Qiagen GmbH H69836WO 24.06.2024 METHOD FOR REMOVING INHIBITORY CONTAMINANTS FROM NUCLEIC ACID- CONTAINING SAMPLES INTRODUCTION The invention provides methods, compositions and kits for removing inhibitory contaminants from nucleic acid-containing samples, e.g. environmental or biological samples. The invention allows to prepare inhibitor contaminant depleted purified nucleic acid samples which can be used in downstream analyses like polymerase chain reaction (PCR) or Next-Generation Sequencing (NGS) analysis, in particular in methods of purifying and/or isolating nucleic acids, followed commonly by amplifying, hybridizing, detecting and/or characterizing the obtained nucleic acids. Providing purified nucleic acids is required for a variety of application fields, including agriculture, forensics, zoology and combating bioterrorism. Particularly, purified nucleic acid samples are required in the areas of molecular biological applications, including, for example, analytical, cloning, diagnostic and detection in thefields of agriculture, horticulture, forestry, forensics, biological research, organism and sample composition identification and characterization. BACKGROUND OF THE INVENTION AND PRIOR ART Nucleic acid sequences have a wide variety of applications in thefield of molecular biology. They are a valuable tool in many analytical and application techniques used in thefield of molecular biology, health and medicine (gene therapy, diagnostics, recombinant protein expression), bioterrorism (agent detection and analysis), forensics, space science, and food science. Some examples of these techniques include genotyping microorganisms, DNAfingerprinting plants and animals, detecting pathogens and beneficial microorganisms in soils, water, plants and animals, forensic identification of biological samples and environmental samples contaminated with different biological entities. All these techniques are based on identifying a specific sequence of nucleic acid in either a biological sample, such as a microorganism, plant tissues or animal tissues, or in any environment capable of supporting life. Identifying target nucleic acid sequences directly in biological samples and in environmental samples has the advantages of speed, accuracy, high-throughput and a low limit of detection to picogram or femtogram quantities of nucleic acids. Thefield of nucleic acid extraction and subsequent amplification of the obtained nucleic acid by PCR has revolutionized the rapid analysis of the genetic composition of several ecosystems. Methods and kits are available for isolating nucleic acids, like DNA, including genomic DNA, and RNA from a wide range of biological entities, and from the environment in which these living entities dwell. The PCR is a very powerful and sensitive analytical technique with applications in many diversefields, including molecular biology, clinical diagnosis, forensic analysis, and population genetics. However, the success rate
in biological and environmental samples strongly depends on the quality and purity of the isolated nucleic acid. Therefore, the target nucleic acid sequence, in order to be used as a diagnostic tool in such applications, should be free of contaminants that inhibit PCR and other downstream applications. Multiple sample types have high levels of such inhibitory contaminants, such as in particular samples being of interest for microbiome research, for example stool, soil, or wastewater. Such inhibitory contaminants are often from the groups that include polyphenols, polysaccharides and humic substances and as examples of such inhibitory substances in biological and environmental samples humic acids, fulvic acids, humin, hymatomelanic acid, polyphenols, bile salts, polysaccharides, and others, can be mentioned to be known to inhibit the analysis of purified nucleic acid with PCR or NGS. For example, in plant genomic DNA analysis, the DNA is invariably co-extracted with other plant components such as polyphenols and polysaccharides which inhibit PCR applications. In thefield of soil ecosystems, nucleic acid extraction methods suffer from compounded inefficiencies of DNA sorption to soil surfaces and co-extraction of enzymatic inhibitors from soils. Both, the clay and organic fractions of soil affect DNA isolation and purification. Clay has a tendency to bind DNA adsorptively, whereas humic polymers found in the organic fraction tend to co-purify with DNA during the extraction procedure. The higher the montmorillonitic clay and organic matter content, the higher the buffering capacity of the soil system and also the greater the amount of DNA adsorbed to the soil particles. Thus, methods developed for a particular soil type with a clay : organic ratio may not work for any other soil type with a different clay : organic ratio. It has been previously reported that phenol extraction of DNA contaminated with humic substances resulted in lowering the DNA recovery efficiency. Moreover, compost may have a variety of additional organic compounds that may co-purify with DNA and inhibit enzymatic manipulations of the DNA. An additional concern for example when isolating microbial DNA from compost is that plant material in various stages of decomposition may be present in significant concentrations in compost. When studying higher organisms such as fungi, plants and animals, direct nucleic acid isolations are still plagued with quality issues. In cyanobacteria, fungi, algae and plants, pigments and cell wall components such as chitins and polysaccharides will inhibit PCR. These cell types are rich in endo- and exonucleases and contain photosynthetic pigments, which can inhibit enzymatic reactions, especially reverse transcription, and PCR. The nature of the contaminants in crude nucleic acid preparations from soils and sediments and their interactions with DNA and RNA have frequently been considered to be humic and fulvic acids and a heterogeneous mixture of phenolic polymers and
oligomers. Humic substances are formed when microbes degrade plant residues and are stabilized against degradation by covalent binding of their reactive sites to metal ions and clay minerals. Humic substances consist of polycyclic aromatics to which saccharides, peptides, and phenols are attached. The predominant types of humic substances in soils are humic acids (HA, molecular weight of 300 kDa and greater) and fulvic acids (FA, molecular weight of as low as 0.1 kDa). Humic acids are soluble at an alkaline pH and precipitate with hydrochloric or sulphuric acids at pH 1.0 to 2.0, while fulvic acids remain in solution even at an acidic pH. Most frequently, DNA extracts from soils showing brown coloration are indicative of contamination with humic like substances. These brown compounds cannot be easily removed from DNA extracts. Solvent extraction of crude DNA extracts with solvents such as phenol, diethyl ether, acetone, methanol and ethanol were not successful in removing the brown coloration, and the DNA was still discolored and resistant to digestion by restriction endonucleases. Some of these compounds also appear to co-migrate with DNA during CsCl-ethidium bromide isopycnic ultracentrifugation, resulting in light brown coloration of the recovered DNA. These observations imply an intimate association between the contaminants and DNA. While the nature of the association between contaminating compounds and DNA has not been elucidated, the reversible and irreversible binding of polyphenols, such as tannins, to proteins is well understood. Direct extraction of total nucleic acid from soils or sediments usually results in co- extraction of other soil components, mainly humic acids or other humic substances, which negatively interfere with DNA transforming and detecting processes. It has been reported that these substances inhibit restriction endonucleases and Taq polymerase, the key enzyme of PCR, and decrease efficiencies in DNA-DNA hybridizations. Generally, such inhibitory substances are not easily separated from nucleic acids during the standard isolation, e.g. using spin columns or magnetic beads, and therefore, additional purification measures must be applied. However, such additional purification measures are usually time-consuming and require a considerable personal and material effort. In particular, separation of humic substances from DNA usually involves time- consuming and tedious steps. Size-exclusion chromatography and polyvinylpolypyrrolidone spin columns have been used. There are further methods, compositions or kits available, which have been developed to remove inhibitory substances by separating them from the nucleic acids after lysis of the sample, but before purification. This is usually done by precipitation and physical separation of the precipitated substances by centrifugation. Many kits for purification of especially stool are available on the market. However, they are either not suitable for special inhibitor removal and are therefore not generally suitable for many applications,
or additional purification steps are required to remove inhibitors, leading to long workflows. WO2019/209597A1 and WO2019/209600A1 describe single-step precipitation methods for removing inhibitory contaminants and isolating nucleic acids from samples by contacting the samples with inhibitor removing agents and separating the resulting mixture into a solid and a liquid phase, followed by isolating the nucleic acids from the liquid phase. WO2006/073472A2 describes methods and compositions, e.g. kits, for removing contaminants from nucleic acids from biological and environmental samples, wherein the method described therein comprises contacting a nucleic acid-comprising sample with a first flocculant to form a flocculant precipitate, separating the nucleic acid containing composition from the first flocculant precipitate and contacting the nucleic acid containing composition with a second flocculant to form a second flocculant precipitate and separating the nucleic acid containing second composition from the second flocculant precipitate. In the process described therein, the separation of the nucleic acid containing compositions from the flocculant precipitates is carried out by centrifugation. However, a centrifugation step is not easily automated, which forces all workflows, even high-throughput ones, into an if at all semi-automated form or even conduct them completely manually. Further, methods are known to immobilize or bind cells on surface-modified magnetic beads, thereby allowing to remove the bead-bound cells from samples together with the magnetic beads from the remainder of the sample by applying a magnetic field. For example, WO2013/067399A1 describes carboxy-modified magnetic beads wherein magnetic bead particles are surface-modified to carry free anionic carboxy-moieties on the particle surface. Further, WO2014/032990A1 describes magnetic beads wherein the anionic carboxy-moieties on the particle surface are additionally functionalized with polyethylenimine. These surface-modified magnetic particles are used for cell separation from cell-containing samples. WO2019214988A1 describes a lysis method for releasing microbial nucleic acids from microorganisms comprised in a plant sample, comprising mechanically disrupting the plant sample in a liquid lysis composition using at least two types of solid disrupting particles, wherein (i) the first type of disrupting particles having a size of at least 1,5 mm and (ii) the second type of disrupting particles having a size of 1 mm or less, as well as a method for isolating nucleic acids from a plant sample and kits.
