CN115165454A

CN115165454A - Glass fiber membrane-based eDNA solid passive sampler and passive sampling method

Info

Publication number: CN115165454A
Application number: CN202210791826.9A
Authority: CN
Inventors: 姚蒙; 陈晓宇
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-07-05
Filing date: 2022-07-05
Publication date: 2022-10-11

Abstract

The invention discloses an eDNA solid passive sampler based on a glass fiber membrane and a passive sampling method, and relates to the field of biology and environmental science.

Description

Glass fiber membrane-based eDNA solid passive sampler and passive sampling method

Technical Field

The invention relates to the field of biology and environmental science, in particular to an eDNA solid passive sampler based on a glass fiber membrane and a passive sampling method.

Background

At present, the global aquatic ecosystem is seriously threatened, and the diversity of aquatic organisms is rapidly reduced ^[1,2] . In 1970 to 2016, the population size of global vertebrates decreased by 68% on average, with a reduction of up to 84% in the population number of freshwater wild animals ^[3] . The aquatic ecosystem provides a plurality of services for human survival and development, including drinking water, food supply, medical and industrial and agricultural water, hydroelectric power generation, water transportation, culture and tourism, climate regulation, material circulation and the like ^[4,5] . Unreasonable and excessive utilization of water resources by human beings, such as lake-surrounding farmland building, substandard sewage discharge, increase of pesticide and fertilizer dosage, over-fishing, random release and the like, directly or indirectly cause the problems of habitat degradation and loss, biological diversity reduction, water body pollution, eutrophication, biological invasion and the like ^[2,6,7] 。

In order to alleviate the threat faced by the ecosystem and realize effective protection of endangered species, biological researchers and environmental managers need to acquire high-resolution time and space distribution information of the species and habitat use conditions, perform long-term monitoring and make effective management and protection countermeasures ^[8,9] . For detection of endangered species or early invasive species, the method faces a dilemma of low detection efficiency due to the characteristics of small population quantity, secret activities and the like. The traditional direct capture or trap trapping method has the defects of damage, low efficiency, high cost and the like ^[10] . The advent of the environmental DNA (eDNA) method has alleviated this dilemma. Monitoring endangered species by utilizing eDNA is a high-cost-benefit and non-destructive investigation method, and can quickly and effectively acquire species distribution information in an aquatic ecosystem ^[11,12] . The eDNA has been successfully applied to researches on vertebrates, invertebrates, algae and the like, including biodiversity investigation, endangered species detection, invasive species monitoring and the like. The research on vertebrates includes fish, amphibians, mammals, birds, etc., and the research on invertebrates includes arthropods, crustaceans, mollusks, etc ^[13-18] . Since various animals and water bodies are also carriers of various pathogens, the eDNA technology is gradually used for detecting various infectious pathogens and parasites of human and animals, such as schistosomiasis, enteronematodes, various viruses and the like, provides information for mastering the transmission path and range of pathogens, and promotes early warning and treatment of infectious diseases ^[19,20] 。

Problems and challenges have also arisen in the rapid development of eDNA technology for applications in biological monitoring. Because the sampling, experiment and analysis methods of various laboratories are not uniform, the eDNA research performed in the same ecosystem is lack of comparability ^[21] . Where eDNA collection is one of the most diverse steps between studies. The eDNA is used for researching non-microbial animals and plants, the development is only about 10 years till now, the related technology is far from reaching the mature stage, and a plurality of technical steps are still in exploration and optimization. The current widely used collection method of eDNA isThe active sampling method is that the collected water sample is actively filtered in a suction filtration or manual pressurization mode, and a large amount of time is consumed for sample collection ^[16] . For example, patent CN114047036A discloses a site collection method of DNA samples in karst cave water environment, which realizes filtration by manually squeezing a syringe. The volume difference of water samples filtered by different researches is large, the used filtering methods (filter membranes, suction filters and the like) are inconsistent, and the water samples are turbid or the suction filtration water samples are easy to block when the volume is large, and impurities are brought into the water samples, so that the eDNA loss is caused. Based on the current filtration and sampling mode, if the water sample cannot be filtered in time, the storage and transportation process of the water sample easily causes eDNA pollution and degradation, and the detection precision and accuracy are seriously influenced ^[22,23] . Therefore, there is a need to optimize and standardize the sampling method of eDNA so as to obtain reliable detection data.

