CN110636903A - Bead impact tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms - Google Patents

Abstract

The disclosure herein provides improved methods for bead impact and bead impact systems useful therefor. The disclosure herein further provides methods of using the bead impact system to extract nucleic acids from nucleic acid-containing cells.

Description

Bead impact tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms

1. Background of the invention

Genetic testing and diagnosis of infectious diseases play an important role in the field of clinical microbiology. To overcome the inherent bias and limitations of culture-based methodologies, genetic testing is increasingly being used to detect microorganisms contained in a sample. In order for such genetic tests to be performed, the cells must be lysed to allow extraction of DNA and/or RNA from the microorganism at as high a concentration as possible.

Zhongtang Yu et al, 2004, "Improved extraction of PCR-quality community DNA from digestas and focal samples," BioTechniques, Vol.36 (5): 808-812 describes methods for isolating DNA from digesta and fecal samples. In this method, 0.25g of a sample liquid taken from the gastrointestinal system of a cow, which is assumed to contain microorganisms (i.e., bacteria), is first mixed with 1mL of a buffer solution containing 500mM sodium chloride, 50mM Tris hydrochloride, pH 8.0, 50mM ethylenediaminetetraacetic acid and 4% sodium dodecylsulfate. In addition, 0.4g of sterile zirconia beads was introduced into the sample liquid. The mixture thus obtained is then placed in a sample tube with a Mini-BeadBeater^TMShaking for three minutes allowed the beads to disrupt the cell walls of the microorganisms. In this procedure, the DNA contained in the cells is released. The buffer may also be used to protect the DNA from degradation by DNase contained in the sample fluid. After the bead impact was performed, impurities were removed from the sample by precipitation with ammonium acetate. The nucleic acid is obtained by precipitation with isopropanol. In a further method step, the DNA is digested sequentially with RNase and proteinase K and subsequently purified by column chromatography.

This method has the following drawbacks: which is relatively complex. Since the sample solution is diluted by the addition of the buffer solution, the DNA concentration in the sample decreases. Thus, this method only allows for limited detection accuracy and sensitivity.

Accordingly, there is a need for improved devices and methods for lysing cells of microorganisms contained in a sample fluid and extracting nucleic acids therefrom.

2. Summary of the invention

The disclosure herein relates to improved bead impact tubes and bead impact systems and methods. The term "bead impact tube" and the term "sample tube" are used interchangeably herein. Without wishing to be bound by theory, the inventors believe that bead impact allows nucleic acids to dissolve into solution. The disclosure herein is based (in part) on the following findings: the use of bead impact in nucleic acid extraction methods can result in loss of nucleic acids due to adsorption on the beads, and such loss can be prevented by blocking the binding sites on the beads with an appropriate amount of a blocking agent. The disclosure herein is also based on the recognition that: although some of the lysis buffers previously used in bead blasting may contain reagents that act as blockers, such reagents may be used in much lower amounts than are commonly used. As used herein, the term "lysis buffer" refers to a buffer solution used to rupture open cells. The lysis buffer may comprise, for example, a buffer (e.g., Tris-HCl) and one or more salts (e.g., NaCl, KCl) and/or a detergent (e.g., sodium dodecyl sulfate).

Further, the disclosure herein is also based on the recognition that: some biological samples (such as blood) inherently contain a blocker and thus can undergo bead impact in the absence of additives (such as lysis buffer). Thus, for example, blood can be bead bumped directly after being collected into a commercially available blood collection tube, e.g., by adding bead-bumped beads to the blood collection tube and subjecting the collection tube to agitation. Examples of commercially available blood collection tubes include a light purple cap tube containing EDTA, a light blue cap tube containing sodium citrate, a gray cap tube containing potassium oxalate, or a green cap tube containing heparin. Alternatively, a portion of the blood may be transferred to a sample tube for bead impact.

In certain aspects, the disclosure herein provides a bead impact system suitable for use when beads impact the following samples: samples that do not inherently contain a blocking agent or contain an amount of a blocking agent that is lower than an amount suitable for effectively blocking adsorption of nucleic acids by the beads. The bead impact system of the present disclosure includes a dry blocker. Surprisingly, it has been found that when the bead impact system of the disclosure herein is used for lysing microorganisms and extracting deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) from microorganisms contained in a liquid sample, a greater extraction rate of DNA and/or RNA can be achieved than when the tube is impacted with corresponding beads that do not contain the blocking agent.

The bead impact system of the disclosure herein may also circumvent the need to combine the sample with the lysis solution prior to bead impact, allowing for recovery of larger amounts of nucleic acids due to no sample dilution.

In another aspect, the disclosure herein provides methods for performing bead blasting to lyse cells and extract nucleic acids from a biological sample. The method comprises performing bead blasting on the biological sample in the absence of a lysis buffer. The bead impact method can utilize the bead impact system of the disclosure herein or a standard bead impact system. When using a standard bead impact system, in some embodiments, one or more blocking agents are added to the bead impact system. In other embodiments, particularly where the biological sample naturally includes one or more blocking agents, no blocking agent is added prior to bead impact.

In certain embodiments, the bead impact system of the present disclosure is comprised of: a sample tube comprising a container member having a lumen, an aperture for filling a sample liquid containing microorganisms into the lumen, and an attached or non-attached closure for closing the aperture, wherein a plurality of macroscopic particles are arranged in the lumen, which are adapted to mechanically disrupt cell walls of microorganisms contained in the sample liquid when the sample liquid is filled into the lumen and the bead impact tube is subjected to mechanical oscillation. Exemplary bead systems are described in section 4.1 below and in numbered embodiments 1-24 and 90-98. Exemplary sample tubes that can be used in the bead impact system are described in section 4.1.1, exemplary blockers that can be used in the bead impact system are described in section 4.1.2, and exemplary beads that can be used in the bead impact system are described in section 4.1.3.

The disclosure herein further relates to a method for lysing microorganisms for extracting deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) from said microorganisms, wherein a sample fluid is provided, which is supposed to contain said microorganisms, wherein a plurality of particles which are movable with respect to each other are introduced into the sample fluid and wherein the sample fluid containing the particles is shaken such that said particles are capable of mechanically disrupting cell walls of the microorganisms contained in the sample fluid. An exemplary sample from which DNA can be extracted is described in section 4.2 and an exemplary sample pretreatment step that can be used to prepare the sample prior to nucleic acid extraction is described in section 4.3. Exemplary methods for extracting nucleic acids from a sample (e.g., from a sample described in section 4.2 or a sample that has been pretreated as described in section 4.3) are described in section 4.4 and numbered embodiments 25-81 and 99-104 below. Kits useful for performing the nucleic acid extraction methods of the disclosure herein are described in section 4.7 below and numbered embodiments 82 through 89.

3. Brief description of the drawings

FIG. 1: side view of an exemplary bead impact system (1) comprising a sample tube (2) and a closure cap (6).

FIG. 2: a top view of the bead impact system is shown in fig. 1.

FIG. 3: a bottom view of the bead impact system is shown in fig. 1.

FIG. 4: by a longitudinal section through the centre plane of the bead impact system shown in fig. 1, wherein the closure cap is removed from the sample tube, an aperture (4) is revealed through which the lumen (3) of the sample tube can be accessed. The beads (7) and freeze-dried blocking agent (8) are shown within the lumen.

FIG. 5: a longitudinal section through the centre plane of the bead impact system shown in fig. 1 filled with sample liquid (5).

FIG. 6: shows the intensity of PCR amplification products using DNA recovered from Candida albicans (C.albicans) as a template after bead impact. Calbi144 and Calbi54 are two different Candida albicans target genes, and HCO1-Rat87 is a negative control gene. This study showed that urea had a blocking effect in bead crashes, facilitating the recovery of candida albicans DNA from PD fluid.

FIG. 7: shows the intensity of PCR amplification products using DNA recovered from Candida albicans as a template after bead impact. Calbi144 and Calbi54 are two different Candida albicans target genes and HCO1-Rat87 is a negative control gene. This study showed that RNA has a blocking effect in bead collisions, facilitating the recovery of candida albicans DNA from PD fluid.

FIG. 8: shows the intensity of PCR amplification products using DNA recovered from Candida albicans as a template after bead impact. Calbi144 and Calbi54 are two different Candida albicans target genes, and HCO1-Rat87 is a negative control gene. The amount of candida albicans DNA corresponding to the maximum theoretical yield of extraction was included as a template. This study showed that candida albicans DNA could be recovered by bead impact blood in the absence of additives.

FIG. 9: shows the intensity of PCR amplification products using DNA recovered from staphylococcus aureus (s. aureus) after bead impact as a template. Genes probed from them were used to detect eubacteria (Eub), gram-negative bacteria (Gng), gram-positive bacteria (Gpo), staphylococcus (alphylococci), enterobacteriaceae (entobacteriaceae) (enttb), staphylococcus aureus (Sau), mycoplasma (Myco). N03 Rat87 is a negative control, and CC is an internal control. An amount of staphylococcus aureus DNA corresponding to the maximum theoretical yield of extraction was included as a template. This study showed that staphylococcus aureus DNA could be recovered by bead impact blood in the absence of additives.

FIG. 10: shows the intensity of the PCR amplification product using DNA recovered from e.coli (e.coli) after bead impact as a template. Genes probed from them were used to detect eubacteria (Eub), gram-negative bacteria (Gng), gram-positive bacteria (Gpo), escherichia coli (Eco), staphylococcus (AllStaph), enterobacteriaceae (Entb). N03 Rat87 is a negative control, and CC is an internal control. Coli DNA in an amount corresponding to the maximum theoretical yield of extraction was included as a template. This study showed that E.coli DNA can be recovered by bead impact blood in the absence of additives.

4. Detailed description of the preferred embodiments

Bead blasting is a homogenization procedure for lysing cells to release their contents, including their nucleic acids (DNA and RNA). The sample is placed in a tube with appropriate abrasive beads and subjected to high energy agitation. The sample is typically then centrifuged and the lysate recovered from the beads. Typically, the sample subjected to bead impact is treated with a lysis buffer to facilitate release of DNA from the cells.

The disclosure herein relates to improved bead impact methods and systems. The bead impact method can be performed without the use of lysis buffers, simplifying the procedure of cell lysis and nucleic acid extraction and avoiding sample dilution while minimizing losses due to adsorption on the beads.

