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WO2016024263A1 - Procédés d'isolement d'adn microbien à partir d'un échantillon de sang - Google Patents

Procédés d'isolement d'adn microbien à partir d'un échantillon de sang Download PDF

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WO2016024263A1
WO2016024263A1 PCT/IL2015/050343 IL2015050343W WO2016024263A1 WO 2016024263 A1 WO2016024263 A1 WO 2016024263A1 IL 2015050343 W IL2015050343 W IL 2015050343W WO 2016024263 A1 WO2016024263 A1 WO 2016024263A1
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dna
microbial
sample
solution
aqueous solution
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PCT/IL2015/050343
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English (en)
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Aryeh Gassel
Yaron SUISSA
Raphael Gassel
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Molecular Detection Israel Ltd.
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Publication of WO2016024263A1 publication Critical patent/WO2016024263A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • a blood sample containing higher eukaryotic cells particularly wherein the DNA-containing microorganisms are present at a concentration significantly lower than the eukaryotic cells in the sample.
  • Sepsis is a life-threatening illness in which inflammatory cytokines are released by the body in response to the presence of infectious bacteria or other pathogens.
  • the worldwide annual incidence of sepsis in 2013 was estimated to be 26 million cases.
  • WO 2014/076706 presents rapid real-time PCR-based assays for differential identification of bacterial and fungal pathogens, and antibiotic -resistance genes from as few as two copies of microbial DNA.
  • Methods for isolating microbial DNA from a whole blood sample available at the time of initial presentation of sepsis symptoms would enable use of these or similar molecular assays to provide data for patient treatment decisions in a clinically relevant time frame.
  • Methods have been developed for extracting DNA from intact microbial cells present in a non-blood sample and for purifying said microbial DNA so as to enable amplification by PCR.
  • none of the previously developed methods are sufficiently efficient to detect the potentially low concentration of a microorganism in a sepsis or pre-sepsis patient.
  • a common method comprises a first step of enzymatic lysis of the microbial cells, followed by extraction of the DNA, binding the extracted DNA to magnetic beads,
  • a 4 ml blood sample from a patient with sepsis may contain as few as 4 colony forming units (CFU) of microbial cells, while also containing about 16,000 to 44,000 white blood cells (leukocytes) and about 600,000 to 1.6 million platelets (thrombocytes).
  • CFU colony forming units
  • the high amount of non-microbial DNA competes with the limited microbial DNA during the bead binding process and increases background in subsequent DNA amplification.
  • the presence of heme from hemoglobin in red blood cells (erythrocytes) strongly decreases the activity of enzymatic lysis and DNA polymerase.
  • a whole blood sample may contain as few as a single copy of pathogen DNA per milliliter, in order to obtain the minimally required 2 copies of microbial DNA from the sample, such DNA isolation methods must be sufficiently sensitive to provide a minimum 50% yield of microbial DNA from a starting sample quantity of 4 ml or more of blood.
  • centrifugation and filtration can have several disadvantages. They are designed for processing microfluidic quantities of a sample and are difficult to apply to samples of 4 ml or larger, discussed above as required for effective identification of blood stream pathogens and antibiotic resistance. Centrifugation also requires a separate instrument that is difficult to incorporate into an automated process, thereby increasing the risk of sample contamination. Filtration entails cells becoming trapped either on the surface of a size exclusion filter or within the structure of a depth filter followed by recovery using elution or a reverse flow washing process. Filtration is also problematic in the case of limited quantities of microbial cells being present in 4ml or more of a blood sample, wherein the yield of microbial cells is impaired by the presence of viscous milliliter volumes of blood fluid.
  • microorganism-specific antibodies to specifically capture microorganisms from a whole blood. This method is both expensive and may not be appropriate for sepsis diagnosis, as antibodies tend to be organism or species- specific and may not be able to capture all varieties of pathogen cells that might be present in the blood.
  • the method involves providing an aqueous solution, which is a diluted or substantially undiluted blood sample from a higher eukaryotic organism; adding a lysis reagent to the solution; incubating the solution for a time period sufficient to lyse the higher eukaryotic cells in the blood sample and release therefrom higher eukaryotic DNA, while still preserving the integrity of microbial cells in the blood sample; adding to the aqueous solution an insoluble magnetic solid surface, and at least one of a water soluble polymer and an inorganic salt in a quantity sufficient to cause the microbial cells to displace onto the magnetic solid surface; separating the microbial cells displaced onto the insoluble magnetic solid surface from the aqueous solution, without the use of centrifugation or filtration; washing the separated microbial cells one or more times with a wash solution that does not extract microbial DNA from the aqueous solution.
  • SEQ ID NOs 1 and 2 are forward and reverse PCR primers for amplification of the Vancomycin-Resistant Enterococcus vanA gene.
  • SEQ ID NO 3 is a vanA probe oligonucleotide.
  • SEQ ID NOs 4 and 5 are forward and reverse PCR primers for amplification of the MRS A mecA gene.
  • SEQ ID NO 6 is a mecA probe oligonucleotide.
  • Amplification When used in reference to a nucleic acid, any technique that increases the number of copies of the entire sequence of a nucleic acid molecule, or a portion thereof, in a sample or specimen.
  • An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
  • Aqueous sample Any sample that is water-based; does not include alcohols or other water soluble organic solvents.
  • an aqueous sample or solution contains a blood sample mixed into it at 10%, 20%, 30%, 40%, 50% or even greater concentration.
  • the aqueous sample is a 100% blood sample that has been minimally processed from the time of extraction from a subject.
  • Blood sample Any blood sample drawn from a subject.
  • a blood sample can be a 100% whole blood sample.