WO2018085817A1 describes methods and devices for reduced biased isolation of genetic materials from mixed microbial samples using beads for disrupting microbial cells and viruses. WO2016183292A1 describes compositions and methods for extracting nucleic acids and/or targeted nucleic acids sequences from biological samples using buffer solutions and magnetic silicon beads, followed by transferring the nucleic acids to magnetic carboxy beads in a binding buffer, as well kits comprising the tools and compositions for performing said methods. US2023/407288A1 describes a method of separating RNA from a sample, wherein magnetic beads are used for purification, as well as kits. EP1154443A1 describes magnetic nanosized particles having a glass surface and the use thereof for the purification of DNA or RNA in particular in automated processes. CN109735532A describes a magnetic bead suspension for nucleic acid purification and screening and application thereof. CN115612685A describes a magnetic bead reagent for purifying nucleic acid, removing small and large fragments, and screening of nucleic acid fragments, Efficient, safe and reliable inhibitor removal workflows which can be implemented into highly automated processes are desired. Providing such inhibitor removal workflows which can be implemented into highly automated sample processing workflows allows to further increase the degree of automation and therefore helps to save time and laboratory capacities in the preparation of such samples. OBJECT OF THE INVENTION It was the object of the invention to provide novel and improved methods for removing inhibitory contaminants from nucleic acid-containing samples. Generally, the new method should be able to solve the problems arising from procedures known in the prior art and should avoid the disadvantages connected with so far known and applied techniques. In particular aspects of the invention, the new methods should be able to solve one or more of the following objects and address one or more of the following aspects. The improved methods should be able to remove a broad variety of inhibitory compounds from a broad variety of sample types to be broadly applicable. The new methods should be suitable for easy and simple implementation and integration into standard working procedures, they should be compatible with standard sample processing techniques and/or they should be scalable. The new method should be accessible to a high degree of automation and therefore allow to increase the degree of
automation compared to currently available methods. The new method should therewith allow to save time and labour in sample preparation, in particular when handling large amounts of samples in high-throughput methods. The new method should allow cutting down hands-on time required in standard protocols. The new methods should be able to remove inhibitory contaminants and provide nucleic acid-containing samples with high yield. The new methods should be able to provide nucleic acid-containing samples with a high degree of purity and high level of inhibitory contaminant depletion, i.e. a low level of inhibition. Accordingly, the new methods should help to increase the accuracy and/or efficiency of nucleic acid amplification, hybridization, detection and/or characterization. The method should be able to provide high quality isolated nucleic acid samples from biological and/or environmental samples with significantly reduced amount up to being free from contaminating substances that inhibit PCR, RT-PCR and other downstream applications in molecular biology. The inventors of the present invention surprisingly found that these objects can be solved with the newly developed method described herein in detail. SUMMARY OF THE INVENTION The inventors of the present invention developed a method comprising at least two inhibitory contaminant precipitation steps and wherein the precipitated contaminants are removed from the precipitate via surface-functionalized magnetic particles comprising negatively charged functional groups on the particle surface. In the method of the invention the contaminants associate to the surface-functionalized magnetic particles and are removed from the precipitate by applying a magnetic field. Further aspects of the invention relate to compositions and kits for such methods. ASPECTS OF THE INVENTION: The present invention includes, without being limited thereto, the following aspects: [1] A method for removing at least a part of one or more inhibitory contaminants (particularly substances that interfere with a reaction involving DNA and/or RNA isolation) from a nucleic acid-containing sample, wherein the method comprises at least a first and a second inhibitory contaminants removal step for precipitating and removing at least a part of the inhibitory contaminants to obtain an inhibitory contaminant depleted liquid mixture, wherein the method comprises: (a) a first inhibitory contaminants removal step comprising subjecting a nucleic acid containing sample (S) to a combined treatment step comprising a lysis treatment for lysing the sample and a first inhibitory contaminants removal treatment by contacting the sample with a lysis composition and a first inhibitor precipitation composition (IP-1), i.e. a composition for precipitating at least a first part of the one or more inhibitory contaminants, thereby lysing the sample
and precipitating at least a first part of the one or more inhibitory contaminants from the sample, and removing the precipitated inhibitory contaminants resulting in a first nucleic acid containing inhibitory contaminant depleted liquid mixture; and (b) a second inhibitory contaminants removal step comprising contacting the nucleic acid-containing liquid mixture obtained in step (a) with a second inhibitor precipitation composition (IP-2), i.e. a composition for precipitating at least a second part of the one or more inhibitory contaminants, and with surface- functionalized magnetic particles, precipitating at least a second part of the one or more inhibitory contaminants and removing the precipitated inhibitory contaminants associated with the surface-functionalized magnetic particles from the liquid mixture by applying a magnetic field, resulting in a second nucleic acid containing inhibitory contaminant depleted liquid mixture, wherein - the surface-functionalized magnetic particles are carrying anionic functional groups on the particle surface employing a negative overall charge, - the surface-functionalized magnetic particles are having an average diameter of < 3.0 µm; and - the surface-functionalized magnetic particles are added to the liquid mixture to result in a concentration of ≥ 190.0 µg particles per ml of liquid mixture. [2] The method according to [1], wherein the surface-functionalized magnetic particles has one or more of the subsequent characteristics: - a carboxylated surface carrying free carboxyl groups, such as carboxylic acid groups, carboxylate anions or carboxylate salts, - a phosphorylated surface carrying free phosphate or phosphonate groups, - a sulfonated surface carrying free sulfonate groups, and - anionic functional groups on the particle surface selected from mixtures of two or more of carboxyl, phosphate, phosphonate and sulfonate groups. [3] The method according to [1] or [2], wherein the surface-functionalized magnetic particles comprise an anionic functional groups content as determined by conductometric titration with sodium hydroxide of at least 0.1 mEq/g. [4] The method according to [1] to [3], wherein the surface-functionalized magnetic particles carrying anionic functional groups are further partly functionalized with linear or branched polyethylenimine (PEI).
[5] The method according to [1] to [4], wherein the surface-functionalized magnetic particles are selected from the group comprising SeraMagTM Carboxylate-Modified Beads, SeraMagTM Carboxylate-Modified SpeedBeads, MagnefyTM COOH Beads, QIAseq® Beads, ProMagTM magnetic spheres (1 µm), DynabeadsTM carboxylic acid (2.8 or 1 µm), and from fluidMAG-ionic particles selected from fluidMAG-CT, fluidMAG-EDTA, fluidMAG-IDA, fluidMAG-NTA, fluidMAG-PA, fluidMAG-P, fluidMAG-GP, fluidMAG-PY, and PEI-functionalized modifications thereof; preferably selected from SeraMagTM Carboxylate-Modified Beads, SeraMagTM Carboxylate-Modified SpeedBeads, MagnefyTM COOH Beads, QIAseq® Beads, ProMagTM magnetic spheres (1 µm), DynabeadsTM carboxylic acid (2.8 or 1 µm), fluidMAG-P, and PEI-functionalized modifications thereof; more preferably selected from SeraMagTM Carboxylate-Modified Beads, SeraMagTM Carboxylate-Modified SpeedBeads, MagnefyTM COOH Beads and QIAseq® Beads. [6] The method according to [1] to [5], wherein the surface-functionalized magnetic particles have an average diameter of ≤ 2.0 µm, preferably ≤ 1.5 µm, more preferably ≤ 1.0 µm. [7] The method according to [1] to [6], wherein the surface-functionalized magnetic particles are added to the liquid mixture to result in a concentration of ≥ 250.0 µg particles per ml of liquid mixture, preferably ≥ 300.0 µg/ml, more preferably ≥ 350.0 µg/ml, more preferably ≥ 400.0 µg/ml, more preferably ≥ 450.0 µg/ml, more preferably ≥ 490.0 µg/ml, more preferably ≥ 500.0 µg/ml, more preferably ≥ 550.0 µg/ml, more preferably ≥ 600.0 µg/ml, more preferably ≥ 650.0 µg/ml, more preferably ≥ 700.0 µg/ml, more preferably ≥ 750.0 µg/ml, more preferably ≥ 800.0 µg/ml, more preferably ≥ 850.0 µg/ml, more preferably ≥ 900.0 µg/ml, more preferably ≥ 930.0 µg/ml. [8] The method according to [1] to [7] wherein the surface-functionalized magnetic particles are superparamagnetic or ferrimagnetic particles. [9] The method according to [1] to [8], wherein the lysis treatment has one or more of the following characteristics: (i) the lysis treatment is carried out before contacting the sample (S) with the first inhibitor precipitation composition (IP-1), (ii) the lysis treatment is carried simultaneously with contacting the sample (S) with the first inhibitor precipitation composition (IP-1), (iii) the lysis treatment is induced chemically, enzymatically, mechanically or by a combination thereof, (iv) the lysis treatment is induced by a combination of chemical and mechanical lysis,
(v) the mechanical lysis is induced by one or more of bead beating, sonication and homogenization, and (vi) the lysis treatment comprises contacting the sample (S) with a lysis composition (L), which comprises one or more agents selected from the group of chaotropic agents, detergents, buffers, homogenizing agents, preservative agents, denaturation agents, chelating agents and combinations and mixtures thereof, preferably the lysis composition comprises at least one chaotropic agent and at least one buffer. [10] The method according to [1] to [9], wherein the first inhibitory contaminants removal treatment comprises subjecting the sample (S) to a combined lysis and inhibitor contaminant precipitation treatment having one or more of the following characteristics: (i) the sample (S) is subjected to a mechanical and chemical lysis by contacting it with a lysis composition (L) comprising one or more lysing components, with disruption particles and with the first inhibitor precipitation composition (IP-1), and conducting a bead beating process, (ii) the sample (S) is subjected to a mechanical lysis by contacting it with disruption particles and the first inhibitor precipitation composition (IP-1) and conducting a bead beating process (iii) the disruption particles are contacted with the sample (S) in the form of a mixture with the lysis composition (L), (iv) the disruption particles are contacted with the sample (S) separately from the first inhibitor precipitation composition (IP-1), (v) the disruption particles are contacted with the sample (S) in the form of a mixture with the first inhibitor precipitation composition (IP-1), (vi) the lysis composition (L) and the first inhibitor precipitation composition (IP- 1) are contacted with the sample (S) separately from each other, (vii) the sample (S) is not subjected to a chemical lysis, (viii) the sample (S) is subjected to a mixture of lysis composition (L) and first inhibitor precipitation composition (IP-1). [11] The method according to [10], wherein the disruption particles of the lysis treatment have one or more of the following characteristics: (i) the disruption particles are made of or comprise glass, ceramic, metal, mineral, plastic or a combination of two or more of such materials, (ii) the disruption particles are yttrium-stabilized zirconium particles, (iii) the disruption particles have an average particle diameter of from 0.05 mm to 3.00 mm, (iv) the disruption particles are a mixture of particles with different sizes,