The method of the present disclosure is directed to performing bead impact using an appropriate amount of a blocking agent. The blocking agent may be exogenous or endogenous to the sample. For example, urine contains urea, a reagent that is found to block binding of nucleic acids in a biological sample to bead-impact beads. Thus, while supplementation of endogenous blockers with exogenous blockers is also contemplated, the disclosure herein provides a method for beads to impact such samples without adding exogenous blockers. For biological samples that do not contain endogenous blocking agents or that contain low amounts of endogenous blocking agents, exogenous blocking agents can be used. The disclosure herein further provides a bead impact system into which a dry blocker is incorporated, which allows the beads to impact a biological sample without the addition of reagents that cause sample dilution.

Accordingly, the disclosure herein provides a bead impact system that includes a dry blocker. The disclosure herein further provides methods for performing bead blasting to lyse cells in a biological sample and extract nucleic acids therefrom. The method comprises performing bead blasting on the biological sample in the absence of a lysis buffer. The bead impact method can utilize the bead impact system of the disclosure herein or a standard bead impact system. When using a standard bead impact system, in some embodiments, one or more blocking agents are added to the bead impact system. In other embodiments, particularly where the biological sample naturally includes one or more blocking agents, no blocking agent is added prior to bead impact.

Also provided herein are kits containing (or suitable for obtaining) the bead impact systems of the disclosure herein.

4.1 bead impact System

The disclosure herein provides bead impact systems that can be used for cell lysis and extraction of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from cells (e.g., microorganisms) contained in a liquid sample. The bead impact system of the present disclosure includes a sample tube and a bead and a dry blocker within the sample tube. Blocking agents that may be used in the bead impact systems of the disclosure include urea, guanidine salts, detergents, nucleotides, polyvinylpyrrolidone (PVP), and oligonucleotides. The blocking agent is described in detail in section 4.1.2. Beads useful in the bead impact systems of the disclosure herein include beads comprising minerals and/or metals. Beads suitable for use in the bead impact system of the disclosure herein are described in section 4.1.3.

4.1.1 sample tube

The bead impact system of the present disclosure includes a sample tube having an internal cavity accessible through an aperture and capable of receiving beads, a blocker, and a liquid sample. The sample tube may be constructed of any biologically inert material, such as plastic or borosilicate, and is preferably constructed of a plastic, such as polypropylene, polypropylene copolymer, or polycarbonate. As used herein, the term "tube" encompasses vials (e.g., grind vials) and tubes (e.g., conical tubes).

The bead impact system can include a closure for covering the aperture and sealing the sample tube. The closure may be fixed to the sample tube (e.g., to the lid of the sample tube by a hinge) or may be separable from the sample tube (i.e., a removable lid). The sample tube may comprise a threaded region adapted to engage a threaded cap. The threads may be on the inner or outer surface of the sample tube (e.g., as shown in fig. 4-5).

Sample tubes useful in the bead impact systems of the disclosure herein are commercially available, such as screw cap polypropylene or microcentrifuge tubes. Standard tube sizes can be used to make the bead impact tubes and systems of the disclosure herein, e.g., 0.5mL, 1.7mL, 2mL, 4.5mL, 7mL, 15mL, 50mL, or 250 mL.

4.1.2 blocking agent

The bead impact system of the present disclosure includes a dry blocker. As used herein, a "dry" or "dry" blocker is a blocker that contains less than 20% moisture. In some embodiments, the dry blocker contains less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% moisture.

The dry blocker is preferably long-term stable, i.e., the bead impact system can be transported and stored without problems for extended periods of time without the blocking efficacy of the blocker being substantially reduced. The dry blocking agent may be loosely disposed within the lumen of the container member, and/or a portion or the entire lumen of the sample may be coated with a layer of blocking agent or a thin film of blocking agent. The blocking agent is preferably freeze-dried. The blocking agent can be freeze-dried before or after the beads are added to the tube.

Examples of blocking agents include one or more dispersants (e.g., urea, guanidinium), one or more detergents, one or more nucleotides, polyvinylpyrrolidone (PVP), one or more oligonucleotides, or any combination thereof. Details of such exemplary blockers are shown in sections 4.1.2.1 through 4.1.2.5, described below. The inclusion of a blocking agent (e.g., a detergent) that facilitates the dissolution of microbial cell pores can improve the yield of nucleic acids prepared using the bead impact systems of the disclosure herein and circumvent the need to combine the sample with a lysis solution prior to bead impact. Generally, the amount of blocking agent used in the bead impact system of the disclosure herein is lower than the amount used in the lysis solution.

The bead impact system can be produced by freeze-drying a lysis solution (also referred to as an extraction buffer or lysis buffer) in a bead impact tube. Cleavage solutions containing blocking agents are known in the art or are commercially available.

In some embodiments, the blocking agent comprises one or more dispersants. In some embodiments, the blocking agent comprises one or more detergents. In some embodiments, the blocking agent comprises one or more nucleotides. In some embodiments, the blocking agent comprises one or more oligonucleotides. In some embodiments, the blocking agent comprises one or more dispersants and one or more detergents. In some embodiments, the blocking agent comprises one or more dispersants and one or more nucleotides. In some embodiments, the blocking agent comprises one or more dispersants and one or more oligonucleotides. In some embodiments, the blocking agent comprises one or more detergents and one or more nucleotides. In some embodiments, the blocking agent comprises one or more detergents and one or more oligonucleotides. In some embodiments, the blocking agent comprises one or more nucleotides and one or more oligonucleotides. In some embodiments, the blocking agent comprises one or more dispersants, one or more detergents, and one or more nucleotides. In some embodiments, the blocking agent comprises one or more dispersants, one or more detergents, and one or more oligonucleotides. In some embodiments, the blocking agent comprises one or more dispersants, one or more nucleotides, and one or more oligonucleotides. In some embodiments, the blocking agent comprises one or more detergents, one or more nucleotides, and one or more oligonucleotides. In some embodiments, the blocking agent comprises one or more dispersants, one or more detergents, one or more nucleotides, and one or more oligonucleotides.

The blocking agent may comprise or further comprise ethylenediaminetetraacetic acid (EDTA) and/or a sodium salt thereof. EDTA binds to calcium, magnesium and iron and thereby inactivates DNase and RNase. This measure also counteracts the degradation of DNA and RNA contained in the sample fluid treated with the bead impact system. Thus, higher detection sensitivity and reproducibility of the measurement results is possible during examination of DNA and/or RNA extracted from cells (e.g., microorganisms) using the bead impact system of the disclosure herein.

The blocking agent may be loosely disposed within the interior cavity of the container member, for example in powder form. For example, the powder may be mixed with said beads in the sample or may be arranged in the sample tube in the form of a layer located above and/or below said beads. Alternatively or in addition, the inner wall of the sample tube may be at least partially coated with a layer of a blocking agent or a thin film of a blocking agent. The layer or film may be prepared by evaporating a liquid from an aqueous mixture comprising the blocking agent that has been added to the sample tube. In other embodiments, some or all of the beads in the bead impact system may be coated with the blocking agent.

4.1.2.1 centrifugal powder

The dispersing agent disrupts the structure of macromolecules such as proteins and nucleic acids and denatures them. Discrete solutes reduce the net hydrophobic effect of the hydrophobic region by disordering the water molecules of the adjacent protein. This dissolves the hydrophobic region in the solution, thereby denaturing the protein. This also applies directly to the hydrophobic regions in the lipid bilayer; if a critical concentration of discrete solutes (in the hydrophobic region of the bilayer) is reached, the membrane integrity is broken and the cells are lysed.

Exemplary dispersants that can be used as blocking agents are provided below.

Urea: when the blocking agent comprises urea (or thiourea; for use herein, unless the context indicates otherwise, the term urea includes thiourea), the amount of urea may be matched to the amount of the sample liquid intended for use with the bead impact system, such that the concentration of urea dissolved in the sample liquid after addition of the sample liquid to the sample tube ranges between 10 and 100 grams/liter, between 50 and 100 grams/liter, between 20 and 50 grams/liter, or between 25 and 35 grams/liter. Thus, with respect to the above embodiments, if a 1mL sample is to be added to a 2mL tube, the amount of urea used for the blocking reagent would be between 10mg and 100mg, between 50mg and 100mg, between 20mg and 50mg, or between 25mg and 35mg (respectively), and if a 0.8mL sample is to be added to a 2mL tube, the amount of urea used for the blocking reagent would be between 8mg and 80mg, between 40mg and 80mg, between 16mg and 40mg, or between 20mg and 28mg (respectively).

Salt: certain salts may function as a chaotropic agent. Salts can have discrete properties by shielding charge and preventing stabilization of salt bridges. Discrete salts include various salts of guanidine, lithium and magnesium.

Guanidine salt: guanidine salts that can be used as blocking agents in the bead impact systems of the present disclosure include guanidine isocyanate, guanidine chloride, and combinations thereof.

Lithium salt: lithium salts that may be used as a blocking agent in the bead impact system of the present disclosure include lithium perchlorate, lithium acetate, and combinations thereof.

Magnesium salt: magnesium salts that may be used as blocking agents in the bead impact systems of the disclosure herein include magnesium chloride.

Cleaning agent: some detergents may function as a dispersant, such as sodium dodecyl sulfate ("SDS"). The use of detergents as blocking agents is described in section 4.1.2.3.

4.1.2.2 Creatinine

When the blocking agent comprises creatinine, the presence of the creatinine in the lumen of the sample tube may adversely affect the degradation of DNA and/or RNA contained in a sample treated using the bead impact system. In addition, creatinine prevents the DNA and/or RNA from non-specifically binding to the inner wall of the beads and/or sample tube. This allows for greater yields when extracting DNA and/or RNA from cells (e.g., microorganisms) treated with the bead-impact tube system.

4.1.2.3 detergent

Detergents that may be used as blocking agents in the bead impact systems of the present disclosure include sodium lauryl sulfate, sodium lauroyl sarcosinate, polyoxyethylene (20) sorbitan monolaurate, and combinations thereof. Polyoxyethylene (20) sorbitan monolaurate is also known as polysorbate 20.

The amount of cleaning agent used for the blocker may be matched to the amount of the sample fluid intended for use with the bead impact system. In some embodiments, the concentration of detergent dissolved in the sample fluid after the sample fluid is added to the sample tube ranges from 1 to 50mg/mL, from 1 to 25mg/mL, or from 25 to 50 mg/mL. Thus, with respect to the above embodiment, if a 1mL sample is to be added to a 2mL tube, the amount of detergent used for the blocking agent may range from 1 to 50mg, 1 to 25mg, or 25 to 50mg (respectively), and if a 0.8mL sample is to be added to a 2mL tube, the amount of detergent used for the blocking agent may range from 0.8 to 40mg, 0.8 to 20mg, or 20 to 40 mg.

