other pollutant concentration, water supersaturation oxygen, water environment DNA and the like, and the effective intake of a water sample with a designated water level is particularly important for obtaining accurate experimental results.
The water body environment DNA detection can help people evaluate the water body environment to obtain relevant parameters such as pollutant types, microbial community distribution conditions and the like, and has high practical significance and economic value, so that the water body sampling has important significance for collecting and detecting the water body environment DNA.
The sampling device of present water environment DNA all is after gathering the water with the sampling tube, filters the sediment and accomplishes water environment DNA collection in leading-in other containers with the water of gathering, and the process is comparatively loaded down with trivial details, makes sampling efficiency reduce, and the filter membrane is great, is difficult for accomplishing centrifugal separation process, and when water environment samples, needs every sample to gather 4 ~ 6, all is through accomplishing one pipe sample after at present at the sampling, carries out the sample of next pipe again, and is very inconvenient.
Disclosure of Invention
In order to solve the technical problems, the invention provides a water body environment DNA sampling device and a method.
The technical key points of the invention are as follows:
a water body environment DNA sampling device mainly comprises: the device comprises a first sampling column for sampling a larger volume of DNA, a plurality of second sampling columns capable of completing sampling of smaller DNA from the first sampling columns, first sampling plates arranged at the middle ends of the first sampling columns in a penetrating manner, second sampling plates arranged at the positions, close to the bottom ends, of the second sampling columns in a penetrating manner, and a driving mechanism for completing simultaneous sampling of the first sampling columns and the second sampling columns;
the first sampling column comprises: the device comprises a first sampling port, a plurality of sampling channels for sucking a sample source, a first sampling piston slidingly arranged in the sampling channels, and a crushing blade arranged below the first sampling plate;
the first sampling plate is inserted on the first sampling column through a spout, the first sampling plate includes: the device comprises a handle arranged on the first sampling plate, a first collecting plate which is equally divided into a plurality of parts and is used for collecting samples, a fixing groove arranged on the first collecting plate, a first filter membrane arranged on the first collecting plate and a first collecting plate cover body used for fixing the first filter membrane;
the second sampling column is disposed at the first sampling column top end, which includes: the liquid outlet is arranged above the second sampling plate, the check valve is arranged on the liquid outlet, the second sampling piston is arranged in the second sampling column in a sliding manner and used for sucking samples, the sample middle rotary column is arranged at two ends in the first sampling column, the sample middle rotary column is communicated with the second sampling column, and a liquid inlet is arranged at the position, close to the top end, of the sample middle rotary column;
the second sampling plate includes: a detachable sampling plate shell and a pull block arranged on the shell;
the driving mechanism includes: the utility model provides a crushing blade, including the crushing blade, the crushing blade is connected on, with the transmission pole is accomplished the second lead screw of mutual transmission through a plurality of drive gears, two drive wheels, with the chain of drive wheel meshing, install on the top drive handle on the drive wheel, fixed mounting on another the drive wheel from the driving wheel, with the driving lever that is used for driving that the driven wheel meshes the second sampling piston, the driving lever includes: the first lead screw is used for driving the second lead screw, and the rack is used for being meshed with the driven wheel.
Further, a plurality of weight columns are arranged around the first sampling column, a plurality of fixing rods are arranged at the bottom end of the first sampling column, an observation window is arranged at the position of the rotating column in each sample, the weight columns enable the device to naturally sink, meanwhile, the fixing rods can be inserted into soil at the water bottom by gravity, and the observation window can observe the precipitation degree of suspended matters.
Further, the first collecting plate cover plate includes: the fixing wall used for being inserted into the fixing groove and the fixing column used for fixing each first filter membrane can facilitate the disassembly of the filter membranes.
Further, the chain is divided into a plurality of chain sections, each section of chain section is connected through a limiting rotating shaft, and adsorption plates are arranged at two ends of each section of chain section, so that sampling of various depths can be facilitated.
Furthermore, a protection box is arranged outside the transmission rod, so that the transmission rod is prevented from rusting, and the service life of the device is prolonged.