(v) the disruption particles are a mixture of beads with a diameter of 0,1 and 0,5 mm. [12] The method according to [1] to [11], wherein the first and second inhibitor precipitation composition (IP-1) and (IP-2) are the same or different and comprise a tri- or tetra-valent salt containing a cation having a valence of three or four, preferably an aluminium salt or an of iron salt. [13] The method according to [1] to [12], wherein the first inhibitor precipitation composition (IP-1) and the second inhibitor precipitation composition (IP-2) are the same or different and independent from each other comprise one or more selected from a. an aluminium salt, including aluminium chloride (AlCl3), aluminium sulfate, aluminium ammonium sulfate, aluminium sulfate dodecahydrate, aluminium ammonium sulfate dodecahydrate, aluminium potassium sulfate, aluminium chlorohydrate and sodium aluminate, b. ammonium acetate (NH4OAc) and ammonium sulfate, c. a calcium salt, including calcium oxide and calcium chloride (CaCl2), d. a magnesium salt, including magnesium chloride (MgCl2), e. an iron salt, including iron (III) chloride (FeCl3) and iron (II) sulfate, f. erbium (III) acetate, erbium (III) chloride, holmium chloride, zirconium (IV) chloride and/or hafnium (IV) chloride, and combinations thereof; preferably the first inhibitor precipitation composition (IP-1) and/or the second inhibitor precipitation composition (IP-2) comprises at least one of aluminium chloride, aluminium chlorohydrate, ammonium acetate, and aluminium sulfate dodecahydrate, and mixtures thereof. [14] The method according to [1] to [13], wherein the first inhibitor precipitation composition (IP-1) and the second inhibitor precipitation composition (IP-2) comprise the same or different compounds in same or different amounts and/or ratios. [15] The method according to [1] to [14], wherein in the second inhibitory contaminants removal step the second inhibitor precipitation composition (IP-2) and the surface-functionalized magnetic particles are contacted with the nucleic acid- containing liquid mixture resulting from step (a) simultaneously as a pre-prepared mixture, or successively, preferably simultaneously as a pre-prepared mixture. [16] The method according to [1] to [15], wherein the sample (S) is an unprocessed, preserved, freshly isolated, crude or unrefined biological or environmental sample,
or, the sample is broken up, denatured or disrupted, having one or more of the following characteristics; (i) the sample (S) is a liquid, solid or semi-solid sample; (ii) it is a biological sample derived from an animal, animal remains, a food, a microorganism, a plant or its components, soil, sediment, rock, reef, sludge, compost, decomposing biological matter, a biopsy, a histological sample, a semen sample, a blood or saliva sample, any bodyfluid sample, a hair sample, a skin sample, a fecal sample, archaeological remains, a peat bog, compost, oil, water, terrestrial water or subterranean water, atmospheric and industrial water, dust, urban dust, commercial potting mixtures or soil amendments, deep sea vents, or air; (iii) it is a liquid, solid or semi-solid biological or environmental sample or a lysate or supernatant derived from a biological or environmental sample; (iv) it is a sample derived from stool, soil, a plant material or wastewater. [17] The method according to [1] to [16], wherein the inhibitory contaminants are selected from one or more of polyphenols, polysaccharides and/or humic substances that interfere with the reaction involving DNA and/or RNA isolation. [18] The method according to [1] to [17], wherein the inhibitory contaminants are selected from one or more of (i) haemoglobin and the metabolites thereof, bilirubin, bile acids and bile acid derivatives, undigested or partially digested fiber, or undigested or partially digested food, and/or polysaccharides; (ii) humic acids, fulvic acids, humic polymers and/or humin; (iii) chitin, decomposing plant materials, organic compounds from compost, phenolics, phenolic polymers or oligomers, polyphenol, polysaccharides, and/or tannin. [19] The method according to [1] to [18], which further comprises the following steps after the at least partial removal of the inhibitory contaminants: (i) adding to the second inhibitory contaminant depleted liquid mixture of step (b) a nucleic acid binding agent, which includes a nucleic acid binding buffer solution, and a solid support for binding the nucleic acid, preferably a DNA binding agent which includes a DNA binding buffer solution and solid support for binding DNA; (ii) applying one or more purification and/or isolation steps selected from one or more of: a) washing, b) centrifugation, c) filtration,
d) magnetic separation , e) decanting, f) elution. [20] The method according to [1] to [19], wherein the nucleic acid in the second nucleic acid containing inhibitory contaminant depleted liquid mixture or the purified and/or isolated nucleic acid is analysed using one or more of: (i) amplification, (ii) hybridization, (iii) quantification, (iv) sequencing, (v) detection, and (vi) characterization. [21] The method according to [1] to [20], which is carried out in an at least partly automated process using one or more of automated sampling, bead beating, centrifugation, magnetic separation, purification, isolation, amplification, characterization and detection, preferably the second inhibitory contaminants removal step and all subsequent purification, isolation and/or analyzing steps are carried out automated. [22] Kit for removing at least a part of one or more inhibitory contaminants (particularly substances that interfere with a reaction involving DNA and/or RNA isolation) from a nucleic acid-containing sample and/or for providing purified nucleic acid samples for PCR or NGS analysis, which comprises (i) a first inhibitor precipitation composition (IP-1), i.e. a composition for precipitating at least a first part of the one or more inhibitory contaminants; (ii) a second inhibitor precipitation composition (IP-2), i.e. a composition for precipitating at least a second part of the one or more inhibitory contaminants; and (iii) surface-functionalized magnetic particles carrying anionic functional groups on the particle surface employing a negative overall charge and having an average diameter of < 3.0 µm in an amount to result in a concentration of ≥ 190.0 µg particles per ml of liquid mixture to be treated; (iv) optionally a lysis composition (L), and/or (v) optionally disruption particles. [23] The kit according to [22], wherein the kit-components are further characterized by one or more of the features as defined in the preceding points.
[24] The kit according to [22] or [23], wherein the kit comprises a cartridge comprising multiple troughs, wherein the individual kit-components are provided in the multiple troughs. [25] The use of surface-functionalized magnetic particles carrying anionic functional groups on the particle surface employing a negative overall charge, which have an average diameter of < 3.0 µm, in a concentration of ≥ 190.0 µg particles per ml liquid mixture to be treated for removing at least a part of one or more precipitated inhibitory contaminants (particularly substances that interfere with a reaction involving DNA and/or RNA isolation) from a nucleic acid-containing sample by associating the precipitated inhibitory contaminants with the surface-functionalized magnetic particles, applying a magnetic field and separating the surface- functionalized magnetic particles associated with the precipitated inhibitory contaminants from the nucleic acid-containing sample. [26] The use according to [25] in a method according to [1] to [21]. The present invention is described in more detail as follows. DETAILED DESCRIPTION OF THE INVENTION The inventors of the present invention developed a new method for removing inhibitory contaminants from nucleic acid-containing samples, being able to solve the above defined objects. In particular, the novel method surprisingly turned out to be suitable for removing not only specific but a broad variety of inhibitory compounds and was able to be used in a broad variety of sample types, thereby offering a broadly applicability. The new method further turned out to be easily and simply implemented and integrated into standard working procedures, being compatible with standard sample processing techniques and being scalable. In particular, the possibility of implementing the new method into highly automated sample processing techniques allows to increase the degree of automation compared to currently available methods of removing inhibitory contaminants from nucleic acid containing samples. This now allows to save time and labour in sample preparation and the efficient handling of large amounts of samples in high-throughput methods. The inventors were able to show that the new methods are able to remove inhibitory contaminants and provide nucleic acid-containing samples with high yield and with a high degree of purity, i.e. a high level of inhibitory contaminant depletion. Therewith, the new method of the invention helps to increase the accuracy and efficiency of nucleic acid amplification, hybridization, quantification, sequencing, detection and characterization, in particular for nucleic acid-containing samples from biological and environmental samples.
The new method of the invention comprises at least two steps of precipitating and removing inhibitory contaminants from the nucleic acid-containing sample and is characterized in that a first inhibitory contaminants precipitation and removal step is carried out in combination with a sample lysis treatment step and a further (second) inhibitory contaminants precipitation and removal step is carried out using specific surface-functionalized magnetic particles effecting association of the precipitated inhibitory contaminants with the surface-functionalized magnetic particles and allowing their removal from the nucleic acid-containing sample by applying a magnetic field. The inventors found out that a specific selection of surface-functionalized magnetic particles and a particular adjustment of precipitation conditions was required to provide sufficiently pure samples and high yields of the nucleic acid-containing inhibitory contaminant depleted sample. Therefore, a first aspect of the invention relates to a method for removing at least a part of one or more inhibitory contaminants from a nucleic acid-containing sample, wherein the method comprises at least a first and a second step of precipitating and removing at least a part of inhibitory contaminants to obtain an inhibitory contaminant depleted sample, wherein the method comprises: a first inhibitory contaminants removal step comprising subjecting a nucleic acid- containing sample (S) to a combined treatment step comprising a lysis treatment for lysing the sample and a first inhibitory contaminants removal treatment by contacting the sample with a lysis composition and a first inhibitor precipitation composition (IP-1), thereby lysing the sample and precipitating at least a first part of the one or more inhibitory contaminants from the sample, and removing the precipitated inhibitory contaminants resulting in a first nucleic acid containing inhibitory contaminant depleted liquid mixture; and a second inhibitory contaminants removal step comprising contacting the nucleic acid-containing liquid mixture from step (a), i.e. the remainder of the sample treated in step (a), with a second inhibitor precipitation composition (IP-2) and with surface- functionalized magnetic particles, precipitating at least a second part of the one or more inhibitory contaminants and removing the precipitated inhibitory contaminants associated with the surface-functionalized magnetic particles from the liquid mixture, i.e. from the remainder of the sample treated in step (a), by applying a magnetic field, resulting in a second nucleic acid-containing inhibitory contaminant depleted liquid mixture. Therein, the surface-functionalized magnetic particles used in the second inhibitory contaminant removal step are carrying anionic functional groups on the particle surface employing a negative overall charge and these surface-functionalized magnetic particles are having an average diameter of < (less than) 3.0 µm and the surface- functionalized magnetic particles are added to the liquid mixture treated in step (b) to result in a concentration of ≥ 190.0 µg particles per ml of liquid mixture to be treated.