The eDNA sampler which is simple and convenient to develop and operate, environment-friendly, high in cost benefit and capable of being produced in a standardized mode has important significance for research of eDNA of an aquatic ecosystem. On one hand, the economical and environment-friendly simple sampler can promote the development of eDNA research, reduce the entrance threshold for eDNA research, reduce eDNA degradation and eDNA pollution among samples, and facilitate the development of wider biological investigation and research based on the eDNA technology; on the other hand, the standardized sampler is helpful for solving the problem that the conventional eDNA research collection method is not uniform, so that the eDNA researches in different countries and regions are easy to perform spatial transverse comparison, and the eDNA researches in the same research place can effectively perform longitudinal comparison on a time scale.

Disclosure of Invention

In order to overcome the defects of the conventional method for enriching the eDNA by using a filtering method, the invention provides a standardized eDNA solid passive sampler which is simple to use, strong in operability and used for realizing the effective adsorption and enrichment of the DNA in a water environment, can be widely applied to the collection of the DNA in an indoor or outdoor natural water environment, is suitable for the rapid adsorption and detection of the DNA of aquatic organisms, water microorganisms and pathogens (bacteria, viruses and the like), and provides a novel efficient passive sampling method for related research or detection.

In order to realize the purpose, the technical scheme adopted by the invention is as follows:

a glass fiber membrane based eDNA solid state passive sampler comprising: the glass fiber filter membrane is arranged in the net bag, the middle part of the suspension line is connected with the net bag, and one end of the suspension line is connected with the lead weight.

Further, the pore size of the glass fiber filter was 0.7. Mu.m.

Furthermore, the mesh bag is made of PET non-woven fabric and passes through a nylon wire bundle opening.

Further, the hanging line is made of nylon.

Further, the lead weight is provided with a hook which is used for connecting a suspension line.

A glass fiber membrane-based eDNA solid passive sampling method comprises the following steps:

when sampling, the eDNA solid passive sampler is used for carrying out eDNA collection on a water body to be sampled, wherein a mesh bag provided with a glass fiber filter membrane is immersed into the water body, a suspension line is fixed on a shoreside object or a buoy, and lead is dropped into the water body to drag the mesh bag;

after sampling, the glass fiber filter membrane is taken out from the mesh bag and is placed in a sterile sealing bag for freezing storage.

Further, the sampling time of the water body is 8-72 hours.

The innovation and the advantages of the invention are illustrated as follows:

1. as seen in the background art, because the eDNA is used for researching non-microbial animals and plants, the method mainly adopts an active sampling method at present, and has a plurality of disadvantages; however, since no external force is provided, the passive sampling method of eDNA in water is still under continuous research. In order to overcome the defects of the prior art, the inventor carries out systematic testing and screening on 12 adsorbing materials, and finds that the passive sampling effect of the eDNA of the glass fiber filter membrane is the best (see comparative data below), and the eDNA passive sampling is selected from the materials which are not shown in documents such as articles, journals and the like.

2. After research, the inventor selects a glass fiber filter membrane as a core component of an eDNA solid-state actuator, and searches published patents based on the core component, and although the most technically similar published patents CN104568680A, CN105368949A, CN114047036A and CN112763284A use the glass fiber filter membrane for sampling, CN104568680A, CN105368949A and CN114047036A are all active sampling, and need a suction filtration device or artificial pressure, and all have the disadvantage of active sampling; while CN112763284A is passive sampling, it samples air, not water. Further, CN104568680A, CN105368949A, and CN112763284A are all sampling microbes or pollutant particles in the air, but not eDNA in the water body, and the visible sampling environment and the sampling object are all completely different from the present invention; CN114047036A is an active sampling method for fish eDNA in karst cave water, and obviously cannot provide technical inspiration for passive sampling.

