WO2016024263A1 - Methods for isolating microbial dna from a blood sample - Google Patents

Abstract

This disclosure provides methods for isolating bacterial and fungal DNA present in 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.

Description

METHODS FOR ISOLATING MICROBIAL DNA FROM A BLOOD SAMPLE

CROSS REFERENCE TO RELATED APPLICATIONS

Benefit is claimed to United States Provisional Patent Application No. 62/037,125, filed August 14, 2014; the contents of which are incorporated by reference in their entirety.

FIELD

Provided herein are methods for isolating bacterial and fungal DNA present in 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.

BACKGROUND

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.

For patients with symptoms of septic shock, current guidelines recommend the administration of antibiotics within one hour after diagnosis. However, in the absence of microbiological information within this time frame, current practice relies on the empiric use of broad- spectrum antibiotics while the pathogen is cultured, identified and then subjected to antibiotic susceptibility testing over the course of several days.

Inadequate and/or delayed empirical antimicrobial therapy is a major cause of mortality, morbidity and increased hospital length of stay for sepsis patients. Mortality from sepsis increases at a rate of 8% for every hour that the patient is not receiving the antimicrobial therapy (Daniels, 2011). Approximately 30-50% of all patients presenting with the clinical symptoms of sepsis receive inappropriate antimicrobial therapy for the first several days, because the causative pathogen and its antibiotic resistance profile is unknown at the time therapy is initiated. The use of inappropriate antibiotics is also discouraged because it increases the burden of antibiotic resistance in general.

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. However 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,

immobilizing the beads with a magnet, washing away the non-DNA components in the sample, and eluting the now purified DNA from the beads into a PCR compatible buffer solution.

However, such methods are not useful for blood samples containing small quantities of microbial pathogens and large amounts of non-microbial cellular material. 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). 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. In addition, the presence of heme from hemoglobin in red blood cells (erythrocytes) strongly decreases the activity of enzymatic lysis and DNA polymerase.

As 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.

Methods have also been developed for the separation of microbial cells present in blood samples using centrifugation or filtration. As applied to pathogen cells found in human blood samples, 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.

Other previously-developed methods use paramagnetic beads coated with

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.

Accordingly, a continuing need exists for methods of processing whole blood for effective diagnosis of sepsis.

SUMMARY

Provided herein are methods for isolating microbial DNA from a blood sample containing or suspected of containing one or more microorganisms. 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 microbial cells, and while at least most of the microbial cells remain bound to the magnetic solid surface; mixing the separated microbial cells with a microbial lysis reagent, lysing the separated microbial cells, thereby releasing microbial DNA therefrom; and separating the released microbial DNA from non- nucleotide components in the solution, thereby isolating the microbial DNA.

Also described herein are methods for eluting DNA bound to a solid magnetic surface by incubating the surface-bound DNA with a solution to produce a pH of about 9.5 or higher, thereby eluting the DNA.

The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The oligonucleotide sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file named 2077_6_2_SeqList.txt, created March 30, 2015, about 2 KB, which is incorporated by reference herein. In the accompanying sequence listing:

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. DETAILED DESCRIPTION

I. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." Additionally, the terms "comprising" and "comprised of are intended to include embodiments encompassed by the terms "consisting essentially of and "consisting of." Similarly, the term "consisting essentially of is intended to include embodiments encompassed by the term "consisting of." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting. 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. In particular embodiments, an aqueous sample or solution contains a blood sample mixed into it at 10%, 20%, 30%, 40%, 50% or even greater concentration. In other particular embodiments, 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. As used herein, a blood sample can be a 100% whole blood sample. In other embodiments, 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. In particular examples, 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.

Contacting: Placement in direct physical association. Includes both in solid and liquid form.

Cycle Threshold (Ct): 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. In a particular embodiment, 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.

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

development, such as those which occur for example in multicellular animal or plant organisms. In some embodiments, 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. In some embodiments, microbial cells can be for example prokaryotic cells, such as bacteria or archaebacteria. In other embodiments microbial cells can be eukaryotic cells, such as yeasts, lower and higher fungi or protozoa.

