DK178664B1 - A system and a method for concentrating traces of tissue from aquatic organisms in a water sample and use thereof - Google Patents

Abstract

Use of a system and a method for concentrating traces of tissue from aquatic organisms (eDNA), in particular aquatic animal species and/or aquatic plant species or nonaquatic organisms, including humans, which may lose tissue to the water environment, in a water sample (2) is disclosed. The method comprises subjecting the water sample (2) to membrane filtration in one or more membrane module(s) (12), preferably microfiltration or ultrafiltration module(s) using tangential flow filtration and repeating the membrane filtration step one or more times on the retentate for concentration of the sample up to 1000 times, preferably up to 100-1000 or more preferred up to 200-1000 times. The retentate is either recycled from a retentate outlet (14) of the membranemodule(s) (12) to a sample inlet (11) of the membrane module(s) (12) or the flow is reversed through the membrane module(s) (12). Hereby a concentration of the retentate to a high degree is obtained and it is possible to extract eDNA in sufficient quantities to enable detection of certain species of animal and/or plant species in the aquatic environment even if the animal or plant species is present in very low numbers in the aquatic environment. The method and the system as used enable DNA extraction from the concentrated retentate by simple standard methods developed for extraction of nucleic acids from suspended samples. Any efforts to release the sample from any filter surfaces during extraction are avoided.

Description

[Use of a system and a method for concentrating traces of tissue from aquatic organisms in a water sample and use thereof]

Field of the Invention

The present invention relates to a method for concentrating mitochondria and/or chlo-roplasts which are present in water samples as traces of partially decomposed tissue from aquatic organisms in a water sample, in particular aquatic animal species and/or aquatic plant species, or non-aquatic organisms, including humans, which may lose tissue to the water environment.

The present invention relates also to use of a system for concentrating mitochondria and/or chloroplasts which are present in water samples as traces of partially decomposed tissue from aquatic organisms (eDNA), in particular aquatic animal species and/or aquatic plant species, or non-aquatic organisms, including humans, which may lose tissue to the water environment.

In addition, the present invention relates to the use of the method and/or the apparatus for concentrating traces of tissue and nucleic acids from aquatic organisms in a water sample from an environmental habitat, in particular aquatic animal species and/or aquatic plant species, or non- aquatic organisms, including humans, which may lose tissue to the water environment.

Background of the Invention

All organisms emit fragments of tissue to the environment they inhabit. It has recently be shown, that aquatic organisms which lose traces of tissue to the water environment, e.g. through faeces, can be detected by DNA techniques (e.g. Dejean et al., 2011; Jer-de et al., 2011; Thomsen et al., 2012a, 2012b). Traces of DNA from tissue residuals are called environmental DNA (eDNA).

Analysis of eDNA is expected to open a new area for biodiversity assessments and analysis in the aquatic environment (the water matrix), with various applications. The concentration of tissue residuals and eDNA in the water will determine the detection limit for a specific organism in a water sample (Thomsen et al. 2012b). Therefore increased eDNA concentration before DNA analysis will increase sensitivity of any technique detecting eDNA. Methods to concentrate eDNA in environmental samples prior to DNA analysis will therefore have a crucial impact on assay performance and are highly needed.

The following articles are all examples which deal with the analysis of the water samples for detecting traces of nucleic acids, particularly mitochondrial DNA (i.e. eDNA), from aquatic animal species for detecting their presence in the environment:

Dejean, T., Valentini, A., Duparc, A., Pellier-Cuit, S., Pompanon, F., Taberlet, P., Miaud, C., 2011. Persistence of Environmental DNA in Freshwater Ecosystems. PLoS ONE 6, e23398.

Jerde, C.L., Mahon, A.R., Chadderton, W.L., Lodge, D.M., 2011. “Sight-unseen” detection of rare aquatic species using environmental DNA. Conserv. Lett. 4, 150-157. Thomsen, P.F., Kielgast, J., Iversen, L.L., Møller, P.R., Rasmussen, M., Willerslev, E., 2012a. Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples. PLoS ONE 7, e41732.

Thomsen, P.F., Kielgast, J., Iversen, L.L., Wiuf, C., Rasmussen, M., Gilbert, M.T.P., Orlando, L., Willerslev, E., 2012b. Monitoring endangered freshwater biodiversity using environmental DNA. Mol. Ecol. 21, 2565-2573.