3. In the active sampling in the prior art, a water sample passes through a filter membrane by utilizing pressure equipment (such as a suction pump or manpower) so as to obtain the eDNA in water, the method is easy to block when used for treating the turbid water sample or the water sample with suspended particles, and the treatment of the water sample with larger volume can not be carried out frequently when the field water body is filtered, so that the eDNA acquisition failure is caused. In addition, the active sampling method involves the collection, transportation, storage, and filtration of water samples, and the eDNA is degraded in these processes, affecting the accuracy of analysis. The passive sampling method adopted by the invention is not influenced by water quality, is also effective in turbid water, and compared with active sampling, the method does not depend on a filter instrument and a power supply, greatly reduces the field sampling time and material consumption cost, furthest reduces the cross contamination among different samples, can realize the effective comparison among eDNA researches at different times and places, and obtains long-term effective ecological monitoring data, thereby more efficiently and accurately researching the diversity of aquatic organisms and the distribution condition of pathogens, and formulating effective protection and management measures.

4. The invention aims to carry out the passive sampling of the eDNA of the water body simply, conveniently and efficiently, and the key point of the invention is the simplification and convenient use of equipment. The invention utilizes the design of the simply assembled filter membrane and the underwater suspension structure, does not depend on complex equipment and a large amount of manpower, can be widely applied to the collection of the eDNA of the water body under various laboratory and field conditions, and greatly reduces the sampling time, the material cost and the personnel investment of a single sample. The inventor compares the passive eDNA sampling effect with the commonly used active filtration method, and finds that the passive eDNA sampling effect and the commonly used active filtration method have the same biodiversity investigation effect (all can obtain DNA information of all fish species in a research water body), but the passive sampling method greatly reduces field work input and water sample treatment time, so that larger-scale investigation and sample acquisition can be realized at the same manpower and material cost, and large-scale, large-scale and highly refined sample collection is promoted, and the environmental and biodiversity research is greatly promoted.

Drawings

FIG. 1A is a schematic diagram of an eDNA solid passive sampler according to an embodiment of the present invention.

FIG. 1B is a schematic diagram of the components of an eDNA solid passive sampler according to an embodiment of the present invention.

FIG. 2 is a cubic graph showing the eDNA adsorption capacities of 12 kinds of adsorbents.

FIG. 3 is a graph of eDNA amount data collected over time for 3 adsorbents.

In the figure: 1-glass fiber filter membrane; 2-a suspension wire; 3-mesh bag; 4-lead weight.

Detailed Description

In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below.

The embodiment specifically discloses an eDNA solid passive sampler based on a glass fiber membrane, which is shown in FIGS. 1A-1B, and comprises a glass fiber filter membrane, a water permeable mesh bag, a suspension wire and a lead weight. In this embodiment, the glass fiber filter membrane is a 0.7 μm glass fiber filter membrane (Merck Millipore, ireland), but the pore size is not limited thereto, and other glass fiber filter membranes can be selected according to the actual detection requirement. The permeable mesh bag is made of 90 × 70mm PET non-woven fabric and has nylon thread. The suspension line is a transparent fishing line. The weight of the lead weight is 50g, and a hook capable of winding is arranged on the lead weight.

The assembly method of the eDNA solid passive sampler comprises the following steps: the filter membrane is put into a mesh bag by wearing sterile clean gloves, and the nylon thread is tied tightly. And (3) sequentially penetrating a fishing line through the lead weight hook and the mesh bag nylon line, fixing the mesh bag at a position about 5-15cm above the lead weight (the visible water depth and the sampling water layer are adjusted), and assembling the eDNA solid passive sampler.

The embodiment also discloses an eDNA solid-state passive sampling method, which uses the eDNA solid-state passive sampler for sampling, and specifically comprises the following steps: during sampling, the mesh bag provided with the filter membrane is immersed into a sampling water body, the lower part of the mesh bag is ensured to be immersed below the water surface, and a mesh bag suspension line is tied to a shoreside object or a buoy for fixing. The sampling time varies according to the research requirements, and usually 8-72 hours can ensure that the eDNA is fully adsorbed. And after sampling is finished, collecting the sampling bag, taking out the filter membrane, and placing the filter membrane in a sterile sealing bag for freezing and storing for subsequent treatment.