As used herein, 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. Thus, 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,

Enterococcus, Streptococcus, Staphylococcus, Salmonella, Legionella, Clamydia, Shigella, Pseudomonas, Listeria, Yersinia, Corynebacterium, Bordetella, Bacillus, Clostridium, Haemophilus, Helicobacter and Vibrio.

In other embodiments, the microbial cells or microorganisms can also include eukaryotic cells. Some embodiments of eukaryotic microbial cells are fungal cells. In more specific embodiments, 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. gregatum) and other pathogenic fungi of the genera Entomophthora, Skopulariopsis, Mucor, Rhizomucor, Absidia, Rhizopus, Altenaria, Stemphylium, Botrytis, Chrysosporium, Curvularia, Helmithosporium, Hemispora, Nigrospora, Paecilomyces, Phoma, Thielavia or Syncephalastrum. In other embodiments, pathogenic yeasts of the genus Candida, e.g. C. albicans, C. guilliermondii, C. kruzei, C. parapsilosis, C. tropicalis are also included. 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. In particular embodiments, a subject can present to a medical professional with symptoms indicating a sepsis-related infection. However, in other embodiments, the infectious microorganism (bacteria or other pathogen) may be present in sufficiently low concentrations that symptoms have not developed. As understood herein, 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 (or substrate): 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.

Under conditions sufficient for [carrying out a desired activity]: A phrase that is used to describe any environment that permits the desired activity.

II. Overview of Several Embodiments

Provided herein are methods for isolating microbial DNA from a blood sample containing or suspected of containing one or more microorganisms. 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 microbial cells, and while the microbial cells remain bound to the magnetic solid surface; mixing the separated microbial cells with a microbial lysis reagent; ysing the separated microbial cells, thereby releasing microbial DNA therefrom; and separating the released microbial DNA from non-nucleotide components in the solution, thereby isolating the microbial DNA.

In particular embodiments of the described methods, the starting volume of the aqueous solution is at least 4 milliliters.

In other embodiments, the aqueous solution is a substantially undiluted blood sample.

In some embodiments, the microorganism is a bacterium or a fungus.

In particular embodiments, the lysis reagent includes a non-ionic detergent. In other embodiments, 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

In particular embodiments, the water soluble polymer is polyethylene glycol (PEG), which can be used in particular examples at a final concentration between 2%-10% w/v.

In other embodiments, 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.

In particular embodiments, 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.

In particular embodiments, separating the microbial cells comprises using a magnet to immobilize the insoluble magnetic solid surface, and removing substantially all of the aqueous solution.

In further embodiments, 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. In other embodiments, the water soluble polymer in the wash solution is polyethylene glycol (PEG). In still other embodiments, 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.

Also described herein are methods for eluting DNA bound to a solid magnetic surface by incubating the surface-bound DNA with a solution to produce a pH of about 9.5 or higher, thereby eluting the DNA.

In particular embodiments, the solution includes NaOH or KOH.

In other embodiments, the surface-bound DNA is incubated at a temperature greater than room temperature, such as at least 65°C for at least five minutes.

III. Methods for Isolating Microbial DNA from a Blood Sample

Provided herein are methods for isolating microbial DNA, such as bacterial or fungal DNA, present in a blood sample. Also presented are 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.

In a particular embodiment of the method, microbial DNA isolation is performed by:

(A) Providing an aqueous solution comprising blood of a higher eukaryotic organism, an anticoagulant agent and also containing or suspected to contain a microorganism;

(B) Adding to the aqueous solution a lysis reagent and incubating the aqueous solution for a time period sufficiently long to lyse the higher eukaryotic cells, and extract higher eukaryotic DNA therefrom, while preserving the integrity of any microbial cells that are in the solution;

(C) Adding to the aqueous solution a magnetic solid surface, a water soluble polymer and/or salt in a quantity sufficient as to cause the intact microbial cells present in the sample to displace onto the magnetic solid surface;

(D) Separating the intact microbial cells displaced onto the magnetic solid surface from the aqueous solution together with the higher eukaryotic DNA, without the use of

centrifugation or filtration;

(E) Washing the intact microbial cells displaced onto the magnetic solid surface one or more times with a wash solution that does not extract microbial DNA from the microbial cells, removes at least some residual material of the higher eukaryotic organism, while enabling, at least most of the microbial cells to remain bound to the magnetic solid surface;

(F) Mixing said intact microbial cells displaced onto the magnetic solid surface with a microbial lysis reagent solution, lysing the microbial cells and extracting therefrom microbial DNA; and

(G) Separating extracted microbial DNA from non-nucleotide components in the solution.