4.1.2.4 nucleotides

Nucleotides that can be used in the bead impact systems of the disclosure include ribonucleotides, deoxyribonucleotides, and combinations thereof.

The nucleotide may be naturally occurring. Naturally occurring ribonucleotides are adenosine monophosphate, guanosine monophosphate, cytosine monophosphate and thymidine monophosphate. Naturally occurring deoxyribonucleotides that can be used as blocking agents are deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate and deoxythymidine monophosphate.

The nucleotide may be a non-naturally occurring analog of a naturally occurring nucleotide. Examples of non-naturally occurring analogs include, but are not limited to, peptide nucleotides, locked nucleic acid ("LNA") nucleotides (which contain a bicyclic sugar moiety in place of a deoxyribose or ribose sugar), those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and those containing pendant moieties. In particular embodiments, the analog is 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxoacetic acid methyl ester, uracil-5-oxoacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxoacetic acid methyl ester, uracil-5-oxoacetic acid, pseudouracil, queosine, 2-thiocytosine and 2, 6-diaminopurine or 7-deazaguanosine.

In some embodiments, the blocking agent comprises a mixture of at least two, at least three, or four naturally occurring ribonucleotides. In other embodiments, the blocking agent comprises a mixture of at least two, at least three, or four naturally occurring deoxyribonucleotides. In yet other embodiments, the blocking agent comprises a mixture of at least two, at least three, four, or even more than four nucleotide analogs. In other embodiments, the blocking agent comprises a mixture of: (a) naturally occurring ribonucleotides and naturally occurring deoxyribonucleotides; (b) naturally occurring ribonucleotides and nucleotide analogs; (c) naturally occurring deoxyribonucleotides and nucleotide analogs; or (d) naturally occurring ribonucleotides, naturally occurring deoxyribonucleotides, and nucleotide analogs.

The amount of ribonucleotide used for the blocking agent may be matched to the amount of the sample fluid intended for use with the bead impact system. In certain embodiments, the concentration of ribonucleotides dissolved in the sample fluid after adding the sample fluid to the sample tube ranges from 200pg/mL to 20 μ g/mL, 1ng/mL to 15 μ g/mL, or 1 μ g/mL to 10 μ g/mL. Thus, with respect to the above embodiment, if 1mL of sample is to be added to a 2mL tube, the amount of ribonucleotide for the blocker can range from 200pg to 20 μ g, 1ng to 15 μ g or 1 μ g to 10 μ g (respectively), and if 0.8mL of sample is to be added to a 2mL tube, the amount of ribonucleotide for the blocker can range from 160pg to 16 μ g, 0.8ng to 12 μ g or 0.8 μ g to 8 μ g.

4.1.2.5 oligonucleotides

Oligonucleotides useful as blocking agents may be produced or synthesized from naturally occurring sources.

Reference herein to an "oligonucleotide" does not denote the size of the molecule and means a polymeric form of nucleotides of any length. Typically, however, the oligomer is no greater than 2,000 nucleotides, 1,000 nucleotides, more typically no greater than 500 nucleotides, and even more typically no greater than 250 nucleotides. The term oligonucleotide includes double-and single-stranded DNA and RNA (but is preferably at least partially single-stranded). It also includes known types of modifications, for example, labels, methylation, "capping", and the substitution of one or more of the naturally occurring nucleotides with an analog (such as those described in section 4.1.2.4) as are known in the art.

The oligonucleotides may be a mixture of different sizes and/or may comprise ribonucleotides, deoxyribonucleotides, nucleotide analogs, or a combination of two or more of the foregoing (e.g., a mixture of ribonucleotides and deoxyribonucleotides, a mixture of ribonucleotides and nucleotide analogs, a mixture of deoxyribonucleotides and nucleotide analogs, or a mixture of ribonucleotides, deoxyribonucleotides and nucleotide analogs). In some embodiments, the blocking agent comprises RNA.

Synthetic oligonucleotides useful in the bead impact systems of the disclosure herein are typically 2 to 120 nucleotides in length. In various embodiments, the oligonucleotide is 2, 5, 8, 10, 15, 18, 25, 40, 50, 80, 100, or 120 nucleotides in length, or an oligonucleotide having a length ranging between any pair of the above values (e.g., 2 to 100 nucleotides in length, 2 to 50 nucleotides in length, 2 to 25 nucleotides in length, 5 to 40 nucleotides in length, 2 to 15 nucleotides in length, 8 to 120 nucleotides in length, 8 to 80 nucleotides in length, 10 to 100 nucleotides in length, 15 to 50 nucleotides in length, 50 to 100 nucleotides in length, 100 to 120 nucleotides in length, etc.).

Naturally occurring oligonucleotides can be prepared by cleaving DNA from one or more naturally occurring sources (e.g., salmon sperm DNA, calf thymus DNA, herring sperm DNA, or a combination thereof).

The amount of oligonucleotide used for the blocker may be matched to the amount of the sample fluid intended for use with the bead impact system. In certain embodiments, the concentration of the oligonucleotide dissolved in the sample fluid after the sample fluid is added to the sample tube ranges from 200pg/mL to 20 μ g/mL, 1ng/mL to 15 μ g/mL, or 1 μ g/mL to 10 μ g/mL. Thus, with respect to the above embodiment, if 1mL of sample is to be added to a 2mL tube, the amount of oligonucleotide used for the blocker can range from 200pg to 20 μ g, 1ng to 15 μ g, or 1 μ g to 10 μ g (respectively), and if 0.8mL of sample is to be added to a 2mL tube, the amount of ribonucleotide used for the blocker can range from 160pg to 16 μ g, 0.8ng to 12 μ g, or 0.8 μ g to 8 μ g.

4.1.3 beads

The bead impact system of the present disclosure comprises beads that are movable relative to each other when they are disposed in the sample fluid. As used herein, the term "bead" encompasses both spherical and non-spherical particles. The beads may be mineral beads, such as those composed of crystalline particles (e.g., zirconium, zircon (zirconium silicate) and zirconia (zirconium dioxide)), quartz, alumina, silicon carbide (also known as silicon carbide), garnet, ceramic particles, glass (e.g., silica glass or silica), or a combination comprising one or more of the foregoing (e.g., zirconia/silica beads or zirconia/gadolinium beads). The beads may also be metal beads, such as stainless steel beads or chrome steel beads.

For the recovery of bacterial DNA, the beads preferably comprise quartz particles and/or zirconium particles. Such beads are commercially available in large quantities and are chemically inert to DNA and RNA. The quartz and/or zirconium particles have a relatively high specific gravity, are hard and may have sharp edges. It is therefore well suited for opening cell membranes when exposed to mechanical shock.

The material and size of the beads of a given bead impact system can be selected by one of ordinary skill in the art based on the nature of the cell type in the liquid sample to be processed. Generally, the bead diameter will range from 50 μm to 3 mM. For the extraction of nucleic acids from bacterial cells, small to medium sized beads can be used, which are typically composed of glass or zirconium and range in diameter from 50 μm to 0.5 mm. For extracting nucleic acids from larger microbial cells, such as yeast, medium-sized or large-sized beads (e.g., beads having a bead diameter of 0.5mm, 1mm, or 1.5mm, which are typically composed of glass or zirconium) can be used. Beads of different sizes and compositions for processing different cell types are commercially available. See, e.g., benchmark scientific, Product Note: benchmark scoring Guide available at www.denvillescientific.com/sites/default/files/lead _ marking _ notes.

The bead impact system of the disclosure herein can include multiple types of beads and/or multiple sizes of the same type of beads, particularly when recovery of nucleic acids from multiple sources (e.g., bacterial and fungal DNA) is desired. Examples of bead impact systems having multiple types of beads include systems having a combination of alumina beads and silicon carbide beads, ceramic beads and silica beads, glass beads and zirconia beads, zirconia beads and alumina beads, or silicon carbide beads and glass beads. The different types of beads may be of the same or different sizes.

4.2 samples

Examples of samples from which nucleic acids can be extracted using the bead impact methods of the disclosure herein include various liquid samples. In some examples, the sample may be a sample of bodily fluid from a subject. The sample may comprise tissue collected from the subject. The sample may include body fluids, secretions, and/or tissues of the subject. The sample may be a biological sample. The biological sample may be a body fluid, exudate and/or tissue sample. Examples of biological samples may include, but are not limited to, blood, serum, saliva, urine, gastric and digestive fluids, tears, feces, semen, vaginal fluids, interstitial fluid from neoplastic tissue, ocular fluids, sweat, mucus, cerumen, oil, glandular secretions, breath, spinal fluid, hair, nails, skin cells, plasma, nasal swabs or nasopharyngeal wash, spinal fluid, cerebrospinal fluid, tissue, throat swabs, wound swabs, biopsies, placental fluid, amniotic fluid, umbilical cord blood, enhanced fluids (emphatic fluid), cavity fluids, sputum, pus or other wound exudates, infected tissue samples from wound debridement or excision, cerebrospinal fluid, lavage, leukogenic samples, peritoneal dialysis fluids, breast milk and/or other secretions, and plant parts.

The sample may be placed into the bead impact tube immediately after isolation from the subject or may have undergone some type of pre-treatment (e.g., as described in section 4.3 below), storage, or transport prior to placement in the bead impact tube. The sample may be added to the bead impact system without experiencing interference or for a long time.

The subject can provide a sample, and/or the sample can be collected from the subject (e.g., a blood sample can be collected in a blood collection tube containing an anticoagulant such as EDTA). The subject may be a human or non-human animal. The sample can be collected from a living or dead subject. The animal may be a mammal, such as a farm animal (e.g., cow, pig, sheep), a sport animal (e.g., horse), or a pet (e.g., dog or cat). The subject may be a patient, a clinical subject or a preclinical patient. The subject may be undergoing diagnosis, treatment and/or disease management or lifestyle or prophylactic care. The subject may or may not be under the care of a health care professional.

In some embodiments, the sample may be an environmental sample. Examples of environmental samples may include air samples, water samples (e.g., ground water, surface water, or wastewater), soil samples, or plant samples.

Other samples may include food products, beverages, manufacturing materials, textiles, chemicals, treatments, or any other sample.

4.3 sample pretreatment

In some instances, it may be beneficial to pre-treat the sample prior to treatment in the bead impact systems of the disclosure herein. Examples of pretreatment steps that can be used prior to placing the sample in the bead impact tube include filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, as discussed herein or otherwise as known in the art.

The following may be particularly advantageous: unwanted cell types and particulate matter are removed from the biological sample prior to placing the biological sample into the bead impact tube of the disclosure herein to maximize recovery of DNA from the cell type of interest.