Further, the sampling plate shell is fixed through the mounting, conveniently dismantles the sampling plate shell, and then makes things convenient for the dismantlement of filter membrane.
The method for collecting the water body environment DNA by the device further comprises the following steps:
s1: the first filter membrane adopts a filter membrane with the diameter of 0.45 mu m for measuring eukaryotes, the second filter membrane adopts a filter membrane with the diameter of 0.22 mu m for measuring bacteria, after the filter membrane is installed, the length of a chain is set according to the measuring depth, the device is stably submerged in water, each section of the chain section is opened by gravity, and the device is fixed by a fixing rod;
s2: the driving handle is rotated to drive the driving wheel, the driving rod drives the second lead screw and the transmission rod to rotate, the crushing blade crushes sundries such as algae and the like to prevent the blockage device, meanwhile, the driving rod can also vertically slide, the first sampling column is filled with water through the first sampling piston until the water flows into the sample rotary column from the liquid inlet through the first filter membrane, and when the driving rod slides upwards, the driving rod drives the second sampling piston to slide, so that the water in the sample rotary column enters the second sampling column through the second filter membrane;
s3: standing for 10min to enable suspended ions in water to be precipitated and adsorbed on a filter screen, and then repeating S2 to enrich suspended particles and precipitates on the filter screen;
s4: and rotating the driving handle to withdraw the device, withdrawing the first sampling plate and the second sampling plate after 30min of precipitation, taking down the first filter membrane and the second filter membrane, placing the filter membrane and the precipitate into a centrifuge tube, and storing in a refrigerator at the temperature.
Compared with the prior art, the invention has the beneficial effects that:
firstly, the DNA in the water body environment is collected in a grading way through the multi-layer sampling columns, and the enrichment of the DNA on the filter membrane is completed during the collection, so that the method is simple and convenient to use, and the collection efficiency is higher.
Secondly, the whole device is controlled to collect and sample suspended matters in the water body environment through the driving handle, liquid is firstly sucked through the first sampling piston, so that a first filter membrane in the first sampling column enriches the sample, liquid storage is completed through the sample rotating column, when the driving handle drives a second sampling piston to enable the liquid in the sample rotating column to pass through the second filter membrane, sample enrichment on the second filter membrane is completed, the water body suspended matter DNA is adopted in a layer-by-layer progressive mode, the collected sample is more complete, and the detected data is more accurate.
Thirdly, the invention enables the filter membrane with the same area to be assembled on one collecting plate by arranging the collecting plates with a plurality of equal components, and the fixing of the filter membrane on the collecting plate is completed by the collecting plate shell and the cover body, so that the collected samples can be quickly separated in the subsequent separation process by a plurality of spliced filter membranes, the separation is more thorough, and the shell and the cover plate enable the filter membrane to be detached and installed more conveniently.
Drawings
FIG. 1 is a front view of a schematic of the internal structure of the present invention;
FIG. 2 is a front view of a schematic of the external construction of the present invention;
FIG. 3 is a right side view of a schematic representation of the drive rod structure of the present invention;
FIG. 4 is a schematic view of the chain extension of the present invention;
FIG. 5 is a schematic view of the chain folding structure of the present invention;
FIG. 6 is a top view of a schematic diagram of a second sampling plate structure of the present invention;
FIG. 7 is a top view of a schematic of a first collector plate structure of the present invention;
fig. 8 is a perspective view showing the structure of the cover body of the first collecting plate of the present invention.
Wherein 1, first sampling column, 11, first sampling port, 12, fixed rod, 13, weighting column, 14, observation window, 15, sampling channel, 16, crushing blade, 2, first sampling plate, 21, handle, 22, first collecting plate, 221, fixed slot, 23, chute, 24, first filter membrane, 25, first collecting plate cover, 251, fixed column, 252, fixed wall, 3, second sampling column, 31, drain port, 311, check valve, 32, second sampling plate, 321, sampling plate housing, 322, fixing piece, 323, second filter membrane, 33, second sampling piston, 34, sample middle rotary column, 341, liquid inlet, 4, driving mechanism, 41, driving wheel, 42, chain, 421, chain section, 422, limit rotary shaft, 423, adsorption plate, 43, driving handle, 44, driven wheel, 45, driving rod, 451, first sampling piston, 452, first, 453, rack, 46, second lead screw, 47, transmission rod, protection box, transmission gear, 48, and gear.