In the inventors’ experiments leading to the present invention, it turned out that using larger anionic surface-functionalized magnetic particles with average particle diameters of 3.0 µm and more and/or using lower concentrations of the surface-functionalized magnetic particles in the liquid mixture to be treated, i.e. concentrations less than (<) 190.0 µg per ml of liquid mixture to be treated, no sufficient inhibitory contaminant removal was possible and the yield of the nucleic-acid containing sample after such purification treatment decreased. Accordingly, the inventors found that in the new method of the invention the size of the surface-functionalized magnetic particles must be sufficiently small and should exhibit an average diameter of less than 3.0 µm. Preferably, the surface-functionalized magnetic particles to be used in the second inhibitory contaminant precipitation and removal step should exhibit an average diameter of not more than (≤) 2.8 µm, more preferably of ≤ 2.5 µm, more preferably of ≤ 2.0 µm, more preferably of ≤ 1.8 µm, more preferably ≤ 1.5 µm, more preferably ≤ 1.2 µm, even more preferably ≤ 1.0 µm. The particle size is not particularly limited with respect to the lowest possible diameter and can be as small as well-known or commercially available particles, e.g. as those described in WO2013/067399A1 or in WO2014/032990A1 having a diameter down to e.g.100 nm. In this respect, the defined sizes of the surface-functionalized magnetic particles generally indicate the longest distance between two opposite points of the respective particle. Accordingly, in the case of round or essentially round particles (beads, balls, spheres), the defined size relates to the average diameter. For determining the particle sizes well- known established methods can be used by the skilled person. The magnetic particles are preferably magnetic beads or magnetic (nano)spheres having an essentially round or spherical appearance with essentially uniform size and shape to provide reliable and reproducible results. The surface-functionalized magnetic particles to be used in the second inhibitor contaminants precipitation and removal step of the method of the present invention can for example have superparamagnetic, paramagnetic, ferrimagnetic or ferromagnetic characteristics. The magnetic particles may comprise a magnetic material that is incorporated in the particles and/or is associated with the particles. To avoid leaching of the magnetic material, the magnetic material may be completely encapsulated e. g. by the material providing the surface such as e.g. silica, polysilicic acid, glass or a polymeric material such as polyacrylate. Often, magnetic particles or beads are designed to comprise a layered structure with an inert core, e.g. a polymer-based core, and one or more magnetic layers, e.g. magnetite layers. Depending on the amount of magnetic layers and the resulting magnetic force, some suppliers distinguish between magnetic beads and so-called magnetic “speed beads”, which exhibit higher magnetic forces. In accordance with the general meaning superparamagnetic particles are magnetic only in a magnetic field, with no residual magnetism once removed. Superparamagnetic and
ferrimagnetic particles are preferred, most preferred are superparamagnetic particles. Exemplary magnetic beads include those which are described in the German patent application DE 102005058979.9. or in the above mentioned WO2013/067399A1 and WO2014032990A1. Further, a variety of magnetic beads are commercially available and can be used as long as they exhibit the characteristics described herein. The surface-functionalized magnetic particles used in the second inhibitory contaminant precipitation and removal step of the method of the present invention are magnetic particles carrying anionic functional groups on their surfaces and are characterized by employing a negative overall charge on their surface. Such overall surface charge can be obtained by surface-modifying the magnetic beads so that they carry anionic functional groups on the particle surface. Such anionic functional groups can be selected from carboxyl, phosphate, phosphonate and sulfonate groups, as well as mixtures of two or more thereof. Accordingly, the surface-functionalized magnetic particles to be used in the inhibitory contaminant precipitation and removal step of the method of the present invention comprise - a carboxylated surface carrying free carboxyl groups, such as carboxylic acid groups, carboxylate anions or carboxylate salts, or - a phosphorylated surface carrying free phosphate or phosphonate groups or - a sulfonated surface carrying free sulfonate groups, or - a surface-functionalization providing mixtures of two or more of carboxyl, phosphate, phosphonate and sulfonate groups. In a further aspect, the surface-functionalized magnetic particles to be used in the method of the invention comprise an anionic functional groups content as determined by conductometric titration with sodium hydroxide of at least 0.1 mEq/g; at least 0.2 mEq/g; at least 0.3 mEq/g; at least 0.4 mEq/g; from about 0.1 to about 0.7 mEq/g; from about 0.1 to about 0.6 mEq/g; from about 0.1 to about 0.5 mEq/g; from about 0.2 to about 0.7 mEq/g; from about 0.2 to about 0.6 mEq/g; from about 0.2 to about 0.5 mEq/g; from about 0.3 to about 0.7 mEq/g; from about 0.3 to about 0.6 mEq/g; from about 0.3 to about 0.5 mEq/g; from about 0.4 to about 0.7 mEq/g; from about 0.4 to about 0.6 mEq/g; or from about 0.4 to about 0.5 mEq/g. In a preferred aspect, the surface-functionalized magnetic particles to be used in the method of the invention comprise a carboxylated surface, a phosphorylated surface or a mixed carboxylated and phosphorylated surface, more preferably a carboxylated or phosphorylated surface, most preferred is a carboxylated surface. Regarding the term “carboxylated surface” reference is made to the definition as described e.g. in WO2013/067399A1, which applies accordingly for the surface-carboxylated magnetic particles described herein. Magnetic particles carrying carboxy groups on their surface
may also be designated as “COOH Beads”, “carboxy beads” or “Carboxylate-Modified Beads”. It is further possible to introduce additional surface-functionalization, such as those described in WO2014/032990A1, provided that the overall surface charge remains a negative charge. In particular, the surface-functionalized magnetic particles as used in the method of the invention carrying anionic functional groups may additionally be partly functionalized with linear or branched polyethylenimine (PEI). In this respect, reference is made to WO2014/032990A1, the teaching of which relating to PEI-functionalization of magnetic beads is included herein by reference. The degree of surface-functionalization is not particularly limited, which means that a complete or just a partial surface-functionalization is possible, provided that the overall surface charge of the magnetic particles is a negative surface charge. In principle, commercially available surface-functionalized magnetic particles with the above defined particle size and the herein defined anionic surface-functionalization can be used in the method of the invention. The following table provides an overview: Commercial Surface Magnetism Size Supplier Name Modification TM carboxylated Polysciences ProMag superparamagnetic 1 µm (COOH) Inc. MagnefyTM carboxylated high-performance Polysciences 1 µm COOH Beads (COOH) superparamagnetic Inc. Sera-MagTM carboxylated Carboxylate- superparamagnetic 1 µm Cytiva (COOH) Modified Beads Sera-MagTM Carboxylate- carboxylated superparamagnetic 1 µm Cytiva Modified (COOH) (2 magnetite layer) SpeedBeads ® carboxylated QIAseq Beads superparamagnetic 1 µm QIAGEN (COOH) DynabeadsTM carboxylated 2.8 µm superparamagnetic ThermoFischer Carboxylic Acid (COOH) 1 µm Citric acid fluidMAG-CT superparamagnetic 0.05 µm (carboxyl) Chemicell 0.1 µm EDTA GmbH fluidMAG- EDTA superparamagnetic 0.2 µm (carboxyl)
Iminodiacetic fluidMAG-IDA acid superparamagnetic (carboxyl) Nitrilotriacetic fluidMAG-NTA acid superparamagnetic (carboxyl) L-ascorbic acid fluidMAG-PA, (phosphate superparamagnetic carboxyl) Diphosphate fluidMAG-P superparamagnetic (phosphate) Glycerol fluidMAG-GP phosphate superparamagnetic (phosphate) Phytic acid fluidMAG-PY superparamagnetic (phosphate) Accordingly, in a further aspect in the method of the invention commercially available magnetic particles selected from SeraMagTM Carboxylate-Modified Beads, SeraMagTM Carboxylate-Modified SpeedBeads, MagnefyTM COOH Beads, QIAseq® Beads, ProMagTM magnetic spheres (1 µm), DynabeadsTM carboxylic acid (2.8 or 1 µm), and from fluidMAG-ionic particles selected from fluidMAG-CT, fluidMAG-EDTA, fluidMAG- IDA, fluidMAG-NTA, fluidMAG-PA, fluidMAG-P, fluidMAG-GP, and fluidMAG-PY, and PEI-functionalized modifications thereof can be used. Preferably, commercially available magnetic particles selected from from SeraMagTM Carboxylate-Modified Beads, SeraMagTM Carboxylate-Modified SpeedBeads, MagnefyTM COOH Beads, QIAseq® Beads, ProMagTM magnetic spheres (1 µm), DynabeadsTM carboxylic acid (2.8 or 1 µm), fluidMAG-P, and PEI-functionalized modifications thereof are used; more preferably commercially available magnetic particles selected from SeraMagTM Carboxylate-Modified Beads, SeraMagTM Carboxylate-Modified SpeedBeads, MagnefyTM COOH Beads and QIAseq® Beads are used. The inventors’ experiments leading to the present invention further showed that the surface-functionalized magnetic particles need to be added to the liquid mixture to be treated and purified in the second inhibitory contaminants removal step in a sufficiently high concentration. In the second inhibitory contaminants removal step the surface- functionalized magnetic particles are added to the liquid mixture to be treated to result in a concentration of at least (≥; equal to or more than) 190.0 µg particles per ml of liquid mixture. That means, the amount of magnetic particles to be added to the liquid mixture
needs to be adapted and selected depending on the volume of the liquid mixture in the test vial containing the nucleic acid containing liquid mixture obtained in the first inhibitory contaminants removal step and the second inhibitory precipitation composition and optional further compounds (also designated as “test volume” or “final volume”). Preferably, the surface-functionalized magnetic particles to be used in the second inhibitory contaminant precipitation and removal step are added to the liquid mixture to be treated to result in a concentration of ≥ 200.0 µg particles per ml of liquid mixture, preferably ≥ 250.0 µg/ml, more preferably ≥ 300.0 µg/ml, more preferably ≥ 350.0 µg/ml, more preferably ≥ 400.0 µg/ml, more preferably ≥ 450.0 µg/ml, more preferably ≥ 490.0 µg/ml, more preferably ≥ 500.0 µg/ml, more preferably ≥ 550.0 µg/ml, more preferably ≥ 600.0 µg/ml, more preferably ≥ 650.0 µg/ml, more preferably ≥ 700.0 µg/ml, more preferably ≥ 750.0 µg/ml, more preferably ≥ 800.0 µg/ml, more preferably ≥ 850.0 µg/ml, more preferably ≥ 900.0 µg/ml, more preferably ≥ 930.0 µg/ml. In this respect it is important to note that both characteristic need to be fulfilled to offer the beneficial and surprising results according to the invention, namely, the small particle size and the sufficiently high particle concentration in the liquid mixture to be treated, each as defined herein. The upper limit of the magnetic particles concentration is not particularly limited and can be as high as technically feasible in the test setting. For example, the magnetic particles can be added to result in a concentration up to 6000 µg particles per ml of liquid mixture or up to 5000 µg particles per ml of liquid mixture or 4000 µg particles per ml of liquid mixture or 3500 µg particles per ml of liquid mixture. The method of the present invention is further characterized by comprising a first inhibitory contaminant removal step (herein also designated as “step (a)”) which is a combined treatment step, comprising a sample lysis treatment and a first treatment with a first inhibitory precipitation composition (IP-1). Such combined lysis and first inhibitory contaminants precipitation and removal treatment in the method of the invention can be carried out successively or simultaneously. In a successive combination treatment step the sample (S) is first subjected to the lysis treatment to lyse and/or disrupt the sample, followed by contacting the sample with the first inhibitor precipitation composition (IP-1) for precipitating and removing at least a first part of the one or more inhibitory contaminants from the sample, resulting in a first nucleic acid-containing inhibitory contaminant depleted liquid mixture. In a simultaneous combination treatment step the sample (S) is subjected to the lysis treatment to lyse and/or disrupt the sample simultaneously with the first inhibitor precipitation composition (IP-1), either by adding the components for the lysis, e.g. the