However, none of the mentioned articles effectively deal with the problem of concentrating eDNA from various sources, e.g. animals and/or plants in the highly diluted water samples collected from the environment in order to prepare DNA extraction and improve subsequent detection. WO 98/30685 A2 discloses concentrating substances and removal of endotoxins by filtering a liquid, e.g. blood or fermentation broth, through an ultrafiltration membrane module e.g. for obtaining vaccines. The method is suitable for a significantly higher concentration of the substances in the liquid, and does not deal with the problem of obtaining a concentrate of e.g. several hundred times the original concentration of an extremely diluted water sample of eDNA in order to be able to perform a subsequent detection of the eDNA present in the sample. US 2011/061474A discloses concentrating dilute biological samples with e.g. DNA by capturing the particles or DNA in or on a filter while eliminating filtrate. The device includes a filter in a disposable pipette tip from which the sample can be dispensed after processing of the entire sample. When the entire sample is processed, an elution foam or liquid is used to elute captured particles/molecules and dispense them into a reduced volume. When used on an environmental water sample, e.g. from a lake, where the eDNA in the sample is present in extremely low concentrations, it will require the operator to filter several litres of the sample manually through the pipette in order to collect sufficient DNA in the filter during the subsequent elution of the filter. This requires a significant amount of manual handling of the sample during pipetting of the sample, which is time consuming and costly. In addition, the extensive manual use of the pipette in order to collect enough material to be able to detect certain species of organisms present in the water sample, e.g. certain species of plants and/or animals, is not ergonomically beneficial to the laboratory staff as such long term use of the pipette may cause injury to the person operating the pipette.

Traces of DNA and/or RNA from tissue residuals, which can be retrieved and analysed from an environmental sample without isolating the organism it originates from, is called environmental DNA (eDNA). In its broadest definition eDNA is thus defined as any nucleic acids (DNA and/or RNA) found in environmental samples. eDNA is DNA or RNA which can be extracted from an environmental samples without isolating the organism it originates from, i.e. eDNA is present in the environmental sample, in particular a water sample, and forms no longer part of the organism it originates from.

The organisms are in particular defined as aquatic or semi aquatic multicellular, i.e. macroscopic, animal species and/or plant species and/or other organisms which may lose DNA and/or RNA, including DNA present in mitochondria and/or chloroplast, to the water environment, e.g. through faeces, litter or other fomites. Such other organisms also include non-aquatic multicellular, i.e. macroscopic, animals, including humans, which have been in contact with the aquatic environment for sufficient time to lose eDNA to the aquatic environment. eDNA may in this application be defined as DNA from mitochondria and/or chloro-plasts originating from tissue residuals of eukaryotic organisms as defined above. The eDNA in water samples may be in solution and/or inside intact mitochondria and/or chloroplast and/or intact but excreted cells.

Object of the Invention

An objective of the present invention is to provide a method and use of a system for concentrating highly diluted eDNA in the water samples from the aquatic environment to increase sensitivity of the assays used for detecting eDNA in order to be able to subsequently detect eDNA more reliable and with increased sensitivity.

In addition, it is an object of the present invention to provide a method and use of a system for concentrating eDNA in a highly diluted aqueous sample which can run in a fully automated or semi-automated way and thus does not require extensive manual intervention.

Description of the Invention

The objects of the present invention are met by a method for concentrating chloro-plasts and/or mitochondria and/or DNA contained herein which are present in a water sample from an aquatic biotope as traces of tissue from aquatic or semi aquatic macroscopic organisms (eDNA) in an water sample, in particular aquatic organisms or macroscopic non-aquatic organisms, including humans, which may lose tissue to the water environment. The method comprises the steps of subjecting the aqueous sample to membrane filtration in at least one membrane module using tangential flow filtration and repeating the membrane fdtration step one or more times on the retentate for the retention and concentration of the sample before DNA extraction. The volume of the sample in the retentate is reduced up to 1000 times, preferably 100-1000, more preferably 200-1000 times.

The objects of the present invention are also met by use of a chloroplast and/or mitochondria in a water sample concentrating system for concentrating traces of tissue from macroscopic organisms (eDNA) in a water sample from an aquatic biotope prior to detection of eDNA from selected species, in particular aquatic organisms or macro scopic non- aquatic organisms, including humans, which may lose tissue to the water environment, comprising one or more tangential flow (also known as cross flow) mi-crofiltration/ultrafiltration modules, wherein the one or more filtration modules each comprises a filtration membrane having a pore size of 0.1-5 pm, and wherein the apparatus comprises means for recycling the retentate to the inlet of module or means for reversing the flow of the retentate through the membrane module from an outlet to an inlet of the membrane module for concentrating eDNA in the retentate flow.

Hereby a concentration of the retentate to a high degree is obtained and it is possible to extract sufficiently eDNA to obtain detection of the presence of species of animals and/or plants in the aquatic environment, e.g. a lake, a river or the sea, from which the sample was taken, even if the animal or plant species is present in very low numbers. The method according to the invention and the system as used according to the present invention enables DNA extraction from the retentate by simple standard methods developed for extraction of nucleic acids from suspended samples. Any efforts to release the sample from any filter surfaces during extraction are avoided.

The method is particularly useful, when the water sample is collected in order to detect very rare animal and/or plant species in the aquatic environment from which the sample was taken, even if the animal or plant species is present in very low numbers in the investigated aquatic environment.

In addition, the method is suitable for investigating variations of quantitative presence of certain species which also may be present in high numbers in an aquatic environment. The variations in abundance of particular species, e.g. fish species, can be relevant for a number of applications, e.g. for evaluating geographical distribution of a population, for water quality assessment, and for detection of low level presence of species, e.g. due to overfishing or diseases in the population.