The eDNA solid passive sampler passively enriches the eDNA in water by means of non-active filtration. The main technical principle of the eDNA sampler is as follows: the glass fiber filter membrane is used as a container for efficiently enriching DNA, and the eDNA existing in various forms in water is intercepted, adsorbed and enriched in the immersion process of the water. The high-permeability mesh bag ensures that water can flow freely in the mesh bag on one hand and is used for fixing and protecting the filter membrane on the other hand. The high-density lead plummet is under the action of gravity, so that the mesh bag part can be always immersed in water, and continuous enrichment of eDNA in the water can be realized.

The beneficial effects of the invention are tested by a series of indoor and outdoor control experiments. The indoor control experiment result of the eDNA solid passive sampler shows that the eDNA adsorption effect of the glass fiber filter membrane is superior to or equal to that of a classical method for filtering and enriching eDNA. Compared with other adsorbing materials or filter membranes, the glass fiber filter membrane has higher concentration of the adsorbed eDNA, and the expression is more consistent and stable in different experimental groups. As shown in FIG. 2, the test results of the passive sampling capability and the active sampling capability of the eDNA of 12 adsorbing materials are shown, and the eDNA collection amount of each material in the water body is detected by using a quantitative fluorescence PCR method. Wherein PAC is powdered activated carbon, GAC is granular activated carbon, CO is absorbent cotton, AER is macroporous weakly-alkaline acrylic anion exchange resin, HA is hydroxyapatite, SG is silica gel, GF is a glass fiber filter membrane, CN is a cellulose nitrate filter membrane, MCE1.2 and MCE0.45 are mixed cellulose acetate and cellulose nitrate filter membranes with different pore diameters, NL is a nylon filter membrane, PC is a polycarbonate filter membrane, and 500mL and 50mL respectively are eDNA obtained by a traditional active water filtration method. The experimental result shows that the eDNA passive sampling performance of the glass fiber filter membrane GF is obviously superior to other 11 adsorbing materials, and the eDNA quantity obtained by filtering 500mL of water by the traditional active sampling method is similar to or even better than that obtained by filtering 500mL of water by the traditional active sampling method. FIG. 3 shows the amount of eDNA collected by 3 adsorbents over time, and the experimental results show that the glass fiber filter membrane can collect eDNA similar to that obtained by treating 1000mL of water by the conventional active sampling method within 24 hours, and can continuously collect a larger amount of eDNA with the increase of the adsorption time, so that the effect is better.

The high efficiency of the glass fiber filter for the enrichment of eDNA was deduced to be related to its own characteristics. The glass fiber filter is considered as a depth filter (depth filter) which can retain particulate matters in the filter membrane, and other filter membranes can more strongly block large particles on the surface of the filter membrane, so that the eDNA can be more stably retained ^[24,25] . Furthermore, the concentration of the adsorbed eDNA on the glass fiber filter is linearly related to time, i.e. gradually accumulates over time. Therefore, in practical applications of practical eDNA solid-state passive samplers, the enrichment of eDNA can be increased by increasing the immersion time of the sampler.

The field experiment result shows that the eDNA solid passive sampler can be applied to investigation of biological diversity of field natural water and can reach species abundance at a level equivalent to that of a method for filtering water to enrich eDNA. Short times, e.g., 0.5h, of adsorption can reach 90% of the number of eDNA-enriched assays in 1L of filtered water, while 8h and longer times can reach a number of species comparable to 1L of water filtered. The enrichment mode of the solid-state passive sampler for eDNA can greatly reduce the workload of field collection, reduce the burden of collecting, transporting and storing water samples on the premise of equal eDNA enrichment efficiency, and reduce the cross contamination between samples and sampling points without purchasing and carrying heavy high-power filters. In addition, the eDNA method was used for comparisonSpecies abundance changes or beta diversity studies are questioned, mainly in relation to the amount of eDNA sampled, and the sampling coverage is not sufficient to reflect the local true species abundance ^[26,27] . This is practically limited by the time and cost of filtering water to enrich eDNA, for example, for fast flowing bodies of flowing water, it is often necessary to filter large volumes of water to enrich a certain amount of eDNA. Comparison of biodiversity in space and time and with natural and man-made environmental changes is central to biodiversity monitoring ^[21,28] . The sampling mode of the eDNA solid passive sampler can solve the dilemma, reduce the time for collecting and filtering water samples, realize large-scale sampling by setting a plurality of sampling repetitions, and further solve the ecological problem of larger space-time scale.