If it is desired to detect bacteria in a biological sample, the following is desired: the biological sample is pre-treated through the filter so that particulates and non-bacterial cells are retained on the filter while bacterial cells (including spores thereof, if desired) pass through. As used herein, a "filter" is a membrane or device that allows particle-to-molecule size-based differentiation to pass through. Typically, this is done by having holes of a particular nominal size in the filter. For example, filters of particular interest for bacterial detection applications have pores that are large enough to allow bacteria present in a sample of interest to pass through but small enough to prevent passage of eukaryotic cells. Generally, bacterial cell diameters range from 0.2 to 2 μm (microns), most fungal cell diameters range from 1 to 10 μm, platelet diameters are about 3 μm and most nucleated mammalian cell diameters are typically 10 to 200 μm. Thus, filter pore sizes of less than 2 μm or less than 1 μm are particularly suitable for removing non-bacterial cells from a biological sample if the detection of bacteria is intended.

In addition to or instead of the filtration step, the biological sample may be subjected to centrifugation prior to bead impact to remove cells and debris from the sample. Centrifugation parameters for precipitating eukaryotic cells but not bacterial cells are known in the art. The supernatant may then be filtered, if desired.

The filtrate resulting from the filtration step may be a "sample" and may be placed into a bead impact tube according to the disclosure herein, or subjected to further pretreatment steps (e.g., concentration or dilution).

4.4 bead impact

As a preliminary step in nucleic acid preparation, the sample is placed into a bead impact tube for bead impact. The bead impact step can utilize the bead impact system of the disclosure herein or a standard bead impact system. In some examples, one or more exogenous blocking agents may be added when using a standard bead impact system, but if the biological sample contains endogenous blocking agents, the addition of exogenous blocking agents is not required. When one or more blocking agents are added, they may be added to the bead impact tube before the sample is added or after the sample is added, or both the sample and the blocking agent may be added simultaneously (e.g., the sample and the blocking agent may be mixed and then transferred to the bead impact tube).

After the sample is placed in the bead impact tube, the tube is subjected to agitation to mechanically lyse the cells. Lysis can be achieved by a typical laboratory vortexer or homogenizer. Although the processing time in a vortexer is 3-10 times longer than in a dedicated homogenizer, vortexing can easily destroy cells and is inexpensive.

Many homogenizers suitable for bead impact are commercially available and may be used with the impact system of the disclosure herein. Exemplary homogenizers are BeadBug and BeadBlaster 24(BenchmarkScientific), PowerLyzer 24(MO Bio Laboratories Inc.), FastPrep-24, FastPrep-245G and SuperFastPrep-1(MP Biomedicals), and Mini-BeadBeater-16(BioSpec Products).

The homogenization parameters may be selected according to the manufacturer's recommendations. In general, the duration of mechanical disruption (e.g., bead impact) can be less than 1 second, 1-5 seconds, 5-10 seconds, 10-25 seconds, 25-60 seconds, 1 minute-2 minutes, 2 minutes-5 minutes, or more. The number of repetitions of mechanical disruption (e.g., the number of bead impact periods) can be 2, 3, 4, 5, 6, 7-10, 10 or more repetitions. The rate of destruction (e.g., the rate or setting of bead impact) may be less than 50, 50-100, 100-.

4.5 nucleic acid purification

After cell lysis, the liquid sample is separated from the beads, e.g. by removing the liquid sample from the sample tube using a pipette or the like, leaving the beads which settle at the bottom of the sample tube.

Impurities that may interfere with the analysis of the extracted DNA may be removed, for example by precipitating the impurities via known methods, such as using ammonium acetate or using commercial kits.

The nucleic acid can be recovered and washed and optionally further purified. Recovery and/or purification of the nucleic acid can be achieved using conventional methods, such as by precipitation with isopropanol or using commercial kits or automated instruments.

4.6 nucleic acid analysis

Sample analysis for the presence of a nucleic acid of interest, such as bacterial or fungal DNA, may be performed using any nucleic acid analysis method including, but not limited to, amplification techniques, Polymerase Chain Reaction (PCR), isothermal amplification, reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (Q-PCR), digital PCR, gel electrophoresis, capillary electrophoresis, mass spectrometry, fluorescence detection, ultraviolet spectroscopy, hybridization analysis, DNA or RNA sequencing, restriction analysis, reverse transcription, NASBA, allele-specific polymerase chain reaction, Polymerase Cycle Assembly (PCA), asymmetric polymerase chain reaction, post-exponential linear polymerase chain reaction (LATE-PCR), helicase-dependent amplification (HDA), hot-start polymerase chain reaction, inter-sequence specific polymerase chain reaction (ISSR), Reverse polymerase chain reaction, ligation-mediated polymerase chain reaction, methylation-specific polymerase chain reaction (MSP), multiplex polymerase chain reaction, nested polymerase chain reaction, solid-phase polymerase chain reaction, or any combination thereof. Such techniques are well known to those skilled in the art. Knowing the sequence of the target nucleic acid would enable scientists to construct primers and/or probes that allow for amplification and/or detection of the target. The PCR amplificate may also be sequenced to confirm its identity.

Using a bead impact system according to the disclosure herein, the following are possible: the DNA and/or RNA is extracted from the microorganisms contained in the sample fluid using methods known per se in concentrations sufficient for direct microbiological examination of the DNA and/or RNA. This can occur in particular by: the sample fluid is contacted with receptors immobilized on a surface, said receptors specifically binding to the DNA and/or RNA and/or to the DNA and/or RNA components contained therein. The binding of the DNA and/or RNA and/or DNA and/or RNA components to the receptors can be detected in a manner known per se with labels, in particular optical labels, such as fluorescent dyes, and if desired quantified. Alternatively, DNA and/or RNA from the microorganisms contained in the sample fluid may be amplified (e.g. by PCR) prior to examination.

4.7 kits

The disclosure herein further provides kits containing (or suitable for use in obtaining) the bead impact systems of the disclosure herein. The kit can comprise a sample tube (such as the sample tube described in section 4.1.1), one or more blocking agents (such as those described in section 4.1.2), and/or one or more types of beads (such as those described in section 4.1.3). The one or more blocking agents may each be pre-freeze dried and may be contained within the tube or separate from the tube. Likewise, the beads may be contained within or separate from the tube.

In some embodiments, the kit comprises a sample tube and one or more blocking agents. In further embodiments, the kit further comprises one or more types of beads.

The kits of the disclosure herein can include one or more components for sample pretreatment, such as saline, one or more buffer solutions, filters (as described in section 4.3), and the like.

The kit may include one or more oligonucleotides for amplifying DNA and/or RNA from one or more pathogens of interest, such as one or more of the following: mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium avium subspecies paratuberculosis (Mycobacterium flavum subspecies), Staphylococcus aureus (Staphylococcus aureus), methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes (Streptococcus pygogenes), Streptococcus pneumoniae (Streptococcus pneumniae), Streptococcus agalactiae (Streptococcus agalactiae), Haemophilus influenzae (Haemophilus fluuenzae), Haemophilus parainfluenzae (Haemophilus parainfluenzae), Moraxella catarrhalis (Moraxella catarrhalis), Klebsiella pneumoniae (Klebsiella pneuma), Escherichia coli (Escherichia coli), Neisseria aeruginosa (Pseudomonas aeruginosa), Acidobacter actinobacillus (Mycobacterium vaccaria), Streptococcus pneumoniae (Streptococcus pneumoniae), Mycobacterium tuberculosis (Streptococcus pneumoniae, Streptococcus pneumoniae (Streptococcus), Mycobacterium tuberculosis (Streptococcus), Streptococcus (Streptococcus pneumoniae), Mycobacterium tuberculosis (Streptococcus), Streptococcus (Streptococcus pneumoniae), Mycobacterium species (Streptococcus), Streptococcus (Streptococcus) and Mycobacterium tuberculosis (Streptococcus) Legionella (Legionella) species, Pneumocystis jejuni (Pneumocystis jiirovirus), influenza A virus, cytomegalovirus, rhinovirus, Enterococcus faecium (Enterococcus faecalis), Acinetobacter baumannii (Acinetobacter baumannii), Corynebacterium amycolatoides (Corynebacterium amycolatum), Enterobacter aerogenes (Enterobacter aeogens), Enterococcus faecalis (Enterococcus faecalis) CI 4413, Serratia marcescens (Serratia marcescens), Streptococcus equi (Streptococcus equi) and Candida albicans (Candida albicans).

The kit may comprise one or more probes for detecting DNA and/or RNA from one or more pathogens of interest, such as one or more of the following: mycobacterium tuberculosis, Mycobacterium avium subspecies paratuberculosis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter species, Bordetella pertussis, Neisseria meningitidis, Bacillus anthracis, Nocardia species, Actinomyces species, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella species, Pneumocystis Jacob, influenza A virus, cytomegalovirus, rhinovirus, enterococcus faecium, Acinetobacter baumannii, Corynebacterium amycolamycolata, Enterobacter aerogenes, enterococcus faecalis CI 4413, Serratia marcescens, Streptococcus equisimilis and Candida albicans. In embodiments, the one or more probes comprise an oligonucleotide labeled with a fluorescent dye. Different probes may be labeled with different dyes to enable simultaneous detection of multiple pathogens of interest.

5. Exemplary bead impact System

An exemplary bead impact system is shown in the drawings. The bead impact system, designated by the reference numeral (1) in fig. 1 to 3, comprises a sample tube (2) having an inner cavity (3) and a closable orifice (4). The sample tube is preferably constructed of plastic, but may also be constructed of another biologically inert material.

A sample fluid (5), which is assumed to contain microorganisms, can be filled into the lumen (3) of the sample tube (2) through the orifice (4) and removed from the lumen through the orifice (4). For closing the aperture (4), the sample tube has a closure (6) adapted to be transported to an open position and a closed position and a closure cap designed to contain an internal thread adapted to be screwed to an external thread provided by an edge region of the outer wall of the sample tube (2) which is integrated with the aperture (4).

In the inner cavity (3) beads (7), for example 2 grams of silica and/or zirconium beads, are further arranged, the largest dimension of which is on average about 100 μm. The beads (7) are available in the form of loose agglomerates, in which they are movable relative to each other. A freeze-dried blocking agent (8) is also disposed in the internal cavity (3).

Said beads (7) are useful for mechanically disrupting cell walls of microorganisms contained in the sample fluid (5) when the sample fluid (5) is filled into the lumen (3) and the bead impact tube is subjected to mechanical oscillations, such as ultrasonic oscillations. Which can be generated and transferable to the bead impact system (1) with a vibration generator not illustrated in detail in the drawings. Such a vibration generator is commercially available from MP Biomedicals under the name MPBio bead impactor. Due to the disruption of the cell wall, nucleic acids (e.g., DNA) contained in the microorganism are released and dissolved in the sample liquid (5).