lysis composition (L) and/or disruption particles, and the inhibitor precipitation composition (IP-1) to the sample individually (independent from each other) or in the form of a pre-prepared mixture and subjecting the sample to the so provided mixture for lysing and precipitating and removing the at least first part of the one or more inhibitory contaminants from the lysed nucleic acid-containing sample, resulting in the first nucleic acid-containing inhibitory contaminant depleted liquid mixture. Such pre-purified or partly depleted liquid mixture is herein also referred to as “first nucleic acid-containing liquid mixture” or “nucleic acid-containing liquid mixture resulting from / obtained in step (a)”. The lysis treatment applied in the method of the present invention can be induced with well-known lysis techniques, including chemical, mechanical and enzymatic lysis techniques and combinations thereof. In one aspect, the lysis treatment is induced by a combination of chemical and mechanical lysis. A mechanical lysis can be induced by one or more of bead beating, sonication and homogenization. In a further aspect the lysis treatment comprises contacting the sample (S) with a lysis composition (L), which comprises one or more agents being suitable for chemically and/or enzymatically disrupting the sample. Suitable components to be included in such a lysis composition (L) can be selected from chaotropic agents, detergents, buffers, preservative agents, reagents with protective effect on the nucleic acids, denaturation agents, anti-foaming agents, osmotic stabilizer, chelating agents, and combinations and mixtures thereof. The lysis composition (L) may also comprise enzymes for enzymatic lysis and/or homogenizing agents, like disruption particles, for mechanical lysis. In principle, the following compounds can be used, without being limited thereto: Chaotropic agents (or chaotropes) are effective to support or increase chemical digestion, may help to reduce nuclease activity and help to denature proteins, which can cause havoc on freshly homogenized samples. In principle all common chaotropes or salts thereof may be used that cause disorder in a protein or nucleic acid by, for example, altering the secondary, tertiary or quaternary structure of a protein or a nucleic acid. Examples of chaotropic agents (and salts thereof) comprise guanidinium, thiocyanate, isothiocyanate, perchlorate, trichloroacetate and/or trifluoroacetate as chaotropic ion, including guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate, sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluroacetate and urea, as well as mixtures thereof.
Preferred chaotropic agents are selected from guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate and sodium thiocyanate, with sodium thiocyanate being most preferred. The lysis composition (L) is preferably a liquid composition and may comprise the chaoptropic agent, which is preferably a chaotropic salt as mentioned above, in a concentration that lies in a range selected from about 0.1M up to the saturation limit, about 0.2M to 8M, about 0.3M to 4M, about 0.4M to 3M, about 0.5M to 2.5M, about 0.6M to about 2M, about 0,6M to about 1.5M and 0.6M to about 1M. Detergents (or surfactants) may support the lysis of the sample and support solubilization of the homogenate. Detergents comprise nonionic, cationic, anionic and zwitterionic detergents, comprising sodium dodecyl sulfate (SDS), sarkosyl, sodium lauryl sarcosinate, cetyltrimethyl ammonium bromide (CTAB), cholic acid, deoxycholic acid, benzamidotaurocholate (BATC), octyl phenol polyethoxylate, polyoxyethylene sorbitan monolaurate, tert-octylphenoxy poly(oxyethylene) ethanol, 1,4-piperazinebis- (ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid, polyethylene glycoltert-octylphenyl ether (Triton®X-100), and (1,1,3,3-tetramethylbutyl)phenyl- polyethylene glycol (Triton®X-114). Preferred detergents are selected from SCS, CTAB and Triton®X-100, with SDS being most preferred. Suitable buffers comprise TRIS, PBS, Good’s buffer, SSC, sodium citrate, sodium acetate, phosphate buffers, and biological buffers, selected from the list comprising for example MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSOPOPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS. Preferred buffers are selected from TRIS and MOPS. Preservative agents may include, for example, sodium azide, 5-chloro-2-methyl-4- isothiazoline-3-on, 2-methyl-4-isothiazoline-3-on and mixtures thereof, including commercially available preservative solutions like ProClinTM preservative solutions (Merck). Further, compounds can be added having a protective effect on the nucleic acids, like sodium phosphate (NaH2PO4,). Denaturation agents may include, for example, organic solvents or alkaline compounds (strong bases). A composition having an alkaline pH value, e.g. a pH value of 10 or more, a pH value of 11 or more, a pH value of 11.5 or more, a pH value of 12 or more, a pH value of 12.5 or more, a pH value of 12.75 or more, a pH value of 13 or more and a pH
value of 13.25 or more achieves or supports denaturation. Instead of or in addition to the denaturation agent denaturation may be employed (or supported) by heating the sample. Further, it is possible to add at least one anti-foaming agent (or defoamer), which may reduce and prevent the formation of foam in the test mixture and thus improve the further processability. Anti-foaming agents may be used to prevent formation of foam or may be added to break an already formed foam. In principle, all commonly used anti-foaming agents can be used, such as for example insoluble oils, polydimethylsiloxanes and other silicones, certain alcohols, stearates and glycols – provided that they do not negatively affect any of the reaction agents, the tissue sample, the isolated analytes or disturb any subsequent isolation, harvesting and analysis methods. A preferred anti-foaming agent is polydimethylsiloxane. Osmotic stabilizers may be added to help bind up water and prevent dissociation of related solutes. Osmotic stabilizers comprise, for example, sucrose or sorbitol. Chelating agents are organic compounds that are capable of forming coordinate bonds with metals through two or more atoms of the organic compound. In preferred aspects of the invention no chelating agent is used as it is usually not necessary. However, if the use of a chelating agent becomes necessary or reasonable in a certain test setting, then suitable chelating agents include and can be selected from diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA). As used herein, the term "EDTA" indicates inter alia the EDTA portion of an EDTA compound such as, for example, K2EDTA, K3EDTA or Na2EDTA. Among those, EDTA is preferred. Preferably, the lysis composition (L) comprises at least one chaotropic agent and at least one buffer. Suitable enzymes for enzymatic lysis can be selected from the group of hydrolases (according to the group EC3 in the EC number classification of enzymes). In particular, enzymes for enzymatic lysis can be selected from the group of proteases (EC 3.4), which are often also designated as peptidases, proteinases, or proteolytic enzymes, including proteinaseK, collagenase, dispase, trypsin, and pepsin. Generally, besides the enzymes for enzymatic lysis (proteolytic enzymes) additional enzymes, other than the enzymes for enzymatic lysis, may be added, e.g. enzymes from the group of hydrolases acting on ester bonds according to enzyme class EC 3.1, including an RNase, for example RNase A. An enzymatic lysis composition may further comprise suitable additive compounds, such as stabilizing agents, solvents, or buffer. In
principle, any buffer, which is suitable for receiving the sample, the enzymes for enzymatic lysis and the optional additional agents in the enzymatic lysis composition, can be used. In particular, the buffer must be compatible with the enzyme for enzymatic lysis and must not completely inhibit the activity of the proteolytic enzyme or of any additional agent of the composition. Regarding additive compounds to be comprised in an enzymatic lysis composition reference is made to the compounds defined above. It is further possible that the lysis composition comprises homogenizing agents, like disruption particles or milling beads for mechanical lysis. Such disruption particles or milling beads are well-known and commercially available and are usually small particles, beads or spheres made of or comprising glass, ceramic, metal, mineral and plastic particles or beads, or a combination of two or more of such materials. Preferred disruption particles are yttrium-stabilized zirconium particles. Such disruption particles for mechanical lysis treatments usually have an average particle diameter of from 0.05 mm to 3.00 mm. It is also possible to use mixtures of disruption particles of different sizes. Further it is possible to mix disruption particles of any form and/or material. Preferably, disruption particles used for mechanical lysis are a mixture of beads with a diameter of 0,1 and 0,5 mm. It is further possible, as described herein, to add the disruption particles to the inhibitor precipitation composition (IP-1) used in the method of the present invention. Disruption particles used in the lysis treatment of the method of the invention may have one or more of the following characteristics: (i) the disruption particles are made of or comprise glass, ceramic, metal, mineral, plastic or a combination of two or more of such materials, (ii) the disruption particles are yttrium-stabilized zirconium particles, (iii) the disruption particles have an average particle diameter of from 0.05 mm to 3.00 mm, (iv) the disruption particles are a mixture of particles with different sizes, (v) the disruption particles are a mixture of beads with a diameter of 0,1 and 0,5 mm. Mechanical disruption can be carried out using a vortex or TLIII. In principle it is possible to carry out the combined lysis and inhibitory contaminants precipitation and removal treatment by subjecting the sample (S) to a mechanical and chemical lysis in a lysis composition (L), which comprises chemical disruption agents and disruption particles, and optionally one or more of the above defined additive components, in particular buffer, and which further comprises the first inhibitor precipitation composition (IP-1). Such lysis composition (L) and first inhibitor precipitation
composition (IP-1) can be added individually (independent from each other) or in the form of a pre-prepared mixture. It is also possible to carry out the combined lysis and inhibitory contaminants precipitation and removal by subjecting the sample (S) to a mechanical lysis by adding disruption particles to the first inhibitor precipitation composition (IP-1). Therein the disruption particles and the first inhibitor precipitation composition (IP-1) can be added to the sample (S) individually (independent from each other) or in the form of a pre-prepared mixture. Preferably, the lysis treatment is induced by a combination of chemical and mechanical lysis. The lysis composition (L) may be added in the form of a composition, a liquid composition or aqueous composition, preferably in the form of a buffered aqueous solution. The lysis composition (L) may be added in an amount of up to 800 µl, e.g. in amounts of up to 200 µl, up to 300 µl, up to 400 µl, up to 500 µl, up to 600 µl, up to 700 µl, or up to 800 µl per sample to be treated. The combined lysis and inhibitor contaminant precipitation treatment of the first inhibitory contaminants precipitation and removal step of the method of the present invention may comprise one or more of the following characteristics: (i) the sample (S) is subjected to a mechanical and chemical lysis by contacting it with a lysis composition (L) comprising one or more lysing components with disruption particles and with the first inhibitor precipitation composition (IP-1) and conducting a bead beating process, (ii) the sample (S) is subjected to a mechanical lysis by contacting it with disruption particles and the first inhibitor precipitation composition (IP-1) and conducting a bead beating process, (iii) the disruption particles are contacted with the sample (S) in the form of a mixture with the lysis composition (L), (iv) the disruption particles are contacted with the sample (S) separately from the first inhibitor precipitation composition (IP-1), (v) the disruption particles are contacted with the sample (S) in the form of a mixture with the first inhibitor precipitation composition (IP-1), (vi) the lysis composition (L) and the first inhibitor precipitation composition (IP- 1) are contacted with the sample (S) separately from each other, (vii) the sample (S) is not subjected to a chemical lysis, (viii) the sample (S) is subjected to a mixture of lysis composition (L) and first inhibitor precipitation composition (IP-1).