The eDNA solid passive sampler has the following broad application and development prospects in future eDNA research:

(1) Monitoring of biodiversity. The eDNA solid passive sampler can be used for simulating an infrared trigger camera which is generally applied to terrestrial biodiversity monitoring at present, realizing large-scale cross-regional standardized grid monitoring on natural water, establishing an eDNA research database and realizing effective long-term ecological monitoring. And the research group is not limited to aquatic organisms, so that the DNA can be effectively monitored only by releasing DNA into water for terrestrial or amphibious species, and the species information with the research vacancy is filled.

(2) Detection of endangered species. Most of aquatic species such as fishes, amphibians and reptiles have secret activities and are difficult to observe directly by naked eyes, and the appearance of the eDNA technology enables people to acquire potential distribution information of the aquatic species without observing biological individuals. The eDNA solid passive sampler has the characteristics of simplicity and convenience in operation, high cost benefit and environmental friendliness, and can be arranged at multiple points on the premise of not damaging endangered species, efficiently detect the endangered species, quickly acquire species distribution information and help to make and execute protective management measures as soon as possible.

(3) Prevention and monitoring of invasive species. Biological invasion is one of the serious threats facing aquatic ecosystems. Similar to the principle of (2), the eDNA solid passive sampler has multiple advantages that the eDNA solid passive sampler can realize early monitoring of invasive species, can quickly acquire time-space distribution information of the invasive species, and makes reasonable countermeasures to prevent further expansion of harm of biological invasion.

(4) And (5) detecting a pathogen. The pathogens of various human and animal infectious diseases can be detected in the environmental water body, and important information such as the transmission range, the environmental concentration, the transmission path and the like is provided. The eDNA solid passive sampler is widely distributed in key monitoring water bodies (such as domestic sewage, farm wastewater and the like) for pathogen eDNA sampling, can realize low-cost, interference-free and wide-periodic monitoring, and is beneficial to public health and management of animal-derived infectious diseases.

(5) And (5) monitoring the water quality. Because the eDNA solid passive sampler has the characteristic of strong adsorbability, the prospect of the sampler on monitoring the physicochemical indexes of water quality can be continuously developed in the future, if the eDNA solid passive sampler is realized, the eDNA solid passive sampler is beneficial to monitoring standardized environmental indexes, and simultaneously monitoring units of biological indexes and environmental indexes are uniformly quantized.

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Although the present invention has been described with reference to the above embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An eDNA solid-state passive sampler based on a glass fiber membrane, comprising: the glass fiber filter membrane is arranged in the net bag, the middle part of the suspension line is connected with the net bag, and one end of the suspension line is connected with the lead weight.

2. The solid-state passive sampler of eDNA of claim 1, wherein the glass fiber filter has a pore size of 0.7 μm.

3. The eDNA solid-state passive sampler of claim 1, wherein the mesh bag is made of PET nonwoven fabric and passes through the nylon thread.

4. The eDNA solid-state passive sampler of claim 1, wherein the hanging string is a nylon string.

5. The eDNA solid-state passive sampler of claim 1, wherein the lead weight is provided with a hook for connecting a hanging wire.

6. An eDNA solid passive sampling method based on a glass fiber membrane is characterized by comprising the following steps:

when in sampling, the eDNA solid passive sampler of any one of claims 1-5 is used for carrying out eDNA collection on a water body to be sampled, wherein a mesh bag provided with a glass fiber filter membrane is immersed into the water body, a suspension line is fixed on a shore object or a buoy, and lead is dropped into the water body to drag the mesh bag;

and after sampling, taking out the glass fiber filter membrane from the mesh bag, and placing the glass fiber filter membrane in a sterile sealing bag for freezing and storing.

7. The method for passively sampling eDNA in solid state according to claim 6, wherein the time for sampling the water is 8-72 hours.