CN111635928A

CN111635928A - Water environment DNA sample collection and normal temperature drying storage method

Info

Publication number: CN111635928A
Application number: CN202010528875.4A
Authority: CN
Inventors: 杨江华; 张效伟
Original assignee: Nanjing Yijinuo Environmental Protection Technology Co ltd
Current assignee: Nanjing Yijinuo Environmental Protection Technology Co ltd
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2020-09-08

Abstract

The invention discloses a method for collecting a water environment DNA sample and storing the sample at normal temperature in a drying way, belonging to the technical field of environment detection. The method for storing the DNA sample in the water environment at normal temperature comprises the steps of collecting the sample in a water body by using a filter membrane, treating the filter membrane by sequentially using isopropanol and ethanol under the condition of negative pressure adsorption after the collection is finished, and finally, further air-drying the filter membrane and sealing for storage, wherein the dosage of the ethanol is 1.2-1.5 times of that of the isopropanol. The method can effectively prolong the storage time of the environmental sample, reduce the storage condition of the environmental water sample, realize the long-term storage of the environmental DNA sample in the filter membrane under the normal temperature condition, obviously reduce the cost, improve the aging, and can not cause interference to the subsequent DNA monitoring.

Description

Water environment DNA sample collection and normal temperature drying storage method

Technical Field

The invention belongs to the technical field of environmental detection, and particularly relates to a method for collecting and storing a water environment DNA sample.

Background

It is well known that species abundance studies are based on environmental DNA concentrations, which are affected by the release and degradation processes of DNA, as well as environmental DNA extraction methods and water sample volumes. The eDNA macro-barcode technology is mainly used for detecting environmental DNA by acquiring plankton (including plant bodies, animal bodies, microorganisms and organisms with different sizes) in a receiving water body, suspended particulate matters in a water sample and the like, so that if the DNA in a corresponding water body environment is truly reflected, the true validity of a collected sample must be ensured. In order to minimize the above-mentioned changes, some protective measures are generally taken on the sample, such as refrigeration, freezing or adding a stabilizer; then, the transportation time is shortened as much as possible and the analysis and measurement are carried out as soon as possible.

The retrieval shows that in the prior art, the storage of a general environmental DNA sample is mainly in the modes of water sample storage and low-temperature freezing storage, and the drying of the environmental sample is mainly performed by a freeze-drying method, the core of the method is that the water is gradually removed by utilizing the desublimation effect of water under vacuum in a low-temperature environment, and the method has the advantages that the environmental sample is always kept at low temperature in the dehydration process, the propagation of microorganisms in the sample is effectively inhibited, and the degradation of the environmental DNA in the environmental sample can be well delayed; the method has the disadvantages that the operation needs to be finished by a freeze dryer, can only be finished in a laboratory, and cannot be operated in the field, in addition, the freeze-drying process of the sample needs to be precooled, the freeze-drying of the sample is slow, and the timeliness is poor.

Through retrieval, related applications are disclosed in the prior art, for example, the publication number CN 105636937B, which is published as 2018, 07, 17.2018, discloses a stabilizer for preserving a biological sample, and the stabilizer is mixed with the biological sample according to a certain ratio during use, so that the integrity and stability of the biological sample can be maintained for a long time under non-freezing conditions; however, the added stabilizer risks interfering with the monitoring program.

Therefore, a novel and effective water environment DNA sample collecting and storing method is developed, and the method plays an important role in improving the analysis and monitoring quality of the water environment species abundance.

Disclosure of Invention

1. Problems to be solved

Aiming at the defects of complicated operation, high cost and poor timeliness of the preservation of an environmental DNA sample obtained by adopting a freeze-drying method in the prior art and the defect of monitoring interference caused by adding a stabilizer, the invention provides the method for collecting the environmental DNA sample and storing the environmental DNA sample at normal temperature.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a method for collecting a water environment DNA sample and storing the sample at normal temperature in a drying way, which comprises the following steps:

1) filtering and collecting a water environment DNA sample by using a filter membrane, wherein the DNA sample is collected to the filter membrane;

2) sequentially filtering the filter membrane by using isopropanol and ethanol under the condition of negative pressure adsorption; the dosage of the ethanol is 1.2-1.5 times of that of the isopropanol;

3) and (3) air-drying the filter membrane treated in the step 2), and sealing and storing.