After the lysis and first inhibitory contaminants precipitation the first removal of contaminants can be achieved by centrifugation and separating the precipitate from the clarified supernatant, which corresponds to the first nucleic acid-containing liquid mixture, which is then subjected to the second inhibitory contaminants precipitation and removal treatment. In the method according to the invention the first and second inhibitor precipitation composition (IP-1) and (IP-2) may be the same or different. In particular, the first inhibitor precipitation composition (IP-1) and the second inhibitor precipitation composition (IP-2) may comprise the same or different compounds in same or different amounts and/or ratios and/or may be added in the same or different amounts to the nucleid acid containing sample or liquid mixture to be treated. Preferably, the first and second inhibitor precipitation composition (IP-1) and (IP-2) comprise a tri- or tetra-valent salt containing a cation having a valence of three or four, such as preferably an aluminium or an iron salt. Generally, the first inhibitor precipitation composition (IP-1) and the second inhibitor precipitation composition (IP-2) may independent from each other comprise one or more compounds selected from (i) an aluminium salt, including aluminium chloride (AlCl3), aluminium sulfate, aluminium ammonium sulfate, aluminium sulfate dodecahydrate, aluminium ammonium sulfate dodecahydrate, aluminium potassium sulfate, aluminium chlorohydrate, sodium aluminate, and mixtures thereof, (ii) ammonium acetate (NH4OAc), ammonium sulfate, or both, (iii) a calcium salt, including calcium oxide and calcium chloride (CaCl2), and mixtures thereof (iv) a magnesium salt, including magnesium chloride (MgCl2), (v) an iron salt, including iron (III) chloride (FeCl3) and iron (II) sulfate, and mixtures thereof (vi) erbium (III) acetate, erbium (III) chloride, holmium chloride, zirconium (IV) chloride and hafnium (IV) chloride, and mixtures thereof, and combinations and/or mixtures of the aforementioned compounds and groups of compounds. Preferably the first inhibitor precipitation composition (IP-1) and/or the second inhibitor precipitation composition (IP-2) independent from each other comprise at least one of aluminium chloride, ammonium acetate, aluminium sulfate dodecahydrate and aluminium chlorohydrate, and mixtures thereof. The first inhibitor precipitation composition (IP-1) and/or the second inhibitor precipitation composition (IP-2) may further (independent from each other) comprise one or more additive compounds, which are preferably selected from the groups and compounds
defined above in context with the lysis composition (L). Particularly, the first and/or second inhibitor precipitation composition (IP-1) and/or (IP-2) comprise a buffer, e.g. a buffer selected from those defined above for the lysis composition (L). In such a preferred aspect of the invention the first inhibitor precipitation composition (IP-1) and/or the second inhibitor precipitation composition (IP-2) can be used in the form of a precipitation buffer solution comprising at least one of aluminium chloride, ammonium acetate, aluminium sulfate dodecahydrate and aluminium chlorohydrate and additionally NaH2PO4 as a nucleic acid protective agent, more preferably in the form of a precipitation buffer solution comprising ammonium acetate, aluminium chloride and NaH2PO4 as a nucleic acid protective agent. In principle, conventional and well-known or commercially available compositions and test kits for precipitating and removing inhibitory contaminants can be used in the method of the present invention, including for example compositions as described in WO2006/073472A2 or in WO2019/209597A1 or in WO2019209600A1 or as commercialized under the tradenames DNeasy® PowerSoil® Pro DNA Isolation Kit (company MoBio Laboratories Inc. / QIAGEN), QIAamp® PowerFecal® Pro DNA Kit (company QIAGEN). The first inhibitor precipitation composition (IP-1) and/or the second inhibitor precipitation composition (IP-2) as defined herein may be added in the form of a composition, a liquid composition or aqueous composition, preferably in the form of a buffered aqueous solution. The first inhibitor precipitation composition (IP-1) and/or the second inhibitor precipitation composition (IP-2) as defined herein may be added in the first and second inhibitory contaminants removal step, respectively, in the same or in different amounts ranging between 20 to 2000 µl, preferably between 50 to 1500 µl per sample or liquid mixture to be treated. Most preferred are amounts of 50, 100, 200, 500, 600, 800 and 1000 µl. In a preferred aspect, the first inhibitor precipitation composition (IP-1) as defined herein is added in an amount of 100 to 1500 µl, preferably 200 to 1000 µl, more preferably 100, 200, 400, 500, 600, 800 or 1000 µl per sample to be treated. In a preferred aspect, the second inhibitor precipitation composition (IP-2) as defined herein is added in an amount of 10 to 200 µl, preferably 20 to 150 µl, more preferably 25, 30, 35, 40, 45, 50, 60, 75 or 100 µl per liquid mixture to be treated. In the second inhibitory contaminants precipitation and removal step of the method of the present invention (herein also designated as “step (b)”) the second inhibitor precipitation composition (IP-2) and the surface-functionalized magnetic particles can be added to the pre-purified or at least partly depleted nucleic acid-containing liquid mixture
resulting from the first inhibitory contaminants precipitation and removal step (“step (a)”) simultaneously, i.e. as a pre-prepared mixture, or successively (independent from each other). It is preferred to add the second inhibitor precipitation composition (IP-2) and the surface-functionalized magnetic particles simultaneously, i.e. in the form of a pre- prepared mixture. Depending on the test setting and samples to be treated using a pre- prepared mixture instead of adding the components one after the other may provide better results with respect to the yields and reduction of DNA loss. Samples (S), which can be treated with the method of the present invention can be selected from unprocessed, preserved, freshly isolated, crude or unrefined biological or environmental samples. Alternatively, the samples can be broken up, denatured or disrupted. It is preferred to use samples (S) which are liquid, solid or semi-solid samples. In a preferred aspect the samples to be treated (purified) in the method of the present invention are selected from biological samples. Biological samples comprise a sample derived from an animal, animal remains, a food, a microorganism, a plant or its components, soil, sediment, rock, reef, sludge, compost, decomposing biological matter, a biopsy, a histological sample, a semen sample, a blood or saliva sample, any body fluid sample, a hair sample, a skin sample, a fecal sample, archaeological remains, a peat bog, compost, oil, water, terrestrial water or subterranean water, atmospheric and industrial water, dust, urban dust, commercial potting mixtures or soil amendments, deep sea vents or air. Preferred is to use a liquid, solid or semi-solid biological or environmental sample or a lysate or supernatant derived from a biological or environmental sample. More preferred is to use samples deriving from stool, soil, plant or wastewater. As used herein, the term “inhibitor” or “inhibitory contaminant(s)” refers to any substance that interferes with a reaction involving DNA and/or RNA isolated from a sample, and has a detrimental effect on DNA and/or RNA manipulation. Inhibitory contaminants include, for example, inhibitors of an enzymatic reaction that uses DNA or RNA as a substrate and a contaminant that disrupts hybridization of DNA or RNA. Depending on the types of samples, inhibitors may vary. For example, inhibitors in stool samples include haemoglobin and the metabolites thereof, bilirubin, bile acids and bile acid derivatives, undigested or partially digested fiber, or undigested or partially digested food, and polysaccharides. Inhibitors / inhibitory contaminants from soil samples include humic substances formed when microbes degrade plant residues and are stabilized to degradation by covalent binding of their reactive sites to metal ions and clay minerals. They comprise polycyclic aromatics to which saccharides, peptides, and phenols are attached. The predominant types of humic substances in soils are humic acids and fulvic
acids. Additional humic substances include humic polymers and humin. Additional exemplary inhibitors include chitin, decomposing plant materials, organic compounds from compost, phenolics, phenolic polymers or oligomers, polyphenol, polysaccharides, and tannin. The method provided herein is capable of substantially removing at least a part of one or more inhibitors / inhibitory contaminants from a sample. An inhibitor / inhibitory contaminant is substantially removed if 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 inhibitors / inhibitory contaminants from the sample remains in the liquid mixture after the second precipitation and removal step carried out in the method of the present invention. Depending on the sample, analytical set-up and/or analysis target the method of the present invention may be used to remove only selected, specific inhibitory contaminants or mixtures of inhibitory contaminants. Inhibitory contaminant depletion in accordance with the present invention does not necessarily require a depletion with respect to all inhibitors present in a sample but may focus on specific (target) inhibitors. The inhibitor precipitation compositions (IP-1) and/or (IP-2) may be composed to specifically target and precipitate selected inhibitors. Therefore, in the sense of the present invention the expression “at least a part of one or more inhibitory contaminants” covers that only specific (selected inhibitory contaminants from all of the contained) inhibitors are targeted, precipitated and removed (fully or to a targeted degree) or that one or both of the inhibitory contaminants precipitation and removal steps achieve a partial (limited or targeted degree of) precipitation and removal of one or more or all inhibitory contaminants contained. The first inhibitory contaminants precipitation and removal step (a) usually provides a partially depleted sample, meaning a first removal to a partial degree. However, it is also possible to precipitate and remove a certain selection of inhibitory contaminants in the first step (a) and a different selection of inhibitory contaminants in the second step (b). In certain embodiments, an inhibitor inhibits PCR amplification of isolated nucleic acids and is referred to as “a PCR inhibitor.” “PCR amplification” as used herein includes various types of PCR reactions, such as qPCR and RT-PCR. The removal of such an inhibitor by a particular inhibitor removal process may be evaluated by comparing certain features (e.g., Ct values) of PCR reactions using nucleic acids isolated with the inhibitor removal process with PCR reactions using nucleic acids isolated without the inhibitor removal process. The degree of reduction in Ct values between the PCR reactions may indicate the effectiveness of the inhibitor removal process in depleting PCR inhibitor(s). In the sense of the present invention the expression “nucleic acid-containing sample” or “nucleic acid-containing liquid mixture” covers any sample, sample vial, test suspension, liquid mixtures, supernatants or remainders obtained at any step in the method of the