In the described embodiment, 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.

Further, it will be appreciated that 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. In particular embodiments, 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. In certain embodiments, the solid surface incorporates material that exhibits permanent magnetic behavior. In other embodiments, the solid surface is composed of material that exhibits magnetic behavior only when subjected to a magnetic field.

It is preferred that 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. In certain embodiments, 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.

4,554,088), Forrest (US. Pat. No. 4,659,678), Liberti et al. (US. Pat. No. 5,186,827), Ownet al. (US. Pat. No. 4,795,698), and Liberti et al. (WO 91/02811), the content of each of which is incorporated by reference herein in its entirety.

According to some embodiments, 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.

Examples of 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.

Examples of eukaryotic microorganisms from which DNA can be isolated using the described methods include, but are not limited to fungal cells. In more specific embodiments, 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. gregatum) and other pathogenic fungi of the genera Entomophthora, Skopulariopsis, Mucor, Rhizomucor, Absidia, Rhizopus, Altenaria, Stemphylium, Botrytis, Chrysosporium, Curvularia, Helmithosporium, Hemispora, Nigrospora, Paecilomyces, Phoma, Thielavia or Syncephalastrum. In other embodiments, pathogenic yeasts of the genus Candida, e.g. C. albicans, C. guilliermondii, C. kruzei, C. parapsilosis, C.

tropicalis are also included.

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. In particular embodiments, the blood sample is 90%- 100% blood extracted from the subject. In other embodiments, 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.

As discussed further in Example 2, prior described methods of microorganism displacement onto solid surfaces are not operable in the context of a blood sample. 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.

Previous methods have been described for selectively lysing higher eukaryotic cells, such as cells in a blood sample, in an aqueous solution, while preserving the integrity of microbial cells present in the same solution. In the Molysis™ DNA isolation kit (Molzym) samples are incubated with chaotropic agents, such as guanidine, and DNase. In contrast, International Patent Publication WO 2011/070507 discloses the use of non-chaotopic non-ionic detergents together with an alkaline buffer rendering the solution with a pH of around 9.5 or higher.

In particular embodiments, 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. In other embodiments, the selective lysis reagent contains instead of a chaotropic agent, an alkaline buffer together with one or more non-ionic detergent.

Chaotropic agents, such as guanidine, denature substances released from higher eukaryotic cells which may increase viscosity of the solution, while incubation in alkaline buffers with a pH of 9.5 or higher and non-ionic detergents risks lysis of microbial cells in the solution. Accordingly, 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. In some embodiments, the non-ionic detergent is Tween-20 at a final concentration of between around 0.1% to 5%. In particular embodiments, the Tween-20 is at a final concentration of 1%, 2%, 3%, or 4%.

Selective lysis of the higher eukaryotic cells (as shown in Step B of the above method) releases proteins into the aqueous solution, which increase viscosity and stickiness within the sample, making it more difficult to subsequently remove inhibitors of microbial lysis and other enzymatic reactions. In particular embodiments therefore, 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. In particular

embodiments, 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.

In some embodiments, 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. In certain embodiments, 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. In particular embodiments, 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.

In particular embodiments, 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.

In a particular embodiment, to maximally displace microorganisms onto a solid surface while limiting displacement onto a solid surface of impurities, contaminants and inhibitors from the blood, PEG is added to a final concentration between 6.5% and 7.5% (w/v), and sodium chloride is added to a final concentration between 0.5M and .75M.

As described herein, following displacement of the microorganism onto a solid surface, such as onto metal beads, 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. In a particular embodiment, microbial lysis of Gram-negative bacteria can be accomplished with heat alone. In other embodiments, cell lysis, including lysis of Gram-positive bacteria and fungi, requires more than heat. In particular embodiments, microbial lysis is accomplished through the use of enzymatic lysis reagents, such as lysozyme and achromopeptidase. In other embodiments, lysis is accomplished with chemical lysis reagents, such as SDS and sodium hydroxide. In still other embodiments, lysis is achieved through mechanical means, for example the beating of the cells with fine glass beads. In a particular example, mechanical lysis is achieved using a device such as the Disrupter Genie® cell disrupter (Scientific Industries, Inc.).

In particular embodiments, wherein chemical, enzymatic and/or mechanical methods of lysing Gram-positive bacteria are used, 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). In such particular embodiments 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.

IV. Elution of Microbial DNA from a Magnetic Surface

Following microbial cellular lysis and release or extraction of microbial DNA from microbial cells, 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.

One factor influencing the yield of microbial DNA in the described methods is the binding of microbial DNA to the solid surface following microbial cell lysis. In some embodiments, 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. In such embodiments, 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.

Previously described methods of 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. As described herein, unmodified Fe₃0₄ 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 Fe₃0₄ 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.

As described herein, 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

65°C. Many bases are suitable for such purposes, including, but not limited to, sodium hydroxide (NaOH) and/or potassium hydroxide (KOH). In particular embodiments, the solution is agitated for a minimum of 2 seconds before, after or both before and after incubation at elevated pH. In a still further embodiment, the solution is agitated throughout most or all of incubation at elevated pH, such as by magnetic stirring.

In an alternative non-limiting embodiment, 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. Particular phosphate-containing reagents include water containing sodium phosphate, water containing potassium phosphate, and the like.

As with elution by elevated pH, agitation of the aqueous DNA-bound magnetic particle suspension can be included in the DNA unbinding process and can further improve isolation yield. In particular embodiments, the solution is agitated for a minimum of 2 seconds before, after, or both before and after incubation at with the phosphate-containing solution. In a still further embodiment, the solution is agitated throughout incubation with the phosphate- containing solution, such as by magnetic stirring.

Using the methods described herein, 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.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: Displacement of Bacteria onto Magnetite Beads

in a Non-Blood Aqueous Solution

An effective test for sepsis requires the ability to detect as few as two copies of pathogen DNA recovered from a 4ml whole blood sample containing as few as four colony forming units (CFU) of pathogen. For this reason the efficiency of bacterial isolation in a sample must be maximized. This example shows how addition of NaCl and/or polyethylene glycol (PEG) influences efficiency of bacterial displacement onto magnetite beads.

To determine displacement efficiency, 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.

With this protocol, the following conditions were tested:

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.

To preserve consistency in bacterial concentration, double the volume was plated in Samples 2-5, as compared to Sample 1.

Results and Discussion

The mean result of the two bacterial culture plates for each sample is shown in Table 1.

Table 1: CFU remaining after displacement

As shown in Table 1, a comparison of Sample 2 to the Non-Displacement Control (Sample 1) suggests that, in the presence of displacement reagents, minor quantities (16%) of bacteria will displace to the solid surface of polypropylene tube walls and thereby be removed from a solution, even in the absence of an insoluble magnetic solid surface. In the presence of PEG and a magnetic solid surface, 79% of bacteria displaces to the solid surface as evident from a comparison of Sample 4 to the Control. By contrast, 95% of bacteria displaces to a magnetic solid surface in the presence of inorganic salt, such as sodium chloride, as evident from a comparison of Sample 3 to the Control. Comparing the results of Samples 3 and 4, it appears that NaCl alone is more effective that PEG alone is displacing bacteria to a magnetic solid surface. Further, comparing the results from Sample 5 to Sample 3 we see no statistically significant improvement in displacement when PEG is added to NaCl in a non-blood solution, suggesting that the presence of a water soluble polymer may be inconsequential to the utility of NaCl in effecting displacement of intact bacterial cells to an insoluble magnetic solid surface.

Example 2: Isolating Bacteria from Whole Blood

Introduction

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. However, the components of a blood sample are very different from sputum and urine, and US 8,603,771 does not demonstrate its method in blood. Moreover, 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.

Method

To determine the utility of applying the method of US 8,603,771 to microorganisms present in whole blood as compared to an aqueous solution not containing blood, an identical quantity of MRSA bacteria was added to a 10ml sample of whole blood and into a 10ml sample of purified water. The blood sample was human blood stored in an EDTA solution to prevent coagulation.

Both samples were incubated at room temperature for 3 minutes in 10ml of binding solution (comprising a final concentration of 7% PEG, 0.5M NaCl and 1% uncoated magnetite particles). The samples were then placed in a magnet (DynaMag-50 Dynal) to immobilize the magnetite particles, and the supernatant was removed and discarded. A wash solution

(comprised of 10% PEG and 0.5M NaCl) was added to each sample. The samples were incubated, the magnetic particles immobilized and the liquid removed and discarded. The wash process was repeated two addition times. When the blood sample was placed in a magnet to draw the magnetic particles from the solution to the tube edge, a gel-like deep red solution of blood material bound to the magnetic particles, moved with the particles to the magnetic edge, and separated from the less turbid supernatant. Repeated washes were unable to substantively separate the residual blood material from the magnetic particles or reduce the turbidity of the solution when removed from the magnet.

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.

The samples were placed in a magnet to immobilize the magnetic beads. The supernatant from each sample was transferred to a separate Zymo-Spin™ 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).

Results

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.

Discussion

As demonstrated herein, 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.

Furthermore, it was also observed that without the pretreatment described further herein, and in sharp contrast to other aqueous solutions, addition of magnetic particles and PEG to a blood sample produces particle aggregates and the solution assumes a gel-like viscosity, further eliminating any utility of the US 8,603,771 method for sepsis detection. Taken together, the observations presented above demonstrate that not only is US 8,603,771 insufficient for applications wherein the aqueous solution contains whole blood, but US 8,603,771 is not applicable to sepsis.

Example 3: Requirements for Isolation of Bacteria in Whole Blood

The previous example demonstrates that microbial DNA cannot be effectively isolated from blood samples using previously described methods; indicating the need for additional sample treatment. This experiment compares the efficacy of various pre-treatment options.

Methods

Four 10ml samples were prepared, each containing 20% whole blood, EDTA and MRSA bacteria. As a first pre-treatment step, selective lysis solution was added to each, comprising Tween-20, Tris and EDTA.

· Sample 1 received no additional pre-treatment.

• Sample 2 received a Proteinase Solution, comprising 20mg/ml Sigma- Aldridge

Proteinase K and 1 mM CaCl₂ buffer.

• Sample 3 received DNase 1 (Sigma- Aldridge).

• Sample 4 Received the Proteinase Solution and DNase 1.

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. Each sample was 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.

All 4 samples were placed in a magnet to immobilize the magnetic beads. The supernatant from each sample was transferred to a separate Zymo-Spin™ Column and prepared for PCR using the reagents and protocol the Genomic DNA Clean & Concentrator Kit from Zymo Research. Duplicates from each sample (labeled A and B) 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).

Results and Discussion

The results for each sample are presented in Table 2.

Table 2

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.

In contrast to the non-pretreated blood sample of the previous example, 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. However, 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.

Example 4: DNA Elution from Magnetic Beads using a Basic Solution

It is known that under certain conditions DNA will bind to magnetite beads in an aqueous solution. In order to subsequently amplify the DNA in the solution by PCR, it is necessary to elute the DNA from the magnetite beads and separate the beads from the solution. This example includes two experiments demonstrating the observation that elution of DNA from magnetite beads is significantly improved when the elution is performed in a high pH solution.

Method - Experiment 1

Three samples were processed; one containing no bacteria and serving as a control and two samples containing vancomycin-resistant Enterococcus bacteria incorporating the vanA gene. 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

immobilize the magnetite particles and the solution was decanted. A microbial lysis solution was added to each sample (comprising 0.25 % SDS + 50mM NaOH) and the samples were incubated at 96°C for 5 minutes and then allowed to cool. The samples were again placed in a magnet, and the solution decanted and discarded.

NaOH was added to the second sample rendering the solution with a pH of around 12.5, while Tris-EDTA was added to the third sample rendering the solution with a pH of around 8.3. The samples were agitated and incubated at 65°C for 5min. The samples were placed in a magnet to immobilize the magnetic beads. The supernatant from each sample was transferred to a separate Zymo-Spin™ Column and prepared for PCR using the reagents and protocol the Genomic DNA Clean & Concentrator Kit from Zymo Research. The samples were assessed in real-time PCR using a primer-probe assay targeting the vanA gene present in the sample bacteria (using SEQ ID Nos. 1-3).