Preferably, in the step 2), the negative pressure adsorption condition is maintained for 0.5 to 1 minute after all the isopropanol is filtered and before the ethanol is filtered.

Preferably, in the step 2), the continuous filtration time of the isopropanol is kept at least 30 seconds, the negative pressure adsorption condition is kept for 0.5-1 minute after the filtration is finished, and then the continuous filtration time of the ethanol is kept at least 1 minute.

Preferably, in the step 3), the sealed storage method specifically comprises: and (5) filling the air-dried filter membrane into a sealed storage tube filled with a molecular sieve for storage.

Preferably, the sealed storage tube is further filled with allochroic silica gel.

The sealed storage tube is filled with allochroic silica gel and the molecular sieve at the same time, the molecular sieve can continuously adsorb water in the filter membrane and keep a dry environment, and the allochroic silica gel is used for indicating the water absorption saturation of the molecular sieve so as to realize long-term storage of environmental DNA in the filter membrane.

Preferably, the air drying temperature in the step 3) is 35-55 ℃, and the air drying time is 2-10 min.

Preferably, the sealed storage tube comprises a tube body for containing the filter membrane, a hollow sleeve arranged inside the tube body and a tube cover for sealing the tube body; the hollow sleeve is filled with a molecular sieve and allochroic silica gel, and the top surface of the hollow sleeve is connected with the tube cover and forms an integrated structure with the tube cover.

Preferably, the isopropanol is anhydrous isopropanol, the ethanol is anhydrous ethanol, and/or the filter membrane is a water-based filter membrane.

Preferably, the operation of the method is realized by using a negative pressure filtering device, the negative pressure filtering device comprises a negative pressure pump, a water environment DNA sample filter with a filter membrane and a communicating pipeline, the negative pressure pump is communicated with the water environment DNA sample filter with the filter membrane through the communicating pipeline, and the communicating pipeline is provided with a pressure release valve.

Preferably, the method for collecting the DNA sample in the water environment and storing the DNA sample in the water environment by drying at normal temperature comprises the following steps:

(1) filtering a water sample by using a negative pressure filtering device, closing a negative pressure pump and opening a pressure release valve after the water sample is filtered;

(2) closing the pressure release valve after the pressure is zero, adding anhydrous isopropanol into the water environment DNA sample filter, starting the negative pressure pump again for filtering, closing the negative pressure pump after the filtering is finished, and opening the pressure release valve;

(3) closing the pressure release valve after the pressure is zero, adding ethanol into the water environment DNA sample filter, starting the negative pressure pump again for filtering, keeping the negative pressure pump in an open state after the filtering is finished, and performing air drying treatment on the filter membrane by using a fan;

(4) after the treatment is completed, the filter membrane is taken down for sealed storage.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the novel water environment DNA collecting and storing method provided by the invention, the filter membrane is firstly filtered by using isopropanol under the negative pressure condition, so that residual moisture in the filter membrane can be removed, moisture in an environment sample can be taken away, and residual active microorganisms in the filter membrane can be effectively killed; the method utilizes the strong bactericidal property of the isopropanol and the volatility of the ethanol to combine the isopropanol and the ethanol to realize the synergistic effect of quickly removing residual moisture in the environmental sample and the filter membrane and effectively killing the active microorganisms, ensures no residue of the isopropanol and realizes drying through quick volatilization, and does not cause interference on the final detection result.

(2) According to the novel water environment DNA acquisition and storage method provided by the invention, after the isopropanol is filtered, negative pressure is continuously pumped for a period of time, so that the isopropanol residue can be more effectively removed, and the detection result cannot be interfered; according to the method, the filter membrane which is dried is directly placed in the sealed storage tube filled with the allochroic silica gel and the molecular sieve for storage, the molecular sieve can be used for further and continuously adsorbing water in the filter membrane, so that the DNA sample is continuously in a dry environment, meanwhile, the allochroic silica gel is used for indicating the water absorption saturation of the molecular sieve, the invalid molecular sieve is conveniently replaced in time, and therefore long-term storage of the environmental DNA in the filter membrane in the normal-temperature environment is achieved.

(3) The novel water environment DNA collecting and storing method provided by the invention can ensure that the concentration of the environment DNA in the collected filter membrane cannot be reduced along with the prolonging of the storage time (the concentration of the DNA after being stored for 30 days is basically not different from that before being stored), and the concentration of the DNA sample stored at normal temperature according to the method is basically not different from that of a result stored by freezing at minus 80 ℃, so that the method can realize the long-term effective storage of the environment DNA sample under the normal temperature condition, and is simple to operate, low in cost and beneficial to popularization.