6. Example study

6.1 recovery of pathogen DNA from sputum Using bead impact

Mycobacterium tuberculosis ("MTB") is an obligate pathogenic bacterial species of the Mycobacteriaceae family and is the cause of most cases of tuberculosis. It is primarily a pathogen of the mammalian respiratory system, which infects the lungs. Evidence suggests that mycobacteria are able to enter a non-replicating or dormant (sporulating) state during stress such as nutrient deprivation or hypoxia. Spore formation makes it challenging to lyse bacterial cells to extract bacterial DNA.

Further complicating the recovery of mycobacterial DNA is that the mycobacterial strain is often isolated from the sputum of infected patients. Sputum is thick, viscous and difficult to handle. Most sputum samples for analysis contain varying amounts of organic debris and a variety of contaminating, normal or short-time bacterial flora. Chemical decontamination/treatment is often used to reduce viscosity and kill contaminants while allowing recovery of the mycobacteria.

Samples suspected of containing MTB are potentially dangerous to the user. Thus, sputum samples may be pretreated to mitigate this risk by heating and/or inclusion of reagents suitable for inactivating microorganisms present in the sample. Inactivation of microorganisms (such as MTB) can be achieved by: heating (e.g., 90 ℃, 5min) to denature active proteins, enzymatically digest cell wall structures, mechanically disrupt to physically disrupt or inactivate cells, chemical treatment, or a combination thereof.

For consistent sample processing, sputum samples (typically collected in volumes between 1-10mL, 5-10mL, or more) may be initially liquefied to reduce their viscosity and non-homogeneity. Sputum samples pose particular challenges. MTB in sputum is one of the most challenging cell and sample types to be processed due to the lipid-rich hydrophobic cell wall of the acid-fast bacilli and the viscous heterogeneous nature of sputum. Standard extraction methods for sputum typically begin with a precipitation process, which often involves treatment with a mucolytic agent such as N-acetyl-L-cysteine (NALC), benzalkonium-phosphate (zephiran) -trisodium phosphate (Z-TSP), or algicidal amine (benzalkonium), followed by centrifugation, decantation, and resuspension.

Thus, when processing a highly viscous sample (such as sputum), the sample may be subjected to a chemical treatment to reduce viscosity so that subsequent processing steps are not hindered. In one embodiment, liquefaction of the sputum sample by chemical treatment with mucolytic agents is performed at 60 ℃ for 20 minutes. Sputum has viscosities ranging from about 100-6,000cP (mPas) with a shear rate at 90 s-1. The viscosity (which is measured in mPas) is determined by the shear strength divided by the shear rate. Preferably, the sample is liquefied to reduce the viscosity of the sputum by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

After resuspension, the sample is added to the bead impact system of the disclosure herein (as described in section 4.1) to lyse cells, as described in section 4.4. After lysis and nucleic acid extraction (see section 4.5), MTB DNA can be analyzed by PCR amplification of MTB sequences and MTB drug resistance can be analyzed by amplification of drug resistance genes. Sputum samples may be analyzed for other bacterial and viral pathogens including (but not limited to): staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter species, Bordetella pertussis, Neisseria meningitidis, Bacillus anthracis, Nocardia species, Actinomyces species, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella species, Pneumocystis jeikeium, influenza A virus, cytomegalovirus, and rhinovirus.

6.2 use of bead impact to recover DNA from veterinary pathogens

Mycobacterium avium subspecies paratuberculosis (MAP) is a veterinary pathogen, which is responsible for paratuberculosis or Johne's Disease (JD), a chronic granulomatous gastroenteritis in domestic and wild ruminants. MAP is transmitted directly in the herd through semen, milk, colostrum and uterus and indirectly through the oral route (the faecal route) through contaminated feed, fodder, forage grass, water and the like. JD has a devastating impact on livestock productivity (premature elimination and reduced milk production) and the livestock industry incurs significant economic losses. JD control work has been hampered by the continued presence of MAP in soil and water and by the discharge of subclinical and clinical livestock.

As mentioned in the previous section, spore formation makes it challenging to lyse mycobacterial cells to extract bacterial DNA. Thus, the bead impact system of the disclosure herein may be used to improve MAP recovery. For example, the bead impact system may be used to recover DNA from MAP present in milk, soil, excrement, semen, etc.

The milk sample may be directly treated using the bead impact system of the disclosure herein or may be pretreated, for example, to increase the concentration of MAP (if present) in the sample. An exemplary pretreatment method includes centrifuging a sample of milk to provide an emulsifiable concentrate fraction, a whey fraction, and a pellet. The cream fraction and the precipitation pellets can be poured together and added to the bead impact system of the disclosure herein for lysing cells.

The fecal sample can be pre-treated, for example, by preparing a fecal suspension, which can then be added to the bead impact system of the disclosure herein for lysing the cells. An exemplary fecal suspension can be prepared by: the fecal sample is mixed with water, saline or a buffer (e.g., sodium phosphate, phosphate buffered saline, Tris, etc.), the mixture is allowed to settle, the supernatant is separated and then centrifuged, and finally the pellet is resuspended in a suitable liquid (such as water, saline or buffer).

The soil sample may be suspended in a liquid (e.g., water, saline, or buffer) and then added to the bead impact system of the disclosure herein for lysis. The semen sample may be similarly pre-treated prior to bead impact by combining the semen sample with a quantity of water, saline, or buffer.

6.3 use of bead impact to recover pathogen DNA from wounds

For detection of wound infection, wound swabs may be cultured in saline solution, cell culture media, or sample preparation solutions from commercial kits. The swab is typically incubated for a period of about 5 minutes to 1 hour and more preferably 0.25 to 1 hour (e.g., 0.25 hour, 0.5 hour, or 0.75 hour). Suitable volumes of the solution are 200 μ L to 10mL and particular embodiments are 500 μ L, 1mL, 2mL, 5mL, 8mL or a range selected with boundaries of any two of the above embodiments (e.g., 1mL to 5mL, 500 μ L to 10mL, 200 μ L to 8mL, etc.). The solution may be periodically shaken or agitated to facilitate release of pathogenic cells from the swab. At the end of the incubation period, the swab can be squeezed and discarded.

This solution can then be placed in the bead impact system of the disclosure herein. Optionally, it is filtered and/or concentrated prior to bead impact.

Common wound-infecting pathogens include, but are not limited to, Escherichia coli, Pseudomonas aeruginosa, enterococcus faecium, Staphylococcus aureus (including MRSA), Klebsiella pneumoniae, Acinetobacter baumannii, Corynebacterium amycolatoides, Enterobacter aerogenes, enterococcus faecalis CI 4413, Serratia marcescens, Streptococcus equi, and Candida albicans. Thus, a wound swab sample may be analyzed for any of the above organisms (alone or in combination).

6.4 use of bead impaction to recover pathogen DNA from peritoneal dialysis solutions

Peritoneal dialysis is a treatment for patients with severe chronic kidney disease. This type of dialysis uses the peritoneum in the patient's abdomen as a membrane across which liquid exchanges self-blood with dissolved substances. The liquid is introduced through a fixed tube in the abdomen and then washed out to remove excess salt, uric acid and other waste products. Solutes are exchanged between the blood and the dialysate by permeation across the peritoneum. After a suitable time, the dialysate is removed from the peritoneum and discarded. In this way, the blood becomes equilibrated and prevents an infinite accumulation of terminal metabolites (such as uric acid) in the blood.

The main complication of peritoneal dialysis is infection due to the presence of a fixed tube in the abdomen. At least one infection site is in the peritoneal space, resulting in abdominal pain, turbid dialysis drainage fluid and finding pathogens on gram stain or on culture from within the catheter. Further, the tissue tract or the outer surface of the catheter is a common infection site from contact contamination. Infection in tissue passages is most often due to movement from the skin site. Infection can lead to peritonitis and in some cases death.

Peritoneal infections can be detected in peritoneal dialysis fluid removed from an individual undergoing peritoneal dialysis. Although Staphylococcus aureus and Pseudomonas aeruginosa are the cause of most infections, other bacteria (diphtheroids, anaerobic organisms, non-fermenting bacteria, streptococci, non-tuberculous mycobacteria, Legionella, yeasts and fungi) may also be involved. Thus, a peritoneal dialysis fluid sample can be analyzed for any of the above organisms (alone or in combination).

The peritoneal dialysis solution can be pretreated by filtration and optionally concentration, as described in section 4.3.

6.5 use of bead impact to recover DNA from wastewater contaminants

Example 1 of U.S. patent No. 9,290,796, which is directed to detecting problematic foaming and swelling bacterial species in biological wastewater treatment processes, describes the extraction of DNA from wastewater collected from wastewater treatment plants using a bead impact protocol. This bead impact scheme may be applicable to the bead impact systems incorporating the disclosure herein.

6.6 use of bead impact to recover DNA from food pathogens

The liquid food sample may be pretreated by filtration and/or centrifugation prior to bead impact, as described in section 4.3 above. For example, the beverage may preferably be pretreated by filtration to collect microorganisms that may be present. Microorganisms collected by filtration may optionally be washed and subjected to centrifugation prior to bead impact. With regard to detecting pathogens from solid food, such as meat or fish, a sample of the food may be wiped to collect microorganisms from the surface of the food. The microorganisms can then be washed from the swab and then subjected to bead impact.

In some examples, the following may be desirable: the food sample is pre-incubated for a period of time to promote the growth of pathogens (e.g., Salmonella) that may be present on or within the sample prior to bead impact. For example, solid food samples may be used with a laboratory mixer (e.g., a laboratory mixer)Circulator, forward Limited, uk) was milled and subsequently cultured to promote microbial growth (e.g. for 8 to 12 hours). A sample of the culture may then be subjected to bead impact.

6.7 use of bead impact to recover pathogen DNA from blood

The development of rapid molecular diagnostic tests for human infection is the highest priority of the world health organization to improve the health of the world population (Daar et al, 2002, nat. Genet.32: 229-. Severe blood infections are one of the leading causes of morbidity and mortality in hospitalized patients worldwide and one of the most important challenges in intensive care. For example, the recent incidence of sepsis in the united states has been estimated to be 240 cases per 100000 cases. The human and economic burden of sepsis is considerable (Grossi et al, 2006, surg. infection. (Larchmt) 7: S87-S91). Despite advances in infectious disease and intensive care management and numerous attempts to develop new treatments, sepsis mortality remains unacceptably high, ranging from 20% to 50%. Identifying signs of severe blood infection and/or severe sepsis and making an early and correct diagnosis are key to improving care and increasing survival. Indeed, rapid diagnosis can increase patient survival by reducing the time interval between sampling blood and administration of antimicrobial therapy.

For molecular techniques for detecting pathogens (e.g., PCR and DNA sequence analysis), proper isolation of pathogen DNA is critical to ensure successful detection. Various methods use lysis buffers, sometimes accompanied by physical disruption methods (such as bead impact) to enhance recovery of pathogen DNA from the blood.

The disclosure herein takes advantage of the presence of naturally occurring blockers in blood and provides a simplified method for isolating pathogen DNA from blood. For example, a commercially available blood collection tube (e.g., BD) may be usedBlood collection tube) collects blood from the subject. Many standard blood collection tubes are commercially available (some of them contain anticoagulants) and are color-coded for identification, for example, light purple cap tubes containing EDTA, light blue cap tubes containing sodium citrate, gray cap tubes containing potassium oxalate and sodium fluoride, or green cap tubes containing heparin are available. For example, blood that has been collected into an EDTA-containing collection tube may be subjected to bead blasting without dilution with any buffer or other additive using the bead blasting systems of the disclosure herein (containing a blocking agent) or conventional bead blasting systems (lacking a blocking agent). Following bead impact, standard methods can be used to recover pathogen DNA, such as those described in section 4.5. Optionally, the recovered pathogen DNA is subsequently analyzed, for example by the methods described in section 4.6.

The pathogens responsible for sepsis are usually bacteria or fungi. The general bacterial etiology of sepsis is gram-negative bacilli (e.g., escherichia coli, pseudomonas aeruginosa, elkenella rodensis (e.corrodens), and haemophilus influenzae). Other bacteria that may also contribute to sepsis are Staphylococcus aureus, Streptococcus (Streptococcus) species, Enterococcus (Enterococcus) species and Neisseria (Neisseria). Candida species are some of the most common sepsis-causing fungi. Thus, a blood sample may be analyzed for any of the above organisms (alone or in combination).

7. Examples of the embodiments

7.1 example 1: bead impact system caused by DNA loss

7.1.1 materials

The materials used in this study were:

bead impact tube: part number: ARY0007 OPS Diagnostics, 4.5mL refrigerated vial, with 2.0gm of 100 μm SiO₂Beads.

PD solution: contains glucose and Na⁺、Ca²⁺、Mg²⁺The balanced peritoneal dialysis solution of (1).

BE buffer: taigen Bioscience. Contains 1. mu.g of RNA/. mu.l.

Urea: an ACS stage.

7.1.2 methods

Two (2) mL of PD solution spiked with EDTA at a level of 10mM and Candida albicans at 5000CFU/mL were the standard substrates. Twelve (12) different combinations of urea and BE buffer were added to the sample, as shown in table 1 below:

all samples were impacted by the beads for 45 seconds. After the impact of the beads, use48 nucleic acid isolation System (Taigen Bioscience Corporation, Taipei, Taiwan) was usedManufacturer's reagents and protocols isolate DNA from the sample. DNA isolated from 2.0mL of each bead impact sample was eluted in 100. mu.l of TE (Tris EDTA) buffer. The isolated DNA was subjected to real-time PCR amplification and quantification to determine the amount of target DNA in each sample.

7.1.3 results

The optical density ratio (which is an index of the purity of the isolated DNA) and the corresponding DNA concentration of each sample are shown in table 2 below:

the yield of DNA reflected in the signal intensity obtained from imaging the signal from the array after PCR amplification and hybridization is illustrated graphically in fig. 6 and 7.

7.1.4 conclusion

In sample 1 (without the blocking agent), the recovery of Candida albicans DNA was only 5.5 ng/mL. The inclusion of urea as a blocking agent increases recovery by a factor of 3-4 and the inclusion of RNA as a blocking agent increases recovery by a factor of approximately 10-20, and yields approach the theoretical maximum. This increase in recovery resulted in an improvement in the amplification of the Candida albicans PCR gene.

7.2 example 2: extraction of DNA Using the bead impact System of the disclosure herein

7.2.1 preparation of the bead impact System of the disclosure herein

Stock blocker solutions containing 250mg/mL urea, 25mg/mL tetrasodium salt of ethylenediaminetetraacetic acid (EDTA), 125mg/mL adenosine monophosphate, 125mg/mL guanosine monophosphate, 125mg/mL cytosine monophosphate, 125mg/mL thymidine monophosphate, and 75mg/mL sodium lauroyl sarcosinate were prepared in TE (Tris/EDTA) buffer.

200 microliters of this stock solution was added to a 4.5-to 5-mL sample tube and dried by vacuum, resulting in a dry blocking agent that precipitated in the lower region of the inner wall of the sample tube. The dry blocker is expected to be stable for at least one year.

2.0 grams of 100 micron silica beads were then added to the sample tube.

7.2.2 bead impact systems Using the disclosure herein

6mL of urine and 6mL of peritoneal dialysis solution were filtered through a 0.4 micron sterile filter and then 10,000 colony forming units/mL of Staphylococcus aureus grown in a culture laboratory were added to the urine filtrate and the peritoneal dialysis solution filtrate.

2.9mL of spiked (spiked) urine and 2.9mL of spiked peritoneal dialysis solution were transferred to a sample tube prepared as described in section 7.2.1 or to a sample tube containing 2.0 grams of 100 micron silica beads but lacking the blocking agent.

The tube was capped and clamped in an MPBio bead impactor and run at full power for 30 seconds. The cap was removed and placed on the sample holder of a commercial DNA separation apparatus. 2mL of each sample was taken from each tube and then DNA extractant was addedDNA extraction is then performed. 100 microliters of DNA extract was obtained for each sample.

The amount of staphylococcus aureus DNA extracted in each sample was determined by real-time polymerase chain reaction according to the following: gillespie, et al, 2005, "Simulanous Detection of Mastitis Pathologens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by Multiplex Reaction-Time Polymerase Chain Reaction," J.Dairy Sci.88: 3510-3518. 1 microliter of each DNA extract was amplified in a 20 microliter reaction.

Samples processed using the sample tube lacking this blocking agent had a circulation threshold of 32 for urine and 38 for peritoneal dialysis fluid, indicating low DNA recovery.

Samples processed using the sample tube with the blocking agent had a circulation threshold of 31 for urine and 32 for peritoneal dialysis solution, indicating improved recovery of peritoneal dialysis solution under these conditions.

7.3 example 3: blood as blocking agent

7.3.1 overview

This study was performed to identify the most ideal bead impact conditions for blood samples. Negative blood samples were cultured by spiking with the pathogens staphylococcus aureus, escherichia coli and candida albicans. Positive PCR controls were performed. The concentration (genomic copy) of this control was set to the theoretical 100% recovery of the introduced pathogen. This study demonstrated that it is possible to extract DNA from blood pathogens using bead impact in the absence of additives (such as buffers, detergents, etc.).

7.3.2 materials

The materials specifically used in this study were:

bead impact tube: part number: ARY0007 OPS Diagnostics, 4.5mL refrigerated vial, with 2.0gm of 100 μm SiO₂Beads.

Blood: culture negative, from healthy donors.

Staphylococcus aureus, escherichia coli and candida albicans cultures: all from ATCC.

7.3.3 methods

A series of thirty-eight (38) spiked blood samples consisting of 3.0mL of blood and 15 μ L of saline broth containing the cultured pathogens were prepared. No material other than the saline broth containing the cultured pathogens was added to each blood sample. Use of-24 homogenizer (MP Biomedicals) at a rate of 6.5 m/s. Is then used48 nucleic acid extraction System (Taigen Bioscience Corporation) DNA was isolated from the sample using the manufacturer's reagents and protocols. DNA was amplified and hybridized to the array in 55 cycles of PCR, followed by washing and imaging. The amount of pathogens and bead impact time are shown in table 3 below:

for the three different pathogens at a given level of incorporation, the genomic output per μ l from the DNA isolation was calculated. This genomic copy value was used as a 100% efficiency target.

7.3.4 results

The results are illustrated graphically in fig. 8 to 10, which show DNA yields reflected in signal intensity obtained from imaging the signal from the array after PCR amplification and hybridization. Fig. 8 presents time series of candida albicans and 100% target. Figure 9 presents the staphylococcus aureus time series and 100% target. FIG. 10 presents E.coli time series and 100% target.

7.3.5 conclusion

For the three different pathogens, there was no evidence of signal loss due to binding of pathogen DNA to the beads. In all examples, at least 100% of theoretical values are exhibited. In 2 cases, more than 100% was recovered, which may be due to the presence of DNA from dead bacteria in the spiked.

8. Detailed description of the preferred embodiments

The disclosure herein is exemplified by the following detailed description.

1. A bead impact system comprising (i) a sample tube having a lumen accessible through an orifice, (ii) a bead, and (iii) a dry blocker, wherein the bead and dry blocker are disposed within the lumen of the sample tube.

2. The bead impact system according to embodiment 1, wherein the blocking agent comprises one or more dispersants, creatinine, one or more nucleotides, one or more oligonucleotides, or a combination thereof.

3. The bead impact system according to embodiment 2, wherein the blocking agent comprises one or more dispersants comprising urea, one or more guanidine salts, one or more lithium salts, one or more magnesium salts, one or more detergents, or a combination thereof.

4. The bead impact system of embodiment 3, wherein the blocking agent comprises one or more guanidine salts comprising guanidine isocyanate, guanidine hydrochloride, or a combination thereof.

5. The bead impact system according to embodiment 3 or embodiment 4, wherein the blocking agent comprises one or more lithium salts comprising lithium perchlorate, lithium acetate, or a combination thereof.

6. The bead impact system according to any one of embodiments 3-5, wherein the blocking agent comprises magnesium chloride.

7. The impact system of any of embodiments 3-6, wherein the blocking agent comprises one or more detergents comprising sodium lauryl sulfate, sodium lauroyl sarcosinate, polyoxyethylene (20) sorbitan monolaurate, or a combination thereof.

8. The bead impact system according to any one of embodiments 2-7, wherein the blocker comprises one or more nucleotides, comprising naturally occurring nucleotides, non-naturally occurring nucleotides, or a combination thereof.

9. The bead impact system according to embodiment 8, wherein the one or more nucleotides comprise one or more naturally occurring deoxyribonucleotides, one or more naturally occurring ribonucleotides, or a combination thereof.

10. The bead impact system according to embodiment 9, wherein the blocking agent comprises one or more deoxyribonucleotides comprising deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, deoxythymidine monophosphate, or a combination thereof.

11. The bead impact system according to embodiment 9 or embodiment 10, wherein the blocking agent comprises one or more ribonucleotides comprising adenosine monophosphate, guanosine monophosphate, cytosine monophosphate, thymidine monophosphate, or a combination thereof.

12. The bead impact system according to any one of embodiments 2-11, wherein the blocker comprises one or more oligonucleotides comprising ribonucleotides, deoxyribonucleotides, nucleotide analogs, or a combination thereof.

13. The bead impact system according to embodiment 12, wherein the blocker comprises one or more oligonucleotides each independently of the one or more oligonucleotides is 2 to 120 nucleotides long, 2 to 100 nucleotides long, 2 to 50 nucleotides long, 2 to 25 nucleotides long, 5 to 40 nucleotides long, 2 to 15 nucleotides long, 8 to 120 nucleotides long, 8 to 80 nucleotides long, 10 to 100 nucleotides long, 15 to 50 nucleotides long, 50 to 100 nucleotides long, or 100 to 120 nucleotides long.

14. The bead impact system according to embodiment 12 or embodiment 13, wherein the oligonucleotide comprises DNA and/or RNA.

15. The bead impact system according to any one of embodiments 1 to 14, wherein the lumen of the sample tube is at least partially coated with the layer or film of the blocking agent.

16. The bead impact system according to any one of embodiments 1 to 15, wherein some or all of said beads are partially or completely coated with a layer or film of the blocker.

17. The bead impact system according to any one of embodiments 1 to 16, comprising a powder comprising the blocking agent.

18. The bead impact system of embodiment 17, wherein the powder is a freeze-dried powder comprising the blocking agent.

19. The bead impact system according to any one of embodiments 1 to 18, wherein the beads are suitable for lysing bacteria, yeast, filamentous fungi, spores, plant cells or animal cells.

20. The bead impact system according to any one of embodiments 1-19, wherein the beads comprise mineral beads, ceramic beads, glass beads, metal beads, or a combination thereof.

21. The bead impact system of embodiment 20, wherein the beads comprise zirconium beads, zircon beads, zirconia beads, quartz beads, alumina beads, silicon carbide beads, ceramic beads, silica glass beads, stainless steel beads, chrome steel beads, or a combination thereof.

22. The bead impact system according to any one of embodiments 1 to 21, wherein the beads have a diameter in the range of 50 μm to 3 mm.

23. The bead impact system according to any one of embodiments 1-22, further comprising ethylenediaminetetraacetic acid (EDTA) and/or a sodium salt thereof disposed within the lumen of the sample tube.

24. The bead impact system according to any one of embodiments 1-22, further comprising creatinine disposed within the lumen of the sample tube.

25. A method for lysing cells contained in a liquid sample (e.g., to extract nucleic acids from the cells), comprising agitating the liquid sample within a sample tube of a bead impact system according to any one of embodiments 1-24 under conditions sufficient to lyse the cells.

26. A method for lysing cells contained in a liquid sample containing an exogenous blocking agent (e.g., to extract nucleic acids from the cells), comprising agitating the liquid sample within a bead impact system comprising a sample tube and beads in the absence of a lysis buffer and/or additive.

27. A method for lysing cells contained in a liquid sample that has not been incubated with a lysis buffer (e.g., to extract nucleic acids from the cells), comprising agitating the liquid sample within a bead impact system comprising a sample tube and beads with one or more blocking agents, optionally wherein the one or more blocking agents are blocking agents present in a sample tube according to any one of embodiments 1 to 24.

28. The method of embodiment 27, wherein the liquid sample comprises an endogenous blocker.

29. The method according to any one of embodiments 26 to 28, wherein the beads are suitable for lysing bacteria, yeast, filamentous fungi, spores, plant cells or animal cells.

30. The method of any one of embodiments 26 to 29, wherein the beads comprise mineral beads, ceramic beads, glass beads, metal beads, or a combination thereof.

31. The method of embodiment 30, wherein the beads comprise zirconium beads, zircon beads, zirconia beads, quartz beads, alumina beads, silicon carbide beads, ceramic beads, silica glass beads, stainless steel beads, chrome steel beads, or a combination thereof.

32. The method according to any one of embodiments 26 to 31, wherein the beads have a diameter in the range of 50 μ ι η to 3 mm.

33. The method of any one of embodiments 25-32, further comprising the step of placing the liquid sample within the sample tube prior to agitating the liquid sample.

34. The method according to any one of embodiments 25-33, wherein the agitating comprises subjecting the bead impact system to an oscillating motion.

35. The method of any one of embodiments 25 to 34, wherein the sample is a biological sample, an environmental sample, or a food product.

36. The method according to embodiment 35, wherein the sample is a biological sample selected from the group consisting of: blood, serum, saliva, urine, gastric fluid, digestive fluid, tears, feces, semen, vaginal fluid, interstitial fluid, fluid derived from neoplastic tissue, ocular fluid, sweat, mucus, cerumen, oil, glandular secretions, breath, spinal fluid, hair, nails, skin cells, plasma, fluid obtained from a nasal swab, fluid obtained from a nasopharyngeal wash, cerebrospinal fluid, a tissue sample, fluid or tissue obtained from a throat swab, fluid or tissue obtained from a wound swab, biopsy tissue, placental fluid, amniotic fluid, peritoneal dialysis fluid, umbilical cord blood, lymph fluid, cavity fluid, sputum, pus, microbiota, meconium, breast milk, or a sample treated, extracted, or fractionated from any of the above.

37. The method of embodiment 36, wherein the biological sample is urine, sputum, or a sample from the processing, extraction, or fractionation of urine.

38. The method of embodiment 36, wherein the biological sample is sputum or a sample from which sputum is processed, extracted, or fractionated.

39. The method of embodiment 36, wherein the biological sample is a wound swab or a sample obtained by processing, extracting or fractionating a wound swab.

40. The method of embodiment 36, wherein the biological sample is blood or a sample from which blood is processed, extracted, or fractionated.

41. The method of embodiment 40, wherein said blood comprises an anticoagulant.

42. The method of embodiment 41, wherein the anticoagulant is EDTA.

43. The method of embodiment 41, wherein the anticoagulant is citrate (e.g., sodium citrate).

44. The method of embodiment 41, wherein the anticoagulant is an oxalate salt (e.g., potassium oxalate).

45. The method according to embodiment 41, wherein the anticoagulant is heparin (e.g., sodium heparin).

46. The method of embodiment 41, further comprising transferring blood from the blood collection tube to a sample tube prior to agitating, optionally wherein all or only a portion of the blood in the blood collection tube is transferred to the sample tube.

47. The method of embodiment 46, wherein the blood collection tube comprises an anticoagulant.

48. The method of embodiment 47, wherein said anticoagulant is EDTA.

49. The method of embodiment 47, wherein the anticoagulant is citrate (e.g., sodium citrate).

50. The method of embodiment 47, wherein the anticoagulant is an oxalate salt (e.g., potassium oxalate).

51. The method of embodiment 47, wherein the anticoagulant is heparin (e.g., sodium heparin).

52. The method of any one of embodiments 46-51, further comprising collecting blood into a blood collection tube prior to transferring the blood to a sample tube.

53. The method of embodiment 52, wherein prior to said collecting, said blood collection tube comprises an anticoagulant.

54. The method of embodiment 52 or embodiment 53, further comprising transporting and/or storing the blood in a blood collection tube prior to transferring the blood to the sample tube.

55. A method for lysing cells contained in a blood sample that has not been incubated with a lysis buffer (e.g., to extract nucleic acids from the cells), comprising (a) combining beads with blood in a blood collection tube containing blood, and (b) agitating the blood in the presence of the beads, thereby lysing the cells contained in the blood sample.

56. The method of embodiment 55, wherein the beads are added to a blood collection tube containing blood.

57. The method of embodiment 55, wherein the blood is collected into a blood collection tube comprising beads.

58. The method according to any one of embodiments 55-57, wherein the blood collection tube comprises an anticoagulant.

59. The method of embodiment 58, wherein said anticoagulant is EDTA.

60. The method of embodiment 58, wherein said anticoagulant is citrate (e.g., sodium citrate).

61. The method of embodiment 58, wherein the anticoagulant is an oxalate salt (e.g., potassium oxalate).

62. The method of embodiment 58, wherein the anticoagulant is heparin (e.g., sodium heparin).

The method of embodiment 36, wherein the biological sample is peritoneal dialysis solution or a sample that is treated, extracted or fractionated from peritoneal dialysis solution.

64. The method according to embodiment 35, wherein the sample is an environmental sample selected from the group consisting of: soil, groundwater, surface water, wastewater, or a sample treated, extracted, or fractionated from any of the above.

65. The method according to any one of embodiments 25 to 64, wherein the cells comprise one or more pathogens.

66. The method of embodiment 65, wherein the one or more pathogens comprise one or more bacterial pathogens, viral pathogens, fungal pathogens, or combinations thereof.

67. The method according to embodiment 65 or embodiment 66, wherein the one or more pathogens comprise one or more of the following: mycobacterium tuberculosis, Mycobacterium avium subspecies paratuberculosis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter species, Bordetella pertussis, Neisseria meningitidis, Bacillus anthracis, Nocardia species, Actinomyces species, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella species, Pneumocystis Jacob, influenza A virus, cytomegalovirus, rhinovirus, enterococcus faecium, Acinetobacter baumannii, Corynebacterium amycolamycolata, Enterobacter aerogenes, enterococcus faecalis CI 4413, Serratia marcescens, Streptococcus equisimilis and Candida albicans.

68. The method of embodiment 65, wherein the sample is sputum or a sample from which sputum is processed, extracted, or fractionated, the one or more pathogens comprising one or more of: mycobacterium tuberculosis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter species, Bordetella pertussis, Neisseria meningitidis, Bacillus anthracis, Nocardia species, Actinomyces species, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella species, Pneumocystis jejuni, influenza A virus, cytomegalovirus, and rhinovirus.

69. The method of embodiment 68, wherein the one or more pathogens comprise mycobacterium tuberculosis.

70. The method of embodiment 65, wherein the sample is breast milk or semen or a sample obtained by treating, extracting or fractionating breast milk, soil, excrement or semen, and the one or more pathogens comprises Mycobacterium avium subspecies paratuberculosis.

71. The method of embodiment 65, wherein the sample is a wound swab or a sample obtained by processing, extracting or fractionating a wound swab, the one or more pathogens comprising one or more of: escherichia coli, Pseudomonas aeruginosa, enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Corynebacterium amycolata, Enterobacter aerogenes, enterococcus faecalis CI 4413, Serratia marcescens, Streptococcus equi and Candida albicans.