Similarly, the method is suitable for investigating whether certain non-aquatic animals, including humans, have been in contact with the water environment either directly or indirectly through emission of waste streams, e.g. manure or sewage, to the environment. Thus, the invention may also be useful for detecting pollution of the aquatic environment and for determining the source of pollution, in particular if it originates from manure or sewage or from animal corpses or parts thereof, which are dumped in the aquatic environment.

Depending on the dilution of eDNA in the investigated environment and the abundance of target species it is often not possible to detect eDNA from such animals and/or plant species by direct DNA extraction and DNA amplification without concentrating the water, e.g. 100 times in relation to the original sample, i.e. removing e.g. 99% of the water in the sample.

Water is removed from the water sample e.g.by passing the sample from a sample reservoir through tubes or pipes to the membrane module for concentration of eDNA in the retentate.

The membrane module as used according to the present invention uses tangential flow filtration and is preferably a spirally wound, a tubular module or a hollow fiber module. The modules may be based on microfiltration and/or ultrafiltration membranes, e.g. modules based on membranes of nylon, cellulose ether, mixed cellulose ether, polysolfone or polyethersulfone resins or mixtures thereof or combinations thereof. Alternatively, the membrane module is a ceramic module, e.g. based on aluminium oxide, 01-AI2O3, which is sterilisable and easy to clean.

The pressure applied to the inlet side of the membrane drives the filtration process, and is e.g. 0.2-4.0 bar and may be constant or gradually increased during concentrating of the environmental water sample in order to provide the highest possible degree of concentration of the retentate without damaging the membrane module.

The permeate, here also called filtrate, which escapes through the pores of the membrane is not used for analytical purpose. The retentate is subjected to further concentration for subsequent analysis of the eDNA present in the sample.

In one preferred embodiment of the present invention the membrane module used according to the present invention (or modules, see below) is a microfiltration or ultrafiltration module, or a combination thereof, having an pore size of 0.01 to 10 pm, preferably 0.1 to 5 pm, or more preferred 0.1-3 pm, in particular 0.1-1.0 pm, which pro- vides sufficient retention of eDNA, while allowing a significant amount of water, dissolved salts and other dissolved or suspended materials of smaller size, e.g. salts, to escape through the membrane pores and into the permeate. Thus, efficient concentration of the water sample is achieved to enable subsequent detection of the eDNA present in the concentrated sample, in particular when using species-specific assays during DNA analysis. In addition, any efforts to release the sample from filter surfaces during extraction are avoided.

In one embodiment of the invention the method comprises recycling the retentate flow from a retentate outlet of the membrane module to a sample inlet of the membrane module, optionally via a retentate reservoir. Likewise, one embodiment of the system as used further comprises means for recycling the retentate flow from a retentate outlet of the membrane module to a sample inlet of the membrane module, optionally via a retentate reservoir.

The retentate may be collected in a retentate reservoir prior to being returned to the inlet of the membrane module. Hereby the entire sample can be subjected to a first concentration step before it is returned to the inlet of the membrane module for further concentration.

In another variant, the flow of the untreated sample may be mixed with recirculating retentate before the mixed flow enters the membrane module. Then, after completing the addition of the required volume of the sample, the flow entering the membrane module will only comprise the recirculated retentate. The result is a process which is quicker and requires less equipment because the retentate reservoir mentioned above can be omitted.

In all variants, the pressure applied in the membrane module during membrane filtration is preferably increased continuously during the filtration for each time the flow is reversed, e.g. by 0.1-1.0 bar, preferably 0.1-0.5 bar or more preferred by 0.2-0.4 bar in order to increase the amount of filtrate and thus also the degree of concentration in each membrane filtration step. Before any increase in pressure the membrane may alternatively be exposed to relaxation (pressure fall) for a limited period of time, e.g. a period of up to 5 minutes, such as 0.5 -4 minutes or preferably 1.5-2.5 minutes to improve filtration efficiency.

In an alternative embodiment of the invention, the method comprises reversing the flow through the membrane module to enable flow of retentate from the retentate outlet towards the sample inlet of the membrane module. Likewise, an alternative embodiment of the system as used further comprises means for reversing the flow through the membrane module to enable flow of retentate from the retentate outlet towards the sample inlet of the membrane module.

When the sample is run through the membrane module, the retentate is collected in a first retentate reservoir. When the entire sample is processed, the flow is reversed from the retentate reservoir, e.g. by activating a retentate pump in the retentate outlet pipe line, or by reversing the flow direction of the retentate pump. The retentate returning through the membrane module is then collected in a second retentate reservoir on the inlet side of the module. Then the flow is again reversed, e.g. by activating the inlet pump, through the membrane module to enable flow of collected retentate from the second retentate reservoir through the membrane module, which is then collected in the first retentate reservoir. This procedure of reversing the flow direction is then repeated until the above mentioned concentration factor of sample is obtained in the retentate. The pressure applied in the membrane module during membrane filtration is preferably increased for each time the flow is reversed, e.g. by 0.1-1.0 bar, preferably 0.1-0.5 bar or more preferred by 0.2-0.4 bar in order to provide a high degree of concentration in each membrane filtration step. Before any increased pressure the membrane may alternatively be exposed to relaxation (pressure fall) for a limited period of time, e.g. periods up to 5 minutes, such as 0.5 -4 minutes or preferably 1.5-2.5 minutes to improve filtration efficiency.

