Plant sample cleavage method

Abstract

The present invention provides a cleavage method for releasing microbial nucleic acids from microorganisms contained in a plant sample, comprising mechanically disrupting the plant sample in a liquid cleavage composition using at least two solid disruption particles wherein: (i) a first type is provided by one or more disruption particles having a size of at least 1.5 mm, and (ii) providing the second type from a plurality of crushed particles having a size of 1 mm or less. Methods and kits for isolating nucleic acids, including microbial nucleic acids, from plant samples are also provided.

Description

BACKGROUND

[0001] Isolation of nucleic acids from plants can be very challenging due to the nature of the plant cells which are difficult to lyse and the presence of a large number of inhibitors including polysaccharides and polyphenolic compounds. Furthermore, these parameters can vary greatly between plant species and different parts of the same plant. This often results in the isolation of small amounts of low quality nucleic acids such as DNA.

[0002] The most commonly used method for plant sample lysis is the mortar and pestle or mechanical disruption with common grinding media, i.e. metal or glass beads. When using this standard procedure, the yield is usually low and depends on the sample species, the presence of DNA including a large number of inhibitors.

[0003] To isolate nucleic acids such as DNA from plants, the method commonly used is the combination of a mortar and pestle with a CTAB lysis buffer for the lysis of the plant material and the removal of inhibitors. The use of a mortar and pestle is very time consuming, inefficient and difficult to use with multiple samples. CTAB is a toxic reagent. Commercial kits use a mortar and pestle or some combination of spherical ceramic or metal beads with chaotropic and detergent-based buffers for lysis. The drawback of these methods is that they are by nature non-standard and cannot be applied to a large number of different plant species with similar success. This often results in a lower yield, an increased presence of inhibitors and (in the case of the mortar and pestle) a time-consuming experiment.

[0004] There is also a need for methods allowing the efficient isolation of microbial nucleic acids from plant samples. Many plant samples contain microorganisms (e.g. on the leaves or within the plant tissue). There is a need to make the microbial nucleic acids contained in plant samples available for analysis. With the rise of next generation sequencing (NGS), and in particular the rise of microbiome studies (bacteria / fungi / viruses), there is an exponential increase in interest in isolating high quality DNA that can be immediately used for these applications. This is also true for the field of plant biology. The main challenge when aiming at isolating microbial nucleic acids contained in plant samples is the mixture of plant host cells and target microbial cells. The nature of the cells which are difficult to lyse and the presence of a large number of PCR and enzyme inhibitors make the use of conventional prior art methods less efficient.

[0005] There is a need for improved methods for isolating nucleic acids, in particular DNA, from plant samples.

[0006] In particular, there is a need for a method which also allows efficient release of microbial nucleic acids from microorganisms comprised in the plant sample and thereby from microorganisms present on, around or inside the plant sample itself, so that these microbial nucleic acids can be isolated from the lysate obtained. In particular, there is a need for an improved lysis method which efficiently releases nucleic acids, such as DNA, from plant samples and which also efficiently releases microbial nucleic acids, such as microbial DNA, from microorganisms comprised in the plant sample, so that they are available for subsequent isolation. In particular, there is a need for an improved lysis and isolation method which increases the amount of microbial nucleic acids, such as microbial DNA, from different plant samples, in particular from various plant roots.

[0007] Furthermore, there is also a need for a solution to increase nucleic acid yield and remove inhibitors for a variety of plant sample types.

[0008] It is an object of the present invention to overcome at least one of the disadvantages of the prior art. In particular, it is an object of the present invention to provide a method which fulfils at least one of the needs. It is a further object of the present invention to provide an improved lysis method which efficiently releases nucleic acids, such as DNA, from various plant samples and which also efficiently releases microbial nucleic acids, such as microbial DNA, from microorganisms comprised in the plant sample, so that they are available for subsequent isolation. SUMMARY

[0009] According to a first aspect, a lysis method for releasing microbial nucleic acids from microorganisms comprised in a plant sample is provided, comprising mechanical disruption of the plant sample in a liquid lysis composition using at least two solid disruption particles, wherein:

[0010] (i) a first type is provided by one or more disruption particles having a size of at least 1.5 mm, and

[0011] (ii) a second type is provided by a plurality of disruption particles having a size of 1 mm or less.

[0012] According to a second aspect, a method for isolating nucleic acids, including microbial nucleic acids, from a plant sample is provided, comprising:

[0013] (a) performing the lysis method according to the first aspect;

[0014] (b) isolating nucleic acids from the lysed and optionally further treated sample; and

[0015] (c) optionally sequencing the isolated nucleic acids, preferably sequencing the isolated DNA.

[0016] According to a third aspect, a lysis system, preferably a kit, for releasing microbial nucleic acids from microorganisms comprised in a plant sample is provided, comprising:

[0017] (a) a liquid lysis composition,

[0018] (b) at least two types of solid breakage particles, wherein:

[0019] (i) the first type is provided by one or more breakage particles having a size of at least 1.5 mm, and

[0020] (ii) the second type is provided by a plurality of breakage particles having a size of less than 1 mm.

[0021] The present invention also relates to the use of such a lysis system in the lysis of a plant sample comprising or suspected of comprising microorganisms.

[0022] According to a fourth aspect, the present disclosure relates to the use of a system according to the third aspect in a method according to the first aspect.

[0023] According to a fifth aspect, the present disclosure relates to the use of a system according to the third aspect in the lysis of a plant sample and the release of microbial nucleic acids, preferably DNA, from microorganisms comprised in the plant sample, wherein the user can use (i) the first and second type, or (ii) the second type of breakage particles for the lysis of the plant sample for the release of microbial nucleic acids, preferably DNA, from microorganisms comprised in the plant sample.

[0024] The use of the first type of breakage particles as disclosed herein can provide high DNA yields and is thus particularly effective in homogenizing and thereby breaking up various plant samples. However, such large breakage particles alone are less effective in mechanically breaking up microorganisms such as bacteria comprised in a plant sample. The combined use of at least one particle according to the first type, e.g. a ball cone, with a plurality of smaller particles of the second type, e.g. zirconium beads, can achieve high total yields of isolated total DNA. Furthermore, the combined use of the first and second type of particles can effectively release microbial nucleic acids comprised in microorganisms contained in a plant sample, as shown by the high percentage of bacterial DNA in the isolated total DNA (see examples). Thus, as taught herein, the combined use of the first and second type of breakage particles is preferred, particularly when used in combination with the lysis chemistry disclosed herein. The present invention can be advantageously used for the release of nucleic acids, including microbial nucleic acids, from various plant samples, including difficult to lyse samples, such as root samples.

[0025] The combined mechanical lysis described herein provides DNA from various plant samples in high yields, wherein the obtained DNA comprises a large amount of microbial DNA and is thus useful for analysis. For analysis, various methods can be used, such as amplification-based procedures (e.g. PCR) and sequencing (e.g. next generation sequencing).

[0026] Other aspects, objectives, features and advantages of the present application will become apparent to those of ordinary skill in the art from the following description and the attached claims. It is to be understood, however, that the following description, the attached claims, and the embodiments are given by way of example only and are not intended to limit the application.

[0027] In the following description, any range provided herein includes all values within the range.

[0028] It is also to be noted that the term "comprising" or "containing" or "including" or "having" or "featuring" or "comprises" or "contains" or "includes" or "has" or "featuring" is used synonymously and is intended to mean that the item(s) in question are present, but does not exclude the presence of other items.

[0029] Also, as used in the description and the appended claims, the singular "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0030] The terms "comprising", "having", "including", and "containing" are synonymous and are used synonymously with each other and are interpreted to be non-limiting.

[0031] As used herein, the term "one of a combination thereof refers to one of all possible combinations of the listed items preceding the term. For example, "A, B, C, or one of a combination thereof is intended to refer to any one of the following: A, B, C, AB, AC, BC, or ABC. Similarly, as used herein, the term "a combination thereof refers to all possible combinations of the listed items preceding the term. For example, "A, B, C, or a combination thereof is intended to refer to all of the following: A, B, C, AB, AC, BC, and ABC. DETAILED DESCRIPTION

[0032] The present application provides an improved lysis method for lysing plant samples that efficiently releases microbial nucleic acids from microorganisms contained in various plant samples. The overall yield is high and the released nucleic acids contain a large amount of microbial nucleic acids and are thus useful for subsequent isolation and analysis. The technology disclosed herein allows obtaining DNA from plant samples in high yield, wherein the obtained DNA not only contains plant DNA in good yield, but also contains a large amount of microbial DNA and is thus useful for analysis.

[0033] Furthermore, the method described herein allows isolating nucleic acids from a plant sample and at the same time removing inhibitors from the isolated nucleic acids, thereby allowing efficient downstream analysis of the isolated nucleic acids.

[0034] Method according to the first aspect

[0035] According to a first aspect, a lysis method for releasing microbial nucleic acids from microorganisms comprised in a plant sample is provided, comprising mechanical disruption of the plant sample in a liquid lysis composition using at least two solid disruption particles, wherein:

[0036] (i) the first type is provided by one or more disruption particles having a size of at least 1.5 mm, and

[0037] (ii) the second type is provided by a plurality of disruption particles having a size of 1 mm or less.

[0038] The individual steps and preferred embodiments will now be described in detail.

[0039] The plant sample is mechanically disrupted in a liquid lysis composition according to the method of the present disclosure. At least two solid disruption particles are used in the mechanical disruption. One or more disruption particles are agitated, e.g. mixed or vortexed, as further described herein, in order to exert a disruptive force on the plant sample and comprised microorganisms upon contact. The plant sample is preferably homogenized.

[0040] The solid disruption particles of the first type and the second type are preferably different from each other not only in size, but also in shape and / or material. Preferably, the one or more disruption particles used as the first type are not spherical and have at least one discontinuity, preferably an edge, and the plurality of particles used as the second type are substantially spherical.

[0041] The solid disruption particles of the first type

[0042] For mechanical disruption of the plant sample, at least one solid disruption particle is used as the first type.

[0043] The at least one disruption particle used as the first type is non-spherical and preferably has an irregular shape. An important advantage is that the one or more disruption particles used as the first type are non-spherical.

[0044] In a particularly preferred embodiment, the surface of the one or more breaking particles used as the first type has at least one discontinuity, particularly preferably an edge or a peak. According to an embodiment, the breaking particles used have one or more beveled edges. The discontinuity provides the advantage that it can be used to exert an irregular, preferably punctiform or linear, impact on the plant material to be broken. It also allows the contact between the particle and the plant material to have random properties. The discontinuity causes the movement of the particle to be more irregular compared to the movement of a spherical particle and can be used to attack the plant material to be broken more randomly. When the discontinuity, such as an edge or a peak, hits the plant material to be broken, the destructive force increases. This even allows very diverse plant materials to be effectively broken, allowing many different plant sample varieties to be broken. As disclosed, a single such breaking particle can be used as the first type.

[0045] In a preferred embodiment, the surface of the particle comprises a first portion and comprises a second portion, whereby the first portion and the second portion meet by forming an edge. Preferably, the edge extends along a line. The line can be a circle or an arc. The line can also be a straight line. In a preferred embodiment, the first portion is the surface of a truncated cone and the second portion is the surface of a truncated cone. Preferably, in the embodiment where the first portion is the surface of a truncated cone and the second portion is the surface of a truncated cone, both cones have their larger bases against each other, the larger bases preferably having the same diameter, meeting at the edge. The particle preferably has one line of symmetry, in a preferred embodiment only one line of symmetry. Preferably, the particle has rotational symmetry around the line of symmetry. The edge can be provided in the form of a tilted central flange, for example Figures 6 to 8 as shown.

[0046] The one or more particles used as the first type can have a sub-portion consisting of a piece or a portion of a sphere or an ellipsoid.

[0047] The particle can have a tip. In an embodiment, the tip is a truncated cone. According to an embodiment, the larger base of the truncated cone providing the tip rests against the smaller base of the truncated cone of the second portion. In this embodiment, the sub-portion consisting of a piece or a portion of a sphere or an ellipsoid can rest against the smaller base of the truncated cone of the first portion. In an embodiment, the sub-portion consisting of a piece or a portion of a sphere or an ellipsoid is a hemisphere. One embodiment of such a breaking particle, which is a spherocone, is shown in Figure 6 as shown. At least one such particle can be used as the first type.

[0048] In one embodiment, the particles comprise at least two tips, wherein preferably both tips are truncated cones. According to one embodiment, the larger base of the truncated cone providing the first tip is resting against the smaller base of the truncated cone of the first section, and the larger base of the truncated cone providing the second tip is resting against the smaller base of the truncated cone of the second section. One embodiment of such a particle having two tips is shown in Figure 7 At least one such particle can be used as the first type.

[0049] In one embodiment, the particles have two sub-sections, wherein each subsection consists of a piece or a portion of a sphere or an ellipsoid. In this embodiment, the first subsection consisting of a piece or a portion of a sphere or an ellipsoid is resting against the smaller base of the truncated cone of the first section, and the second subsection consisting of a piece or a portion of a sphere or an ellipsoid is resting against the smaller base of the truncated cone of the second section. One embodiment of such a particle having two hemispheres is shown in Figure 8 At least one such particle can be used as the first type.

[0050] The non-spherical particles used for disruption as the first type can have one or more discontinuities, such as edges. They can be solid cones, cylinders, cubes, triangles, rectangles forms and similar suitable geometric forms. Another example is a diagonal with beveled edges. According to one embodiment, at least one disruptive particle used as the first type has an irregular shape and can be selected from a spherocone and a satellite-like (shape like Saturn, a planet or a UFO) for achieving disruption of plant tissue material upon application of a mixing or grinding force to a plant sample in the composition of the present application. The use of spherocone is particularly effective for disrupting plant samples and is therefore preferred. The selection of the solid disruptive particles should take into account not affecting or damaging the released cellular components or analytes.

[0051] In order to achieve sufficient disruption and homogenization of the plant material and the desired release of nucleic acids without damage for isolation, it is preferred that the one or more solid disruption particles used as first type are solid inert particles, i.e. particles made of a material which does not react with the tissue material, does not react with any reagents of the composition and in any case does not react with the desired nucleic acids released based on the disruption. It is particularly preferred that the released nucleic acids cannot adsorb or adhere to the inert solid disruption particles used as first type under the employed lysis conditions. Suitable inert materials include for example inert metals, steel, stainless steel, plastics and ceramics. Preferably, the at least one disruption particle used as first type is made of a metal. Preferably, it is made of steel, stainless steel, tungsten or other heavy metals. Other examples are metals and alloys derived from tantalum, platinum and the like. Steel materials include but are not limited to carbon steel, stainless steel and chromium steel. Steel like stainless steel is preferred. Other suitable inert disruption materials are known from commercially available inert disruption particles. It is also possible to use a mixture of one or more disruption particles, i.e. to use disruption particles of different form and / or made of different inert materials as first type.

[0052] It is further preferred that the one or more disruption particles used as first type exhibit a sufficient hardness so that no wear occurs during the milling or grinding process.

[0053] In one embodiment, the density of the one or more non-spherical disruption particles used as first type is in the range selected from 5.0 g / cc to 20 g / cc, 5.5 g / cc to 15 g / cc, 6 g / cc to 12 g / cc and 6 g / cc to 10 g / cc.

[0054] In order to achieve sufficient disruption power to effectively disrupt, in particular homogenize, the plant sample, the disruption particles used as first type should preferably have a relatively large size. This is advantageous and allows the isolation of nucleic acids, such as DNA, from a variety of plant sample species with high yield.

[0055] The size of the one or more solid disruption particles used as first type is at least 1.5 mm. It can be at least 2 mm, at least 2.5 mm or at least 3 mm in size. Further, the one or more disruption particles used as first type can have a size of at least 3 mm (> 3 mm), at least 3.5 mm or preferably at least 4 mm. The at least one solid disruption particle used as first type can have a size of at least 4.5 mm or at least 5 mm.

[0056] The one or more disruption particles used as first type can have a size of at most 15 mm, for example at most 12 mm, at most 10 mm or at most 8 mm.

[0057] The one or more breaking particles used as the first type can have a size of 1.5 mm to 15 mm, such as 2 mm to 15 mm, 2.5 mm to 15 mm, 3 mm to 15 mm, or 4 mm to 15 mm. The particles can also have a size of 1.5 mm to 12 mm, such as 2 mm to 12 mm, 2.5 mm to 12 mm, 3 mm to 12 mm, or 4 mm to 12 mm. The one or more breaking particles used as the first type can also have a size of 1.5 mm to 10 mm, such as 2 mm to 10 mm, 2.5 mm to 10 mm, 3 mm to 10 mm, or 4 mm to 10 mm. The one or more breaking particles used as the first type can also have a size of 1.5 mm to 7 mm, such as 2 mm to 7 mm, 2.5 mm to 7 mm, 3 mm to 7 mm, 3.5 mm to 7 mm, or 4 mm to 7 mm. Most preferred is a size of 3 mm to 7 mm or 4 mm to 7 mm. In case more than one breaking particle is used as the first type, a mixture of breaking particles of different sizes within this range can also be used.

[0058] A given size of the one or more breaking particles used as the first type refers to the longest distance between two opposing points of the respective particle. As discussed, it is preferred to use irregularly shaped particles, such as satellites or spherocylinders, having at least one discontinuity at their surface as the first type. Here, the longest distance between two opposing points is typically the diameter of the "Saturn-like ring" around the spherical or spherocylindrical part of such a particle.

[0059] Depending on the size of the one or more breaking particles used as the first type, one or more breaking particles can be used. In case of very large particles, the ideal results of breaking and preserving of the analyte can be achieved with only one particle, in particular one spherocylinder. It is particularly preferred to use one, i.e. a single breaking particle as the first type. As described herein, it is preferred to use a single spherocylinder for breaking the plant sample material.

[0060] Examples of preferred commercially available irregularly shaped particles, spherocylindrical or satellite-shaped particles have the following sizes:

[0061] Table I

[0062]

[0063] wherein half of the steel spherocylinder is hemispherical (a ball) and the other half is a cone, both separated by an inclined central flange (ring). An example of a spherocylinder is provided in the figure.

[0064] As discussed herein, the at least one solid breaking particle used as the first type is preferably a heavy solid device. According to one embodiment, the weight of the solid breaking particle is at least 300 mg, such as at least 400 mg, at least 500 mg, at least 600 mg or at least 700 mg. In embodiments, the weight of the solid breaking particle is in the range of 300 mg - 1500 mg, 400 mg - 1250 mg, 500 mg - 1000 mg and 600 mg - 900 mg.

[0065] Preferably, the weight is in the range of 500 mg - 1000 mg or 600 mg - 900 mg. This is especially true if individual breaking particles of irregular shape, such as a spherocone, are used as the first type for mechanical breaking. Breaking particles of such weight can also have a size in the range of 1 mm - 10 mm, such as 1.5 mm - 9 mm, 2 mm - 8 mm, 2.5 mm - 7 mm, 3 mm - 7 mm or 4 mm - 7 mm. Most preferred is a size in the range of 3 mm - 7 mm, preferably 4 mm - 7 mm, and a weight in the range of 500 - 1000 mg, preferably 600 mg - 900 mg. The breaking particles of irregular shape used as the first type are preferably sphericones. The use of individual sphericones is advantageous as discussed herein.

[0066] As disclosed, it is preferred to use individual non-spherical breaking particles, such as a sphericone, having a size in the range of 3 mm - 7 mm, preferably 4 mm - 7 mm, and a weight in the range of 500 - 1000 mg, preferably 600 mg - 900 mg, as the first type.

[0067] Second type of solid breaking particles

[0068] The second type of solid breaking particles used in combination with the first type is provided by a plurality of breaking particles having a size of 1 mm or less. Thus, the particles of the second type are smaller than the particles of the first type. Furthermore, a plurality of such particles is used as the second type. The second type of breaking particles particularly supports efficient lysis of microorganisms contained in the plant sample, such as bacteria and / or fungi that can be present on, around or inside the plant sample. As further demonstrated and explained in the examples, the combined use of the particles of the first and second type provides for a high nucleic acid yield, wherein in turn, the amount of contained microbial nucleic acid is improved.

[0069] The particles used as the plurality of particles of the second type are substantially spherical. Conventional beads used in the art are often described as being "substantially" spherical, as those beads are not necessarily mathematically perfect spheres, but can include minor imperfections that affect their shape. As discussed herein, the use of one or more larger non-spherical breakage particles as the first type (as described above, e.g. a spherocone or the like) in combination with a plurality of smaller substantially spherical breakage particles provides particularly advantageous results for total DNA yield and yield of microbial nucleic acids (e.g. bacterial DNA).

[0070] According to one embodiment, the particles used as the plurality of particles of the second type are crystalline particles.

[0071] According to one embodiment, the plurality of particles used as the second type comprises or consists of zirconium, zircon (zirconium silicate), zirconium oxide (zirconium dioxide), yttrium stabilized zirconium, quartz, aluminum oxide, silicon carbide, ceramic, glass (e.g. silica glass or silica) or a combination thereof. According to one embodiment, the particles used as the plurality of particles of the second type are substantially spherical and comprise or consist of zirconium, zircon (zirconium silicate), zirconium oxide (zirconium dioxide) or yttrium stabilized zirconium. According to one embodiment, the particles used as the plurality of particles of the second type are made of the same material.

[0072] The particles used as the plurality of breakage particles of the second type are smaller than the first type, with a size of 1 mm or less. A given size of a particle used as the second type refers to the longest distance between two opposing points of the respective particle. As the particles of the second type are substantially spherical, this is the diameter.

[0073] According to one embodiment, the plurality of particles used as the second type has at least two different sizes, wherein (i) the average of the first particle size is in a range selected from 0.05 mm to 0.25 mm and (ii) the average of the second particle size is in a range selected from 0.3 mm to 0.9 mm.

[0074] According to one embodiment, the plurality of particles used as the second type has at least two different sizes, wherein (i) the average of the first particle size is in a range selected from 0.05 mm to 0.25 mm and (ii) the average of the second particle size is in a range selected from 0.3 mm to 0.9 mm.

[0075] According to one embodiment, the plurality of particles used as the second type have at least two different sizes, wherein (i) the average size of the first particle is selected from the range of 0.05 mm to 0.25 mm, 0.07 mm to 0.2 mm, 0.08 mm to 0.175 mm, and 0.9 mm to 0.15 mm, and (ii) the average size of the second particle is selected from the range of 0.3 mm to 0.9 mm, 0.35 mm to 0.8 mm, 0.4 mm to 0.7 mm, and 0.45 mm to 0.6 mm. Suitable and preferred embodiments are as described above. As discussed, the plurality of particles used as the second type can be made of the same material. In one embodiment, two different sizes of zirconia beads are used as the second type. According to one embodiment, the particles of the first size and the particles of the second type are mixed in a ratio of 1:2 to 2:1, preferably 1:1.

[0076] According to one embodiment, the plurality of particles used as the second type are substantially spherical and contain or are composed of zircon, zircon (zirconium silicate), zirconium oxide (zirconium dioxide), or yttrium-stabilized zirconium, and have an average size in the range of 0.08 mm to 0.7 mm, preferably 0.09 mm to 0.6 mm. Preferably, zirconium beads are used.

[0077] According to one embodiment, the density of the particles used as the second type of multiple particles is at least 2.0 g / cc, for example at least 2.5 g / cc, at least 3.0 g / cc, at least 3.5 g / cc, at least 4.0 g / cc, at least 4.5 g / cc, at least 5.0 g / cc, or at least 5.5 g / cc. Its density can be selected from the range of 2.0 g / cc to 15 g / cc, for example 2.5 g / cc to 12 g / cc, 3.0 g / cc to 10 g / cc, 3.5 g / cc to 9 g / cc, 4.0 g / cc to 8 g / cc, 4.5 g / cc to 7.5 g / cc, and 5 g / cc to 7 g / cc.

[0078] The appropriate amount of the multiple particles used as the second type can be determined by those skilled in the art in accordance with the guidance given herein and in the examples. According to one embodiment, 5 mg to 500 mg of the second type of particles is used relative to each milligram of plant material.

[0079] According to one embodiment, a combination of the following crushing particles is used to mechanically crush plant samples:

[0080] (i) At least one non-spherical broken particle is used as the first type, which has the following characteristics:

[0081] - It has a surface comprising a first part and a second part, whereby the first part and the second part meet by forming an edge, wherein the first part is the surface of a truncated cone, the second part is the surface of a truncated cone, wherein both cones abut against each other with their larger bases, forming an edge where the larger bases meet, the larger bases having the same diameter, and wherein at least one non-spherical particle is selected from the group consisting of particles having the following characteristics:

[0082] (aa) The particle includes at least one tip, which is a truncated cone, wherein the larger base of the truncated cone providing the tip abuts against the smaller base of the truncated cone of the second portion, and wherein the particle includes a sub-portion consisting of a piece or portion of a sphere or ellipse abutting against the smaller base of the truncated cone of the first portion, wherein preferably, the sub-portion consisting of a piece or portion of a sphere or ellipse is a hemisphere.

[0083] (bb) The particle comprises at least two tips, both of which are truncated cones, wherein the larger base of the truncated cone abuts against the smaller base of the first truncated cone, and the larger base of the truncated cone abuts against the smaller base of the second truncated cone.

[0084] (cc) The particle comprises two sub-parts, each sub-part consisting of a piece or portion of a sphere or ellipse, wherein the first sub-part consisting of a piece or portion of a sphere or ellipse abuts against the smaller base of a truncated cone of the first part, and the second sub-part consisting of a piece or portion of a sphere or ellipse abuts against the smaller base of a truncated cone of the second part, and / or

[0085] (dd) The particle comprises two hemispheres, wherein the first hemisphere abuts against the smaller base of the truncated cone of the first portion, and the second hemisphere abuts against the smaller base of the truncated cone of the second portion;

[0086] - Its weight is at least 300 mg, preferably at least 400 mg, more preferably at least 500 mg; and

[0087] - Its size is at least 1.5mm, preferably at least 2mm, and more preferably at least 3mm;

[0088] as well as

[0089] (ii) The second type is provided by a plurality of substantially spherical zirconia beads, preferably with a size in the range of 0.08 mm to 0.7 mm, more preferably 0.09 mm to 0.6 mm. At least one non-spherical crushed particle used as the first type is preferably made of steel, stainless steel, tungsten, or other heavy metals, as discussed above. As disclosed herein, the plurality of particles used as the second type can have at least two different sub-sizes falling within this wide range. This has been described in detail above.

[0090] According to one implementation, no other types of particles are used besides the first and second types of crushed particles.

[0091] Mechanical crushing using first and second type particles

[0092] Plant samples and the microorganisms contained in the plant samples can be broken down sequentially or simultaneously using the first and second types of breaking particles. It is preferred that they are broken down simultaneously.

[0093] The first and second types of crushing particles can be contained in a container, which preferably also contains a lysis solution. In one embodiment, the lysis solution and the first and / or second type of crushing particles are contained in the same compartment of the container and provided as a composition. A plant sample from which nucleic acids are to be isolated can be added to the container. The container is then closed, and mechanical crushing can begin. If the first and second types are not provided in the same container, the missing one can be added subsequently to allow crushing with both types of particles simultaneously or sequentially.

[0094] The container for receiving tissue material can be any suitable container or reaction vessel, preferably inert relative to the reagents used in the lysis process, exhibiting sufficient mechanical stability to withstand the forces of the lysis particles without damage or wear, having dimensions suitable for receiving plant sample material, lysis solution, and one or more selected lysis particles, and still providing suitable space to allow for the agitation and movement of inserted components to lysis and further cleavage of the plant material, and can be suitably used with apparatus for grinding or milling tissue material by means of cleavage particles. Suitable containers or reaction vessels (tubes) are known and generally available.

[0095] According to one embodiment, plant samples are homogenized to provide lysates. As demonstrated in the examples, this technique effectively homogenizes a variety of plant samples.

[0096] Mechanical crushing using first and / or second type crushed particles may include the use of bead-based pulping and / or homogenizing devices. Suitable devices may include, but are not limited to, high-performance or high-speed mixers, as well as low-power mixers such as standard laboratory vortex mixers, benchtop vortex mixers, or standard laboratory shakers (e.g., horizontal shakers). Crushing can be performed using vortex mixers with bead adapters or stirring devices, such as the TissueLyzer II (QIAGEN) or AMBION. TM Vortex adapters (Thermo Fisher Scientific, Waltham, MA) and Omini Bead Rupter homogenizers (OMNI Int'l, Kennesaw, Georgia), as well as various homogenizers from OPS Diagnostics. High-power or high-performance mixers typically operate at frequencies of 15–60 Hz. Low-power mixers, especially common vortex mixers, typically operate at forces from 150 to a maximum of 3200 rpm. For example, applying reduced mechanical power by a low-power mixer or vortex mixer can help maintain the quality of the released nucleic acids and prevent damage or deterioration. In one embodiment, a high-speed oscillator (e.g., 15–60 Hz) is used. In this embodiment, it achieves oscillations per minute in the range of 150–2500, for example, 180–1800. The appropriate and advantageous duration for mechanical disruption can be determined by a technician. For example, a crushing cycle may include mechanical crushing with one or more crushing particles for 30 sec to 20 min, 1 min to 15 min, 1.5 min to 10 min, and 2 min to 7 min. If necessary, two or more crushing cycles may be performed to achieve good homogenization of the pyrolyte.

[0097] Preferred pyrolysis conditions

[0098] The liquid pyrolysis composition preferably contains at least one liquid release agent. According to one embodiment, the liquid pyrolysis composition is a solution, preferably an aqueous solution. Solid pulverized particles may be contained in the solution.

[0099] According to one implementation, the liquid release agent is a liquid release salt.

[0100] According to one embodiment, the liquid release agent is selected from sodium thiocyanate, sodium carbonate, ammonium thiocyanate, potassium thiocyanate, lithium thiocyanate, lithium perchlorate, guanidine sulfate, and combinations thereof. This liquid release agent can be used to generate pyrolysis products.

[0101] According to one embodiment, the liquid release agent is selected from sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, lithium thiocyanate, and combinations thereof. This liquid release agent is particularly suitable for producing pyrolysis products.

[0102] According to one embodiment, the liquid dissociation agent is a liquid dissociation salt. According to one embodiment, the pyrolysis composition contains only one liquid dissociation agent, and preferably only NaSCN as the liquid dissociation agent.

[0103] NaSCN, Na2CO3, KSCN, NH4SCN, LiSCN, LiClO4, and guanidine sulfate are relatively mild liquefaction agents, which is advantageous for combining this mild pyrolysis with mechanical crushing using first and second types of crushing particles in the embodiments. Preferably, the relatively mild liquefaction agent is NaSCN.

[0104] Relatively mild dissociation agents that can be used as dissociation agents in liquid lysis compositions may include those with a weaker affinity for soluble proteins than Mg. 2+ strong anions SCN with cation pairing - Salts; possessing a weaker affinity for soluble proteins than Mg. 2+ The strong anion ClO4 with cation pairing - Salts; and those with a stronger affinity for soluble proteins than NH4+. + The weak anion CO3 with cation pairing 2- Salt.

[0105] Relatively mild ionizing agents (e.g., NaSCN) achieve an ideal balance between stronger ionizing agents (e.g., GuSCN or GuCl) and weaker ionizing agents (e.g., RbSCN). Less aggressive ionizing agents can effectively dissolve biomolecules with the fragmented particles during the disruption process, making them usable for downstream separation. On the other hand, strong ionizing agents and detergents (e.g., SDS) can achieve complete cell lysis, but at the cost of biomolecule degradation (e.g., nucleic acid degradation). The less aggressive ionizing agent preferably used in conjunction with this method is unique in its ability to minimize nucleic acid degradation while dissolving biomolecules (e.g., nucleic acids). Therefore, combining such a mild ionizing agent with a mechanical sample disruption process using the two types of fragmented particles described herein is particularly advantageous and provides an improvement over existing methods.

[0106] The concentration of at least one clinker in the liquid pyrolysis composition and / or pyrolysis mixture (containing plant samples) can be 2.5 M or less, for example 2 M or less, 1.75 M or less, 1.5 M or less, 1.3 M or less, 1.2 M or less, or 1.125 M or less. A suitable concentration of at least one clinker in the liquid pyrolysis composition (preferably a pyrolysis solution and / or pyrolysis mixture) can be in the range selected from 0.5–2.5 M, for example 0.6 M–2 M, 0.7 M–1.75 M, 0.75 M–1.5 M, and preferably 0.8–1.25 M. If multiple clinkers are present in the liquid pyrolysis composition (preferably a pyrolysis solution), the total concentration of the liquid pyrolysis composition and the clinkers in the pyrolysis solution can and preferably is within the above ranges. The same applies to pyrolysis mixtures. Solid fragments are not considered when determining the concentration.

[0107] The ionizing agent is preferably a thiocyanate as described above, more preferably NaSCN. The above concentrations have been found to be particularly suitable for such mild thiocyanates, such as NaSCN. Particularly preferred is that the NaSCN concentration in the liquid pyrolysis composition and / or the pyrolysis mixture is in the range of 0.7M to 1.75M, for example 0.75M to 1.5M, and preferably 0.8M to 1.25M.

[0108] According to one embodiment, the method further includes the addition of at least one phosphate. If such an inhibitor remover is used, at least one phosphate is added (see below) before the lysed sample is contacted with the inhibitor remover. Not wishing to be bound by theory, it is believed that free phosphate groups (PO4) 3- This prevents or reduces the formation of complexes between subsequently used inhibitor removers (e.g., AlCl3) and the phosphodiester groups of nucleic acids by competitively interacting with inhibitor removers.

[0109] Preferably, at least one phosphate is present during the mechanical crushing process. Preferably, at least one phosphate is contained in the liquid pyrolysis composition (preferably a pyrolysis solution, as described above). Therefore, in an advantageous embodiment, the liquid pyrolysis composition comprises at least one dissociating agent and at least one phosphate. According to one embodiment, the liquid pyrolysis composition comprises sodium thiocyanate and phosphate.

[0110] Exemplary phosphates include dihydrogen phosphates, hydrogen phosphates, and phosphates, as well as other compounds containing one or more free phosphate groups, such as sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, lithium dihydrogen phosphate, dilithium hydrogen phosphate, lithium phosphate, trisodium phosphate, sodium poly(vinylphosphonate), sodium hexametaphosphate, sodium pyrophosphate, sodium triphosphate, sodium polyphosphate, other phosphorus-containing oxyanions, and combinations thereof. The cationic portion of the phosphate includes, but is not limited to, ammonium, sodium, potassium, and lithium. In one embodiment, the cationic portion is provided by an alkali metal ion, preferably selected from sodium, potassium, and lithium, more preferably sodium. Preferably, the phosphate is a hydrogen phosphate, more preferably disodium hydrogen phosphate.

[0111] The concentration of at least one phosphate in the liquid lysis composition, lysis mixture (containing plant samples), and / or lysis sample may be selected from 0.05–0.75 M, 0.06 M–0.6 M, 0.075 M–0.5 M, 0.1 M–0.3 M, and 0.1 M–0.25 M, or may be 0.125 M–0.2 M. As disclosed herein, it is preferred that the liquid lysis composition contains at least one phosphate. The concentration of at least one phosphate in the liquid lysis composition (preferably a lysis solution) is preferably in the range of 0.1 M–0.3 M or 0.1 M–0.2 M.

[0112] According to one embodiment, the liquid pyrolysis composition comprises sodium thiocyanate and at least one phosphate, preferably disodium hydrogen phosphate.

[0113] According to one embodiment, the liquid pyrolysis composition and / or liquid pyrolysis mixture contains sodium thiocyanate at a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M, and preferably 0.8M to 1.25M, and contains at least one phosphate, preferably disodium hydrogen phosphate, at a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M, and 0.1M to 0.2M. Preferably, the concentration of at least one phosphate, preferably disodium hydrogen phosphate, is in the range of 0.1M to 0.3M.

[0114] According to one embodiment, the liquid pyrolysis composition and / or liquid pyrolysis mixture contains sodium thiocyanate at a concentration of 0.7M to 1.75M and at a concentration of 0.075M to 0.3M, containing at least one phosphate, preferably disodium hydrogen phosphate.

[0115] According to one embodiment, the liquid pyrolysis composition and / or liquid pyrolysis mixture contains sodium thiocyanate at a concentration of 0.75M to 1.5M and at least one phosphate, preferably disodium hydrogen phosphate, at a concentration of 0.1M to 0.3M.

[0116] According to one embodiment, the liquid pyrolysis composition and / or liquid pyrolysis mixture contains sodium thiocyanate at a concentration of 0.8M to 1.25M and at least one phosphate, preferably disodium hydrogen phosphate, at a concentration of 0.1M to 0.25M.

[0117] The lysis composition (e.g., lysis reagent) can be combined with the cleavage particles before the plant sample is added. However, the plant sample can also be contacted with the lysis reagent before the addition of the first and / or second type of cleavage particles.

[0118] The lysis reagent (preferably a lysis solution) may contain:

[0119] (i) one or more dissociating agents selected from sodium thiocyanate, sodium carbonate, ammonium thiocyanate, potassium thiocyanate, lithium thiocyanate, lithium perchlorate, guanidine sulfate, and combinations thereof; and

[0120] (ii) One or more phosphates.

[0121] The details of the dissociating agent and at least one phosphate have been described above, with reference to the corresponding disclosures. The concentrations described above for the liquid pyrolysis composition also apply to the pyrolysis reagent (which is preferably a pyrolysis solution). Thus, according to one embodiment, the concentration of at least one dissociating agent in the pyrolysis reagent can be 2.5 M or less, for example 2 M or less, 1.75 M or less, 1.5 M or less, 1.3 M or less, 1.2 M or less, or 1.125 M or less. Suitable concentrations of at least one dissociating agent in the pyrolysis reagent can be selected from the range of 0.5 to 2.5 M, for example 0.6 M to 2 M, 0.7 M to 1.75 M, 0.75 M to 1.5 M, and preferably 0.8 to 1.25 M. If multiple dissociating agents are present in the pyrolysis reagent, the total concentration of the dissociating agents in the pyrolysis reagent can and preferably is within the above-described range. The dissociating agent is preferably thiocyanate as described above, more preferably NaSCN. Particularly preferred is that the NaSCN concentration in the lysis reagent is in the range of 0.7M to 1.75M, for example 0.75M to 1.5M, and preferably 0.8M to 1.25M. The concentration of at least one phosphate in the lysis reagent can be selected from 0.05M to 0.75M, for example 0.06M to 0.6M, 0.075M to 0.5M, 0.1M to 0.3M, and 0.1M to 0.25M. Refer to the above disclosure.

[0122] According to one embodiment, the lysis reagent comprises sodium thiocyanate at a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M, and preferably 0.8M to 1.25M, and comprises at least one phosphate, preferably disodium hydrogen phosphate, at a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M, 0.1M to 0.2M, or 0.125M to 0.2M. Preferably, the concentration of at least one phosphate, preferably disodium hydrogen phosphate, is in the range of 0.1M to 0.3M. Refer to the above disclosure.

[0123] Preferably, the cleavage reagent comprises disodium hydrogen phosphate and sodium thiocyanate.

[0124] One or more solid fragments may be contained in the lysis reagent. As described herein, the lysis reagent may be contained in a container that further contains first and / or second type solid fragments. The solid fragments may be contained, for example, impregnated in the lysis reagent. This embodiment is advantageous because a plant sample can be added to a liquid lysis composition containing at least one release agent and first and / or second type fragments, and mechanical lysis can be initiated.

[0125] In some other embodiments, the liquid pyrolysis composition does not contain any detergents, such as SDS.

[0126] The liquid pyrolysis composition (which may be a pyrolysis solution) may optionally further contain one or more buffering substances.

[0127] The pH of the liquid pyrolysis composition can be at least 3, for example at least 4 or at least 5. For example, the pH of the liquid pyrolysis composition can be in the range of pH 3 to pH 10, for example pH 4 to pH 9 and pH 5 to pH 8.0.

[0128] The liquid pyrolysis composition (preferably a pyrolysis solution) may comprise one or more dissociating agents and one or more phosphates, consist substantially of one or more dissociating agents and one or more phosphates, or consist of one or more dissociating agents and one or more phosphates, wherein both the dissociating agent and the phosphate can be aqueous solutions as described above. For mechanical fragmentation, solid fragments as described above are included in the liquid pyrolysis composition (which may be a pyrolysis solution). Preferably, one or more relatively mild dissociating agents comprise NaSCN or NaSCN. One or more phosphates preferably comprise disodium hydrogen phosphate or disodium hydrogen phosphate.

[0129] An exemplary preferred lysis solution comprises 0.5–2 M NaSCN and 0.1–0.2 M Na2HPO4, is substantially composed of 0.5–2 M NaSCN and 0.1–0.2 M Na2HPO4, or is composed of 0.5–2 M NaSCN and 0.1–0.2 M Na2HPO4.

[0130] Other preferred steps for processing pyrolytes

[0131] Other optional but preferred steps for treating the pyrolysis products are described below:

[0132] Clarified pyrolysis products

[0133] This method may further include clarifying the lysate. As discussed herein, the lysed plant sample provides a lysate mixture that may contain a solid component from the plant sample and a liquid fraction containing the released nucleic acids. As disclosed herein, mechanical lysis supported by the lysis chemistry method used advantageously allows for efficient homogenization of different plant species. It is preferable to separate the solid component from the liquid fraction and further process the liquid fraction as a lysate sample.

[0134] The lysate clarification step may include separating the lysate mixture obtained after pulverizing the plant sample into solid and liquid fractions. The liquid fraction contains nucleic acids (and may still contain some plant particles) and can be further processed as a lysate sample. The solid components may be discarded. Separation of the liquid fraction can be aided by sedimentation, centrifugation, or filtration, preferably by centrifugation. A combination of these methods may also be used. The separated liquid fraction (e.g., supernatant) can then be further processed as a lysate sample.

[0135] The lysed sample is contacted with at least one precipitant and at least one inhibitor removal agent, and a mixture is provided.

[0136] This method may also include contacting the (optionally clarified) lysed sample with at least one protein precipitant and at least one inhibitor removal agent and providing a mixture.

[0137] The lysed sample may be contacted with at least one protein precipitant and at least one inhibitor removal agent to provide a mixture. This step may include, for example, agitating the mixture by vortexing.

[0138] Protein precipitant

[0139] According to one embodiment, at least one protein precipitant is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate.

[0140] Some precipitants (e.g., ammonium acetate) can act as protein precipitants at relatively high concentrations (e.g., 1–2 M in a mixture containing a lysed sample, the precipitant, and one or more inhibitor removal agents as described below), but act as molecular sieves at relatively low concentrations (e.g., 5 to 15 times lower than the concentration at which they act as protein precipitants). Ammonium acetate is preferred.

[0141] According to one embodiment, the concentration of at least one precipitant in the mixture is selected from the range of 0.1-4M, for example 0.2M-3M, 0.3M-2.5M, 0.4M-2.25M, 0.5M-2M, and 0.6M-1.75M. According to one embodiment, ammonium acetate is used within such a concentration range, preferably present in the mixture of step (b) at a concentration falling within the range of 0.5M-2M or 0.6M-1.75M.

[0142] Inhibitor removal agent

[0143] Exemplary inhibitor removers include ammonium aluminum sulfate, ammonium aluminum sulfate dodecahydrate, ammonium sulfate, potassium aluminum sulfate, hydrated aluminum chloride, calcium oxide, ferric chloride (III), ferric sulfate (II), sodium aluminate, sodium silicate, magnesium chloride, aluminum chloride, aluminum sulfate, erbium acetate (III), erbium chloride (III), holmium chloride, zirconium chloride (IV), hafnium chloride (IV), and combinations thereof.

[0144] According to one embodiment, the inhibitor removal agent comprises a trivalent cation. Preferably, the inhibitor removal agent comprises aluminum chloride, aluminum sulfate, erbium acetate (III), erbium chloride (III), holmium chloride, zirconium chloride (IV), hafnium chloride (IV), and combinations thereof.

[0145] Therefore, according to one embodiment, at least one inhibitor removal agent is selected from aluminum chloride, erbium acetate (III), erbium chloride (III), holmium chloride, hafnium chloride (IV), zirconium chloride (IV), guanidine sulfate, and combinations thereof, and preferably, the inhibitor removal agent is aluminum chloride.

[0146] Trivalent aluminum salts, such as aluminum chloride, are particularly preferred. The use of aluminum chloride is advantageous because it can be used over a wide pH range.

[0147] As discussed in this article, at least one phosphate may be added, preferably during sample lysis. This serves to prevent the precipitation of nucleic acids, particularly DNA, from the mixture, thus preventing the loss of nucleic acid material.

[0148] The pH of the mixture can be at least 3, for example at least 4 or at least 5. For example, the pH in step (b) can be in the range of pH 3 to pH 10, for example pH 4 to pH 9 and pH 5 to 8.0.

[0149] According to one embodiment, the concentration of at least one inhibitor removal agent in the mixture is selected from the range of 1–150 mM, for example 5 mM–125 mM, 10 mM–100 mM, 15 mM–75 mM, and 20 mM–65 mM. As discussed above, trivalent aluminum salts, such as aluminum chloride, are particularly preferred, and in one embodiment, they are used at such concentrations. A concentration of aluminum chloride selected from 15 mM–75 mM, for example 20 mM–65 mM, or 25 mM–55 mM is particularly preferred.

[0150] Lysed samples contain contaminants or inhibitors that form complexes with one or more inhibitor removers, which are then precipitated and removed by the removers. As described herein, plant samples typically contain a significant number of inhibitors, including polysaccharides and polyphenolic compounds. These inhibitors remain in the lysed samples. This method allows for the efficient removal of inhibitors, thereby enabling the separation of high-quality nucleic acids, such as DNA.

[0151] According to one embodiment, the precipitant is ammonium acetate and the inhibitor removal agent is aluminum chloride.

[0152] The protein precipitation step and the inhibitor removal step can be performed sequentially. However, it is preferable that they are performed simultaneously.

[0153] According to a preferred embodiment, in step (b), the pyrolysis sample is contacted with a composition comprising at least one precipitant and at least one inhibitor removal agent. As discussed, the pyrolysis sample is preferably a clarified pyrolysis product.

[0154] One or more precipitants and one or more inhibitor removal agents can be added in the form of a composition, which can be in solid or solution form, preferably in solution form. Preferably, the composition is an aqueous solution. It can be added to the lysed sample.

[0155] According to one embodiment, the composition comprises, is substantially composed of, or is composed of the following components:

[0156] (i) One or more precipitating agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof.

[0157] (ii) One or more inhibitor removal agents selected from aluminum chloride, erbium acetate (III), erbium chloride (III), holmium chloride, hafnium chloride (IV), zirconium chloride (IV), and combinations thereof; and

[0158] (iii) Water of choice.

[0159] In embodiments where the composition is a solution, the total concentration of one or more precipitants in the added solution is in the range of 0.5M–10M, 1–8M, or 1.5–7.5M, preferably 1M–6M, 1.5M–5.5M, 2M–5M, 2.5–4.5M, and 3M–4M. This is particularly suitable when the precipitant acts as a protein precipitant. The precipitant can be ammonium acetate, and the concentration is particularly suitable when ammonium acetate is used. The composition can be added to the lysed sample.

[0160] In embodiments where the composition is a solution, the total concentration of one or more inhibitor scavengers in the added solution is in the range of 10-500 mM, for example 25 mM-400 mM, 50 mM-350 mM, 75 mM-300 mM, 90 mM-250 mM, preferably 50 mM or 100 mM-200 mM, for example 50 mM-175 mM or 75 mM-150 mM. As discussed above, trivalent aluminum salts, such as aluminum chloride, are particularly preferred as inhibitor scavengers, and in one embodiment are contained in the solution at such concentrations. According to one embodiment, the added solution contains aluminum chloride at a concentration of 50 mM-250 mM. Particularly preferred aluminum chloride concentrations include 50 mM-200 mM, 50 mM-175 mM, and 75 mM-150 mM.

[0161] Exemplary preferred solutions comprising a precipitant and an inhibitor removal agent include:

[0162] (1) A solution containing 1-8 M (preferably 2.5-5 M) ammonium acetate and 20-200 mM aluminum chloride;

[0163] (2) A solution containing 1-10 M (preferably 1-8 M) sodium acetate and 20-200 mM aluminum chloride;

[0164] (3) A solution containing 1-8 M (preferably 1-5 M) of cesium acetate and 20-200 mM of aluminum chloride;

[0165] (4) A solution containing 1-8 M (preferably 2.5-5 M) ammonium acetate and 20-200 mM erbium(III) acetate;

[0166] (5) A solution containing 1-10 M (preferably 1-8 M) sodium acetate and 20-200 mM erbium(III) acetate;

[0167] (6) A solution containing 1-8 M (preferably 1-5 M) of cesium acetate and 20-200 mM of erbium(III) acetate;

[0168] (7) A solution containing 1-8 M (preferably 2.5-5 M) ammonium acetate and 20-200 mM erbium(III) chloride;

[0169] (8) A solution containing 1-10 M (preferably 1-8 M) sodium acetate and 20-200 mM erbium(III) chloride;

[0170] (9) A solution containing 1-8 M (preferably 1-5 M) of cesium acetate and 20-200 mM of erbium(III) chloride;

[0171] (10) A solution containing 1-8 M (preferably 2.5-5 M) ammonium acetate and 20-200 mM holmium chloride;

[0172] (11) A solution containing 1–10 M (preferably 1–8 M) sodium acetate and 20–200 mM holmium chloride; and

[0173] (12) A solution containing 1-8 M (preferably 1-5 M) of cesium acetate and 20-200 mM of holmium chloride.

[0174] According to one embodiment, the precipitant in the composition added to the pyrolysis sample is selected from ammonium acetate, sodium acetate, cesium acetate, or combinations thereof, preferably ammonium acetate, and the inhibitor removal agent is aluminum chloride.

[0175] According to one embodiment, no precipitation, centrifugation, or filtration is performed between contacting the lysed sample with at least one precipitant and contacting the lysed sample with at least one inhibitor removal agent. As disclosed herein, it is preferable to add the precipitant and the inhibitor removal agent simultaneously, for example, by adding a liquid composition comprising at least one precipitant and at least one inhibitor removal agent.

[0176] As used herein, the term "inhibitor" specifically refers to any substance that interferes with reactions involving the isolation of DNA and / or RNA from a sample and has a detrimental effect on the manipulation of DNA and / or RNA. Inhibitors include, for example, inhibitors of enzymatic reactions using DNA or RNA as substrates, as well as contaminants that disrupt the hybridization of DNA or RNA. Inhibitors may include humic substances. These contain polycyclic aromatic hydrocarbons linked to sugars, peptides, and phenols. Other exemplary inhibitors include decomposing plant material, organic compounds from compost, phenols, phenolic polymers or oligomers, polyphenols, polysaccharides, and tannins. Examples of polysaccharide inhibitors include, but are not limited to, pectin and xylan. As discussed herein, the methods of the present invention improve sample lysis, thereby advantageously increasing the release of nucleic acids, particularly DNA, into the lysate. This improved lysis also releases more inhibitors into the lysate, thus allowing a DNA-containing supernatant to be obtained from the lysate as described herein. Therefore, this is advantageous and important for efficiently removing inhibitors to provide high-quality nucleic acids.

[0177] Inhibitor removal agents are capable of substantially removing one or more inhibitors from lysed samples. Once a liquid phase is obtained from the mixture (see below), the inhibitors have been substantially removed.

[0178] For example, after separating the mixture into solid and liquid phases, 20% or less, preferably 18% or less, 15% or less, 13% or less, or 10% or less, more preferably 5% or less, 3% or less, 2% or less, or 1% or less of the inhibitor from the sample is retained in the liquid phase.

[0179] Obtaining the liquid phase from the mixture

[0180] This method may also include obtaining a liquid phase from the mixture.

[0181] During or after protein precipitation and inhibitor removal, solid components are generated, for example, through precipitation and complexation processes. Therefore, it is preferable to implement a step that includes obtaining a liquid phase from the mixture. This can be aided by precipitation, filtration, or preferably centrifugation. Combinations of these techniques may also be used.

[0182] According to one embodiment, the step therefore includes removing the solid components contained in the provided mixture to obtain a liquid phase containing nucleic acids. The liquid phase may be obtained and provided separately in the form of supernatant.

[0183] The mixture can be centrifuged, filtered, precipitated, or otherwise treated to separate its solid phase from its liquid phase, wherein one or more inhibitor removers are predominantly (more than 50%) located in the solid phase. The solid phase can be provided in granular form. One or more inhibitor removers form a complex with inhibitors and other contaminants from the sample, which is precipitated or removed from the liquid phase in the step described.

[0184] In some embodiments, more than 60%, 70%, or 80%, preferably more than 90%, or more preferably more than 95% of one or more inhibitor removers are removed from the liquid phase in this step.

[0185] Nucleic acids, preferably DNA, can be selectively isolated from the liquid phase.

[0186] The resulting liquid phase can then be used to isolate nucleic acids.

[0187] As used herein, the term "nucleic acid" includes single-stranded or double-stranded nucleic acids and may be selected from DNA and RNA. Any method suitable for isolating DNA, RNA, or both from solution may be used. Suitable methods are well known to those skilled in the art and therefore need not be described in detail. Preferably, the nucleic acid isolated from the liquid phase is DNA.

[0188] The improved lysis and inhibitor removal achieved in this embodiment provides a liquid phase containing a large amount of nucleic acids, including microbial nucleic acids (due to improved lysis), and advantageously depletes inhibitors (due to the use of a precipitant and an inhibitor removal agent). Therefore, nucleic acids, such as DNA, can be separated from the provided liquid phase in high yield and with high purity. Essentially any nucleic acid separation method can be used to separate nucleic acids, preferably DNA, from the provided liquid phase. An exemplary method is described in conjunction with the method according to the second aspect thereof.

[0189] As will be appreciated from this disclosure, this method does not require the use of phenol and / or CTAB. Therefore, in embodiments, the method does not involve the use of phenol and / or CTAB. In embodiments, no detergent is added to assist lysis. In embodiments, the method does not involve the use of proteolytic enzymes such as proteinase K to assist lysis.

[0190] According to the second method

[0191] According to the second aspect, a method for isolating nucleic acids, including microbial nucleic acids, from plant samples is provided, comprising:

[0192] (a) Perform the pyrolysis method according to the first aspect;

[0193] (b) Isolation of nucleic acids from lysed samples that have optionally undergone further processing; and

[0194] (c) Optionally, the isolated nucleic acids are sequenced, preferably the isolated DNA is sequenced.

[0195] The details and preferred embodiments of the pyrolysis method implemented in step (a) have been described above, and reference is made to the relevant disclosures, which also apply here.

[0196] Furthermore, suitable and preferred embodiments for further processing of the lysed sample have been described above, with reference to the corresponding disclosures. Preferably, step (a) includes:

[0197] - Contact the (optionally clarified) lysed sample with at least one protein precipitant and at least one inhibitor removal agent and provide a mixture; and

[0198] - Obtain the liquid phase from the mixture.

[0199] Nucleic acids, preferably DNA, can be separated from the liquid phase. Refer to the above disclosure.

[0200] As used herein, the term "nucleic acid" includes single-stranded or double-stranded nucleic acids and may be selected from DNA and RNA. Any method suitable for isolating DNA, RNA, or both from solution may be used. Suitable methods are well known to those skilled in the art and therefore need not be described in detail. Preferably, the isolated nucleic acid is DNA.

[0201] Nucleic acids, such as DNA, can be separated from the provided liquid phase in high yield and with high purity. Basically, any nucleic acid separation method can be used in step (b) to separate nucleic acids, preferably DNA.

[0202] Preferably, a nucleic acid-binding solid support is used in nucleic acid isolation. Exemplary solid supports include silica matrices, glass particles, diatomaceous earth, magnetic beads, nitrocellulose, nylon, and anion exchange materials. The solid support may be in the form of loose particles, filters, membranes, fibers or fabrics, or grids, and is contained in a container including tubes, columns, and preferably centrifuge columns.

[0203] To promote or enhance the binding of nucleic acids to solid supports, binding solutions can be used. The binding solution can be added during sample lysis (e.g., after mechanically breaking up the sample in the presence of a lysis agent), followed by contacting the sample material with the protein precipitant and inhibitor remover during inhibitor removal. Alternatively, the binding solution can be added to the liquid phase obtained after the inhibitor removal process.

[0204] Exemplary DNA binding solutions may contain a dissociation agent (such as GuSCN or GuHCl), an alcohol (such as ethanol or isopropanol), or both. They may also contain a buffer substance, such as Tris HCl.

[0205] In embodiments for separating both DNA and RNA from a sample, DNA separation and RNA separation can be performed in parallel. In other words, the liquid phase of step (b) is divided into at least two parts: one for DNA separation and one for RNA separation. DNA and RNA can also be separated sequentially. When the purpose is to separate RNA, an RNase inhibitor can be used in the lysis step (a) to protect the released RNA.

[0206] Methods for sequentially separating DNA and RNA are known (see, for example, U.S. Patent No. 8,889,393 and WO 2004 / 108925). Preferably, a solid support for DNA binding and a solid support for RNA binding are used. The solid support for DNA binding and the solid support for RNA binding can be the same or different. When using the same solid support to separate DNA and RNA, differential binding of DNA and RNA to the solid support can be achieved by adjusting the composition and / or concentration of the binding mixture. For example, a silica gel centrifuge column can be used first for DNA binding, and the effluent can be mixed with ethanol, and then the resulting mixture can be added to a second silica gel centrifuge column for RNA binding (Triant and Whitehead, Journal of Heredity 100:246-50, 2009).

[0207] After binding to the solid phase, the bound DNA or RNA can be washed away and then eluted from the solid phase. The DNA washing solution may contain a dissociating agent (e.g., GuHCl), an alcohol (e.g., ethanol, isopropanol), or both. It may also contain a buffer (e.g., Tris HCl), a chelating agent (e.g., EDTA (ethylenediaminetetraacetic acid)), and / or a salt (e.g., NaCl). The DNA elution solution may be a buffer (e.g., Tris buffer) or water.

[0208] RNA binding solutions may contain alcohols (e.g., ethanol, isopropanol) and optionally another organic solvent (e.g., acetone). RNA washing solutions may contain one or more of the following: buffers (e.g., Tris HCl and Tris bases), chelating agents (e.g., EDTA), alcohols, and salts (e.g., NaCl). RNA can be eluted from the solid support using DEPC-treated water or other RNase-free water.

[0209] According to one embodiment, at least DNA is isolated. According to one embodiment, DNA is isolated and RNA is consumed simultaneously during the implementation of the method. Alternatively, RNA can be destroyed by using RNase.

[0210] This method may also include analysis of the nucleic acids isolated in the steps. Such analysis may include any conventional analytical techniques, such as PCR, qPCR, RT-PCR, or nucleic acid sequencing. The DNA provided by this method is particularly suitable for sequencing applications, such as next-generation sequencing. For example, sequencing can be performed to identify plant microbiomes. Plant microbiomes can be identified, for example, by sequencing to observe symbiotic and / or pathogenic species.

[0211] Plant samples

[0212] This method is particularly suitable for processing a wide variety of plant samples. Advantageously, this method can be used for different types of plant samples while ensuring good results in terms of yield and purity.

[0213] The term "plant" specifically refers to the whole plant, plant organs, plant tissues, roots, seeds, plant cells, and their offspring. Plant material includes, but is not limited to: seeds, embryos, meristematic regions, callus, leaves, roots, young shoots, gametophytes, sporophytes, pollen, and microspores. Plant parts include differentiated and undifferentiated tissues, including but not limited to roots, stems, young shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells, as well as fruits and flowers.

[0214] As mentioned above, the term "plant" also refers to parts of a plant, such as leaves (bladder (base, midrib, veins, margin, apex) and petiole (leaf stem), stipules), stems, roots (taproot, lateral roots, root hairs, root tip, root crown), needles, flowers (calyx, filaments, anthers, pollen, petals, stigma, style, ovary, ovules), fruits, buds (axillary buds, terminal buds), nodes, and internodes. For example, leaves include any type of leaf, such as A) simple pinnately venated leaves (oak, birch); B) simple palmately venated leaves (gum); C) pinnately compound leaves (walnut); D) palmately compound leaves (horse chestnut); E) parallel veins (grass); F) opposite leaves (maple); G) alternate leaves (elm); H) needles: spruce (4-needle pine – Sitka spruce), pine (2, 3 or 5 needle bundles – yellow pine), fir (flat needles – hemlock), scale leaves (redwood).

[0215] According to one embodiment, the plant sample from which nucleic acids are isolated is selected from leaves, needles, roots, stems, and seeds. Additionally, the plant sample may be selected from fruits and flowers. According to one embodiment, the plant sample is obtained from plants selected from agricultural crops such as wheat, rice, apple, coffee, tobacco, corn, sunflower, grass, etc. Another common plant sample is cotton.

[0216] Exemplary common samples from which nucleic acids, particularly DNA, can be isolated include, but are not limited to: leaf tissue, such as soft or fibrous leaf tissue, such as grape leaves, strawberry leaves, cotton leaves, grass leaves, rice leaves and / or mint leaves; stems, such as tomato stems; needles, such as pine needles; and seeds.

[0217] If the plant sample contains a large amount of phenolic compounds, other compounds can be added during the lysis process to remove the phenolic compounds, which falls within the scope of this method. A suitable example is PVP. This may be advantageous for samples such as pine needles or strawberry leaves.

[0218] Plant samples contain or are suspected of containing microorganisms. The microorganisms contained in a plant sample can be selected from bacteria and fungi, such as Gram-positive bacteria, Gram-negative bacteria, fungi, molds and spores, or combinations thereof. In one embodiment, the microorganisms are bacteria. In a plant sample containing microorganisms, the microorganisms can be present on, around, or inside the plant sample. Microorganisms, such as bacteria, can optionally be contained in root samples, on leaf surfaces, and / or on lesions or tumors in plant tissue. The method of the present invention, using a combination of first and second types of breaking particles, is particularly effective in breaking various plant samples, thereby also releasing the microorganisms contained in the plant sample so that they can be effectively lysed. Suitable plant samples have been described above.

[0219] According to the third aspect of the system

[0220] According to a third aspect, a lysis system, preferably a kit, is provided for releasing microbial nucleic acids from microorganisms contained in a plant sample, comprising:

[0221] (a) Liquid pyrolysis composition,

[0222] (b) At least two types of solid crushed particles, wherein:

[0223] (i) The first type is provided by one or more crushed particles with a size of at least 1.5 mm, and

[0224] (ii) The second type is provided by multiple crushed particles with a size of less than 1 mm.

[0225] The details and preferred combinations of the first and second types of solid fragmentation particles have been described above in conjunction with the method according to the first aspect, and reference is made to the corresponding disclosures, which also apply here. Details of the liquid pyrolysis composition have also been described in conjunction with the first aspect and are hereby referenced.

[0226] The first and second types of crushed particles can be contained in separate containers or in the same container, and preferably in the same container.

[0227] According to one embodiment, the liquid pyrolysis composition comprises at least one dissociating agent, preferably selected from sodium thiocyanate, sodium carbonate, ammonium thiocyanate, potassium thiocyanate, lithium thiocyanate, lithium perchlorate, guanidine sulfate, and combinations thereof. The dissociating agent is preferably a dissociating salt. It can be selected from sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, lithium thiocyanate, and combinations thereof. NaSCN is particularly preferred.

[0228] According to one embodiment, the pyrolysis composition has one or more of the following characteristics:

[0229] (i) The concentration of at least one ionizing agent in the liquid pyrolysis composition is selected from 2.5M or less, for example 2M or less, 1.75M or less, 1.5M or less, 1.3M or less, 1.2M or less and 1.125M or less;

[0230] (ii) The concentration of at least one ionizing agent in the liquid pyrolysis composition is selected from the range of 0.5–2.5 M, for example 0.6 M–2 M, 0.7 M–1.75 M, 0.75 M–1.5 M, and preferably 0.8–1.25 M; and / or

[0231] (iii) The liquid ionizer is NaSCN, and the concentration of NaSCN in the liquid pyrolysis composition is in the range of 0.7M to 1.75M, for example 0.75M to 1.5M or preferably 0.8M to 1.25M;

[0232] The pyrolysis system may also include at least one phosphate. The phosphate is preferably included in the pyrolysis composition containing at least one liquid release agent. According to one embodiment, the phosphate has one or more of the following characteristics:

[0233] (i) It is a hydrogen phosphate salt;

[0234] (ii) The cationic portion of the phosphate is selected from ammonium, sodium, potassium or lithium;

[0235] (iii) It is disodium hydrogen phosphate.

[0236] The concentration of at least one phosphate in the liquid pyrolysis composition can be 0.05–0.75 M. In one embodiment, the concentration of at least one phosphate in the liquid pyrolysis composition is selected from 0.05–0.75 M, 0.06 M–0.6 M, 0.075 M–0.5 M, 0.1 M–0.3 M, and preferably 0.1–0.25 M, 0.15 M–0.2 M, or 0.125 M–0.2 M.

[0237] In one embodiment, the liquid pyrolysis composition comprises sodium thiocyanate and at least one phosphate, preferably disodium hydrogen phosphate.

[0238] In one embodiment, the liquid pyrolysis composition contains sodium thiocyanate at a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M, and preferably 0.8M to 1.25M, and contains at least one phosphate, preferably disodium hydrogen phosphate, at a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M, and 0.1M to 0.2M.

[0239] In one embodiment, the liquid pyrolysis composition contains sodium thiocyanate at a concentration of 0.7M to 1.75M, and contains at least one phosphate, preferably disodium hydrogen phosphate, at a concentration of 0.075M to 0.3M, preferably 0.1M to 0.25M, and more preferably 0.1M to 0.2M.

[0240] The pyrolysis system may also include at least one precipitant. According to one embodiment, the precipitant is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate, with ammonium acetate being preferred.

[0241] The pyrolysis system may also include at least one inhibitor removal agent, preferably selected from aluminum chloride, aluminum sulfate, erbium acetate (III), erbium chloride (III), holmium chloride, zirconium chloride (IV), hafnium chloride (IV), ammonium aluminum sulfate, ammonium aluminum sulfate dodecahydrate, potassium aluminum sulfate, hydrated aluminum chloride, calcium oxide, ferric chloride (III), ferric sulfate (II), sodium aluminate, sodium silicate, magnesium chloride, and combinations thereof.

[0242] According to one embodiment, at least one inhibitor removal agent is selected from aluminum chloride, erbium acetate (III), erbium chloride (III), holmium chloride, hafnium chloride (IV), zirconium chloride (IV), guanidine sulfate, and combinations thereof, wherein preferably, the inhibitor removal agent is a trivalent aluminum salt, such as aluminum chloride.

[0243] According to one embodiment, the pyrolysis system includes ammonium acetate as a precipitant and trivalent aluminum salt, preferably aluminum chloride, as an inhibitor removal agent.

[0244] According to one embodiment, the precipitant and the inhibitor removal agent are contained in the same composition, preferably a solution, more preferably an aqueous solution. According to one embodiment, the composition has one or more of the following characteristics:

[0245] (aa) The total concentration of one or more precipitants in the solution added in step (b) is in the range of 0.5M to 10M, for example 1 to 8M or 1.5 to 7.5M, preferably 1M to 6M, 1.5M to 5.5M, 2M to 5M, 2.5 to 4.5M and 3M to 4M;

[0246] (bb) The total concentration of one or more inhibitor removal agents in the solution added in step (b) is in the range of 10 to 500 mM, for example 25 mM to 400 mM, 50 mM to 350 mM, 75 mM to 300 mM, 90 mM to 250 mM, preferably 50 mM or 100 mM to 200 mM, for example 50 mM to 175 mM or 75 mM to 150 mM;

[0247] (cc) It contains, is essentially composed of, or is composed of the following components:

[0248] (i) One or more precipitating agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof.

[0249] (ii) One or more inhibitor removal agents selected from aluminum chloride, erbium acetate (III), erbium chloride (III), holmium chloride, hafnium chloride (IV), zirconium chloride (IV), and combinations thereof, and

[0250] (iii) Water of choice.

[0251] Other embodiments are also described in conjunction with the method according to the first aspect.

[0252] According to one embodiment, (i) a first type is provided by a single solid crushed particle, and (ii) a second type is provided by a plurality of zirconia beads with a size preferably in the range of 0.08 mm to 0.7 mm, more preferably 0.09 mm to 0.6 mm.

[0253] The lysis system may also include a nucleic acid-binding solid support.

[0254] The lysis system may also include one or more solutions selected from DNA binding solution, DNA washing solution, DNA elution solution, RNA binding solution, RNA washing solution and RNA elution solution.

[0255] The present invention also relates to the use of such a lysis system (preferably a kit) in lysing plant samples containing or suspected of containing microorganisms.

[0256] According to the uses of the fourth and fifth aspects

[0257] According to the fourth aspect, this disclosure relates to the use of the system according to the third aspect in the method according to the first aspect. Refer to the above disclosure. Suitable plant samples have also been described above, and refer to the above disclosure.

[0258] According to the fifth aspect, this disclosure relates to the use of the system according to the third aspect in lysing plant samples and releasing microbial nucleic acids from microorganisms contained in the plant samples, wherein the user can use (i) first and second types, or (ii) second type of fragmentation particles to lyse the plant samples to release microbial nucleic acids, preferably DNA, from microorganisms contained in the plant samples. This allows for differential lysis according to the method.

[0259] This invention is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of the invention. Numerical ranges include the endpoints defining the range. The headings provided herein are not intended to limit the various aspects or embodiments of the invention, and the various aspects or embodiments of the invention can be understood in their entirety with reference to the specification. Suitable and preferred embodiments of aspects of the invention, such as the various steps and the components and reagents used, have been described in detail above, and those skilled in the art will understand that the disclosures regarding the various steps, components, and reagents used in different aspects can be combined with each other. Subject matter arising from corresponding combinations of the various features also falls within the scope of this disclosure.

[0260] As used herein, the term "solution" specifically refers to a liquid composition, preferably an aqueous composition. A solution can be a homogeneous mixture with only one phase, but solutions containing solid components, particularly small amounts of solid components, also fall within the scope of this invention.

[0261] References to “this disclosure” and “this invention”, etc., include one or more aspects taught herein, etc. The aspects taught herein are covered by the term “invention”.

[0262] According to one embodiment, the subject matter described herein, in the case of a method, includes certain steps, or in the case of a composition, solution, and / or buffer, includes certain components, meaning that the subject matter is constituted by the corresponding steps or components. Preferred embodiments described herein are preferably selected and combined, and specific subject matter resulting from corresponding combinations of preferred embodiments also falls within the scope of this disclosure. Attached Figure Description

[0263] Figure 1 The figure shows the total DNA yield (in µg) obtained from pine needle samples by mechanically breaking down different solid fragments and their combinations.

[0264] Figure 2 The figure shows the total DNA yield (in µg) obtained from root samples by using individual cone or zirconia beads or combinations thereof for mechanical disruption of plant samples.

[0265] Figure 3 shows the microbial readings obtained from rose leaf samples by using individual cone or zirconia beads or combinations thereof for mechanical disruption of plant samples. Figure 3a ) and bacterial counts ( Figure 3b The percentage of ).

[0266] Figure 4 shows the microbial readings obtained from maple leaf samples by using individual cone or zirconia beads or combinations thereof for mechanical disruption of plant samples. Figure 4a ) and bacterial counts (Figure 4b The percentage of ).

[0267] Figure 5a A sparsity curve is typically used to indicate the amount of species richness in a sample. Figure 5a The results show that the method of using the particle combination according to the invention for lysis yielded the highest curve, indicating that more bacterial species were detected compared to other methods. Figure 5b Further support, especially in deeper sequencing, will be provided by this combination, which will offer more information than individual zirconium beads.

[0268] Figure 6 One embodiment of the crushed particles is shown here, which are spherical-conical particles. This design combines the abrasive capabilities of a sphere and a cone. Exemplary dimensions A and B that can be applied to spherical-conical beads are listed in Table II below (in inches).

[0269] Table II

[0270]

[0271] Figure 7 and 8 Other exemplary shapes of solid non-spherical broken particles are shown, having surfaces comprising a first part and a second part, such that the first part and the second part meet by forming an edge, here in the form of an inclined central flange. Figure 7 This illustrates an implementation with two tapered tips. Figure 8 This illustrates an implementation with two hemispheres.

[0272] Figure 9 The invention demonstrates the use of the lysis chemistry method of the present invention and individual cones or zirconia beads, or combinations thereof, for the mechanical disruption of plant samples, thereby yielding a total DNA yield (Qubit) from apple leaves (50 mg). Results show that the amount of total DNA (plant and microbial) isolated from apple leaf samples is significantly increased when the lysis chemistry method according to this disclosure is used, along with zirconia beads and cones, for tissue disruption compared to using individual zirconia beads.

[0273] Figure 10 The total DNA yield obtained from apple roots using the lysis chemistry method described in this application, along with a mixture of cones and zirconia beads, or zirconia beads alone, is shown. Total DNA quantification was performed using Qubit. Furthermore, the yield of microbial DNA, assessed using the QuantiTect SYBR green assay, is also shown. The data indicate that combining zirconia beads and cones for the lysis of plant sample materials improves the yield of microbial DNA.

[0274] Figure 11The diagram shows operational taxonomic unit (OTU) clusters based on 16S RNA marker genes, which are operational definitions used to classify closely related microbial species. DNA was isolated from apple tree roots. The figure shows the percentage of reads from different microbial species. It can be seen that using cones is more efficient at lysing plant cells, while zirconia beads are more efficient at lysing bacterial cells. Combining these advantages is achieved using a combination.

[0275] Example

[0276] It should be understood that the following embodiments are for illustrative purposes only and should not be construed as limiting the invention in any way.

[0277] I. Materials and Methods

[0278] In the following embodiments, the effectiveness of different solid crushing particles used as grinding media in aiding the lysis of various plant samples through mechanical crushing was tested. Among other things, the following crushing particles and combinations of crushing particles were tested:

[0279] (1) Two different sizes of zirconia beads (0.1 mm and 0.5 mm (diameter); 0.75 g in each sample formulation). The zirconia beads are essentially spherical.

[0280] (2) Spherical stainless steel beads (approximately 2.4 mm; 3 beads per sample).

[0281] (3) Ball cone. The size of the ball cone is in the range of 4 mm to 7 mm, and the weight is in the range of 600 mg to 900 mg. The ball cone used is made of steel.

[0282] (4) Zirconia beads and spherical steel beads (a combination of (1) and (2)).

[0283] (5) Zirconia beads and cones (a combination of (1) and (3)).

[0284] Unless otherwise specified, the following standard procedures are used to isolate plant DNA:

[0285] 1. Plant sample lysis

[0286] Collect up to 50 mg of plant samples from different sources (e.g., pine needles, roots, rose leaves, maple leaves).

[0287] Each sample was placed into a collection tube (tissue fragmentation tube, QIAGEN) containing 500 µl of lysis buffer. The lysis buffer contained NaSCN and Na₂HPO₄. Preferred concentrations are as described herein. For example, NaSCN may be present in the lysis buffer at a concentration in the range of 0.8 M to 1.25 M. Na₂HPO₄ may be present at a concentration in the range of 0.1 M to 0.25 M or 0.15 M to 0.2 M. Na₂HPO₄ is preferably included in the lysis buffer, but it may also be added separately. Such a lysis buffer was used in the following examples.

[0288] In addition, the collection tube contains the crushed particles or combinations of crushed particles as described above for mechanical crushing (see (1) to (5)). The sample is briefly vortexed to mix and then homogenized by two 2-minute (@24Hz) homogenization cycles (TissueLyzer II, QIAGEN) for lysis.

[0289] When processing plant samples rich in phenolic compounds (e.g., pine needles, see Example 1), 450 µl of lysis solution and 50 µl of phenolic inhibition (PSS) buffer containing PVP can be used optionally.

[0290] Centrifuge the lysate at 12,000 × g for 2 minutes to clarify it, and transfer the supernatant to a clean tube (approximately 350–450 µl). The supernatant may still contain some plant particles. Centrifugation can be performed in tissue disruption tubes.

[0291] 2. Inhibitor removal

[0292] Add 200 µl of IRT solution to the supernatant and briefly vortex the sample for 5 seconds. When treating plants rich in phenolic compounds, PSS buffer can be added at this step instead of the lysis step. As disclosed herein, the use of this buffer is optional.

[0293] Centrifuge the sample at 12,000 g at room temperature for 1 minute. Avoiding particles, transfer the supernatant (liquid phase) to a clean tube. The volume of supernatant is approximately 400–500 µl.

[0294] The inhibitor removal solution (IRT) contains ammonium acetate as a precipitant and AlCl3 as an inhibitor removal agent. Preferred concentrations of both reagents are as described herein. For example, ammonium acetate can be included in the IRT solution at a concentration in the range of 3M to 4M. Aluminum chloride can be included in the IRT solution at a concentration in the range of 100mM to 150mM. Such pyrolysis solutions were used in the following examples.

[0295] 3. Isolation of nucleic acids

[0296] As described herein, virtually any nucleic acid isolation protocol can be used to isolate and thus recover nucleic acids contained in the resulting liquid phase (supernatant). Below, a nucleic acid isolation protocol is used to recover DNA, where the DNA binds to a solid silica support in the presence of a dissociation salt. A commercially available buffer (buffer AVL, Qiagen) containing a dissociation agent is added in a volume approximately corresponding to the volume of the supernatant. The DNA in the lysate is bound to a silica gel centrifuge column (e.g., QIAGEN), the tube containing the sample is centrifuged, and the eluent is discarded. Two washing steps are performed on the DNA bound to the column before eluting the sample into elution buffer (QIAGEN). The following protocol is followed:

[0297] Add 500 µl of AVL solution and vortex for 5 seconds. Load 650 µl of the lysate onto an MB column and centrifuge at 12,000 × g for 1 minute. Discard the eluent and repeat the steps to ensure all lysate has passed through the MB column (MOBIO). Carefully place the MB column into a clean 2 ml collection tube. Avoid splashing any eluent onto the MB column.

[0298] Add 500 µl of AW1 (wash buffer, QiAGEN) to the MB centrifuge column. Centrifuge at 12,000 × g for 1 minute. Discard the eluent and transfer the MB centrifuge column back to the same 2 ml collection tube. Add 500 µl of AW2 (wash buffer, QiAGEN) to the MB centrifuge column. Centrifuge at 12,000 × g for 1 minute. Discard the eluent and transfer the MB centrifuge column back to the same 2 ml collection tube. Centrifuge at up to 16,000 × g for 2 minutes. Carefully transfer the MB centrifuge column into a new 1.5 ml elution tube (provided).

[0299] Add 50–100 µl of solution EB (elution buffer, QIAGEN) to the center of the white filter membrane. Centrifuge at 12,000 × g for 1 minute. Discard the MB column. The eluent contains the eluted DNA.

[0300] The eluted DNA was then analyzed.

[0301] 4. DNA quantification

[0302] The isolated DNA was quantified using a fluorescence method (Qubit dsDNA, HS or BR detection kit, Ingenium Technologies), using 5 µl of elution from 4 independent replicates, thus processing 4 independent samples.

[0303] Microbial DNA-specific quantification was also performed using the QuantiTect SYBR green assay (QIAGEN), which used primers specific to the 16S rRNA gene and a standard reference sample for quantification according to the instructions in the manual. Eight µl eluents from four independent replicates were applied to the assay, resulting in triplicate for each sample. Concentrations were determined by comparison with a standard curve employing linear regression.

[0304] 5. Next-generation sequencing

[0305] Library construction was performed using 400 ng of isolated DNA. Library construction followed the manufacturer's instructions for the QIASeq FX DNA Library Kit. The library was sequenced on an Illumina MiSeq and analyzed using the CLC Microbial Genomics Workbench. The library was localized to all available bacterial genomes. The percentage of bacterial reads was determined by dividing the number of reads localized to this reference microbial database by the total number of reads in the library.

[0306] II. Results

[0307] 1. DNA production

[0308] The results are shown in Figure 1 and Figure 2 Each column represents the mean of four independent replicates, showing the standard deviation.

[0309] pine needle samples

[0310] Figure 1 The image shows the DNA yield obtained from a pine needle sample (50 mg). It can be seen that the cone-shaped beads, a preferred example of the irregularly shaped fragmentation particles used in this invention, provided the highest DNA yield and were therefore most effective in fragmenting plant sample tissue. Conversely, individual zirconia beads provided only a very low DNA yield. Therefore, individual zirconia beads are insufficient to adequately fragment plant tissue, which is reflected in the reduced DNA yield.

[0311] While individual spherical steel beads are effective at lysing samples, DNA yield is significantly reduced when using a combination of spherical steel beads and zirconium beads (4). The yield of the combination is even lower than that obtained by individual zirconium beads. Therefore, spherical steel beads and spherical zirconium beads significantly impair each other's efficiency in mechanolytic cell lysis. The spherical steel beads may hinder the movement of zirconium beads around the spherical surface, thereby reducing the efficiency of zirconium bead polishing.

[0312] Conversely, as demonstrated by the high total yield of isolated total DNA, the combination of cones and zirconia beads (5) is highly effective in lysing samples. The irregular cone shape allows for efficient sample mixing and free movement of the zirconia beads, thereby ensuring effective mechanical lysis of plant samples as well as microorganisms. Therefore, the cones used according to the invention as preferred examples of cleavage particles are particularly suitable for use in combination with multiple small particles such as zirconia beads. Notably, the zirconia beads exhibit consistently high efficiency in the mechanical lysis of bacterial cells in the presence of cones. Thus, the combination of irregular cone shapes and zirconia beads provides very high DNA yields and effectively lyses plant samples as well as the microorganisms contained therein (e.g., bacteria and fungi) (see also below).

[0313] root sample

[0314] High total DNA yields can also be achieved when using a combination of cones and zircon beads to process difficult-to-lyse plant samples. This is demonstrated by the processing of root samples. Roots are plant organs particularly rich in microorganisms such as bacteria. To effectively release the microbial nucleic acids contained in root samples, it is important to achieve thorough disruption and lysis of the root samples, as microorganisms such as bacteria may also be contained within them. Figure 2 This shows the DNA yield obtained when treating a root sample (50 mg).

[0315] The reduced DNA yield clearly demonstrates that zirconia beads alone cannot effectively lyse and homogenize root samples. This is crucial because zirconia beads alone cannot reach microorganisms such as bacteria inside plant tissue (in this case, roots). Therefore, when using zirconia beads alone, microbial nucleic acids originating from microorganisms present within the plant sample may be lost in the analysis.

[0316] Conversely, the combination of cones and zirconia beads provides high DNA yield, indicating that root samples are effectively lysed when these two fragmentation particles are used in combination. The results also show that root samples can be effectively lysed when cones are used alone. However, cones alone are not very effective at lysing the microorganisms contained in the root samples; that is, less microbial nucleic acid is released when cones are used alone compared to the combination of cones and zirconia beads (see below).

[0317] summary

[0318] Using cone-shaped particles as lysis particles provides high DNA yield and is therefore particularly effective in homogenizing and thus disrupting a wide variety of plant samples. However, these large lysis particles alone are less effective at mechanically disrupting microorganisms such as bacteria contained in plant samples (see Figures 3–5 discussed below). Large particles are generally not effective enough for lysing microorganisms.

[0319] The combined use of cones and zirconium beads achieves the same high total yield of isolated DNA as using cones alone. The total DNA yield obtained by mechanical lysis using this combination is significantly higher than that obtained by using zirconium beads alone or a combination of zirconium oxide beads and spherical metal beads in the lysis step. Furthermore, the combined use of cones and zirconium beads effectively releases microbial nucleic acids contained in microorganisms within plant samples, as evidenced by the high percentage of bacterial DNA in the isolated total DNA (see Figures 3–5 discussed below). Therefore, the combined use of cones and zirconium beads is preferred, particularly when used in conjunction with the lysis chemistry methods disclosed herein. This invention can be advantageously used to release nucleic acids, including microbial nucleic acids, from a variety of plant samples, including those that are difficult to lyse, such as root samples.

[0320] 2. Percentage of microbial readings

[0321] While a ball cone alone can effectively lyse various plant samples, it is insufficient for effectively lysing microorganisms (such as bacteria contained within plant samples). Therefore, when a ball cone is used alone to lyse plant samples, there will be some loss of microbial nucleic acids.

[0322] Therefore, in addition to plant DNA, the combination of cone and zirconia beads is advantageous for the efficient release of microbial nucleic acids such as bacterial DNA through lysis. As can be seen from Figures 3 and 4, this combination achieved a high percentage of microorganisms (bacteria and fungi) and bacterial reads, demonstrating efficient plant sample lysis as well as efficient microbial lysis. The higher microbial DNA release is reflected in the higher percentage of microbial and / or bacterial reads obtained in next-generation sequencing after DNA isolation.

[0323] The results showed that using a combination of cones and zirconia beads yielded higher microbial DNA production compared to using cones alone. Furthermore, the results indicated that the total amount of microbial DNA released and thus recoverable increased when using cones and zirconia beads in combination compared to using zirconia beads alone. As discussed above... Figure 1 and 2 As shown, individual zirconia beads cannot effectively lyse plant samples, thus resulting in the loss, for example, of microorganisms, such as bacteria, contained in the plant sample. It should be noted in this regard that if a large amount of plant-derived DNA is present, an increase in the total amount of bacterial or microbial DNA in the isolated DNA can still lead to a lower microbial / bacterial read percentage.

[0324] Because the combination of particles provides a significantly higher total DNA yield and provides available microorganisms, for example, within the plant sample (e.g., in the case of roots), the total amount of microbial DNA is improved compared to using zirconia beads alone. Microbial nucleic acids contained in the microorganisms within the plant sample are additionally released and can therefore be subsequently isolated when using the method of the present invention. This is reflected in the sequencing results, which show that samples lysed using the combination of particles according to the present invention have higher 16S sequence diversity compared to individual zirconia beads or cones (see...). Figure 5a ).

[0325] summary

[0326] Mechanical lysis using a combination of non-spherical fragmentation particles (e.g., cones) and multiple small spherical particles (e.g., zircon beads) as described herein provides high yields of DNA from plant samples, where the obtained DNA contains a large amount of microbial DNA and is therefore available for analysis. For analysis, various methods can be used, such as amplification-based procedures (e.g., PCR) and sequencing (e.g., next-generation sequencing).

[0327] The sequencing results provided also indicate a significantly increased percentage of microbial (e.g., bacterial) reads compared to using cones alone. The percentage of bacterial reads essentially corresponds to using zirconium beads alone, but with an increased total DNA yield (see above). Achieving a high percentage of bacterial DNA in the total DNA obtained through mechanical lysis using a combination of solid, non-spherical fragmentation particles (e.g., cones) and zirconium beads is important because zirconium beads alone cannot adequately disrupt plant cells, thus failing to effectively reach bacteria present within plant cells (see above). Therefore, the combined use of cones and zirconium beads is highly advantageous, especially when employing the lysis chemistry methods disclosed herein.

[0328] 3. Total DNA production and microbial DNA production

[0329] Use other samples to analyze the effects of using cone or zirconia beads alone or in combination (see I. Materials and Methods).

[0330] Apple leaf and apple root samples

[0331] DNA was isolated from an apple leaf sample (50 mg) by combining the lysis method according to the invention with mechanical lysis provided by a ball-and-cone, a ball-and-cone with zirconia beads, or zirconia beads alone. Total DNA yield was determined using a Qubit assay. Results are shown in... Figure 9 This indicates that using both cone and zirconium oxide can increase total DNA yield (plants and microorganisms) compared to zirconium oxide alone.

[0332] for Figure 10 The yields of total DNA (Qubit) and microbial DNA in apple root samples were determined using two different tests. Figure 10 This study demonstrated that the combination of cones and zirconia beads resulted in increased microbial DNA yield from microorganisms contained in apple root samples compared to zirconia beads alone. Microbial DNA was identified using QuantiTect-based qPCR assays. These data highlight the thorough disruption and lysis of the apple root samples and the microorganisms contained within them, which can significantly increase the overall yield in some cases. The data suggest that the mixture of cones and zirconia beads effectively releases intracellular microorganisms from the sample, thereby increasing the yield of microbial DNA, compared to using zirconia beads alone.

[0333] summary

[0334] High yields of DNA containing abundant microbial DNA are released from plant samples through mechanical lysis using a combination of non-spherical fragmentation particles (e.g., cones) and multiple small spherical particles (e.g., zirconia beads) as described herein. For analysis, various methods, such as amplification-based procedures (e.g., PCR) and sequencing (e.g., next-generation sequencing), are used to further quantify the amplification-based microbial DNA contained in the sample.

[0335] 4. Root-associated plant microbiota

[0336] To investigate the diversity of microbial communities in plant samples and the disruption efficiency of plant-associated bacteria, DNA was isolated from 50 mg of apple tree roots using the method of this invention, employing conical spheres, a mixture of conical spheres and small zirconia beads, or zirconia beads alone (see Example IV). A 16S rRNA gene library was prepared using the QIAseq FX DNA Library Kit, sequenced using the Illumina MiSeq system (running 2 × 250 bp), and the resulting reads were analyzed using the CLC Genomic Workbench (QIAGEN Microbial Genomics Pro Suite). Based on the results, operational taxonomic unit (OTU) clustering was performed. Figure 11 The results showed that using a cone cleavage device (CPD) was more effective at lysing plant cells, while small zirconia beads were more effective at lysing bacterial cells. For certain applications, the combined use of CPD and zirconia beads offers significant advantages. The combined mechanical lysis using the non-spherical cleavage particles (e.g., CPD) and multiple small spherical particles (e.g., zirconia beads) described herein provides high yields of DNA from plant samples, with the obtained DNA containing a large amount of microbial DNA and high microbial diversity.

Claims

1. A lysis method for releasing microbial nucleic acids from microorganisms comprised in a plant sample, comprising mechanical disruption of the plant sample in a liquid lysis composition using at least two types of solid disruption particles, wherein: (i) the first type is provided by one or more disruption particles having a size of at least 1.5 mm, and (ii) the second type is provided by a plurality of disruption particles having a size of 1 mm or less.

2. The method of claim 1, wherein the solid disruption particles of the first and second type differ from each other in shape and / or material, and wherein preferably the first type is not spherical and has at least one discontinuity, preferably has an edge, and the second type is provided by a plurality of substantially spherical particles.

3. The method of claim 1 or 2, wherein the first type is provided by one or more non- spherical disruption particles, and wherein the surface of the one or more disruption particles comprises a first portion and comprises a second portion, whereby the first portion and the second portion meet by forming an edge.

4. The method of claim 3, wherein the first type is provided by one or more non- spherical disruption particles having one or more of the following characteristics: (i) the first portion is the surface of a truncated cone and the second portion is the surface of a truncated cone, whereby both cones have their larger bases against each other, forming an edge at the meeting of the larger bases, the larger bases preferably having the same diameter; (ii) the disruption particle has a sub-portion consisting of a piece or a part of a sphere or an ellipsoid; (iii) the disruption particle has at least one tip, which is preferably a truncated cone; (iv) the disruption particle has at least two sub-portions consisting of a piece or a part of a sphere or an ellipsoid; (v) the one or more disruption particles have a shape selected from the group consisting of a cone, a cylinder, a cube, a triangle, a rectangle, a spherocone, and a satellite.

5. The method of claim 3 or 4, wherein the first type is selected from the group of particles having the following characteristics: (aa) the particle comprises at least one tip, which is a truncated cone, whereby the larger base of the truncated cone providing the tip is against the smaller base of the truncated cone of the second portion, and whereby the particle comprises a sub-portion consisting of a piece or a part of a sphere or an ellipsoid, which is against the smaller base of the truncated cone of the first portion, whereby preferably the sub-portion consisting of a piece or a part of a sphere or an ellipsoid is a hemisphere; (bb) the particle comprises at least two tips, whereby both tips are truncated cones, whereby the larger base of the truncated cones is against the smaller base of the truncated cone of the first portion and the larger base of the truncated cones is against the smaller base of the truncated cone of the second portion; (cc) the particle comprises two sub-portions, whereby each sub-portion consists of a piece or a part of a sphere or an ellipsoid, whereby the first sub-portion consisting of a piece or a part of a sphere or an ellipsoid is against the smaller base of the truncated cone of the first portion and the second sub-portion consisting of a piece or a part of a sphere or an ellipsoid is against the smaller base of the truncated cone of the second portion; (dd) the particle comprises two hemispheres, wherein a first hemisphere rests against the smaller base of the frusto-cone of the first portion and a second hemisphere rests against the smaller base of the frusto-cone of the second portion.

6. The method of one or more of claims 1 to 5, wherein the first type is provided by one or more non-spherical breaking particles having a weight in the range of 500 mg to 1000 mg, optionally 600 mg to 900 mg, and having a size of 3 mm to 10 mm, optionally 3 mm to 7 mm or 4 mm to 7 mm.

7. The method of one or more of claims 1 to 6, wherein the first type is provided by a single solid breaking particle, preferably as defined in any one of claims 3 to 6, preferably as defined in claim 5 or 6.

8. The lysis method of one or more of claims 1 to 7, wherein the second type has one or more of the following characteristics: (i) the plurality of particles are crystalline particles; (ii) the plurality of particles comprise or consist of zirconium, zircon (zirconium silicate), zirconium oxide (zirconium dioxide), yttrium stabilized zirconium, quartz, aluminium oxide, silicon carbide, ceramic, glass (e.g. silica glass or silica) or a combination thereof; (iii) the plurality of particles are substantially spherical; (iv) the plurality of particles have a size in the range selected from 0.05 mm to 0.9 mm, 0.07 mm to 0.8 mm, 0.08 mm to 0.75 mm and 0.09 mm to 0.7 mm; (v) the plurality of particles are substantially spherical and comprise or consist of zirconium, zircon (zirconium silicate), zirconium oxide (zirconium dioxide) or yttrium stabilized zirconium having an average size in the range of 0.08 mm to 0.7 mm, preferably 0.09 mm to 0.6 mm, wherein preferably zirconium beads are used; (vi) the plurality of particles have a density of at least 2.0 g / cc, at least 2.5 g / cc, at least 3.0 g / cc, at least 3.5 g / cc, at least 4.0 g / cc, at least 4.5 g / cc, at least 5.0 g / cc or at least 5.5 g / cc; (vii) the plurality of particles have a density in the range selected from 2.0 g / cc to 15 g / cc, 2.5 g / cc to 12 g / cc, 3.0 g / cc to 10 g / cc, 3.5 g / cc to 9 g / cc, 4.0 g / cc to 8 g / cc, 4.5 g / cc to 7.5 g / cc and 5 g / cc to 7 g / cc; (viii) the plurality of particles has at least two different sizes, wherein (i) the average of a first particle size is in the range selected from 0.05 mm to 0.25 mm, 0.07 mm to 0.2 mm, 0.08 mm to 0.175 mm, and 0.9 mm to 0.15 mm, and (ii) the average of a second particle size is in the range selected from 0.3 mm to 0.9 mm, 0.35 mm to 0.8 mm, 0.4 mm to 0.7 mm, and 0.45 mm to 0.6 mm.

9. The lysis method according to one or more of claims 1 to 8, wherein (i) the first type is provided by a single solid breaking particle as defined in any one of claims 3 to 6, preferably as defined in claim 5 or 6, wherein preferably the single breaking particle is a ball cone, and (ii) the second type is provided by a plurality of substantially spherical zirconium oxide beads having a size in the range of 0.08 mm to 0.7 mm, more preferably 0.09 mm to 0.6 mm.

10. The lysis method according to one or more of claims 1 to 9, wherein the breaking with the first and second type of breaking particles is performed sequentially or simultaneously, preferably simultaneously.

11. The lysis method according to one or more of claims 1 to 10, wherein the liquid lysis composition comprises at least one chaotropic agent.

12. The lysis method according to claim 11, wherein (i) the chaotropic agent is selected from the group consisting of sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, lithium thiocyanate, and combinations thereof, wherein preferably the chaotropic agent is sodium thiocyanate; and / or (ii) the concentration of the at least one chaotropic agent in the liquid lysis composition and / or the lysis mixture is in the range of 0.75 M to 1.5 M and preferably 0.8 to 1.25 M, wherein preferably the chaotropic agent is NaSCN.

13. The lysis method according to one or more of claims 1 to 12, further comprising: - clarifying the lysate.

14. The lysis method according to one or more of claims 1 to 13, further comprising: - contacting the optionally clarified lysate sample with at least one protein precipitant and at least one inhibitor removal agent and providing a mixture; and - obtaining a liquid phase from the mixture; - optionally isolating nucleic acids, preferably DNA, from the liquid phase.

15. The lysis method according to claim 14, having one or more of the following features: (i) the precipitant is selected from the group consisting of ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate, wherein preferably ammonium acetate is used, and / or wherein the concentration of the at least one precipitant in the mixture is in the range selected from 0.1 to 4 M, 0.2 M to 3 M, 0.3 M to 2.5 M, 0.4 M to 2.25 M, 0.5 M to 2 M, and 0.6 M to 1.75 M. ​ ​ (ii) the at least one inhibitor removal agent is selected from the group consisting of aluminum chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, færdium (IV) chloride, zirconium (IV) chloride, guanidine sulfate, and combinations thereof, wherein preferably the inhibitor removal agent is a trivalent aluminum salt, more preferably aluminum chloride, and / or wherein the concentration of the at least one inhibitor removal agent in the mixture is in the range selected from the group consisting of 1-150 mM, 5 mM-125 mM, 10 mM-100 mM, 15 mM-75 mM, and 20 mM-65 mM; (iii) the precipitant is ammonium acetate and the inhibitor removal agent is a trivalent aluminum salt, preferably aluminum chloride; (iv) the precipitant and the inhibitor removal agent are comprised in the same composition, preferably a liquid solution, which is contacted with the lysed sample to provide the mixture; and / or (v) the method comprises adding at least one phosphate salt prior to contacting the lysed sample with the at least one inhibitor removal agent, wherein preferably the at least one phosphate salt is comprised in the lysing composition, and wherein optionally the phosphate salt has one or more of the following characteristics: (aa) it is a hydrogen phosphate salt; (bb) the cationic part in the phosphate salt is selected from the group consisting of ammonium, sodium, potassium, or lithium; (cc) it is disodium hydrogen phosphate.

16. The lysing method according to one or more of claims 1-15, wherein: (aa) the plant sample is selected from the group consisting of leaves, needles, roots, stems, seeds, fruits, and flowers, and wherein preferably the plant sample is a root sample; and / or (bb) the microorganism comprised in the plant sample has one or more of the following characteristics: (i) the microorganism is selected from the group consisting of bacteria and fungi, such as gram-positive bacteria, gram-negative bacteria, fungi, molds, and spores, or combinations of the foregoing; (ii) the microorganism is a bacterium; (iii) the microorganism is present on, around, or inside the plant sample, and optionally comprised in a lesion or tumor in a root sample, on a leaf surface, and / or in plant tissue.

17. The method according to one or more of claims 1-16, - wherein the first type of solid disruption particles is provided by one or more non- spherical disruption particles, and preferably the second type of solid disruption particles is provided by a plurality of substantially spherical particles; and - wherein the liquid lysing composition comprises at least one chaotropic agent at a concentration of 1.5 M or less, and wherein the chaotropic agent is selected from the group consisting of sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, lithium thiocyanate, and wherein preferably the chaotropic agent is sodium thiocyanate; and - wherein the method further comprises: o clarifying the lysate, wherein clarifying the lysate comprises separating the lysing mixture obtained after disruption of the plant sample into a solid fraction and a liquid fraction, wherein the liquid fraction is subjected to subsequent processing as the lysed sample; o contacting the lysed sample with at least one protein precipitant and at least one inhibitor removal agent and providing a mixture; and o obtaining a liquid phase from the mixture; wherein optionally the method further comprises adding at least one phosphate salt prior to contacting the lysed sample with the at least one inhibitor removal agent.

18. The method of claim 17, wherein the at least one protein precipitation agent is selected from the group consisting of ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate, and wherein preferably the protein precipitation agent is ammonium acetate, and wherein the at least one inhibitor removal agent is selected from the group consisting of aluminum chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, guanidine sulfate, and combinations thereof, and wherein preferably the inhibitor removal agent is a trivalent aluminum salt, such as more preferably aluminum chloride.

19. The method of one or more of claims 14 to 18, in particular of claim 17 or 18, wherein the liquid lysis composition comprises sodium thiocyanate as chaotropic agent, and wherein the method comprises contacting the lysed sample with ammonium acetate as precipitation agent and a trivalent aluminum salt, preferably aluminum chloride, as inhibitor removal agent.

20. The method of one or more of claims 1 to 19, in particular of any one of claims 17 to 19, wherein the liquid lysis composition comprises sodium thiocyanate at a concentration of 0.7 M to 1.5 M, and at least one phosphate salt, preferably disodium hydrogen phosphate, at a concentration of 0.075 M to 0.3 M.

21. The method of one or more of claims 1 to 20, in particular of any one of claims 17 to 19, wherein the liquid lysis composition comprises sodium thiocyanate at a concentration of 0.8 to 1.25 M, and at least one phosphate salt, preferably disodium hydrogen phosphate, at a concentration of 0.1 M to 0.25 M.

22. The method of one or more of claims 14 to 21, in particular of any one of claims 17 to 20, wherein the method comprises contacting the lysed sample with ammonium acetate as precipitation agent and a trivalent aluminum salt, preferably aluminum chloride, as inhibitor removal agent, wherein in the provided mixture the concentration of ammonium acetate is in the range of 0.5 M to 2 M and the concentration of the trivalent aluminum salt is in the range of 15 mM to 75 mM.

23. The method of one or more of claims 14 to 22, in particular of any one of claims 17 to 22 when dependent on claim 14, further comprising isolating nucleic acids, preferably DNA, from the liquid phase.

24. A method for isolating nucleic acids, including microbial nucleic acids, from a plant sample, comprising: (a) performing a lysis method as defined in one or more of claims 1 to 23: (b) isolating nucleic acids from the lysed and optionally further processed sample; and (c) optionally sequencing the isolated nucleic acids, preferably sequencing the isolated DNA.

25. A lysis system, preferably a kit, for releasing microbial nucleic acids from microorganisms comprised in a plant sample, comprising: (a) a liquid lysis composition, (b) at least two solid disruption particles, wherein: (i) a first type is provided by one or more disruption particles having a size of at least 1.5 mm, and (ii) a second type is provided by a plurality of disruption particles having a size of less than 1 mm, wherein preferably the first type of solid disruption particles have one or more features as defined in one or more of claims 2 to 4 and the second type of solid disruption particles have one or more features as defined in claim 5 or 6, and wherein the first and second type of disruption particles are contained in separate containers or in the same container, preferably in the same container.

26. The lysis system of claim 25, wherein the liquid lysis composition comprises at least one phosphate salt, optionally as defined in claim 15 (v) (aa), (bb) or (cc) and / or as defined in one or more of claims 11, 12, 17, 20 and 21.

27. The lysis system of claim 25 or 26, comprising at least one protein precipitation agent and at least one inhibitor removal agent.

28. The lysis system of claim 27, wherein the at least one protein precipitation agent is selected from the group consisting of ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, and wherein preferably the protein precipitation agent is ammonium acetate, and wherein the at least one inhibitor removal agent is selected from the group consisting of aluminum chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, guanidine sulfate and combinations thereof, and wherein preferably the inhibitor removal agent is a trivalent aluminum salt, such as more preferably aluminum chloride.

29. The lysis system of claim 27 or 28, wherein the liquid lysis composition comprises sodium thiocyanate as a chaotropic agent, and wherein the at least one protein precipitation agent is ammonium acetate, and wherein the inhibitor removal agent is a trivalent aluminum salt, preferably aluminum chloride.

30. Use of the system of any one of claims 25 to 29 in the method of any one of claims 1 to 24.

31. Use of the system of any one of claims 25-29 in lysing a plant sample and releasing microbial nucleic acids from microorganisms contained in the plant sample, wherein, The user optionally performs plant sample lysis using (i) the first and second type, or (ii) the second type of disruption particles to release microbial nucleic acids, preferably DNA, from microorganisms comprised in the plant sample. wherein preferably the first type of solid disruption particles have one or more features as defined in one or more of claims 2 to 4 and the second type of solid disruption particles have one or more features as defined in claim 5 or 6, and wherein the first and second type of disruption particles are contained in separate containers or in the same container, preferably in the same container.

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