Detailed Description
Example 1:
as shown in fig. 1 and 2, a water environment DNA sampling device mainly includes: a first sampling column 1 for sampling a larger volume of DNA, a plurality of second sampling columns 3 for sampling a smaller volume of DNA from the first sampling column 1, a first sampling plate 2 interposed at the middle end of the first sampling column 1, a second sampling plate 32 interposed at the position of the second sampling column 3 near the bottom end, and a driving mechanism 4 for sampling the first sampling column 1 and the second sampling column 3 simultaneously;
as shown in fig. 1, the first sampling column 1 includes: a first sampling port 11, a plurality of sampling channels 15 for sucking a sample source, a first sampling piston 451 slidably disposed in the sampling channels 15, and a pulverizing blade 16 disposed below the first sampling plate 2;
as shown in fig. 1 and 7, the first sampling plate 2 is inserted on the first sampling column 1 through a chute 23, and the first sampling plate 2 includes: a handle 21 provided on the first sampling plate 2, a first collecting plate 22 equally divided into a plurality of parts for collecting a sample, a fixing groove 221 provided on the first collecting plate 22, a first filter membrane 24 provided on the first collecting plate 22, and a first collecting plate cover 25 for fixing the first filter membrane 24;
as shown in fig. 2 and 6, the second sampling column 3 is disposed at the top end of the first sampling column 1, and includes: a liquid outlet 31 arranged above the second sampling plate 32, a check valve 311 arranged on the liquid outlet 31, a second sampling piston 33 arranged in the second sampling column 3 in a sliding manner and used for sucking samples, and sample middle rotary columns 34 arranged at two ends in the first sampling column 1, wherein the sample middle rotary columns 34 are communicated with the second sampling column 3, and a liquid inlet 341 is arranged near the top end of the sample middle rotary columns;
the second sampling plate 32 includes: a detachable sampling plate housing 321, a pull block 35 provided on the housing 321;
as shown in fig. 1 and 3, the driving mechanism 4 includes: a transmission rod 47 connected to the crushing blade 16, a second screw 46 which is mutually transmitted with the transmission rod 47 through a plurality of transmission gears 48, two driving wheels 41, a chain 42 engaged with the driving wheels 41, a driving handle 43 mounted on the driving wheels 41 at the top end, a driven wheel 44 fixedly mounted on the other driving wheel 41, and a driving rod 45 engaged with the driven wheel 44 for driving the second sampling piston 33, the driving rod 45 comprising: a first screw 452 for driving the second screw 46, and a rack 453 for meshing with the driven wheel 44.
As shown in fig. 2, a plurality of weighting columns 13 are disposed around the first sampling column 1, a plurality of fixing rods 12 are disposed at the bottom end of the first sampling column 1, and an observation window 14 is disposed at the position of a rotating column 34 in each sample.
As shown in fig. 8, the first collecting plate cover plate 25 includes: a fixing wall 251 for being inserted into the fixing groove 221, and a fixing column 252 for fixing each first filter membrane 24.
As shown in fig. 1, a protection case 471 is provided outside the transmission rod 47.
As shown in fig. 2, the sampling plate housing 321 is fixed by a fixing member 322.
The chain 42 may be fixed by providing a channel in the driving wheel 41 or by providing a guide plate.
Example 2:
the embodiment 2 is different from the embodiment 1 in that:
as shown in fig. 4 and 5, the chain 42 is divided into a plurality of chain sections 421, each of the chain sections 421 is connected by a limiting rotating shaft 422, and two ends of each of the chain sections 421 are provided with adsorption plates 423.
The method for collecting the DNA of the water body environment by the device of the embodiment 2 is characterized by comprising the following steps:
s1: the first filter membrane 24 adopts a filter membrane with the diameter of 0.45 mu m for measuring eukaryotes, the second filter membrane 323 adopts a filter membrane with the diameter of 0.22 mu m for measuring bacteria, after the filter membrane is installed, the length of the chain 42 is set according to the measuring depth, the device is stably submerged in water, each section of the chain section 421 is opened by gravity, and the device is fixed by the fixing rod 12;
s2: the driving handle 43 is rotated to drive the driving wheel 44, the driving rod 45 drives the second lead screw 46 and the transmission rod 47 to rotate, the crushing blade 16 crushes sundries such as algae and the like to prevent the blockage device, meanwhile, the driving rod 45 also vertically slides, the first sampling column 1 is filled with water through the first sampling piston 33 until the water flows into the sample middle rotary column 34 from the liquid inlet 341 through the first filter membrane 24, and the driving rod 45 drives the second sampling piston 451 to slide when sliding upwards, so that the water in the sample middle rotary column 34 enters the second sampling column 3 through the second filter membrane 323;
s3: standing for 10min to enable suspended ions in water to be precipitated and adsorbed on a filter screen, and then repeating S2 to enrich suspended particles and precipitates on the filter screen;
s4: the driving handle 43 is rotated to withdraw the device, after 30min of sedimentation, the first sampling plate 2 and the second sampling plate 32 are extracted, the first filter membrane 24 and the second filter membrane 323 are taken down, the filter membrane and the sediment are placed into a centrifuge tube, and the centrifuge tube is placed in a refrigerator at 4 ℃ for storage.
The above embodiments can all perform on-site measurement on water temperature, pH, heavy metals, chromaticity, turbidity, smell, conductivity, chlorophyll and dissolved oxygen of a water sample, and the specific implementation method comprises the following steps:
when detecting chlorophyll in a water sample: filtering 1L of water through a 0.45 μm first filter membrane 24; and the filtered water can be used for measuring heavy metals.
The 1L filtered water filtered by the first filter membrane 24 with the thickness of 0.45 μm is acidified to pH1-2 by adding 10mL of concentrated nitric acid, and can be used for measuring heavy metal 1 (chromium, manganese, iron, nickel, copper, zinc, cadmium and lead).
The 1L filtered water filtered by the first filter membrane 24 with the thickness of 0.45 μm is added with 10mL of concentrated hydrochloric acid to acidify to pH1-2, and can be used for measuring heavy metal type 2 (ferrous iron and mercury):
1L of filtered water filtered through the 0.45 μm first filter membrane 24 was added with sulfuric acid to a pH of 1-2 for detection of COD, permanganate index, total nitrogen, total phosphorus, ammonia nitrogen.
The nitrate nitrogen, nitrite nitrogen, orthophosphate, sulfate and suspended matters in the water sample are detected without filtering and adding a fixing agent.
Claims (4)
1. The utility model provides a water environment DNA sampling device which characterized in that mainly includes: a first sampling column (1) for sampling a larger volume of DNA, a plurality of second sampling columns (3) capable of sampling a smaller volume of DNA from the first sampling column (1), a first sampling plate (2) interposed at the middle end of the first sampling column (1), a second sampling plate (32) interposed at the position, close to the bottom end, of the second sampling column (3), and a driving mechanism (4) for simultaneously sampling the first sampling column (1) and the second sampling column (3);
the first sampling column (1) comprises: a first sampling port (11), a plurality of sampling channels (15) for sucking a sample source, a first sampling piston (451) slidably arranged in the sampling channels (15), and a crushing blade (16) arranged below the first sampling plate (2);
the first sampling plate (2) is inserted on the first sampling column (1) through a chute (23), and the first sampling plate (2) comprises: a handle (21) provided on the first sampling plate (2), a first collecting plate (22) equally divided into a plurality of parts for collecting a sample, a fixing groove (221) provided on the first collecting plate (22), a first filter membrane (24) provided on the first collecting plate (22), and a first collecting plate cover (25) for fixing the first filter membrane (24);
the second sampling column (3) is provided at the top end of the first sampling column (1), and comprises: the liquid outlet (31) is arranged above the second sampling plate (32), the check valve (311) is arranged on the liquid outlet (31), the second sampling piston (33) is arranged in the second sampling column (3) in a sliding manner and used for sucking samples, the sample middle rotary column (34) is arranged at two ends inside the first sampling column (1), the sample middle rotary column (34) is communicated with the second sampling column (3), and a liquid inlet (341) is arranged at the position, close to the top end, of the sample middle rotary column;
the second sampling plate (32) includes: a detachable sampling plate housing (321), a pull block (35) arranged on the housing (321);
the drive mechanism (4) includes: a transmission rod (47) connected to the crushing blade (16), a second screw (46) which is mutually transmitted with the transmission rod (47) through a plurality of transmission gears (48), two driving wheels (41), a chain (42) meshed with the driving wheels (41), a driving handle (43) arranged on the driving wheels (41) at the top end, a driven wheel (44) fixedly arranged on the other driving wheel (41), and a driving rod (45) meshed with the driven wheel (44) and used for driving the second sampling piston (33), wherein the driving rod (45) comprises: a first screw (452) for driving the second screw (46), a rack (453) for meshing with the driven wheel (44);
a plurality of weighting columns (13) are arranged around the first sampling column (1), a plurality of fixing rods (12) are arranged at the bottom end of the first sampling column (1), and an observation window (14) is arranged at the position of a rotating column (34) in each sample;
the first collecting plate cover (25) includes: a fixing wall (251) for being inserted into the fixing groove (221), and a fixing column (252) for fixing each first filter membrane (24);
the chain (42) is divided into a plurality of chain sections (421), each section of chain section (421) is connected through a limiting rotating shaft (422), and adsorption plates (423) are arranged at two ends of each section of chain section (421).
2. A water environment DNA sampling device according to claim 1, characterized in that a protective box (471) is arranged outside the transmission rod (47).
3. A water environment DNA sampling device according to claim 1, wherein the sampling plate housing (321) is secured by a securing member (322).
4. A method for collecting DNA from a water environment using the apparatus of any one of claims 1-3, comprising the steps of:
s1: the first filter membrane (24) adopts a filter membrane with the diameter of 0.45 mu m for measuring eukaryotes, the second filter membrane (323) adopts a filter membrane with the diameter of 0.22 mu m for measuring bacteria, after the filter membrane is installed, the length of a chain (42) is set according to the measurement depth, the device is stably submerged in water, each section of chain section (421) is opened by gravity, and the device is fixed by a fixing rod (12);
s2: the driving handle (43) is rotated to drive the driving wheel (41), the driving rod (45) drives the second lead screw (46) and the transmission rod (47) to rotate, the crushing blade (16) crushes algae impurities to prevent the blockage device, meanwhile, the driving rod (45) can also vertically slide, the first sampling column (1) is filled with water through the first sampling piston (451) until the water flows into the sample middle rotary column (34) from the liquid inlet (341) through the first filter membrane (24), and the driving rod (45) can drive the second sampling piston (33) to slide when sliding upwards, so that the water in the sample middle rotary column (34) enters the second sampling column (3) through the second filter membrane (323);
s3: standing for 10min to enable suspended ions in water to be precipitated and adsorbed on a filter screen, and then repeating S2 to enrich suspended particles and precipitates on the filter screen;
s4: and (3) rotating a driving handle (43) to withdraw the device, withdrawing the first sampling plate (2) and the second sampling plate (32) after 30min of precipitation, taking down the first filter membrane (24) and the second filter membrane (323), placing the filter membrane and the precipitate into a centrifuge tube, and storing in a refrigerator at 4 ℃.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010930987.2A CN112111384B (en) | 2020-09-07 | 2020-09-07 | Water body environment DNA sampling device and method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010930987.2A CN112111384B (en) | 2020-09-07 | 2020-09-07 | Water body environment DNA sampling device and method |
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| CN112111384A CN112111384A (en) | 2020-12-22 |
| CN112111384B true CN112111384B (en) | 2023-08-22 |
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| CN112111384A (en) | 2020-12-22 |
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