In yet another embodiment the water sample is subjected to concentration in a series of membrane modules in which the retentate of the upstream membrane module is fed to the sample inlet of the subsequent membrane module. Likewise an alternative embodiment of the system as used further comprises a series of membrane modules in which the retentate of the upstream membrane module is fed to the sample inlet of the subsequent membrane module. This is done to increase the filter surface and to further improve filtration efficiency and speed.

The number of membrane modules as used according to the present invention in the series is e.g. 2, 3, 4, 5, 6, 7, 8 or even a higher number. Hereby a high concentration factor is obtained in a single run through the series of modules. The pressure applied in the membrane module during membrane filtration is preferably increased in each module, e.g. by 0.1-1.0 bar, preferably 0.1-0.5 bar or more preferred by 0.2-0.4 bar in order to provide an optimal and high degree of concentration in each membrane filtration step.

Alternatively a lower number, e.g. 2, 3, or 4, of serially connected modules are used and the retentate of the last module is recycled to the inlet of the first membrane module, or the flow is reversed through the series of membrane modules as discussed above.

In a preferred embodiment of the method the membrane module or modules are subjected to a periodically cleaning process in which the filtration process is interrupted by reversing the filtrate flow backwards across the membrane and/or by enabling flow of a cleaning solution from the sample inlet port through the membrane module.

Using tangential flow membrane filtration causes flow of the sample to continuously flush the surface of the membrane and keep the surfaces clear of particulates etc. adhering to the surface. Reversing the flow of filtrate through the membrane for a short period, e.g. 5-10 seconds during membrane filtration procedure causes a backflush of the membrane module, which may loosen particulates and/or colloidal material adhered in the pores and/or on the surface of the membrane and return these to the retentate side of the membrane, whereby clogging of the membrane can be prevented.

Before filtration of each new sample a cleaning procedure is carried out in order to avoid contamination of a subsequent sample with eDNA present in the system as used, i.e. in the membrane modules, pipes pumps, reservoirs etc. The cleaning procedure comprises flushing the entire system using water, and relevant detergents and/or steri- lizing agents if necessary. The detergents and/or sterilizing agents are selected in order to be compatible with the membrane material.

The object is also met by use of the method according to the invention for preparing samples from an aquatic biotope for subsequent analysis of eDNA present in the sample by concentrating a sample from a an aquatic environment up to 1000 times, preferably up to 100-1000 or more preferred up to 200-1000 times.

Description of the Drawing

The present invention will be described in detail with reference to the drawings in which

Fig. la shows the principle of concentrating eDNA in an water sample using tangential flow filtration

Fig. lb shows the principle of detecting eDNA in an water sample from an aquatic environment

Fig. 2a-c show results of detection of various species of aquatic animals using detection of eDNA in water samples from an aquatic environment without concentration and with concentration to different degrees using tangential flow membrane filtration on a membrane filtration module

Fig. 3a shows a diagram of a first embodiment of an eDNA sample concen trating system

Fig. 3b shows a diagram of a second embodiment of an eDNA sample con centrating system

Fig. 3c shows a diagram of a third embodiment of an eDNA sample concen trating system

Fig. 4a shows a manual water sample concentration setup using a tangential flow microfiltration module as used in the examples, and Fig. 4b shows a setup for water sample concentration setup using a tangen tial flow microfiltration module operated by a peristaltic pump as used in the examples.

Detailed Description of the Invention

The present invention relates to tangential flow microfiltration or ultrafiltration as a tool to significantly increase concentration of eDNA in a water sample from an aquatic environment 1. The principle of the analysis is shown in fig. lb. A sample of the water matrix is taken from an aquatic environment 1, such as ponds, lakes, a stream of water, rivers, seas, oceans, groundwater or drinking water. The water sample is to be used to detect whether certain species of aquatic organisms, in particular aquatic or semi aquatic animals species and/or aquatic plant species are present in the aquatic environment 1 or whether certain non- aquatic animals, including humans, have been in contact with the water environment either directly or indirectly through emission of waste streams to the environment, e.g. manure or sewage. This can be done by extracting DNA present in the water sample and then subjecting the extracted DNA to DNA analysis. DNA analysis may include general quantification of all extracted DNA and detection and/or quantification of specific nucleotide sequences e.g. using PCR based methods.

Such water samples represent extremely dilute composition in relation to traces of eDNA, in particular mitochondrial DNA, from the animals and/or plants present in the aquatic environment 1. In order to be able to extract sufficient DNA from the water sample it is necessary to concentrate the water sample in respect of DNA present in the water sample.