72. The method according to embodiment 65, wherein the sample is peritoneal dialysis solution or a sample that is treated, extracted or fractionated from peritoneal dialysis solution, the one or more pathogens comprising Staphylococcus aureus and/or Pseudomonas aeruginosa.

73. The method of any one of embodiments 25-72, wherein the liquid sample comprises cells from a plurality of species.

74. The method according to any one of embodiments 25 to 73, further comprising recovering the liquid sample from the sample tube after cell lysis.

75. The method of embodiment 74, further comprising purifying the nucleic acid from the recovered liquid sample.

76. The method according to any one of embodiments 25 to 75, further comprising analyzing one or more of the nucleic acids.

77. The method according to embodiment 76, wherein the analyzing is performed using a microarray or by sequencing the one or more nucleic acids.

78. The method according to embodiment 76 or embodiment 77, wherein the target sequence is amplified before performing the assay.

79. The method of embodiment 78, wherein the target sequence is amplified by PCR.

80. The method according to any one of embodiments 25 to 79, wherein the nucleic acid comprises DNA.

81. The method according to any one of embodiments 25 to 80, wherein the nucleic acid comprises RNA.

82. A kit for lysing cells contained in a liquid sample (e.g., to extract nucleic acids from the cells), comprising a bead impact system according to any one of embodiments 1 to 24 and optionally: (i) one or more components for preparing the liquid sample, (ii) one or more oligonucleotides for amplifying said nucleic acids, (iii) one or more probes for detecting one or more of said nucleic acids, or (iv) any combination of (i) to (iii).

83. The kit of embodiment 82, comprising one or more components for preparing the liquid sample, and wherein the one or more components for preparing the liquid sample comprise water, saline, a buffer, a filter, or a combination thereof.

84. A kit according to embodiment 82 or embodiment 83, comprising one or more oligonucleotides for amplifying one or more of said nucleic acids.

85. A kit according to any one of embodiments 82 to 84 comprising one or more probes for detecting one or more of said nucleic acids.

86. A kit for obtaining a bead impact system according to any one of embodiments 1 to 24, comprising a sample tube, a bead and a dry blocking reagent.

87. A kit according to embodiment 86, wherein said bead and/or dry blocking reagent are contained in the kit separately from the sample tube.

88. A kit according to embodiment 86, wherein said beads and/or dry blocking reagent are contained in the kit within the sample tube.

89. The kit of any one of embodiments 86 to 88, further comprising one or more components for preparing a sample containing cells for extracting nucleic acids using the bead impact system, one or more oligonucleotides for amplifying one or more nucleic acids, one or more probes for detecting one or more nucleic acids, or a combination thereof.

90. A bead impact system (1) comprising a sample tube comprising a container means (2), the container member (2) having an inner cavity (3), an orifice (4), the orifice (4) being for filling the inner cavity (3) with a sample liquid (5) potentially containing microorganisms, and a closure (6), the closure (6) being for closing the orifice (4), wherein a plurality of macroscopic mineral particles (7) are arranged in the inner cavity (3), said mineral particles (7) being adapted to mechanically disrupt cell walls (5) of microorganisms contained in the sample liquid (5) when the sample liquid (5) is filled into the inner cavity (3) and the bead impact system (1) is subjected to mechanical oscillations, characterized in that a blocking agent (8) comprising urea and/or at least one guanidine salt and/or at least one detergent is arranged in dry form in the lumen (3) of the sample tube.

91. The bead impact system (1) according to embodiment 90, characterized in that the blocking agent (8) comprises deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, deoxythymidine monophosphate or a mixture of at least two of these monophosphates.

92. Bead impact system (1) according to embodiment 90 or 91, characterized in that the blocking agent (8) comprises adenosine monophosphate, guanosine monophosphate, cytosine monophosphate, thymidine monophosphate or a mixture of at least two of these monophosphates.

93. Bead impact system (1) according to any one of embodiments 90 to 92, characterized in that the monophosphate comprised in at least one of the blocking agents (8) is an oligonucleotide of at least 2 and at most 120 nucleotides.

94. The bead impact system (1) according to any one of embodiments 90 to 93, characterized in that the at least one cleaning agent comprises sodium lauryl sulfate, sodium lauroyl sarcosinate and/or polyoxyethylene (20) sorbitan monolaurate.

95. Bead impact system (1) according to any one of embodiments 90 to 94, characterized in that the dry blocker is loosely arranged in the inner cavity (3) of the container member (2), preferably in powder form.

96. The bead impact system (1) according to any one of embodiments 90 to 95, characterized in that the inner wall of the container member (2) is at least partially coated with a layer of a blocking agent or a thin film of a blocking agent.

97. A bead impact system (1) according to any one of embodiments 90 to 96, characterized in that ethylenediaminetetraacetic acid (EDTA) and/or a sodium salt thereof is arranged in the lumen (3) of the sample tube.

98. Bead impact system (1) according to any one of embodiments 90 to 97, characterized in that creatinine is arranged in the lumen (3) of the sample tube.

99. Use of a bead impact system (1) according to any one of embodiments 90 to 98 for extracting deoxyribonucleic acid and/or ribonucleic acid from a microorganism.

100. A method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms, wherein a sample liquid (5) is provided which is supposed to contain said microorganisms, wherein a plurality of mineral particles (7) which are movable with respect to each other are introduced into the sample liquid (5), and the sample liquid (5) in which the particles (7) are contained is shaken so that the particles (7) are capable of mechanically disrupting the cell walls of the microorganisms contained in the sample liquid (5), characterized in that a blocking agent (8) comprising urea and/or at least one guanidinium salt and/or at least one cleaning agent is introduced into the sample liquid (5) before the particles (7) are shaken and/or while the particles (7) are shaken.

101. The method according to embodiment 100, characterized in that the amount of urea is matched to the amount of the sample liquid (5) such that the concentration of urea dissolved in the sample liquid (5) ranges between 10 and 100 g/l, in particular between 20 and 50 g/l, and preferably between 25 and 35 g/l.

102. The method according to embodiment 100 or 101, characterized in that before and/or while the particles (7) are being shaken, at least one of the following monophosphates is introduced into the sample liquid (5): deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate and deoxythymidine monophosphate.

103. The method according to any one of embodiments 100 to 102, characterized in that before and/or while the particles (7) are being shaken, at least one of the following monophosphates is introduced into the sample fluid (5): adenosine monophosphate, guanosine monophosphate, cytosine monophosphate, thymidine monophosphate.

104. The method according to any of embodiments 100 to 103, characterized in that creatinine and/or ethylenediaminetetraacetic acid (EDTA) and/or a sodium salt thereof is introduced into the sample liquid (5) before and/or while the particles (7) are shaken.

9. Citations of documents

All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document were individually indicated to be incorporated herein by reference for all purposes. In the event of a discrepancy between the teachings of one or more of the documents incorporated herein and the disclosure herein, the teachings of this specification should be used.

Claims (33)

1. A method for lysing cells contained in blood, comprising agitating the blood or a sample treated, extracted, or fractionated from the blood within a bead impact system comprising (i) a sample tube or blood collection tube and (ii) beads in the absence of a lysis buffer.

2. The method of claim 1, wherein the beads are suitable for lysing bacteria, yeast, filamentous fungi, spores, plant cells, or animal cells.

3. The method of claim 1 or claim 2, wherein the beads comprise mineral beads, ceramic beads, glass beads, metal beads, or a combination thereof.

4. The method of claim 1 or claim 2, wherein the beads comprise zirconium beads, zircon beads, zirconia beads, quartz beads, alumina beads, silicon carbide beads, ceramic beads, silica glass beads, stainless steel beads, chrome steel beads, or a combination thereof.

5. A method according to any one of claims 1 to 4, wherein the beads have a diameter in the range 50 μm to 3 mm.

6. The method of any one of claims 1 to 5, further comprising the step of collecting blood into a blood collection tube.

7. The method of any one of claims 1 to 6, comprising placing the blood or a sample processed, extracted or fractionated from the blood in a sample tube prior to agitation.

8. The method of any one of claims 1 to 7, wherein the agitating comprises subjecting the bead impact system to an oscillating motion.

9. The method of any one of claims 1 to 8, wherein the blood comprises an anticoagulant.

10. The method of claim 9, wherein the anticoagulant is EDTA.

11. The method of claim 9, wherein the anticoagulant is citrate (e.g., sodium citrate).

12. The method of claim 9, wherein the anticoagulant is an oxalate salt (e.g., potassium oxalate).

13. The method of claim 9, wherein the anticoagulant is heparin.

14. The method of any one of claims 1-13, wherein the blood is agitated.

15. The method of any one of claims 1-13, wherein the sample is agitated from blood processing, extraction, or fractionation.

16. The method of any one of claims 1 to 15, wherein the bead impact system comprises a sample tube and beads.

17. The method of any one of claims 1-15, wherein the bead impact system comprises a blood collection tube and beads.

18. The method of any one of claims 1 to 17, wherein the cells comprise one or more pathogens.

19. The method of any one of claims 1 to 18, wherein the blood comprises cells from a plurality of species.

20. The method of any one of claims 1 to 19, further comprising recovering the lysate from the sample tube after cell lysis.

21. The method of claim 20, further comprising purifying nucleic acids from the lysate.

22. The method of claim 21, further comprising analyzing the nucleic acid, optionally wherein:

a) the analysis is performed using a microarray or by sequencing one or more nucleic acids;

b) the target sequence is amplified before performing the analysis, optionally by PCR.

23. The method of claim 21 or claim 22, wherein the nucleic acid comprises DNA and/or RNA.

24. A kit for lysing cells present in a blood sample comprising a blood collection tube and beads suitable for bead impact.

25. The kit of claim 24, wherein the blood collection tube comprises an anticoagulant.

26. The kit of claim 25, wherein the anticoagulant is EDTA.

27. The kit of claim 25, wherein the anticoagulant is citrate.

28. The kit of claim 25, wherein the anticoagulant is oxalate.

29. The kit of claim 25, wherein the anticoagulant is heparin.

30. The kit of any one of claims 24 to 29, wherein the beads are packaged separately from the blood collection tube.

31. A method of lysing cells contained in blood, comprising combining blood with the beads and blood collection tube in the kit of claim 30, and agitating the tube until the cells contained in the blood are lysed.

32. The kit of any one of claims 24 to 29, wherein the beads are packaged in the blood collection tube.

33. A method of lysing cells contained in blood, comprising combining blood with the beads and blood collection tube in the kit of claim 32, and agitating the tube until the cells contained in the blood are lysed.