Drawings

FIG. 1 is a schematic view of a sealed storage tube according to the present invention;

FIG. 2 is a comparison of DNA concentrations extracted after storage at different temperatures using the filters obtained by the collection and storage methods of example 3 and comparative example;

FIG. 3 is a comparison of DNA concentrations extracted after storage of the filters obtained by the collection and storage methods of example 3 and comparative example at different storage times;

in the figure: 1. a tube cover; 2. hollowing out the sleeve; 3. molecular sieves and allochroic silica gels; 4. filtering the membrane; 5. a tube body.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or the two elements can be directly connected together; when an element is referred to as being "connected" to another element, it can be directly connected to the other element or the two elements may be directly integrated. In addition, the terms "upper", "lower", "left", "right" and "middle" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the relative positions may be changed or adjusted without substantial technical changes.

The "negative pressure filtration device" described herein may be in the patent number: the intelligent DNA acquisition device for the water body environment in CN209400274U can also be other acquisition devices capable of generating negative pressure acquisition effects.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

In this embodiment, utilize negative pressure filter equipment to filter the water sample, negative pressure filter equipment includes the negative pressure pump, and the negative pressure pump is linked together through communicating pipe and the water environment DNA sample filter cup that has the filter membrane, be equipped with the relief valve on the communicating pipe.

The test water sample of the water environment DNA sample is collected from the Jiuxiang river (northern latitude 32.113681, east longitude 118.945127).

The filtration membrane adopts a Jinteng water system filtration membrane, the aperture of the filtration membrane is 0.45 mu m, and the diameter of the filtration membrane is 50 mm.

In this embodiment, the method for collecting and storing the water environment DNA sample comprises the following steps:

(1) filtering a water sample by using a negative pressure filtering device, and closing a negative pressure pump and opening a pressure release valve after the water sample is filtered;

(2) after the pressure is zero, closing a pressure release valve, adding 250 ml of anhydrous isopropanol into the water environment DNA sample filter, starting the negative pressure pump again until the anhydrous isopropanol is completely filtered (the filtering time is about 0.5min), keeping the negative pressure condition for continuous filtering for 0.5-1 min, and closing the negative pressure pump to open the pressure release valve;

(3) after the pressure is zero, closing the pressure release valve, adding 300 milliliters of absolute ethyl alcohol (the volume of the absolute ethyl alcohol is 1.2 times of that of the absolute isopropanol) into the water environment DNA sample filter, starting the negative pressure pump again until the absolute ethyl alcohol is completely filtered (the filtering time is about 1min), keeping the negative pressure pump in an open state, and carrying out air drying treatment on the filter membrane by using a fan, wherein the air drying temperature is 35 ℃, and the air drying time is 10 minutes;

Example 2

In this embodiment, a negative pressure filtration device is used to filter a water sample, and the negative pressure filtration device includes a negative pressure pump, and the negative pressure pump is communicated with a water environment DNA sample filter cup with a filter membrane through a pipeline (provided with a pressure release valve thereon).

The test water sample was collected from the Jiuxiang river (northern latitude 32.113681, east longitude 118.945127).

The filtration adopts a Jinteng water system filter membrane, the aperture of the filter membrane is 0.45 mu m, and the diameter of the filter membrane is 50 mm.

In this embodiment, the method for drying and storing the water environment DNA sample comprises the following steps:

(2) closing a pressure release valve after the pressure is zero, adding 350 ml of anhydrous isopropanol into the water environment DNA sample filter, standing for at least 30 seconds, then starting a negative pressure pump again, continuing to filter for 0.5-1 minute until the anhydrous isopropanol is completely filtered (the filtering time is about 1min), and closing the negative pressure pump to open the pressure release valve;

(3) closing the pressure release valve after the pressure is zero, adding 525 ml of absolute ethyl alcohol (the volume of the absolute ethyl alcohol is 1.5 times of that of the absolute isopropanol) into the water environment DNA sample filter, standing for at least 1 minute, then starting the negative pressure pump again until the absolute isopropanol is completely filtered (the filtering time is about 1min), keeping the negative pressure pump in an open state, and carrying out air drying treatment on the filter membrane by using a fan, wherein the air drying temperature is 55 ℃, and the air drying time is 2 minutes;