Results and Discussion - Experiment 1

The results for each sample are presented in Table 3.

Table 3

The positive finding of PCR amplification demonstrates that bacteria bound to the magnetic particles, DNA was extracted using the microbial lysis and the DNA then bound to the magnetic particles. However, as evident from the results table, elution with NaOH significantly increased the DNA yield in comparison to eluting the DNA with Tris-EDTA at a pH level of around 8.3.

Method - Experiment 2

The effects of high pH on DNA elution from magnetite beads was tested in a separate experiment, using NaOH or KOH to elute DNA bound to magnetite beads. The same strain of vancomycin-resistant Enterococcus bacteria incorporating the vanA gene was used as in the previous experiment. The bacteria was subjected to an enzymatic microbial lysis procedure; using achromopeptidase incubation for 15 minutes at 37°C followed by 5 minutes of incubation at 95°C.

Three samples containing the bacterial DNA were prepared. 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.

Into Sample 2 was added 60μ1 of 50mM NaOH and into sample 3 was added 60μ1 of 50mM KOH. To elute the DNA from the magnetic particles, the samples were incubated for 8 minutes at 65°C, with 4x mixing by a very low-speed vortex. The magnetic particles were then immobilized in a magnet and 60μ1 from each sample withdrawn into a separate new test tube and neutralized with ΙΟμΙ of 1M Tris-HCL buffer. All three samples were diluted 30-fold with Tris-EDTA at a pH level of around 8.3 (to dilute salts) and 25 μΐ from each prepared for PCR. The samples were assessed in real-time PCR using a primer-probe assay targeting the vanA gene present in the sample bacteria (using SEQ ID Nos. 1-3).

Results and Discussion - Experiment 2

The results for each sample are presented in Table 4.

Table 4

Sample Ct Value

2A. Elution with 50Mm NaOH 26.11

2B. Elution with 50Mm NaOH 26.14

3A. Elution with 50Mm KOH 26.47

3B. Elution with 50Mm KOH 26.87

Discussion

As evident from the table, elution with KOH is equally effective to elution with NaOH and both appear to detach from magnetite particles virtually all bound DNA and render it available for subsequent PCR analysis.

Example 5: Effective Isolation of Microbial DNA from Whole Blood

This example demonstrates positive performance of a preferred embodiment of the described methods.

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 μΐ Tween-20, 123mM Tris and 50mM EDTA) plus 0.1ml Proteinase Solution (comprising 20mg/ml Sigma- Aldridge Proteinase K and 1 mM CaCl₂ buffer). The blood sample was then incubated at 60°C for 15 minutes and incubated at room temperature for an additional 2 minutes.

Bead Binding: To the blood sample was added 11ml biding solution (final

concentration of 7% PEG-8000, 0.5M NaCl and 1% uncoated magnetite particles). The blood sample was then stirred at room temperature for 4 minutes and incubated without stirring for an additional 2 minutes. The sample was then placed in a magnet to immobilize the magnetite particles (DynaMag-50 Dynal) while the supernatant was removed and discarded. Removal of Residual Blood Material: To the blood sample was added 20ml of Wash Solution (comprising 7% PEG-8000 and 0.5ml NaCl) and the sample was stirred at room temperature for 4 minutes and incubated without stirring for 2 minutes. The blood sample was then placed in a magnet to immobilize the magnetite particles while the supernatant was removed and discarded. The wash process was repeated a second time using the identical quantity of Wash Solution. The wash process was repeated a third time using 10ml of the Wash Solution.

Microbial Lysis: To the microbial cells bound to the magnetite particles was added 300 μΐ 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.

Purification: Following 2 minutes of incubation on ice to reduce the temperature to less than 50°C, a 1ml solution was added comprising Tris-HCl to reduce the pH level and 4% PEG-8000 to reinforce the DNA-particle bond. The sample was placed in a magnet to immobilize the magnetite particles while the supernatant was removed and discarded. The sample was then washed a first time with 1ml of ethyl alcohol (EtOH) and gently stirred for 5 minutes to remove residual PCR inhibitors. The sample was then placed in a magnet to immobilize the magnetite particles, while the supernatant was removed and discarded. The sample was then incubated at 75°C for 7 minutes to evaporate any residual EtOH. 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 300μ1 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 55μ1 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.

All three samples were then assessed in duplicates (labeled A and B) in Real-Time PCR with a primer-probe assay targeting the MRSA mecA gene (using SEQ ID Nos. 3-6).

Results and Discussion

The results are presented in Table 5.

Table 5

Positive results were evident for both the blood samples and the positive control samples. The absence of amplification in the negative samples indicates that the experiment was free of DNA contamination. These findings demonstrate the efficiency of an embodiment of the described method to provide microbial DNA that can be detected by PCR from a starting sample consisting of minute quantities of pathogen in 10ml of whole human blood.

References

Daniels, R, Journal of Antimicrobial Chemotherapy, 2011, 66 (suppl 2): iil l-H23.

Saiyed, Z.M. et al 2008, Journal of Physics; Condensed Matter, 20: 204153, 1-5.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for isolating microbial DNA from a blood sample containing or suspected of containing one or more microorganisms, the method comprising:

providing an aqueous solution comprising a blood sample from a higher eukaryotic organism and at least one anticoagulant agent;

adding a lysis reagent to the aqueous solution;

incubating the aqueous solution for a time period sufficient to lyse higher eukaryotic cells in the blood sample and release therefrom higher eukaryotic DNA, wherein the integrity of microbial cells in the blood sample is preserved;

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 microbial cells, wherein at least most of the microbial cells remain bound to the magnetic solid surface;

mixing the separated microbial cells with a microbial lysis reagent;

lysing the separated microbial cells, thereby releasing microbial DNA therefrom; and separating the released microbial DNA from non-nucleotide components in the solution, thereby isolating the microbial DNA.

2. The method of claim 1, wherein the starting volume of the aqueous solution is at least 4 milliliters.

3. The method of claim 1 or claim 2, wherein the aqueous solution is a substantially undiluted blood sample.

4. The method of any one of claims 1-3, wherein the microorganism is a bacterium or a fungus.

5. The method of any one of claim 1-4, wherein the lysis reagent comprises a non-ionic detergent.

6. The method of any one of claims 1-5, wherein the lysis reagent further comprises an agent capable of digesting protein, and wherein incubating the aqueous solution comprises a time period sufficient to digest at least some proteins released from the higher eukaryotic cells into the aqueous solution.

7. The method of claim 6, wherein the agent capable of digesting protein is Proteinase

K.

8. The method of any one of claims 1-7, wherein incubating the aqueous solution comprises incubation at a temperature between about 20°C to about 60 °C, inclusive.

9. The method of any one of claims 1-8, wherein the water soluble polymer is polyethylene glycol (PEG).

10. The method of claim 9, wherein the final concentration of PEG is between 2%-10% w/v.

11. The method of any one of claims 1-10, wherein 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.

12. The method of any one of claims 1-11, wherein the magnetic solid surface comprises uncoated magnetite particles, wherein the particles are spherical or irregular in nature, and wherein the particles are 0.1 micron to 10 micron in diameter.

13. The method of any one of claims 1-12, wherein separating the microbial cells comprises using a magnet to immobilize the insoluble magnetic solid surface, and removing substantially all of the aqueous solution.

14. The method of any one of claims 1-13, wherein the wash solution comprises at least one of a water soluble polymer and an inorganic salt.

15. The method of claims 14, wherein the inorganic salt in the wash solution 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.

16. The method of claim 14, wherein the inorganic salt in the wash solution comprises sodium chloride.

17. The method of any one of claims 14-16, wherein the water soluble polymer in the wash solution is polyethylene glycol (PEG).

18. The method of any of the preceding claims, wherein 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.

19. A method for eluting DNA bound to a solid magnetic surface, comprising: agitating the surface-bound DNA sample and incubating said sample with a solution to produce a pH of about 9.5 or higher at a temperature of between around 60°C to 80°C, thereby eluting the DNA.

20. The method of claim 19, wherein the solution comprises NaOH or KOH.

21. The method of claim 19 or claim 20, wherein the surface-bound DNA is incubated for at least five minutes.

22. The method of claim 21, wherein the temperature is 65°C.