invention which contains the target nucleic acids to be purified and isolated for further analysis. For clarification, the initially provided sample (S) can be a nucleic acid- containing sample and the remainder (supernatant) obtained from such sample (S) after lysis and first inhibitory contaminant precipitation and removal treatment in the first inhibitory contaminants removal step is designated as the nucleic acid-containing liquid mixture. The term "nucleic acid(s)" as used herein refers to one or more nucleic acids of any kind, including single- or double-stranded forms. A nucleic acid can be DNA and/or RNA. In the method of the invention, nucleic acid can be isolated, detected and/or analyzed from one or more organisms present in a sample (S), e.g., such as defined above. Examples include but are not limited to bacteria (e.g., Gram positive or Gram negative), yeast, fungi, algae, viruses (e.g., HIV) and nematodes. Nucleic acid, e.g., RNA and DNA, isolated, detected and/or analyzed using a kit or method of the invention can be from any organism, including, but not limited to viruses, bacteriophage, plasmids, spores, yeast, fungi, algae, nematodes, protozoa, eukaryotic cells, prokaryotic cells and in general, single- and multicellular forms. DNA or RNA isolated, detected and/or analyzed using a kit or method of the invention is not necessarily located within a specific organelle among prokaryotic members, but may be found in the cytoplasm, chloroplasts, mitochondria and nuclei of eukaryotic and multicellular organisms. RNA isolated, detected and/or analyzed using a kit or method of the invention is found in a variety of organisms, including, but not limited to viruses, eukaryotic cells, prokaryotic cells and in general, single- and multicellular forms. RNA isolated, detected and/or analyzed using a kit or method of the invention includes forms found in a multitude of biological forms, including but not limited to, messenger RNA in protein translation, ribosomal RNA in ribosomal protein translation, transfer RNA in protein translation, small interfering RNA and microRNA in gene regulation. Examples of Gram negative bacteria that can be analyzed or detected and/or whose nucleic acid can be isolated using the kits and methods of the invention include but are not limited to Gram negative rods (e.g., anaerobes such as bacteroidaceae (e.g., Bacteroides fragilis), facultative anaerobes, enterobacteriaceae (e.g., Escherichia coli), vibrionaceae (e.g., Vibrio cholerae), pasteurellae (e.g., Haemophilus influenzae), and aerobes such as pseudomonadaceae (e.g., Pseudomonas aeruginosa); Gram negative cocci (e.g., aerobes such as Neisseriaceae (e.g., Neisseria meningitidis) and Gram negative obligate intracellular parasites (e.g., Rickettsiae (e.g., Rickettsia spp.). Examples of Gram negative bacteria families that can be analyzed or detected and/or whose nucleic acid can be isolated include but are not limited to Acetobacteriaceae, Alcaligenaceae, Bacteroidaceae, Chromatiaceae, Enterobacteriaceae, Legionellaceae, Neisseriaceae, Nitrobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Rickettsiaceae and Spirochaetaceae. Examples of Gram positive bacteria that can be analyzed or detected and/or whose nucleic acid can be isolated using the kits and methods of the invention include but are not limited to A.
globiformis, B. subtilis, C. renale, M. luteus, R. erythropolis, Ea39, Ben-28 and S. lividans. Gram positive bacteria that can be detected and/or whose nucleic acid can be isolated also are in groups that include, for example, Corynebacterium, Mycobacterium, Nocardia; Peptococcus (e.g., P. niger); Peptostreptococcus (e.g., Ps. anaerobius; some species in the group form clumps and clusters; some species in the group form diplococci (the latter of which are distinguished by their ability to form butyrate); and some species in the group are capable of fermentation, reduction of nitrate, production of indole, urease, coagulase or catalase); Ruminococcus; Sarcina; Coprococcus; Arthrobacter (e.g., A. globiformis, A. citreus or A. nicotianae); Micrococcus (e.g., M. luteus (previously known as M. lysodeikticus), M. lylae, M. roseus, M. agilis, M. kristinae and M. halobius); Bacillus (e.g., B. anthracis, B. azotoformans, B. cereus, B. coagulans, B. israelensis, B. larvae, B. mycoides, B. polymyxa, B. pumilis, B. stearothormophillus, B. subtilis, B. thuringiensis, B. validus, B. weihenstephanensis and B. pseudomycoides); Sporolactobacillus; Sporocarcina; Filibacter; Caryophanum and Desulfotomaculum. Other Gram positive bacteria that can be analyzed or detected and/or whose nucleic acid can be isolated fall into the group Clostridium, which often include peritrichous flagellation, often degrade organic materials to acids, alcohols, CO2, H2 and minerals (acids, particularly butyric acid, are frequent products of clostridial fermentation), and in one aspect form ellipsoidal or spherical endospores, which may or may not swell the sporangium. Species of Clostridium that can be analyzed or detected and/or whose nucleic acid can be isolated include psychrophilic, mesophilic or thermophilic species, saccharolytic species, proteolytic species and/or specialist species, and those that are both saccharolytic and proteolytic species. Saccharolytic species of Clostridium that can be analyzed or detected and/or whose nucleic acid can be isolated include but are not limited to Cl. aerotolerans, Cl. aurantibutyricum, Cl. beijerinckii, Cl. botulinum B,E,F*, Cl. butyricum, Cl. chauvoei, Cl. difficile, Cl. intestinale, Cl. novyi A, Cl. pateurianum, Cl. saccharolyticum, Cl. septicum, Cl. thermoaceticum, and Cl. thermosaccharolyticum. Proteolytic species of Clostridium that can be analyzed or detected and/or whose nucleic acid can be isolated include but are not limited to Cl. argeninense, Cl. ghoni, Cl. limosum, Cl. putrefaciens, Cl. subterminale and Cl. tetani. Species that are proteolytic and saccharolytic that can be analyzed or detected and/or whose nucleic acid can be isolated include but are not limited to Cl. acetobutylicum, Cl. bifermenans, Cl. botulinum A, B, F (prot.)*, Cl. botulinum C,D*, Cl. cadaveris, Cl. haemolyticum, Cl. novyi B,C,*, Cl. perfringens, Cl. putrefaciens, Cl. sordelli and Cl. sporogenes. As indicated by an asterisk, Cl. botulinum is subdivided into a number of types according to the serological specificities of the toxins produced. Specialist Clostridium species that can be detected and/or whose nucleic acid can be isolated include but are not limited to Cl. acidiurici, Cl. irregularis, Cl. kluyveri, Cl. oxalicum, Cl. propionicum, Cl. sticklandii and Cl. villosum. These specificities are based on neutralization studies. Other Clostridium species that can be detected and/or whose nucleic acid can be isolated include those that produce botulinum toxins. Examples of fungi that can be analyzed, detected and/or whose nucleic
acid can be isolated using the kits and methods of the invention include but are not limited to Halocyphina villosa, Hypoxylon oceanicum, Verruculina enalia, Nia vibrissa, Antennospora quadricornuta, Lulworthia spp. and Aigialus parvus. Examples of algae that can be detected and/or whose nucleic acid can be isolated include but are not limited to brown algae (e.g., Phylum Phaeophycota Dictyota sp. (Class Phaeophyceae, Family Dictyotaceae); green algae (e.g., Phylum Chlorophycota Chaetomorpha gracilis (Class Chlorophyceae, Family Cladophoraceae); and red algae (e.g., Phylum Rhodophycota, Catenella sp. (Class Rhodophyceae, Family Rhabdoniaceae). Organisms that can be analyzed or detected by the kits and methods of the invention in a sample, e.g., an agricultural soil, include but are not limited to Pseudomonas spp., Serratia spp., Bacillus spp., Flavobacterium spp., Actinomycetes and fungi; in polluted soils include but are not limited to Pseudomonas spp. and Xanthomonas spp.; in marsh/sediments include but are not limited to Escherichia spp., Proteus spp., Methanogens and Actinomycetes; and in forest soils include but are not limited to Mycorrhizae, Fungi and Actinomycetes. An example of a bacterium detected in soil samples for use in combating bioterrorism using methods and kits of the invention is Bacillus anthracis. Thus, the methods and kits of the invention have many medical and veterinary applications, e.g., for diagnosis, prognosis, epidemiology, inspection of contamination of materials (e.g., drugs, dressing, instruments, implants), foods (e.g., inspections of meat, vegetables, seafood, etc.), including medical and veterinary analysis of feces (including manure analysis for animals). Medical and veterinary applications include detection of soils, e.g., for bioterrorism purposes, e.g., anthrax, viruses, nematodes, and the like. Virus detection using the kits and methods of the invention can also analyze manure and soil, water, air and the like. Viruses that can be detected by kits and methods of the invention include variola, varicella, reovirus, retroviruses (e.g., HIV), viral hemorrhagic fevers (e.g., Ebola, Marburg, Machupo, Lassa), Variola major, viral encephalitis and the like, as listed in Table 1, below. The kits and methods of the invention can also be used to detect spores, toxins and biologically produced poisons, for example, by detecting Bacillus anthracis, anthrax spores are also detected (albeit, indirectly), detection of Clostridium perferinges implies presence of toxin, etc. Thus, pathogens and toxins that can be detected by kits and methods of the invention includes those listed in Table 1, below: Table 1
The method for precipitating and removing inhibitory contaminants from samples according to the present invention provides an efficient method of purifying and preparing samples for nucleic acid analysis. Accordingly, following the treatment steps to achieve inhibitory contaminant removal as described above, a further aspect of the invention relates to the subsequent analysis of the purified and inhibitory contaminant depleted liquid mixtures (remainders), wherein the method of the present invention further comprises the following steps after the at least partial removal of the inhibitory contaminants: (i) adding to the second inhibitory contaminant depleted liquid mixture of step (b) a nucleic acid binding agent, preferably a DNA binding agent; (ii) applying one or more purification and/or isolation steps, selected from washing, centrifugation, filtration, magnetic separation, decanting and elution, and combinations thereof.
Regarding nucleic acid binding agents according to the above point (i) reference is made to conventional nucleic acid binding agents, including a nucleic acid (preferably DNA) binding buffer solutions and solid supports for binding the nucleic acid (preferably DNA). In a further aspect of the invention the method further comprises the analysis of the nucleic acids in the second nucleic acid-containing inhibitory contaminant depleted liquid mixture or after purification and/or isolation using one or more of: (i) amplification, (ii) hybridization, (iii) quantification, (iv) sequencing, (v) detection and (vi) characterization. In principle, the purification, isolation, preparation and analysis of the nucleic acids in the inhibitory contaminants depleted liquid mixtures obtained with the method of the present invention are well-known and established techniques. For example, in this respect reference can be made to the respective description in WO2019/209594A1. In any case, the subsequent nucleic acid analysis is not particularly limited. In a further aspect, the method for removing at least a part of one or more inhibitory contaminants from a nucleic acid-containing sample according to the present invention comprises the following steps: (I) providing a sample (S); (II) a first inhibitory contaminants precipitation and removal step (a) with a combined lysis and inhibitory contaminant precipitation and removal treatment, comprising i. adding to the sample (S) (a) a lysis composition (L) and (b) disruption particles, either independent from each other or in the form of a pre-prepared mixture, and (c) a first inhibitor precipitation composition (IP-1); ii. subjecting the sample (S) to a lysis treatment, which combines chemical and/or enzymatic with mechanical disruption for sample lysis, and to precipitation of at least a first part of one or more inhibitory contaminants; iii. removal of the first inhibitory contaminant precipitate and the disruption particles, e.g. by centrifugation; (III) a second inhibitory contaminants precipitation and removal step (b), comprising i. adding to the at least partly inhibitory contaminant depleted remainder (first nucleic acid-containing liquid mixture) resulting from step (II / (a))
(a) a second inhibitor precipitation composition (IP-2), and (b) surface-functionalized magnetic particles carrying anionic functional groups on the particle surface employing a negative overall charge and having an average diameter of < 3.0 µm to result in a concentration of ≥ 190.0 µg particles per ml of liquid mixture, wherein the inhibitor precipitation composition (IP-2) and the magnetic particles are added either individually (independent from each other) or in the form of a pre-prepared mixture; and ii. subjecting the mixture obtained in step (III)-i. to precipitation of at least a second part of one or more inhibitory contaminants; iii. subjecting the mixture obtained in step (III)-ii. to a magnetic field, iv. removing the precipitated inhibitory contaminants associated with the surface-functionalized magnetic particles; and v. obtaining an inhibitor contaminant depleted supernatant. In a further aspect, the method as defined above comprises at least a further step (IV) comprising i. adding a nucleic acid binding agent, ii. purification, and iii. isolation of the nucleic acids, each as described above. In a further aspect, the method as defined above comprises at least a further step (V) comprising i. amplification, ii. hybridization, iii. quantification, iv. sequencing, v. detection, and/or vi. characterization of the nucleic acids. The method of the present invention and as described herein may comprise further steps usually applied in the handling of analytical samples not explicitly mentioned herein. In particular, in the method of the invention one or more of the described treatment steps, in particular the inhibitory contaminant precipitation steps can be repeated under identical or different conditions, if necessary, e.g. to further increase inhibitory contaminant depletion. In accordance with the teaching of the present invention a further aspect relates to a method for providing purified nucleic acid samples for PCR or NGS analysis, the method
comprising removing at least a part of one or more inhibitory contaminants from a nucleic acid-containing sample with the method described herein. A specific benefit of the new method of the present invention is the possibility to implement the new two-step inhibitory contaminants removal treatment into at least partly automated established analysis processes. Such established automated processes can comprise one or more of automated sampling, bead beating, centrifugation, magnetic separation, purification, isolation, amplification, characterization and detection. Preferably, at least the second inhibitory contaminants removal step of the method of the present invention and all subsequent purification, isolation and/or analyzing steps are carried out automated Such automated processes can be designed to be carried out as high-throughput assays. Therefore, in a preferred aspect the method of the invention as described herein is carried out in an at least partly automated process using one or more of automated sampling, bead beating, centrifugation, magnetic separation, purification, isolation, amplification, characterization and detection, and is more preferably carried out as a high-throughput assay. The invention comprises in a further aspect kits (kit-of-parts compositions) for removing at least a part of one or more inhibitory contaminants from a nucleic acid-containing sample and/or for providing purified nucleic acid samples for PCR or NGS analysis, which comprise (a) a first inhibitor precipitation composition (IP-1); (b) a second inhibitor precipitation composition (IP-2); and (c) surface-functionalized magnetic particles carrying anionic functional groups on the particle surface employing a negative overall charge and having an average diameter of < 3.0 µm in an amount to result in a concentration of ≥ 190.0 µg particles per ml of liquid mixture to be treated; (d) optionally a lysis composition (L), and/or (e) optionally disruption particles. Regarding these kit-components (a) to (e) reference is made to the above provided definitions, which may further characterize one or more of the above-mentioned kit- components. Such kits may comprise a cartridge comprising multiple troughs, wherein the individual kit-components are provided in the multiple troughs. It goes without saying that such kits may further comprise usual laboratory parts, consumables, handling instructions, leaflets etc., which are usually required to provide a complete test kit for laboratory analysis.
A further aspect relates to the use of such kits for removing at least a part of one or more inhibitory contaminants from a nucleic acid-containing sample and/or for providing purified nucleic acid samples for PCR or NGS analysis. A further aspect of the invention relates to the use of surface-functionalized magnetic particles carrying anionic functional groups on the particle surface employing a negative overall charge, which have an average diameter of < 3.0 µm, in a concentration of ≥ 190.0 µg particles per ml liquid mixture to be treated for removing at least a part of one or more precipitated inhibitory contaminants from a nucleic acid-containing sample by associating precipitated inhibitory contaminants with the surface-functionalized magnetic particles, applying a magnetic field and separating the surface-functionalized magnetic particles associated with the precipitated inhibitory contaminants from the nucleic acid- containing remainder of a sample. In this respect reference is made to the definition and description of suitable surface-functionalize magnetic particles, inhibitory contaminants and samples, which apply accordingly. DESCRIPTION OF THE FIGURES Fig.1 Schematic Illustration of the process steps according to the invention and as carried out in Example 1 Fig.2 Results of Example 1 (REF = Reference; IRT = Inhibitor Removal Treatment) EXAMPLES The invention is described further by the following examples, without being limited thereto. Example 1 Inhibitory Contaminants Removal from Soil The potential to gather aluminium-precipitated inhibitor flocks with carboxylated magnetic beads (COOH magnetic beads) was investigated. The test procedure is illustrated in Figure 1. Sample (S): 150 mg soil Lysis solution (L) / DNA extraction: DNeasy® PowerSoil® Pro DNA Isolation Kit (company QIAGEN) disruption particles: PowerBead Pro Tube (company QIAGEN) bead beating: Vortex or TL II Inhibitory Contaminant Removal: QIAamp PowerFecal® Pro DNA Kit (company QIAGEN). The following surface-functionalized magnetic particles (beads) were investigated and compared:
Sera-MagTM Sera-MagTM MagnefyTM MagAttract Magnetic Dyna- ProMagTM Carboxylate- COOH Carboxylate- Suspension Beads beadsTM Modified Modified Beads G Beads Beads Poly- Poly- Thermo Supplier sciences Cytiva sciences Cytiva QIAGEN Fischer Inc. Inc. surface- functionali- COOH COOH COOH COOH COOH silica zation magnetism superparamagnetic diameter 1 µm 3 µm 1 µm 1 µm 1 µm > 3 µm 186.9 476.3 934.6 934.6 18.7 1401.9 Concentration µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml Clear Clear Observations eluates eluates Total DNA appr. appr. appr. appr. appr. appr. Yield 4 µg 4.6 µg 5.5 µg 6 µg 3.6 µg 4.7 µg [µg] Inhibition Assay > 12 > 12 < 2 < 4 > 12 > 12 [∆ Cq to IC DNA] The sample testing was carried out with the treatment steps according to the present invention, including a first combined lysis and inhibitory precipitation and removal step (a) and a subsequent inhibitory precipitation and removal step (b) with surface- functionalized magnetic particles. In a comparative test approach the same samples with the same surface-functionalized magnetic particles were treated without applying two inhibitory removal steps but only a lysis step followed by a single inhibitory contaminant removal treatment with surface- functionalized magnetic particles. In such comparative approach no inhibition depletion was achieved for neither of the tested samples. The results are shown in Figure 2. From the results the inventors concluded – without being bound to this theory – that there appears to occur an interaction in the form of a nonspecific COOH association to the complex flock which allows the solid inhibitory material to be concentrated by applying a magnet to the magnetic beads. However, the inventors’ investigations showed that this association effect alone is not sufficient to achieve suitable purification and yield results. The inventors observed that by using too large beads the inhibitory contaminant pellet becomes too large and results in a smear or slurry that cannot be separated. Also, using too low amounts of magnetic beads did not provide sufficient purification.
By separating the inhibitor removal treatment into multiple steps, such that a portion of the inhibitory contaminants are precipitated and removed already in the lysis step, the size of the solid inhibitory contaminant pellet could be reduced and the remaining inhibitory contaminant solids could be precipitated and removed with the surface- functionalized magnetic particles to achieve suitable depletion and yield results. Example 2 Determination of Yield The determination of the yield was carried out using UV-Vis technology (Nanodrop) and fluorometric technique (Qubit). Conventional analysis and test protocols were applied. Example 3 Fluorimetric Inhibition Assay The determination of the inhibition was carried out using QuantiFast Pathogen IC DNA Assay. Conventional analysis and test protocols were applied. Eluate spike: undiluted eluate_1 µl & 2 µl & 5 µl