Example 3

(3) closing the pressure release valve after the pressure is zero, adding 455 milliliters of absolute ethyl alcohol (the volume of the absolute ethyl alcohol is 1.3 times of that of the absolute isopropanol) into the water environment DNA sample filter, standing for at least 1 minute, then starting the negative pressure pump again until the absolute isopropanol is completely filtered (the filtering time is about 1min), keeping the negative pressure pump in an open state, and carrying out air drying treatment on the filter membrane by using a fan, wherein the air drying temperature is 35-55 ℃, and the air drying time is 2-10 minutes;

The volume of the filtered water sample for each filter was 200 mL. To calculate the dry weight of the filter, 10 filters were weighed together and then divided by 10 to give the dry weight of each filter. And after the water sample is filtered and dried, weighing 10 dried filter membranes together to obtain the dry weight of the water sample after being collected and dried by one filter membrane.

Comparative example

In this comparative example, the water sample was filtered by using the negative pressure filter device, and the conditions of the negative pressure filter device and the negative pressure filter device were the same as those of example 3.

The test water samples were also collected from the Jiuxiang river (northern latitude 32.113681, east longitude 118.945127).

The filtration membrane is also a Jinteng water system filtration membrane, the aperture of the filtration membrane is 0.45 μm, and the diameter of the filtration membrane is 50 mm.

In the comparative example, the steps of the method for drying and storing the DNA sample in the water environment are as follows:

(2) closing the pressure release valve after the pressure is zero, opening the negative pressure pump again, filtering for 0.5-1 min, keeping the negative pressure pump in an open state, and performing air drying treatment on the filter membrane by using a fan, wherein the air drying temperature is 35-55 ℃, and the air drying time is 2-10 min;

(3) after the treatment is completed, the filter membrane is taken down for sealed storage.

Example 4

This example was conducted to test the water absorption of the filter membrane, and the water sample was filtered by the negative pressure filtration apparatus under the same conditions as in example 3.

In this comparative example, the procedure for performing the filter membrane water uptake test was as follows:

(2) subsequently, the filter was removed and weighed.

The volume of the filtered water sample for each filter was 200 mL. To calculate the dry weight of the filter, 10 filters were weighed together and then divided by 10 to give the dry weight of each filter. After water samples are filtered (not dried), 10 water samples are also adopted and filtered, and then filter membranes are weighed together, so that the wet weight of each filter membrane after the water samples are collected is obtained. As a result, the water absorption capacity of the filter membrane is about 0.196g after water sample collection; after the drying and sampling method in embodiment 3 of the invention is used for dehydration and drying, the dehydration rate reaches 94.4%, and the filter membrane is almost completely dried; after dehydration drying by the method in the comparative example, the dehydration rate of the filter membrane was only 4.7%.

TABLE 1 Wet weight of filter membranes after Water sampling and Dry weight after dehydration drying respectively

Example 5

Storing the filter membrane (containing the DNA sample in the filter membrane) dehydrated and dried in the embodiment 3 and the comparative example for a period of time, and then extracting DNA to verify the storage effect;

the sealed storage tube used in this embodiment, as shown in fig. 1, includes a tube body 5 for containing a filter membrane, a hollow sleeve 2 disposed inside the tube body 5, and a tube cover 1 for sealing the tube body 5; the top surface of the hollow sleeve 2 is connected with the tube cover 1 and forms an integrated structure with the tube cover 1, and the hollow sleeve 3 is filled with a molecular sieve and allochroic silica gel 3 for keeping the interior of the tube dry.

The 6 filter membranes (named as A1-A6) dried and dehydrated in the embodiment 3 are respectively put into 6 different sealed storage tubes, the sealed storage tube containing A1-A3 is put into a refrigerator at minus 80 ℃ for storage, and the sealed storage tube containing A4-A6 is put into a refrigerator for storage at normal temperature; taking 6 filter membranes (named as B1-B6) dried and dehydrated in the comparative example, respectively putting the 6 filter membranes into 6 different sealed storage tubes, placing the sealed storage tube containing B1-B3 in a refrigerator at-80 ℃ for storage, and placing the sealed storage tube containing B4-B6 in a refrigerator at normal temperature for storage;

after being stored for 7 days, the DNA of all samples is extracted by using an OMEGA water sample DNA extraction kit. As shown in FIG. 2, it was found that the concentration (average value) of the environmental DNA extracted from the filter membrane of B4-B6 in the storage state at ordinary temperature was significantly lower than that extracted from the filter membrane of B1-B3 in the storage state at a low temperature after dehydration-drying by the method of comparative example, and the rate of decrease in the DNA concentration was about 50%.