It is possible to provide sufficient concentration of the water sample in respect of eDNA present in the sample by subjecting the water sample to membrane filtration using a tangential flow microfiltration or ultrafiltration setup. The principle in concentrating the water sample is shown in fig. la. The sample 2 is passed to a filtration membrane 8 having pores 9 of a size that enables passage of water, soluble components such as salts, and to some extent also suspended particles or colloids and some vira and/or microorganisms present in the water sample, depending on the size of the pores. The filtration membrane 8 retains larger particles such as entire cells or fragments 3, e.g. mitochondria and flocculated nucleic acid 4 (i.e. chromosomal DNA or fragments thereof) in the retentate 5. The retentate 5 collected in the process, in fig. la shown as poured into laboratory vial 5a, represents a concentrated stream of some of the components present in the water sample, including flocculated nucleic acid 4 (i.e. chromosomal DNA or fragments thereof) and in particular eDNA and/or mitochondrial DNA and/or chloroplast DNA of cells from animal and/or plant species present in the aquatic environment. Detection of eDNA and/or mitochondrial DNA and/or chloroplast DNA, and optionally of chromosomal DNA, can be used for analysis and as means for detecting the relevant species of plants or animal from which the eDNA, chromosomal DNA, chloroplast DNA or mitochondrial DNA comes from, or for determining whether a certain species of plant or animal is present in the aquatic environment from which the sample was taken.

As demonstrated in the example below, the filtrate did not contain any significant amount of target eDNA, and can be discarded or used as a sample for other analytic purposes.

The membrane filtration concentration of the retentate is performed using tangential flow (cross flow) filtration on microfiltration or ultrafiltration membranes, having an pore size of 0.01 to 10 pm, preferably 0.1-5 pm, more preferred 0.1-3 pm, in particular 0.1-1.0 pm. The retentate is collected and subjected to multiple filtration steps for concentrating the sample up to 1000 times, e.g .100-1000 or 200-1000 times.

The membrane module 12 (Fig. 3a) is a microfiltration or ultrafiltration module having a pore size of 0.01 to 10 pm, preferably 0.1 to 5 pm, or more preferred 0.1-3 pm, in particular 0.1-1.0 pm.

The pressure applied to the inlet side of the membrane module 12 drives the filtration process, and forces the filtrate 7 through the membrane pores 9 and is e.g. 0.1-4.0 bar and may constant or may be varied during the concentration procedure as discussed further below. Before any increased pressure the membrane may alternatively be exposed to relaxation (pressure fall) for a limited period of time, e.g. periods up to 5 minutes, such as 0.5 -4 minutes or preferably 1.5-2.5 minutes to improve filtration efficiency.

Fig. 3a shows a first possible embodiment of the system as used according to the invention. The water sample 2 is transferred to the system through an inlet tube or pipeline 11. An inlet pump 15 provides the necessary pressure to the water sample in order to drive the filtration process in the membrane module 12. The filtrate exits through a filtrate outlet line 13 and may be collected in a filtrate reservoir 16. The retentate exits the membrane module 12 through a retentate outlet line 14 and is recycled to the inlet side of the tangential flow membrane module 12. The retentate is optionally collected in a retentate reservoir 17 prior to being recycled to the inlet side of the membrane module 12. A retentate pump 18 (optional) recycles the retentate to the inlet side of the module 12. The retentate line 14 comprise an outlet 19 for extracting a concentrated sample, from the retentate line 14 once the retentate is concentrated to a certain degree, preferably up to 1000 times, such as up to 100-1000 or more preferred up to 200-1000 times.

The water sample is concentrated by removing water, dissolved salts, colloids etc. as a filtrate through the filtration membrane and concentrating eDNA, and other materials in the retentate. The water sample, e.g. 1-5 litres of a water sample from an aquatic environment, is passed to the concentration system via the inlet line 11. The retentate 14 is recycled to the inlet line 11 in order to further concentrate the sample and thereby increase the concentration of eDNA in the retentate and thus increase sensitivity of subsequently used DNA/RNA detection techniques.

The retentate may be collected in a retentate reservoir 17 prior to being returned to the inlet of the membrane module 12. Hereby the entire sample can be subjected to a first concentration step before it is returned to the inlet line 11 of the membrane module 12 for further concentration.

In another variant, the flow of the untreated sample in the inlet line 11 may be mixed with recirculating retentate 14 before the mixed flow enters the membrane module 12. Then, after completing the addition of the required volume of the sample, the flow entering the membrane module 12 will only comprise the recirculated retentate. The result is a process which is quicker and requires less bulky equipment because the retentate reservoir mentioned above can be dispensed with.

The pressure to the water sample in the inlet line 11 by the inlet pump 15 is preferably 0.1-4.0 bar in order to drive filtrate through the filtration membrane. In one variant, the pressure applied by the inlet pump 15 is constant. This may cause the flow of fil- trate to decrease gradually during circulation of the retentate which gradually increases in concentration.

Alternatively, the initial pressure is preferably in the lower end of this interval, and pressure applied in the membrane module during membrane filtration is gradually increased during concentration of the retentate. When the concentration of the circulating retentate is gradually increasing, it may require an increased pressure in order to drive a filtrate through the membrane for even further concentration. In both variants described above, the pressure applied in the membrane module 12 during membrane filtration is preferably increased continuously or in a stepwise during concentrating the retentate, e.g. by 0.1-1.0 bar, preferably 0.1-0.5 bar or more preferred by 0.2-0.4 bar in each step, in order to increase the amount of filtrate and thus also the degree of concentration in each membrane filtration step and in order to speed up the concentration process significantly. The concentration of the retentate thus aims at minimising the amount of circulating retentate. Before any increased pressure is applied, the membrane may alternatively be exposed to relaxation (pressure fall) for a limited period of time, e.g. periods up to 5 minutes, such as 0.5 -4 minutes or preferably 1.5-2.5 minutes to improve filtration efficiency.

When the amount of retentate is reduced to e.g. 1/100 of the water sample volume, preferably 1/10-1/1000, more preferred to between 1/100-1/1000 or more preferred between 1/200-1/1000 of the volume of the water sample, the concentrated retentate is ready for further treatment and/or analysis. Then a sample of the retentate may be tapped from the system 10 at the sample outlet 19 for further treatment and/or analysis, e.g. extraction of eDNA and subsequent DNA/RNA analysis, e.g. by qPCR. DNA extraction from the retentate may follow simple standard methods developed for extraction of nucleic acids from suspended samples. Any efforts to release the sample from filter surfaces during extraction are avoided.

When using tangential flow membrane modules, the flow on the inlet /retentate side of the membrane flows essentially parallel to the membrane surface, which prevents or at least reduces the build-up of deposits on the membrane surface. In order to loosen any solids which, despite the tangential flow used in the membrane module 12, may adhere to the membrane surface and/or in the pores 9 of the membrane 8 the filtrate out let line 13 is optionally equipped with means for enabling backflush of the membrane 8 with filtrate 7 from the filtrate side of the membrane to the retentate side of the membrane 8. The means may e.g. comprise a backflush pump 20 in a filtrate return line 21 from the filtrate reservoir 16 to the fdtrate outlet 13 of the membrane module 12. The backflush means may be activated at relevant intervals during the retentate concentration process, e.g .at intervals of 1-15 minutes.

The sample concentration system is equipped with standard cleaning means for clean-in-place (CIP) of the entire system between processing different samples. This reduces or eliminates the risk of contaminating a subsequent sample with eDNA form the previous sample and thus reduces the risk of false positive results in the subsequent analytical procedures.

Fig. 3b shows an alternative layout of the system as used for concentrating the water sample. The details of this system, which are identical to the above mentioned system, e.g. the backflush and CIP systems, will not be discussed in detail. The system 10 of fig. 3b differs from the system in fig. 3a in that the retentate is subjected to multiple filtration steps in the membrane module 12 by reversing the retentate flow from the retentate outlet line 14 to the sample inlet line 11 and vice versa for a number of times which is sufficient for obtaining a retentate volume corresponding to up to 1/100 of the original water sample volume, preferably 1/10-1/1000, more preferred to between 1/100-1/1000 or more preferred between 1/200-1/1000 of the volume of the original water sample.

In this embodiment the system as used 10 comprises a first retentate reservoir 17a for collecting retentate from the retentate line 14 on the retentate outlet of the membrane module 12. A second retentate reservoir 17b is connected via a second retentate line 23 to the inlet line 11 for collecting retentate when the flow direction through the membrane module 12 is reversed.

When the water sample is injected into the system 10, through the inlet line 11 it passes the membrane module 12 as discussed above and retentate is collected in the first retentate reservoir 17a. When the entire water sample has passed the membrane module 12, flow through the membrane module 12 is then reversed, i.e. the retentate in the first retentate reservoir is passed to the retentate outlet of the membrane module 12 and exits through the inlet line where it is collected in the second retentate reservoir 17b via a second retentate line 23. The retentate is passed to the membrane module 12 from the first reservoir by means of the retentate pump 18. The retentate pump 18 arranged in the retentate line 14 may be a pump, which is able to reverse the flow direction, or alternatively the retentate pump is placed in a branch 14a of the retentate line 14, which connects the first retentate reservoir 17a with the retentate outlet of the membrane module 12. When the retentate is collected in the second retentate reservoir 17b, the flow is again reversed in order to let the retentate flow from the second reservoir 17b which is connected to the inlet line 11 and thereby to the inlet pump 15 and into the membrane module 12. The flow is reversed a number of times so that the retentate flows from the retentate outlet towards the inlet and vice versa multiple times, e.g 10-200 times, or 10-100 times, preferably 15-60 times in order to obtain a concentrated retentate sample containing eDNA, to achieve the above mentioned concentration of the retentate in relation to the volume of the original water sample. The number of times the flow is reversed depends on the pressure level in the membrane module, since a low pressure results in a lower volume of filtrate, and thus a lower concentration factor, than a higher pressure.

The membrane module 12 may be operated the same level of pressure during all passages of water sample/retentate through the membrane module at a pressure level of 0.1-4 bar, which is sufficient for providing a sufficiently high volume of filtrate and thereby obtain a high level of concentration of retentate in each passage.

Alternatively, the initial pressure is preferably in the lower end of this interval, and pressure applied in the membrane module during membrane filtration is increased in a stepwise manner for each time the flow is reversed, e.g. by 0.1-1.0 bar, preferably 0.ΙΟ.5 bar or more preferred by 0.2-0.4 bar, in order to provide an optimal and high degree of concentration in each membrane filtration step of the concentration cycle. Before any increased pressure is applied the membrane may alternatively be exposed to relaxation as discussed above.

In yet another embodiment, the water sample is subjected to multiple filtration steps in a series of serial tangential flow microfiltration and/or ultrafiltration modules, see fig. 3c. The water sample is subjected to concentration in a series of membrane modules 12a, 12b, 12c, 12d etc., in which the retentate of the upstream membrane module, e.g. 12a, is fed to the sample inlet of the subsequent membrane module, e.g. 12b. Likewise an alternative embodiment of the system as used further comprises a series of membrane modules in which the retentate of the upstream membrane module is fed to the sample inlet of the subsequent membrane module.

The number of membrane modules 12a, 12b, 12d, 12e in the series is e.g. 2, 3, 4, 5, 6, 7, 8 or even a higher number. Hereby a high concentration factor is obtained in a single run through the series of modules 12a 12b etc.

The pressure applied in the membrane module during membrane filtration is either of the same level in all membrane modules 12a, 12b, 12c, 12d etc., i.e. between 0.2 -4 bar or the pressure may vary through the series of membrane modules.

Preferably the pressure in the first module is relatively low, e.g. 0.2-1 bar and the pressure is increased in each subsequent module, e.g. by 0.1-1.0 bar, preferably 0.ΙΟ.5 bar or more preferred by 0.2-0.4 bar in order to provide an optimal and high degree of concentration in each membrane filtration step and in order to speed up the concentration process. The membranes may be subjected to relaxation (pressure fall) as discussed above.

In an alternative embodiment, a lower number, e.g. 2, 3, or 4, of serially connected modules are used and the retentate of the last module 12d is recycled to the inlet of the first membrane module 12 via a retentate circulation line 14. This is similar to the system as discussed in relation to fig. 3a, except that a series of two or more membrane modules 12a- 12d are used instead of the single membrane module 12 shown in fig. 3a.

Alternatively, a series of e.g. 2, 3, or 4 serially connected modules 12a, 12b, 12c, 12d, are used, instead of the single module 12 shown in Fig. 3b in which the flow is reversed through the series of membrane modules 12a-12d as discussed above.

Examples:

Proof of principle was performed using the MidiKros® Hollow Fiber module (Spectrum Labs, Part no. D02-E20U-05-N) for tangential flow filtration (Fig. la). Tangential Flow filtration was tested on water samples collected from a freshwater pond, brackish water from Limfjorden and seawater from Kattegat. All samples were subjected to DNA extraction prior to quantification of DNA (Fig. lb). As a model for DNA extraction this setup uses EtOH precipitation (500 pL water sample, 1166 pL absolute EtOH and 50 pL 3M Sodium acetate) prior to DNA purification using the DNeasy Blood &Tissue Kit (Qiagen).

Tangential flow filtration on the microfiltration membrane module is used for concentration of eDNA in water samples 2. Fig. la shows the principle of tangential flow filtration, where the pores 9 allow for removal of water and minor particles 6 across the membrane. Larger particles such as entire cells or cell fragments, e.g. mitochondria and/or chloroplast 3 and flocculated nucleic acids 4 or fragments thereof are concentrated in the retentate 5. We have shown that eDNA is concentrated in the retentate 5. Fig. lb illustrates the overall principle in the experimental setup. Different volumes of water samples 2 are subjected to tangential flow filtration, followed by DNA extraction and analysis by qPCR and measurements of DNA concentration. These results are compared to the results of water samples that have not been concentrated. DNA quantification was performed with two different approaches. Species-specific quantification were performed by qPCR as described by (Thomsen et al., 2012a, 2012b), using different detection systems for the widespread fish species Platichthys flesus (Thomsen et al., 2012a) and for the rare and endangered newt Triturus cristatus (Thomsen et al., 2012b). The total DNA content in the samples was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

The principle will apply to the detection of any aquatic organism in freshwater or marine ecosystems using similar and/or other analysis methods.

The results obtained here show that it is possible to use tangential flow filtration to concentrate samples intended for detection of eDNA in aquatic samples, no matter the water type or detection system (Fig. 2a-2c):

Tangential flow filtration was performed both manually (Fig. 4a) and by a peristaltic pump (Fig. 4b) using a tangential flow micro filtration module having a maximal pore size of 0.2 pm. The module used is a MidiKros® Hollow Fiber module provided by Spectrumlabs. The MidiKros® Hollow Fiber module 12 is marked on the figures 4a-4b along with sample inlet 11, retentate 17 and filtrate outlet 13. Filtration was performed using either A) manual setup with three 50 mL syringes, 2, 16, 17 for pond freshwater samples or B) peristaltic pump 18 for brackish water and sea water samples.

For manual filtration the procedure involved pressing 50 mL water sample back and forth between syringes 2 and 17 (Fig. 4a) until clean water was collected in the filtrate outlet syringe 16. The filtrate outlet syringe 16 was then emptied and sample syringe 2 was then filled again with sample, and the procedure repeated until a suitable volume had been processed. For filtration using the peristaltic pump 18 the retentate 17 was cycled, and continuously added inlet sample 11 and repetitively passed through the MidiKros® Hollow Fiber module 12, until a suitable volume of filtered water had been removed through the filtrate outlet 13.

Figs. 2a-2c show the DNA quantification plotted against the volume concentration factor. The results of qPCR targeting species specific DNA are seen as dots, and the total DNA concentrations are seen as bars (depicted on the secondary y-axis). The three charts represent different water samples and detection systems: Fig. 2a shows the results from a freshwater pond using the Triturus cristatus qPCR analysis system and figs. 2b and 2c shows results using the Platichthys flesus qPCR analysis system on water samples from Limfjorden (brackish water) and Kattegat (seawater) respectively.

It is clear that the use of tangential flow filtration has been successful for concentration of eDNA for the different water samples, which in turn provides more reliable results in the subsequent quantification and identification of species-specific eDNA using a qPCR based assay for analysis and detection of target species. In one case (Fig. 2b) a high concentration factor was necessary in order to detect eDNA traces of the relevant species in Limfjorden.

Corresponding qPCR tests on the filtrates from each of the samples showed no detectable traces of species specific eDNA in the analysed samples. A lack of perfect correlation between sample input (or concentration factor) and DNA output in the retentate may mainly relate to the DNA extraction protocol, and is subject to further optimization.

Although the tests described here used qPCR assays for detection of mitochondrial DNA from animal cells, it is believed, that similar results are obtainable using detection of DNA from chloroplasts from plant cells for detecting various species of plants as well.

Claims (12)

A process for concentrating chloroplasts and / or mitochondria and / or DNA contained herein, which is contained in a water sample from an aquatic biotope as traces of aquatic or semi-aquatic macroscopic organisms (eDNA), in particular aquatic or semi-aquatic animal species and / or aquatic plant species, or macroscopic non-aquatic organisms, including humans, which may lose tissue to the aquatic environment, comprising the steps of subjecting the water sample to membrane filtration in at least one membrane module using tangential flow filtration and repeating the membrane filtration step once or more on the retentate of concentration. the sample up to 1000 times, preferably up to 100-1000 or more preferably up to 200-1000 times. The method of claim 1, further comprising recycling the retentate stream from a retentate outlet of the membrane module to a sample inlet of the membrane module, optionally via a retentate reservoir. The method of claim 1, further comprising reversing the flow through the membrane module to allow flow of retentate from the retentate outlet toward the membrane inlet sample. A method according to any one of the preceding claims, comprising subjecting the sample to a series of membrane modules in which the retentate of the upstream membrane module is fed to the sample inlet of the subsequent membrane module. A method according to any one of the preceding claims, wherein the membrane module (s) is a microfiltration or ultrafiltration module having a pore size of 0.01 to 10 μιη, preferably 0.1-5 μιη, more preferably 0.1-3 μιη, in particular 0.1-1.0 μιη or a combination thereof. A method according to any one of the preceding claims, wherein the membrane module (s) is subjected to a periodic purification process in which the filtration process is interrupted by A: reversing the filtrate flow back across the membrane, and / or B: enabling flow of a cleaning solution from the sample inlet port through membrane module. Use of a chloroplast and / or mitochondria in a water sample concentration system to concentrate traces of tissue from macroscopic aquatic organisms in a water sample from an aquatic biotope prior to registration of DNA from the species, especially aquatic or semi-aquatic animal species and / or aquatic plant species, or macroscopic non-aquatic organisms, including humans, which may lose tissue to the aquatic environment, which system comprises one or more tangential flow filtration modules, wherein one or more filtration modules each comprises a filter membrane having a pore size of 0.1-5 µm, and wherein the apparatus comprises means for recirculating the retentate to the module inlet or means for directing the flow of retentate through the membrane module from an outlet to an inlet of the membrane module for concentrating the retentate volume up to 1000 times, preferably up to 100-1000 or more preferably up to 200 times. 1000 times. Use according to claim 7, wherein the system further comprises means for recycling the retentate stream from a retentate outlet of the membrane module to a sample inlet of the membrane module, optionally via a retentate reservoir. The use of claim 8, wherein the system further comprises means for reversing the flow through the membrane module to enable flow of retentate from the retentate outlet toward the sample inlet of the membrane module. Use according to any one of claims 7-9, wherein the system further comprises a series of membrane modules in which the retentate of the upstream membrane module is supplied to the sample inlet of the subsequent membrane module. Use according to any one of claims 7-10, wherein the membrane module (s) is a filtration module having a pore size of 0.1-3 µm, or preferably 0.1-1.0 µm. Use of a method according to any one of claims 1-6 for the preparation of water samples from an aquatic biotope for subsequent analysis of eDNA contained in the water sample by concentrating a water sample from an aquatic environment up to 1000 times, preferably up to 100 -1000 or more preferably up to 200-1000 times.