  • a blood sample can be diluted in a suitable aqueous solution to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% of the starting sample concentration.
  • a blood sample need not be whole blood, so long as a microbial-containing component of the original whole blood is retained.
  • the blood sample is "substantially undiluted" such as those examples wherein the sample is collected in or transferred to a collection receptacle containing small quantities of a reagent such as heparin, required for maintenance of the sample in liquid state and/or preservation of the blood sample.
  • Placement in direct physical association Includes both in solid and liquid form.
  • Cycle Threshold The cycle number in a real-time PCR reaction at which the slope of detected fluorescence passes a fixed threshold. Used in the art for determining a positive indication of a detectable quantity of nucleic acid in a sample. In particular embodiments Ct can be used for comparing the relative quantity of nucleic acid in a series of samples amplified in parallel during a real-time PCR run. The lower the Ct value, the more nucleic acid is presumed to have been present in the sample.
  • DNA Purification The separation of DNA from other cellular components. The process of DNA purification does not require 100% purity; however, the end-product of DNA purification is DNA that may be used in downstream applications such as DNA sequencing, PCR, and the like.
  • DNA purification involves binding DNA released and/or extracted from a cell to a specially coated magnetic bead, a silica membrane, or a similar agent that binds DNA to an immobilized support; washing away or otherwise removing other components of the sample that was with the DNA; and (c) separating the isolated DNA with an elution buffer to separate the DNA from the support and draw it into a buffer solution in preparation for DNA amplification and/or identification, as for example, real- time PCR.
  • Detect To determine if an agent (such as a signal or particular nucleotide nucleic acid probe) or a cell (such as a microbial cell) is present or absent. In some examples, this can further include quantification.
  • an agent such as a signal or particular nucleotide nucleic acid probe
  • a cell such as a microbial cell
  • Determining expression of a nucleic acid Detection of a level of expression in either a qualitative or a quantitative manner. In one example, it is the detection nucleic acid specific to a particular microbial pathogen. Similar procedures, such as PCR, can also be used to detect the quantity of DNA isolated from a sample.
  • Higher eukar otic cell A eukaryotic cell of a higher state of evolutionary
  • the higher eukaryotic tissue is from a mammal. In more specific embodiments, the higher eukaryotic tissue is from a human, and includes those cells found within a human blood sample.
  • Infectious disease A disease caused by a pathogen, such as a fungus, parasite, bacterium or virus.
  • Isolated An "isolated" biological component (such as a nucleic acid, protein, cell (or plurality of cells), tissue, or organelle) has been substantially separated or purified away from other biological components of the organism in which the component naturally occurs for example other tissues, cells, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. Such isolated materials need not be 100% pure, but must be sufficiently pure from inhibitors of enzymes and other reagents used in downstream detection processes, such as PCR in the case of DNA.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule.
  • Specific, non-limiting examples of labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
  • Lysis The breaking down of a cell, often by chemical, enzymatic or mechanical mechanisms that compromise its integrity and allow for the extraction of DNA contained therein.
  • Microbe, Microbial cell, Microorganism refers to a diverse group of organisms, which exist in nature autonomously as a single cell or as a cell cluster, and therefore differ from higher eukaryotic cells (such as animal cells in a tissue, e.g. tissue cells of a mammal), that do not occur in nature as a single cell, but exclusively as constituents of multicellular organisms.
  • eukaryotic cells such as animal cells in a tissue, e.g. tissue cells of a mammal
  • microbial cells can be for example prokaryotic cells, such as bacteria or archaebacteria.
  • microbial cells can be eukaryotic cells, such as yeasts, lower and higher fungi or protozoa.
  • a prokaryotic cell or prokaryote can mean any cell or any organism belonging to the phylogenetic group of the Archaea or Bacteria (cf. Balows, Truper, Dworkin, Harder, Schleifer: The Procaryotes Chapter 142, pages 2696-2736 (1992)).
  • Prokaryotic cells have clear differences from eukaryotic cells, which are reflected in structural characteristics of cellular organelles, a cell wall and the like. These characteristics are well known to a person skilled in the art, and also form the basis for distinctions between prokaryotic cell types.
  • microbial cell or microorganism as used herein includes various genera of Gram-positive and Gram-negative bacteria, for example pathogenic bacteria of the genera Mycobacterium,
  • the microbial cells or microorganisms can also include eukaryotic cells.
  • eukaryotic microbial cells are fungal cells.
  • the fungi include pathogenic fungi of the genera Aspergillus (e.g. A. fumigatus, A. niger, A. flavus, A. nidulans), Basidiobolus (e.g. B. microsporus, B. ranarum), Cephalosporium (e.g. C. chrysogenum, C. coremioides, C. diospyri, C.
  • Aspergillus e.g. A. fumigatus, A. niger, A. flavus, A. nidulans
  • Basidiobolus e.g. B. microsporus, B. ranarum
  • Cephalosporium e.g. C. chrysogenum, C. coremioides, C. diospyri, C.
  • Pathogen A microorganism capable of causing a disease condition in a host, which is in some embodiments a human host and is in other embodiments a mammalian host, or in other embodiments an avian host.
  • Preserving the integrity Indicates that the structural integrity of at least the majority of the microbial or pathogen cells is preserved to the degree that microbial DNA remains within the cells.
  • a microbial cell whose integrity is preserved can also be referred to as an intact microbial cell. In certain embodiments, essentially all the microbial or pathogen cells in a sample remain intact.
  • Quantitative real time PCR A method for detecting and measuring products generated during each cycle of a polymerase chain reaction (PCR), which products are proportionate to the amount of template nucleic acid present prior to the start of PCR.
  • the information obtained, such as an amplification curve, can be used to quantitate the initial amounts of template nucleic acid sequence.
  • Sepsis A life-threatening illness in which inflammatory cytokines are released by the body in response to the presence of infectious bacteria or other pathogens.
  • a subject can present to a medical professional with symptoms indicating a sepsis-related infection.
  • the infectious microorganism bacteria or other pathogen
  • the infectious microorganism may be present in sufficiently low concentrations that symptoms have not developed.
  • such a patient “has" sepsis, and the methods described herein are provided for the isolation of the microorganism and its DNA to allow for use of sepsis detection methods.
  • Solid support Any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • Numerous and varied solid supports are known to those in the art and include, without limitation, nitrocellulose, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, membranes, and microparticles (such as latex particles). Except as otherwise physically constrained, a solid support may be used in any suitable shapes, such as beads, films, sheets, strips, or plates.
  • the method involves providing an aqueous solution, which is can be a diluted or substantially undiluted blood sample from a higher eukaryotic organism; adding a lysis reagent to the solution; incubating the solution for a time period sufficient to lyse the higher eukaryotic cells in the blood sample and release therefrom higher eukaryotic DNA, while still preserving the integrity of microbial cells in the blood sample; adding to the aqueous solution an insoluble magnetic solid surface, and at least one of a water soluble polymer and an inorganic salt in a quantity sufficient to cause the microbial cells to displace onto the magnetic solid surface; separating the microbial cells displaced onto the insoluble magnetic solid surface from the aqueous solution, without the use of centrifugation or filtration; washing the separated microbial cells one or more times with a wash solution that does not extract microbial DNA from
  • the starting volume of the aqueous solution is at least 4 milliliters.
  • the aqueous solution is a substantially undiluted blood sample.
  • the microorganism is a bacterium or a fungus.
  • the lysis reagent includes a non-ionic detergent.
  • the lysis reagent further includes an agent capable of digesting protein, such as Proteinase K, and wherein incubating the aqueous solution includes a time period sufficient to digest at least some proteins released from the higher eukaryotic cells into the aqueous solution; for example, an incubation at a temperature between about 37 °C to about 60 °C, inclusive
  • the water soluble polymer is polyethylene glycol (PEG), which can be used in particular examples at a final concentration between 2%-10% w/v.
  • PEG polyethylene glycol
  • the inorganic salt is at least one salt selected from the group consisting of: sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.
  • the magnetic solid surface includes uncoated magnetite particles, and can be particles that are spherical or irregular in nature, and which can be 0.1 micron to 10 micron in diameter.
  • separating the microbial cells comprises using a magnet to immobilize the insoluble magnetic solid surface, and removing substantially all of the aqueous solution.
  • the wash solution comprises at least one of a water soluble polymer and an inorganic salt, including at least one salt selected from the group consisting of: sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.
  • the water soluble polymer in the wash solution is polyethylene glycol (PEG).
  • washing the separated microbial cells includes incubating the separated cells in the wash solution at a temperature between about 37 °C to about 60 °C, inclusive.
  • the described methods can be particularly useful in diagnostic assays to determine the presence of sepsis-producing microorganisms in a subject. Accordingly, in particular embodiments of the described methods, the quantity of higher eukaryotic cells in the aqueous solution comprising a blood sample is at least 100 fold greater than the quantity or suspected quantity of microbial cells.
  • the solution includes NaOH or KOH.
  • the surface-bound DNA is incubated at a temperature greater than room temperature, such as at least 65°C for at least five minutes.
  • microbial DNA such as bacterial or fungal DNA
  • methods for isolating microbial DNA such as bacterial or fungal DNA
  • methods for preparing the isolated microbial DNA for DNA amplification The described methods enable early and accurate detection and identification of a pathogen in a sample.
  • the methods described herein are particularly useful for detecting low copy number polynucleotides such as those of pathogens in higher eukaryotic cell-containing whole blood samples of subjects suspected to have sepsis. Often in such samples, the volume of blood that can be drawn is limited, a large number of polynucleotide targets need to be queried, and the amount of non-pathogen DNA is much greater than the amount of pathogen DNA. In these circumstances, it is highly beneficial to remove the non-pathogen DNA and PCR inhibiting substances while isolating at least approximately 50% of the pathogen DNA in the sample.
  • microbial DNA isolation is performed by:
  • Step B and Step C entail adding to an initial aqueous solution, such as a 100% blood solution or dilution thereof, one or more additional chemicals or reagents. While it is critical to the method that the water soluble polymer and/or salt added to the solution in Step C be preceded by Step B, the sequence with which the chemical components of the reagent of Step B and the magnetic solid surface of Step C are added to the initial solution is not critical to the successful performance of the method. Accordingly, in some embodiments, the reagent of Step B is added to the initial solution before adding the solid surface containing compound of Step C. In other embodiments, the solid surface of Step C is added to the initial solution before the reagent of Step B is added. In still further embodiments, some of the chemical components of the reagent of Step B are added separately from others in various sequence.
  • adding a reagent to an initial aqueous solution involves having the initial aqueous solution present in one container and having the reagent present in a second container.
  • the process of "adding" one to the other equally includes transferring some or all of the contents of the first container to the second container or alternatively, transferring some or all of the contents of the second container to the first container.
  • the magnetic solid surface may be a mesh, or filter that permits aqueous solutions to pass through and yet contains solid surface onto which microbial cells can bind.
  • the solid surface incorporates material that exhibits permanent magnetic behavior.
  • the solid surface is composed of material that exhibits magnetic behavior only when subjected to a magnetic field.
  • the magnetic solid surface has a high specific surface area and hence, preferably the solid surface is provided by beads.
  • the term 'beads' includes, but is not limited to, insoluble magnetic particles that are spherical or irregular in nature and of size ranging from 0.1 micron to 10 micron in diameter. These include both (a) particles that are permanently magnetizable, being particles that exhibit bulk ferromagnetic properties, such as magnetic iron oxide or iron platinum, as well as (b) magnetically responsive particles, sometimes termed superparamagnetic particles, being particles that demonstrate magnetic behavior only when subjected to a magnetic field.
  • the particles are nanoparticles that incorporate magnetic materials, or magnetic materials that have been functionalized, or may contain from 10% to 95% superparamagnetic particles or other configurations as are known in the art.
  • a particular example of a magnetic solid surface or bead are uncoated magnetite particles that are highly susceptible to an external magnetic field. Production of magnetic particles is shown for example in Giaever (US. Pat. No. 3,970,518), Senyi et al. (US. Pat. No. 4,230,685), Dodin et al. (US. Pat. No. 4,677,055), Whitehead et al. (US. Pat. No. 4,695,393), Benjamin et al. (US. Pat. No. 5,695,946), Giaever (US. Pat. No. 4,018,886), Rembaum (US. Pat. No. 4,267,234), Molday (US. Pat. No. 4,452,773), Whitehead et al. (US. Pat. No. No.
  • microorganisms or microbial cells that are the subject of the invention include both prokaryotic microorganisms, such as Gram-positive bacteria and Gram-negative bacteria, and eukaryotic microorganisms, such as fungi.
  • prokaryotic microorganisms from which DNA can be isolated using the described methods include, but are not limited to pathogenic bacteria of the genera
  • Mycobacterium Enterococcus, Streptococcus, Staphylococcus, Salmonella, Legionella, Clamydia, Shigella, Pseudomonas, Listeria, Yersinia, Corynebacterium, Bordetella, Bacillus, Clostridium, Haemophilus, Helicobacter and Vibrio.
  • fungi examples include pathogenic fungi of the genera Aspergillus (e.g. A. fumigatus, A. niger, A. flavus, A. nidulans), Basidiobolus (e.g. B. microsporus, B. ranarum), Cephalosporium (e.g. C. chrysogenum, C. coremioides, C. diospyri, C.
  • Aspergillus e.g. A. fumigatus, A. niger, A. flavus, A. nidulans
  • Basidiobolus e.g. B. microsporus, B. ranarum
  • Cephalosporium e.g. C. chrysogenum, C. coremioides, C. diospyri, C.
  • pathogenic yeasts of the genus Candida e.g. C. albicans, C. guilliermondii, C. kruzei, C. parapsilosis, C.
  • the sample for use herein is an aqueous solution that is entirely or in part a blood sample.
  • the blood sample can be from a human or a non-human subject.
  • the blood sample is 90%- 100% blood extracted from the subject.
  • the blood sample is diluted to 10%-90% of the starting sample concentration, such as 90%, 80%, 70%, 60%, 50%, 40%,. 30%, 20%, 10%, or any dilution in between.
  • the blood sample can be a whole blood sample; or it can be a fraction of a blood sample, so long as a microorganism-containing fraction of the blood sample is used.
  • a blood sample must be treated with one or more anticoagulant agents so as to prevent activity that will lead to calcification or particle aggregation at a later stage in the procedure, and then the blood sample must be pre-treated.
  • Anticoagulant agents are well known and include, but not limited to EDTA, heparin and citric acid.
  • the described methods therefore require that prior to adding a water soluble polymer to the blood sample, the blood solution must be pre-treated and incubated with a quantity of a selective lysis formulation that (a) lyses the blood cells, (b) does not lyse most or all of the microbial cells and (c) does not interfere with later precipitation when a water soluble polymer is added to the blood sample.
  • the eukaryotic cells in the blood sample are selectively lysed with a reagent containing chaotropic agents and/or non-ionic detergents.
  • Non-ionic detergents include, but are not limited to Tween-20, Tween-40, Tween-60, Tween-80, Nonidet- P40, Deoxycholate, Brijj, Igepal, Triton, Octyl-beta-Glucoside, Digitonine, and Dodecyl-beta- D-maltoside.
  • the selective lysis reagent contains instead of a chaotropic agent, an alkaline buffer together with one or more non-ionic detergent.
  • a preferred embodiment for selectively lysing higher eukaryotic cells comprises incubation in a solution containing one or more non-ionic detergent, and containing neither any chaotropic agent nor any alkaline buffer.
  • the non-ionic detergent is Tween-20 at a final concentration of between around 0.1% to 5%.
  • the Tween-20 is at a final concentration of 1%, 2%, 3%, or 4%.
  • the described methods include the addition of an agent capable of digesting the released proteins followed by incubating the aqueous solution for a time period sufficiently long and at a temperature sufficiently high to enable digestion of at least some of said released proteins, thereby reducing the viscosity of the solution. Reduction in viscosity makes it easier to mix the solution in order to remove residual blood material.
  • Agents that may be used to digest proteins include, but are not limited to at least one of Proteinase K, Brofasin, OB Protease and Qiagen Protease.
  • Proteinase K is used to digest released eukaryotic proteins.
  • Protocols for digestion of eukaryotic proteins using a solution containing Proteinase K are well known and include, but are not limited to, incubation for around 30 minutes at 37°C and incubation for around 15 minutes at 60°C.
  • the digestion of proteins is completed as part of Step B, prior to adding at least one of a water soluble polymer and salt to the blood solution. In other embodiments, the digestion of proteins is completed following the addition of at least one of a water soluble polymer and salt into the blood solution as part of Step C or part of the washing process of Part E.
  • the described methods capture microorganisms by adsorption/binding to a solid surface. It is well known that the addition to an aqueous solution of polyethylene glycol (PEG) and/or sodium chloride (NaCl) enhances the displacement of biological materials onto magnetic particles (US 5,705,628; Saiyed et al. 2008). Such materials likewise can be used in the currently described methods.
  • PEG polyethylene glycol
  • NaCl sodium chloride
  • water soluble polymers such as PVP or dextran can be used in place of PEG; and in other embodiments alternative inorganic salts can used in place of sodium chloride, including magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.
  • the concentration of the water soluble polymer and/or salt added to the blood sample should be adjusted according to the nature of the polymer so as to produce the desired maximum displacement of the microbial cells from suspension in the blood onto a solid surface, such as magnetic beads, while limiting displacement onto the solid surface of impurities, contaminants and inhibitors from the higher eukaryotic cells, which are also present in the suspension, particularly after eukaryotic cell lysis.
  • the final polymer concentration is from 2% to 15% (w/v), such as 4% to 9% (w/v), and preferably about 7% (w/v).
  • the water-soluble polymer can be a non-ionic hydrophilic polymer, for example a dextran, a PVP or a PEG.
  • the PEG can be polydisperse in molecular weight or monodisperse, branched or straight chain, and may be of a star type.
  • the water soluble polymer can have an average molecular weight of from 200 to 100,000, such as from 1,000 to 20,000, and more preferably 5,000 to 13,000, (e.g. about 8000). These molecular weights are particularly suitable for PEG or PVP.
  • a water-soluble inorganic salt either alone or in conjunction with a water soluble polymer, is used to raise the ionic strength of the aqueous liquid that includes or is a blood sample.
  • the inorganic salt raises the ionic concentration of the liquid to a final concentration of 150mM to 6M, and more preferably from 0.25 to 0.75M, e.g. 0.5M.
  • PEG is added to a final concentration between 6.5% and 7.5% (w/v)
  • sodium chloride is added to a final concentration between 0.5M and .75M.
  • the beads can be immobilized, such as through use of a magnet.
  • the beads are then washed at least one time with one or more wash solution that (a) primarily removes blood components, (b) reduces particle aggregation, (c) does not primarily disrupt the bond formed between the beads and the microbial cells and (d) primarily does not lyse the intact microbial cells.
  • the wash solution can include the same components as the displacement or binding solution. Accordingly in particular embodiments, the wash solution can contain the same concentration of water soluble polymer and/or salt as in the binding solution. In other embodiments, the concentrations of polymer and/or salt in the wash solution are different than in the binding solution. In still further embodiments, the concentrations of polymer and/or salt in a first wash solution, may be different from the concentrations in a second, third or fourth wash solution. For example, a final concentration of 15% PEG and 1.5M sodium chloride, while less than optimal for the binding solution, may be appropriate for the solution used for one or more washes. In particular embodiments, the final polymer concentration is from 2% to 15% (w/v) alone and/or together a final concentration of between 150mM of inorganic salt.
  • the described methods of microbial DNA isolation include the process of microbial cell lysis following the washing of the bead-bound intact microbial cells.
  • microbial lysis of Gram-negative bacteria can be accomplished with heat alone.
  • cell lysis including lysis of Gram-positive bacteria and fungi, requires more than heat.
  • microbial lysis is accomplished through the use of enzymatic lysis reagents, such as lysozyme and achromopeptidase.
  • lysis is accomplished with chemical lysis reagents, such as SDS and sodium hydroxide.
  • lysis is achieved through mechanical means, for example the beating of the cells with fine glass beads.
  • mechanical lysis is achieved using a device such as the Disrupter Genie® cell disrupter (Scientific Industries, Inc.).
  • DNA yield can be reduced in bead/microbial cells derived from a whole blood sample (as compared to non-blood-derived microbial cells attached to beads).
  • DNA yield can be improved with the following additional step or steps: in one embodiment, the container in which the bead/ microbial cells are lysed is mildly or moderately physically agitated. In other embodiment, sound energy (e.g. by sonication) is used to agitate the particles. In another embodiment, both physical agitation and sonic energy are used to agitate the particles.
  • the microbial DNA is separated from the non-DNA components of the aqueous suspension.
  • Multiple methods are known to the art for isolating and purifying DNA in a suspension. However, given the requirements (for effective sepsis diagnosis) for optimizing yield of any microbial DNA isolated, many standard isolation methods are not suitable for use in the described methods.
  • yield can be particularly effected when microbial cells bound to magnetic particles are lysed and microbial DNA is extracted from the cells in the presence of the magnetic particles.
  • the DNA binds to the magnetic particles, leaving said DNA unavailable for subsequent analysis, such as by PCR.
  • US Patent No. 6,433,160 describes methods for binding double stranded DNA to paramagnetic iron particles by suspending the DNA and the particles in an acidic solution. US 6,433,160 further states that DNA bound using an acidic solution may later be eluted from the particles "by heating the environment of the particles with bound nucleic acids and/or raising the pH of such environment". As described in Example 4, reflecting embodiments where intact microbial cells bound to magnetic particles are incubated at 96°C in a solution comprising SDS and NaOH at a pH above 12.0, microbial DNA is extracted from the cells and is single stranded due to the high temperature, said DNA will bind to the magnetic particles, rather than elute from the particles, despite the presence of a highly heated environment and an elevated pH. Simply raising the pH level of the environment and/or heating the environment to a level above 90°C are insufficient to elute DNA bound to magnetic iron particles.
  • unbinding DNA from magnetic particles have often relied on the use of surface modified magnetic particles, such as magnetite derivatised with carboxyl groups, beads coated with silica, or polymer magnetic particles, such as those marketed as Dynabeads® magnetic beads (Life Technologies). Such surface modified magnetic particles often require laborious and time consuming production methods.
  • unmodified Fe 3 0 4 magnetic particles have advantages over modified magnetic particles for isolating microbial cells from clinical samples.
  • Previous methods were described for unbinding DNA bound to unmodified Fe 3 0 4 magnetic particles by suspension in sterile water or Tris-EDTA (pH 7.8), and incubation at 65°C for 5 minutes with agitation (Saiyed et al., 2008). However such methods are insufficient to produce the DNA yield necessary for effective sepsis detection. Accordingly, methods are provided herein to improve unbinding of microbial DNA that has bound to magnetic particles following microbe lysis.
  • At least most DNA bound to magnetic particles can be separated from the particles by first incubating said DNA bound particles at a temperature below 50°C, then incubating the aqueous DNA-bound magnetic particle suspension with a buffer rendering the solution with a pH of about 9.5 or higher for at least several minutes at an temperature of between around 60°C and around 75°C, and agitating said solution; for example five minutes at
  • the solution is agitated for a minimum of 2 seconds before, after or both before and after incubation at elevated pH.
  • the solution is agitated throughout most or all of incubation at elevated pH, such as by magnetic stirring.
  • DNA bound to magnetic particles can be separated from the particles by addition of a phosphate containing reagent to the aqueous solution containing magnetic particles to which DNA are bound, followed by incubation for least several minutes at an elevated temperature; for example five minutes at 65°C.
  • a phosphate containing reagent include water containing sodium phosphate, water containing potassium phosphate, and the like.
  • agitation of the aqueous DNA-bound magnetic particle suspension can be included in the DNA unbinding process and can further improve isolation yield.
  • the solution is agitated for a minimum of 2 seconds before, after, or both before and after incubation at with the phosphate-containing solution.
  • the solution is agitated throughout incubation with the phosphate- containing solution, such as by magnetic stirring.
  • DNA can be unbound from the magnetic particles and may be separated from the magnetic particles from the solution by use of a magnetic field which immobilizes the magnetic particles while the DNA remains in solution. DNA can then be further processed for PCR.
  • Example 1 Displacement of Bacteria onto Magnetite Beads
  • Methicillin-Resistant Staphylococcus aureus (MRSA) bacteria were first incubated in polypropylene tubes at room temperature with and without magnetite beads, NaCl (final concentration 0.5M), and/or PEG (final concentration 10% w/v). Following the incubation, each sample tube was placed into a magnet, and a liquid portion of the sample was spread onto culture plates to determine the number of colony forming units (CFU) remaining in solution (i.e. that were not displaced onto beads, if present in the sample). Three bacterial culture plates were prepared from each sample and the colonies were counted after 24 hour incubation providing an average CFU count with a presumed 10 CFU margin of error.
  • MRSA Methicillin-Resistant Staphylococcus aureus
  • Sample 1 10ml solution: water, MRS A bacteria;
  • Sample 2 20ml solution: water, MRSA bacteria, 0.5 M NaCl, 10% PEG;
  • Sample 3 20ml solution: water, MRSA bacteria, 1% magnetite beads, 0.5 M NaCl;
  • Sample 4 20ml solution: water, MRSA bacteria, 1% magnetite beads, 10% PEG; and
  • Sample 5 20ml solution: water, MRSA bacteria, 1% magnetite beads, 0.5 M NaCl, 10% PEG.
  • US Patent No. 8,603,771 (US 8,603,771) describes capturing onto a solid surface microorganisms present in an aqueous liquid by adding to the liquid a sufficient quantity of a water soluble polymer to displace said microorganisms from the liquid to the solid surface.
  • US 8,603,771 indicates that PEG is the preferred water soluble polymer for this purpose, and describes the method as a first step in isolating and purifying a microorganism to remove inhibitors to downstream assays such as PCR.
  • US 8,603,771 demonstrates the method using sputum or urine samples having a high concentration of microbial cells.
  • US 8,603,771 does not demonstrate its method in blood.
  • US 8,603,771 does not demonstrate isolation of bacteria from even a simple aqueous solution in which a low concentration of micro-organisms is present in comparison to higher eukaryotic cells present in the solution, such as would be necessary for effective sepsis detection.
  • the samples were then subjected to a microbial lysis procedure consisting of incubation with a lysis solution (comprising 0.25% SDS and 50mM NaOH) for 5 minutes at 96°C, followed by a return to room temperature and subsequent agitation for 5 minutes at 65°C to elute DNA from the magnetic particles.
  • a lysis solution comprising 0.25% SDS and 50mM NaOH
  • the samples were placed in a magnet to immobilize the magnetic beads.
  • the supernatant from each sample was transferred to a separate Zymo-SpinTM Column and prepared for PCR using the reagents and protocol the Genomic DNA Clean & Concentrator Kit from Zymo Research.
  • Duplicates from each sample were then assessed in real-time PCR using a primer-probe assay targeting the mecA gene present in the sample MRSA bacteria (using SEQ ID Nos. 4-6).
  • MRSA microbial DNA was detected by PCR in both of the duplicates from the water solution and no PCR amplification was evident in either of the duplicates from the blood sample.
  • Post microbial lysis the blood samples continued to appear turbid with viscous residue from the blood remaining attached to the magnetic particles. While it can be assumed that the binding reagents succeeded in displacing microbial cells to the magnetic solid support in the blood solution, they also appeared to have displaced blood material that could not be washed away.
  • the method of US 8,603,771 cannot effectively isolate and purify bacteria from blood. Instead, it was observed that in the context of a blood solution, the US 8,603,771 method displaces to the solid surface higher eukaryotic cells, impurities, contaminants, and inhibitors from the blood, thereby eliminating the utility of the method for detecting sepsis in a patient.
  • Sample 2 received a Proteinase Solution, comprising 20mg/ml Sigma- Aldridge
  • Samples 2, 3 and 4 were incubated for 15 minutes at 37°C. Samples 2 and 4, containing Proteinase K, were then incubated additionally for 15 minutes at 60°C.
  • Each sample was then incubated for 5 minutes with a binding solution comprising a final concentration of 7% PEG, 0.5M NaCl and 0.75% magnetite particles.
  • the samples were then placed in a magnet to immobilize the magnetite particles and the supernatant removed and discarded.
  • a wash solution (comprised of 10% PEG and 0.5M NaCl) was added to each sample. The samples were incubated, and the liquid removed and discarded. The wash process was repeated two additional times.
  • a microbial lysis procedure consisting of incubation with a lysis solution (comprising 0.25% SDS and 50mM NaOH) for 5 minutes at 96°C, followed by a return to room temperature and subsequent agitation for 5 minutes at 65°C to elute DNA from the magnetic particles.
  • a lysis solution comprising 0.25% SDS and 50mM NaOH
  • All four pre-treatment methods succeeded in reducing the quantity of blood material that displaced onto the magnetite particles during the binding process and succeeded in providing isolated microbial DNA that could be detected by PCR.
  • the pre-treated blood did not form a gel-like turbid solution that would not separate during washes.
  • Proteinase K alone, DNase 1 alone and the combination of both reduced the viscosity of the blood solution evident during the binding process.
  • the higher Ct values for those samples treated with DNase 1 suggests that this enzyme reduces the quantity of microbial DNA available to the PCR reaction.
  • the combination of Tween-20 plus Proteinase K provided a better result than Tween-20 alone.
  • the samples were incubated in an aqueous Binding Solution (comprising a final concentration of 10% PEG, 1.5M NaCl, and 2% magnetite particles).
  • the samples were gently agitated for 3 minutes at room temperature.
  • the samples were placed in a magnet to
  • a Binding Solution (comprising a final concentration of 13%PEG, 1M NaCl and magnetite particles) was added to samples 2 and 3, leaving the first sample as a positive control. The samples were gently agitated for 5 minutes at room temperature. The samples were placed in a magnet to immobilize the magnetite particles and the solution was decanted. A washing solution (comprising a final concentration of 10%PEG and 0.5M NaCl) was added, the samples were gently mixed, then immobilized in a magnet and the liquid decanted and discarded. The wash process was repeated a second time.
  • This example demonstrates positive performance of a preferred embodiment of the described methods.
  • a blood sample was prepared comprising 10ml of human whole blood transferred from a larger sample stored in EDTA, and approximately 14 CFU of MRSA bacteria.
  • Pre- Treatment To the blood sample was added 0.9ml Selective Lysis Solution (comprising 12.3 ⁇ Tween-20, 123mM Tris and 50mM EDTA) plus 0.1ml Proteinase Solution (comprising 20mg/ml Sigma- Aldridge Proteinase K and 1 mM CaCl 2 buffer). The blood sample was then incubated at 60°C for 15 minutes and incubated at room temperature for an additional 2 minutes.
  • Selective Lysis Solution comprising 12.3 ⁇ Tween-20, 123mM Tris and 50mM EDTA
  • Proteinase Solution comprising 20mg/ml Sigma- Aldridge Proteinase K and 1 mM CaCl 2 buffer.
  • Microbial Lysis To the microbial cells bound to the magnetite particles was added 300 ⁇ of Microbial Lysis Solution (comprising 0.125% SDS and 50mM NaOH, pH level around 12.5) and the sample was incubated with magnetic stirring for 5 minutes at 96°C.
  • the sample was washed a second time to further remove PCR inhibitors, using a wash solution comprising 50mM Tris-HCl (pH 7) and 4% PEG-8000.
  • the sample was placed in a magnet to immobilize the magnetite particles, while the supernatant was removed and discarded.
  • Elution of Microbial DNA from Beads To the sample was added 300 ⁇ 1 of 50mM KOH and the sample was continually agitated for 5 minutes at 65°C. The sample was placed in a magnet to immobilize the magnetite particles and the supernatant containing the microbial DNA was transferred to a clean tube.
  • volume Reduction To the microbial DNA was added a low volume 1 ml binding solution comprising 0.1% magnetite particles, 4% PEG and 50mM HC1 (pH 7.0). The sample was gently stirred at room temperature for 4 minutes, incubated without stirring for 2 minutes and then placed in a magnet to immobilize the magnetite particles while the supernatant was removed and discarded. To the sample was added 55 ⁇ 1 of 50mM KOH and the sample was continually agitated for 5 minutes at 65°C. The sample was placed in a magnet to immobilize the magnetite particles and the supernatant containing the microbial DNA was transferred to a clean tube. A positive control sample was prepared with TE buffer and a comparable quantity of the same MRSA bacteria. DNA was extracted from the control sample using an enzymatic microbial lysis solution (comprising achromopeptidase, TE, and sucrose) and incubation for 15 minutes at 37°C and for 7 minutes at 96°C.
  • a negative control sample was prepared comprising TE.

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Abstract

L'invention concerne des procédés d'isolement d'ADN bactérien et fongique présent dans un échantillon de sang contenant des cellules eucaryotes supérieures, en particulier lorsque les microorganismes renfermant l'ADN sont présents à une concentration sensiblement inférieure à celle des cellules eucaryotes dans l'échantillon.
PCT/IL2015/050343 2014-08-14 2015-03-31 Procédés d'isolement d'adn microbien à partir d'un échantillon de sang WO2016024263A1 (fr)

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WO2017042819A1 (fr) 2015-09-11 2017-03-16 Molecular Detection Israel Ltd. Procédés d'isolement de celllules microbiennes présentes dans un échantillon de sang
WO2018125865A1 (fr) 2016-12-29 2018-07-05 Shoreline Biome, Llc Protocole de lyse combiné pour lyse cellulaire complète
CN110199021A (zh) * 2016-12-14 2019-09-03 东安格利亚大学企业有限公司 用于消除核酸的方法
CN110272898A (zh) * 2019-07-11 2019-09-24 上海市浦东新区疾病预防控制中心 一种通用型微生物病原体裂解液及其应用
CN110438120A (zh) * 2019-09-12 2019-11-12 卓源健康科技有限公司 一种从血液中提取微生物基因组dna的试剂盒及其方法
WO2019193332A3 (fr) * 2018-04-03 2019-11-21 Momentum Bioscience Limited Séparation et détection de micro-organismes
CN111073886A (zh) * 2020-01-16 2020-04-28 中国农业科学院农业基因组研究所 一种基于超顺磁性纳米颗粒的dna提取吸附液、试剂盒以及dna提取方法
CN113474465A (zh) * 2019-02-28 2021-10-01 零日诊断有限公司 制备用于核酸扩增的临床样品的改进方法
US11149246B2 (en) 2016-12-29 2021-10-19 Shoreline Biome, Llc Methods for cell lysis and preparation of high molecular weight DNA from modified cells
CN114276932A (zh) * 2022-02-12 2022-04-05 合肥巅峰生物科技有限公司 一种微生物细胞裂解液

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WO2010005444A1 (fr) * 2008-07-11 2010-01-14 Handylab, Inc. Matériaux de capture de polynucléotides, et procédés d’utilisation de ces matériaux
WO2012168003A1 (fr) * 2011-06-06 2012-12-13 Biocartis S.A. Lyse sélective des cellules par des tensioactifs ioniques selective lysis of cells by ionic surfactants

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US12221647B2 (en) 2015-09-11 2025-02-11 Bacteria Detection Ltd Methods for isolating microbial cells from a blood sample
WO2017042819A1 (fr) 2015-09-11 2017-03-16 Molecular Detection Israel Ltd. Procédés d'isolement de celllules microbiennes présentes dans un échantillon de sang
CN110199021A (zh) * 2016-12-14 2019-09-03 东安格利亚大学企业有限公司 用于消除核酸的方法
US11149246B2 (en) 2016-12-29 2021-10-19 Shoreline Biome, Llc Methods for cell lysis and preparation of high molecular weight DNA from modified cells
WO2018125865A1 (fr) 2016-12-29 2018-07-05 Shoreline Biome, Llc Protocole de lyse combiné pour lyse cellulaire complète
CN110312570A (zh) * 2016-12-29 2019-10-08 海岸线生物群有限责任公司 用于全面细胞裂解的组合裂解方案
EP3562576A4 (fr) * 2016-12-29 2020-07-22 Shoreline Biome, LLC Protocole de lyse combiné pour lyse cellulaire complète
US10774322B2 (en) 2016-12-29 2020-09-15 Shoreline Biome, Llc Combined lysis protocol for comprehensive cell lysis
JP7476466B2 (ja) 2018-04-03 2024-05-01 モメンタム・バイオサイエンス・リミテッド 微生物の分離及び検出
WO2019193332A3 (fr) * 2018-04-03 2019-11-21 Momentum Bioscience Limited Séparation et détection de micro-organismes
CN111936633A (zh) * 2018-04-03 2020-11-13 动力生物科学有限公司 微生物分离和检测
JP2021519591A (ja) * 2018-04-03 2021-08-12 モメンタム・バイオサイエンス・リミテッドＭｏｍｅｎｔｕｍ Ｂｉｏｓｃｉｅｎｃｅ Ｌｉｍｉｔｅｄ 微生物の分離及び検出
EP3931347A4 (fr) * 2019-02-28 2022-11-30 Day Zero Diagnostics, Inc. Procédé amélioré de préparation d'échantillons cliniques pour amplification d'acide nucléique
CN113474465A (zh) * 2019-02-28 2021-10-01 零日诊断有限公司 制备用于核酸扩增的临床样品的改进方法
JP7535312B2 (ja) 2019-02-28 2024-08-16 デイ ゼロ ダイアグノスティックス， インコーポレイテッド 核酸増幅用の臨床サンプルを調製する改善された方法
CN113474465B (zh) * 2019-02-28 2025-04-22 零日诊断有限公司 制备用于核酸扩增的临床样品的改进方法
CN110272898A (zh) * 2019-07-11 2019-09-24 上海市浦东新区疾病预防控制中心 一种通用型微生物病原体裂解液及其应用
CN110438120A (zh) * 2019-09-12 2019-11-12 卓源健康科技有限公司 一种从血液中提取微生物基因组dna的试剂盒及其方法
CN111073886A (zh) * 2020-01-16 2020-04-28 中国农业科学院农业基因组研究所 一种基于超顺磁性纳米颗粒的dna提取吸附液、试剂盒以及dna提取方法
CN111073886B (zh) * 2020-01-16 2023-08-29 中国农业科学院农业基因组研究所 一种基于超顺磁性纳米颗粒的dna提取吸附液、试剂盒以及dna提取方法
CN114276932A (zh) * 2022-02-12 2022-04-05 合肥巅峰生物科技有限公司 一种微生物细胞裂解液

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