After dehydration and drying according to the method of example 3 of the present invention, the concentration (average) of the environmental DNA extracted from the filters A4-A6 in the storage state at room temperature was almost the same as the concentration (average) of the environmental DNA extracted from the filters A1-A3 in the storage state at a low temperature. This demonstrates that the dry sampling and storage method of the present invention can better preserve environmental DNA at ambient temperature conditions.

Example 6

Storing the filter membranes obtained after dehydration and drying according to the methods in the embodiment 3 and the comparative example respectively, and extracting DNA after a period of time to verify the storage effect;

the sealed storage tube for storing the DNA sample used in this example was the same as that used in example 5.

6 filter membranes (named as C1-C6) dried and dehydrated according to the method of the embodiment 3 are respectively put into 6 different sealed storage tubes, the sealed storage tube containing C1-C3 is put into a refrigerator at minus 80 ℃ for storage, and the sealed storage tube containing C4-C6 is put into a refrigerator for storage at normal temperature; 6 filter membranes (named as D1-D6) dried and dehydrated according to the method of the comparative example are respectively put into 6 different sealed storage tubes, and the sealed storage tubes containing D1-D3 are put into a refrigerator at minus 80 ℃ for storage; storing the sealed storage tube containing D4-D6 at normal temperature;

after being stored for 7 days, 14 days and 30 days, the DNA of all samples is extracted by using an OMEGA water sample DNA extraction kit. The results are shown in FIG. 3.

The results show that the dry storage mode of the invention does not cause the reduction of the DNA concentration due to long-term storage, thereby maintaining the stability of the DNA sample, while the DNA concentration of the sample in the comparative example is reduced along with the change of the storage time.

The above-mentioned embodiments only express a certain implementation mode of the present invention, and the description thereof is specific and detailed, but not construed as limiting the scope of the present invention; it should be noted that, for those skilled in the art, without departing from the concept of the present invention, several variations and modifications can be made, which are within the protection scope of the present invention; therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for collecting a water environment DNA sample and storing the sample at normal temperature in a drying way is characterized in that: the method comprises the following steps:

2. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 1, which is characterized in that: in the step 2), after all the isopropanol is filtered, the negative pressure adsorption condition is kept for 0.5-1 minute until the ethanol is filtered.

3. The method for collecting and storing the DNA sample in the water environment according to claim 1 or 2, which is characterized in that: in the step 2), the continuous filtration time of the isopropanol is kept at least 30 seconds, the negative pressure adsorption condition is kept for 0.5-1 minute after the filtration is finished, and then the continuous filtration time of the ethanol is kept at least 1 minute.

4. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 3, which is characterized in that: in the step 3), the sealed storage method specifically comprises the following steps: and (5) filling the air-dried filter membrane into a sealed storage tube filled with a molecular sieve for storage.

5. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 4, which is characterized in that: the sealed storage tube is also filled with allochroic silica gel.

6. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 5, which is characterized in that: the air drying temperature in the step 3) is 35-55 ℃, and the air drying time is 2-10 min.

7. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 5, which is characterized in that: the sealed storage tube comprises a tube body (5) for containing the filter membrane (4), a hollow sleeve (2) arranged inside the tube body (5) and a tube cover (1) for sealing the tube body (5); the hollow sleeve (2) is filled with a molecular sieve and allochroic silica gel (3), and the top surface of the hollow sleeve (2) is connected with the tube cover (1) and forms an integrated structure with the tube cover (1).

8. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 7, which is characterized in that: the isopropanol is anhydrous isopropanol, the ethanol is anhydrous ethanol, and/or the filter membrane is a water system filter membrane.

9. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 8, which is characterized in that: the operation of the method is realized by using a negative pressure filtering device, wherein the negative pressure filtering device comprises a negative pressure pump, a water environment DNA sample filter with a filter membrane and a communicating pipeline, the negative pressure pump is communicated with the water environment DNA sample filter with the filter membrane through the communicating pipeline, and a pressure release valve is arranged on the communicating pipeline.

10. The method for collecting and storing the DNA sample in the water environment at normal temperature according to claim 9, which is characterized in that: the method comprises the following specific steps: