A rapid milk sample preparation method compatible with molecular testing

Abstract

The present invention provides a method of preparing a milk sample for downstream nucleic acid amplification process, comprising: contacting a milk sample containing at least one suspected bacterial pathogen involved in mastitis with uncoated magnetic beads; incubating the milk sample in the presence of uncoated magnetic beads to allow bacterial cells in the milk sample to bind to the beads, thereby forming bead-bacteria complexes; applying the bead-bacteria complexes to a magnet so that the bead-bacteria complexes are separated from the milk sample supernatant; removing the milk sample supernatant; resuspending the separated bead-bacteria complexes directly in an aqueous solution to release the bead-bound bacteria into solution; and using an aliquot of the bead resuspension or the bead-free supernatant after separation of the beads as a template for primer-specific downstream nucleic acid amplification.

Description

[Technical field]

[0001] The present invention relates to molecular tests that allow dairy farmers to determine pathogens in milk. More particularly, the present invention relates to a rapid milk sample preparation method for point-of-care molecular detection of etiologic pathogens associated with mastitis in dairy cows. [Background technology]

[0002] Bovine mastitis is the most prevalent and costly disease in dairy cows worldwide. Mastitis is an inflammation of the mammary gland and udder tissue resulting from microbial infection or physical trauma. Lack of early diagnosis of bovine mastitis and associated pathogens has a negative economic impact on dairy farmers. In the United States, mastitis costs the dairy industry approximately $1.7-2 billion annually. Although a wide variety of bacterial pathogens have been implicated, the most common pathogens are Staphylococcus aureus, Streptococcus sp., coagulase-negative staphylococci (CNS), Escherichia coli, and Mycoplasma sp. Diagnosis of bovine mastitis is routinely performed utilizing the California Mastitis Test (CMT, cow-side test), somatic cell count (SCC), and milk culture (reference laboratory test). Typically, if mastitis is diagnosed based on high somatic cell count (SCC), the cow is milked and treated with intramammary infusion of antibiotics. Judicious use of antibiotics can reduce the likelihood of the emergence of resistant bacteria and shorten the length of treatment that cows may require, thus reducing operational costs.

[0003] Accurate bacterial identification is therefore essential to enable appropriate therapeutic interventions and to plan different management strategies. Except for time-consuming bacterial culture methods, all other available on-farm tests do not detect the etiologic agent. Routine bacterial culture in reference laboratories requires shipping of milk samples, usually with a turnaround time of days to weeks. Furthermore, culture tests may show negative results even from truly sub-clinically infected glands due to the presence of very low bacterial loads when the samples are collected. Although molecular methods have proven to be proficient methods of bacterial identification, rapid and reliable on-farm molecular testing to identify the etiologic agent could bring significant savings to dairy farmers. Furthermore, rapid milk sample preparation methods are needed to support point-of-care (PoC) molecular mastitis testing that can be used by untrained dairy farmers. The present invention describes a novel and rapid milk sample preparation method compatible with downstream real-time PCR for the simultaneous detection of bacterial pathogens in milk derived from mastitis quarters of cows.

[0004] A simple and effective milk sample preparation method is a key step in the development of PoC molecular tests for bovine mastitis. Conventional DNA extraction methods from milk limit the application of molecular diagnostics in farm environments. Standard methods of nucleic acid extraction can be broadly divided into two categories: (a) solid-phase extraction (column-based extraction), and (b) nucleic acid capture using magnetic beads. The standard workflow for column-based extraction involves lysis of the components of the sample by lysis buffer, mechanical disruption, heating, or a combination of these techniques. Nucleic acids are released in the lysed sample and bound to a silica surface in a purification column. Several wash steps remove contaminants of the silica-bound nucleic acid, and a final elution step releases the purified DNA / RNA from the silica into an aqueous buffer. This method is the most commonly used throughout molecular labs, as many commercial kits and instruments are available for this type of nucleic acid extraction. The extraction process can be performed manually for a handful of samples or can be automated for high-throughput extraction. Magnetic bead-based nucleic acid extraction kits follow similar principles to column-based extraction (i.e., lysis, capture, wash, and elution), but use magnetic beads to capture DNA / RNA instead of a silica column. During the washing steps, a magnet is used to capture the magnetic beads. After the final elution step, the nucleic acids are released into solution and the magnetic beads can be discarded. Although both of these extraction methods are widely used in research and diagnostic laboratories, they have drawbacks, especially when used in a PoC setting: 1) expensive instrumentation (e.g., centrifuges) is required even for some manual methods, 2) multiple washing and elution steps, 3) pre-training in basic lab techniques is required to perform the procedure, 4) time consuming, 5) some reagents require refrigeration for storage, and 6) space requirements to accommodate additional equipment such as centrifuges and heating blocks. Thus, the current driving force in molecular diagnostics is to develop easier and faster methods of nucleic acid preparation, which should involve only a few steps, for example, without the need for any additional equipment and training.Towards this goal, a simple and easy-to-use milk sample preparation method was developed that allows for the detection of pathogens directly from clinical milk samples without the need for nucleic acid purification or additional equipment such as centrifuges or multiple elution steps. Summary of the Invention

[0005] The present invention provides a method of preparing a milk sample for downstream nucleic acid amplification process, comprising: a) contacting a milk sample containing at least one suspected bacterial pathogen involved in mastitis with uncoated magnetic beads; b) incubating the milk sample in the presence of uncoated magnetic beads to allow bacterial cells in the milk sample to bind to the beads, thereby forming bead-bacteria complexes; c) applying the bead-bacteria complexes to a magnet so that the bead-bacteria complexes are separated from the milk sample supernatant; d) removing the milk sample supernatant; e) resuspending the separated bead-bacteria complexes directly in an aqueous solution to release the bead-bound bacteria into the solution; and f) using an aliquot of the bead resuspension from e) as a template for primer-specific downstream nucleic acid amplification. In one embodiment, the method further comprises applying the bead resuspension from f) to a magnet to separate the beads from the bead-free supernatant, and using an aliquot of the bead-free supernatant as a template for primer-specific downstream nucleic acid amplification.

[0006] In one embodiment the milk sample is a raw milk sample, hi another embodiment the raw milk sample is from a mastitis quarter of a dairy cow.

[0007] In a further embodiment, the bead-bacteria complexes separated in step e) of the method are directly resuspended in an aqueous buffer solution that provides a suitable chemical environment for the activity of the DNA polymerase. In one embodiment, the pH of the aqueous buffer solution is between 8.0 and 9.5. In a further embodiment, the aqueous buffer solution is stabilized by Tris-HCl, Tris-H3PO4, MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or a combination thereof.

[0008] In one embodiment, the aqueous buffer used to resuspend the separated bead-bacteria complexes is stabilized with Tris-HCl or Tris-H3PO4 present at about 10 to about 50 mM and a pH of about 8.0 to about 9.0. In a particular embodiment, the aqueous buffer is 25 mM Tris-HCl, pH 8.5.

[0009] In some embodiments, the buffered aqueous solution further comprises a chelating agent, a salt, a detergent, or a combination thereof. In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), or sodium 4-aminosalicylate. In a further embodiment, the detergent is tricosaethylene glycol dodecyl ether (Brij L23), octoxynol-1 (Triton X-100), polyoxyethylenesorbitan monolaurate (Tween-20), triethanolamine (TEA), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), sodium dodecyl sulfate (SDS), or a combination thereof. In another embodiment, the salt is selected from sodium chloride (NaCl), magnesium chloride (MgCl2), potassium chloride (KCl), or a combination thereof.

[0010] In one embodiment of the method, the milk sample and the uncoated magnetic beads are mixed prior to the incubation step, hi another embodiment, the incubation is for about 5 to about 10 minutes at room temperature.

[0011] In a further embodiment, the step of preparing the milk sample is performed in a tube, hi one embodiment, the tube is placed in a magnetic stand to capture the magnetic beads.

[0012] In one embodiment, the uncoated magnetic beads comprise a silica-like surface chemistry, hi another embodiment, the uncoated magnetic beads are surface derivatized with lectins, free amine groups, or streptavidin.

[0013] In one embodiment, the templates of step f) are analyzed by real-time PCR using primers specific for a bacterial pathogen involved in mastitis. In one embodiment, the bacterial pathogen involved in mastitis is selected from Staphylococcus aureus, Streptococcus sp., or coagulase-negative staphylococci (CNS). In a further embodiment, an aliquot of the bead resuspension or an aliquot of the bead-free supernatant is transferred to a PCR tube or a microfluidic PCR cartridge.

[0014] The present invention also provides a point-of-care (PoC) molecular test that allows the identification of an etiologic agent in a milk sample, the molecular test comprising the use of a template for primer-specific nucleic acid amplification prepared according to any one of the above-mentioned embodiments of the milk sample preparation method. In one embodiment, the milk sample is from a mastitis quarter of a dairy cow. In another embodiment, the molecular test is a real-time PCR. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of a workflow embodiment for rapid milk sample preparation according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] definition Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0017] The term "uncoated magnetic beads" as used throughout this specification refers to magnetic beads or particles that are not coated with immunoglobulins or peptides specific for target bacterial pathogens involved in mastitis, such as Staphylococcus aureus, Streptococcus sp. or coagulase-negative staphylococci (CNS). However, it is understood that the magnetic beads or particles can be surface-derivatized with other moieties that allow the beads to bind to bacterial cells and form bead-bacteria complexes. For example, in one embodiment, the surface of the beads can be derivatized with silica-like chemistry (silanol groups). Without wishing to be bound by any one theory, it is believed that magnetic beads with such silica-like chemistry bind to bacteria in the milk sample via weak Vander Waals forces. In another embodiment, the magnetic beads or particles are lectin-derivatized. Lecithin binds to carbohydrates present on the surface of bacterial cells in the milk sample. Alternatively, streptavidin can be immobilized on the surface of the magnetic beads. Streptavidin binds to extracellular biotin on the surface of bacterial cells in the milk sample. Streptavidin-biotin binding is a protein-ligand interaction. Alternatively, the surface of the magnetic beads can be functionalized with free amine groups ready to bind with ligands. For example, the amine groups interact with proteins located on the surface of bacterial cells in milk samples.

[0018] As used herein, the term "directly resuspending the separated bead-bacteria complexes" means that after removal of the milk sample supernatant, the magnetic beads with bound bacteria are resuspended in an aqueous buffer in the absence of prior washing of the bead-bacteria complexes.

[0019] The terms "nucleic acid", "polynucleotide", "nucleic acid molecule", and the like, may be used interchangeably herein and refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA. Nucleic acids can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. The term "nucleic acid" includes, for example, single-stranded and double-stranded molecules. Nucleic acids can be, for example, genes or gene fragments, exons, introns, DNA molecules (e.g., genomic DNA), RNA molecules (e.g., mRNA), recombinant nucleic acids, plasmids and other vectors, primers and probes. Both 5' to 3' (sense) and 3' to 5' (antisense) polynucleotides are included.

[0020] As mentioned above, the present invention provides a method of preparing a milk sample for a downstream nucleic acid amplification process. Now referring to FIG. 1, a milk sample is contacted with uncoated magnetic beads. The milk sample is suspected to contain at least one suspected bacterial pathogen involved in mastitis, such as Staphylococcus aureus, Streptococcus sp., or coagulase-negative staphylococci (CNS). The milk sample is incubated in the presence of uncoated magnetic beads in a tube for a sufficient period of time to allow bacterial cells in the milk sample to bind to the beads, thereby forming bead-bacteria complexes. The bead-bacteria complexes thus formed are then subjected to a magnetic force, for example by placing on a magnetic rack. This allows the beads with bound bacterial cells to be pelleted and separated from the milk sample supernatant, which can then be removed from the tube. The separated bead-bacteria complexes in the tube are then directly resuspended in the absence of any prior washing step in an aqueous solution to release the bead-bound bacteria into solution. One of two options can then be selected. Option 1 is that an aliquot of the bead resuspension can be used directly as a template for primer-specific downstream nucleic acid amplification (e.g., real-time PCR) to detect the presence of bacterial pathogens. Option 2 is that the bead resuspension is applied to a magnet to separate the beads from the bead-free supernatant, and an aliquot of the bead-free supernatant is used as a template for primer-specific nucleic acid amplification (e.g., real-time PCR). It is understood that the "bead-free supernatant" may still contain residual amounts of beads, i.e., substantially free of beads or free of beads. In one embodiment, less than 0.1% v / v of residual beads may be present in the "bead-free supernatant."

[0021] The present invention provides a point-of-care (PoC) molecular test that allows for the identification of an etiologic agent in a milk sample, using templates for primer-specific nucleic acid amplification prepared according to the milk sample preparation methods described herein.

[0022] As shown in Figure 1, a user in a point-of-care setting (e.g., dairy farmer, veterinarian, or veterinary technician) performs the milk sample preparation method in only 3-4 steps. Also, no special instrumentation other than a magnet or a magnetic separation rack is required to perform the method. Furthermore, the method does not require a nucleic acid purification step. Furthermore, the sample preparation method can be performed in a single tube, such as a microfuge tube. In contrast, in commercially available magnetic bead-based kits, after the lysis step, magnetic particles are used to bind the nucleic acids (DNA / RNA) followed by washing steps to remove any unbound impurities. In such kits, the final elution step involves the addition of a buffer to elute the nucleic acids bound to the magnetic beads. This workflow is labor intensive, involves multiple washing steps with centrifugation between each wash, and requires a high degree of technical expertise to minimize loss of magnetic beads during the multiple washing steps, making it difficult to use these kits at the point-of-care.

[0023] There is a need for point-of-care (PoC) molecular tests that allow dairy farmers or veterinarians to rapidly determine the etiologic pathogen in milk collected from mastitis quarters of cows. The milk sample preparation method of the present invention supports such PoC molecular mastitis testing by allowing direct detection of pathogens from clinical milk samples without the need for a nucleic acid purification step. In one embodiment, the method includes mixing magnetic beads with a clinical milk sample to capture bacterial cells, and then incubating the mixture at room temperature for about 5 minutes. In one embodiment, after incubation, the magnetic beads are pelleted and the milk sample is aspirated using a magnetic separation rack. In another embodiment, the magnetic bead pellet is resuspended in Tris buffer (pH 8.5) and an aliquot of the bead suspension or bead-free supernatant is used directly as a template for downstream amplification by real-time PCR.

[0024] In one embodiment, the magnetic beads used in the methods of the present invention comprise any suitable magnetic material oxide or combination of materials, such as magnetite, ulvospinel, hematite, ilmenete, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrosite, ferroxyhite, iron, nickel, cobalt, awaruite, and wairauite.

[0025] In one desired embodiment, the magnetic beads comprise divalent oxides, trivalent oxides, or combinations thereof. In one preferred embodiment, the magnetic beads comprise gamma Fe2O3 and Fe3O4 throughout the bead.

[0026] The magnetic material may further be combined with a polymer to form beads. For example, magnetic polymer beads are composed of magnetic nanoparticles or microparticles embedded in a polymer matrix. The size of the beads may vary from 100 nanometers to several millimeters. The synthesis of magnetic polymer beads can be carried out by three general methods. In the first one, the magnetic particles are synthesized within the polymer matrix. In the second one, the polymer is synthesized in the presence of the magnetic particles. In the third one, the beads are prepared from preformed polymer and magnetic particles. The beads can have different structures. In one type of beads, the magnetic particles are uniformly distributed in the volume of the polymer matrix. Other types of beads are characterized by a core-shell structure (polymer core magnetic shell or magnetic core polymer shell). Also, mixed systems can be prepared in which the core-shell particles are uniformly dispersed in the polymer matrix.

[0027] As mentioned above, the magnetic beads used in the milk sample preparation method according to the present invention are uncoated magnetic beads or particles, meaning that their surface is not coated with antibodies or peptides specific for target bacterial pathogens involved in mastitis, such as Staphylococcus aureus, Streptococcus sp., or coagulase-negative staphylococci (CNS). However, it is understood that the magnetic beads or particles may be surface derivatized with other moieties that allow the beads to bind to bacterial cells and form bead-bacteria complexes.

[0028] In one embodiment, the magnetic beads are uniform mono-sized ferrimagnetic beads of about 1 μm in diameter, composed of cross-linked polystyrene combined with uniformly distributed magnetic material and silica-like surface chemistry (silanol groups). Such beads are commercially available. For example, the exemplary section describes the use of Dynabeads® MyOne™ Silane (Thermo Fisher Scientific, Waltham, MA). However, the invention is not limited to this embodiment.

[0029] In another embodiment, the magnetic beads may contain streptavidin on their surface. Such beads are commercially available. For example, Dynabeads® MyOne Streptavidin T1 beads (Thermo Fisher Scientific, Waltham, MA) are superparamagnetic beads with a diameter of 1 μm, with a monolayer of covalently bound recombinant streptavidin and a hydrophobic surface. Also, Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific, Waltham, MA) are 2.8 μm magnetic beads with covalently bound recombinant streptavidin and a hydrophobic surface. However, the present invention is not limited thereto.

[0030] In yet another embodiment, the magnetic beads are lectin-derivatized. Such beads are commercially available. For example, the inventors used two types of lectin-derivatized beads from GlycoMatrix (Dublin, Ohio): 1) Concanavalin A (Jackbean) Lectin (Con A)-MagneZoom™ beads, and 2) Triticum vulgaris (Wheat) Lectin (WGA)-MagneZoom™ beads, but the present invention is not limited thereto.

[0031] In yet a further embodiment, the magnetic beads are derivatized with free amine groups. Such beads are commercially available. For example, MagnaBind™ amine-derivatized beads are available from Thermo Fisher Scientific, Waltham, MA, but the invention is not limited thereto.

[0032] In one embodiment, the milk sample is used at a concentration of about 95% v / v to about 98.75% v / v. The magnetic beads are used at a concentration of about 1.25% v / v to about 5.0% (v / v). The term "(v / v)" shall mean volume per volume according to its ordinary meaning.

[0033] The milk sample can be a raw milk sample from a mastitis quarter of a dairy cow. In one embodiment, uncoated magnetic beads are mixed with the milk sample and then incubated at room temperature for about 5 minutes to allow the beads to bind to the bacterial cells in the milk sample, thus forming bead-bacteria complexes. In one embodiment, the milk sample is present at about 95% v / v and the magnetic beads are present at about 5% v / v during mixing and incubation.

[0034] After incubating the milk sample with the magnetic beads, the beads are contacted with a magnet. In one embodiment, the magnet is in the form of a magnetic stand. Magnetic stands are commercially available. For example, the Ambion® Single Tube Magnetic Stand (Thermo Fisher Scientific, Waltham, MA) accommodates one 1.5 mL microfusing tube, and the Invitrogen™ MagnaRack™ Magnetic Separation Rack accommodates 24 x 1.5 mL microfusing tubes. In these stands / racks, as in any magnetic stand / rack, centrifugation is replaced by an attractive force between the captured beads in solution and the magnet in the stand, resulting in the beads being collected on one side of the microfusing tube. Thus, the magnetically captured beads containing the bound bacterial cells from the milk sample can be quickly and efficiently separated from the milk sample supernatant containing the other components of the milk sample. The milk sample supernatant is removed from the tube by decanting or aspiration, which can be done manually using a clean disposable pipette with a clean tip, such as a 200 μL tip, or an automated lab pipette.

[0035] The magnetically separated beads with bound bacterial cells may be referred to herein as bead-bacteria complexes. After removing the milk sample supernatant from the tube, the bead-bacteria complexes are then resuspended in an aqueous solution, which the inventors may simply assume to be distilled water. However, in one embodiment, the bead-bacteria complexes are resuspended in a buffered aqueous solution that provides a suitable chemical environment for the activity of DNA polymerase.

[0036] In one embodiment, the aqueous buffer solution used to resuspend the bead-bacteria complexes has a pH of 8.0 to 9.5. In another embodiment, the aqueous buffer solution is stabilized with Tris-HCl, Tris-H3PO4, MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or combinations thereof. In a further embodiment, the aqueous buffer solution is stabilized with Tris-HCl or Tris-H3PO4 present at about 10 to about 50 mM, and a pH of about 8.0 to about 9.0. In yet a further embodiment, the aqueous buffer solution is 25 mM Tris-HCl, pH 8.5.

[0037] In still further embodiments, the buffered aqueous solution used to resuspend the bead-bacteria complexes further comprises a chelating agent, a salt, a detergent, or a combination thereof. Such components are typically included in PCR buffers. In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), or sodium 4-aminosalicylate. In another embodiment, the detergent is tricosaethylene glycol dodecyl ether (Brij L23), octoxynol-1 (Triton X-100), polyoxyethylenesorbitan monolaurate (Tween-20), triethanolamine (TEA), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), sodium dodecyl sulfate (SDS), or a combination thereof. In yet another embodiment, the salt is selected from sodium chloride (NaCl), magnesium chloride (MgCl2), potassium chloride (KCl), or a combination thereof.

[0038] As described herein and shown in Figure 1, an aliquot of the bead resuspension can be used as a template for primer-specific downstream nucleic acid amplification. Alternatively, as also shown in Figure 1, the bead resuspension can be subjected to a magnet to separate the beads from the bead-free supernatant, and an aliquot of the bead-free supernatant is used as a template for primer-specific downstream nucleic acid amplification. Although acceptable results were obtained with each of these two options, the inventors found that the performance of the bead-free supernatant was superior when compared to the bead suspension as a template (Example 6, Table 2).

[0039] Templates obtained using the sample preparation process of the present invention may be amplified in a polymerase chain reaction (PCR) that specifically amplifies the target sequence to detectable amounts. In one desired embodiment, a suitable nucleic acid detection method for use in conjunction with the present invention is a real-time quantitative PCR method that may be dye-based or probe-based. Probe-based quantitative PCR (qPCR) uses real-time fluorescence from 5' to 3' exonuclease cleavage of a fluorescently labeled target-specific probe to measure DNA amplification at each cycle of PCR. Probe-based qPCR is typically more specific than dye-based qPCR and is therefore often the foundational technology used in qPCR diagnostic assays. Probe designs vary, but the most common type of hydrolysis (e.g., TaqMan®) probe incorporates a 5' reporter fluorophore and a 3' quencher on a short oligonucleotide complementary to the target sequence. Fluorescence resonance energy transfer (FRET) prohibits release of the fluorophore while the oligo probe is intact. During each PCR cycle, the 5' flap endonuclease domain of Taq DNA polymerase hydrolyzes the probe as the primer is extended and the target sequence is amplified. This cleavage event separates the reporter fluorophore from the quencher, resulting in an amplification-dependent increase in fluorescence. Probe-based qPCR allows for the quantification of multiple targets in a single reaction (multiplexing) by using a unique fluorescent dye for each amplicon-specific probe. Software exists for designing PCR primers. For example, PCR primers can be designed using Primer Express© software (Applied Biosystems) or PrimerQuest™ tools (Integrated DNA Technologies).

[0040] The following examples and the tables therein are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. EXAMPLES

[0041] Example 1 -Milk sample collection Milk samples were collected aseptically from mastitis quarters of dairy cows (n=68) in the milk houses of different dairy farms. The sampling method followed the standard recommendations by the National Mastitis Council's Laboratory Handbook on Bovine Mastitis. Briefly, the first flow of milk from each quarter was discarded for lactation, after which the teats were immersed in iodine tincture. The teats were then washed and disinfected using 70% ethanol, the first three flows were discarded, and milk samples were collected in sterile plastic tubes without preservatives. Samples were kept on ice until transport to the laboratory, aliquots were separated for culture and MALDI-TOF analysis, and the remaining samples were stored at -80°C for real-time PCR analysis using the milk sample preparation method according to the present invention.

[0042] Example 2 - Microbial Culture To identify samples suitable for downstream molecular analysis, an agar plate culture system was used to detect target bacterial pathogens commonly associated with mastitis in each milk sample. Aliquots of milk samples (100 μL) were plated on the surface of blood agar plates, incubated aerobically overnight at 37°C, and subsequently read to estimate the bacterial load (CFU / mL). Bacterial populations from all tested samples were then further classified at genus and species level by MALDI-TOF analysis.

[0043] Example 3 - Rapid milk sample preparation method In one embodiment, the milk sample preparation method according to the invention involved mixing 10 μL of magnetic beads with silica-like surface chemistry (Dynabeads™ MyOne™ silane, Thermo Fisher Scientific, Waltham, MA) with 200 μL of clinical milk sample, followed by incubation at room temperature for about 5 minutes. After incubation, the magnetic bead pellet was captured using a magnetic separation rack and the milk supernatant was aspirated. The magnetic bead pellet was then resuspended in 100 μL of 25 mM Tris buffer, pH 8.5. An aliquot (10 μL) of the bead suspension or otherwise bead-free supernatant, again separated from the beads using the magnetic rack, was used directly as a template for amplification by real-time PCR. Figure 1 shows an embodiment of the workflow of this novel method.

[0044] Example 4 - Pathogen-specific real-time PCR assay In a previous study, the following primers and probes were verified to be specific for Gram-positive bacteria: forward primer (FP): 5'CAACGCGAAGAACCTTAC C3' (SEQ ID NO: 1), reverse primer (RP): 5'ACGTCATCCCCACCTTCC3' (SEQ ID NO: 2), and probe: 5'FAM-ACGACAACCATGCACCACCTG BHQ13' (SEQ ID NO: 3) (Wu et al., 2008 Journal of Clinical Microbiology. 46(8):2613-2619) were selected to evaluate the rapid milk sample preparation method according to the present invention. These Gram-positive specific primers and probes used in downstream real-time PCR contained 16S rRNA genes that allow for the simultaneous detection and identification of clinically relevant Gram-positive and Gram-negative bacteria. Gram-positive bacterial pathogens involved in mastitis include Staphylococcus aureus and coagulase-negative staphylococci (CNS) and should be detected in milk samples taken from mastitis quarters of dairy cows in downstream real-time PCR.

[0045] Prior to assay development, both primers and probes were synthesized (Integrated DNA Technologies, IA) and a 20X stock solution was prepared by mixing 20 μL of forward primer (FP), 20 μL of reverse primer (RP), and 10 μL of probe with 150 μL of IDT DNA buffer (Integrated DNA Technologies, Inc., Coralville, Iowa).

[0046] Real-time PCR assays were performed on 10 μL of target magnetic bead suspension or supernatant without magnetic beads prepared as template by the rapid milk sample preparation method in a reaction mixture containing 1× TaqPath ProAmp master mix (Applied Biosystems, CA) with 1.5× or 1.0× primer-probe mix, respectively. The final volume was made up to 25 μL with DNase-RNase free water. The reaction mixture was thermocycled once at 60° C. for 30 s and 95° C. for 10 min, followed by 45 cycles at 95° C. for 15 s and 60° C. for 60 s. These amplicons were then detected in real time using a CFX96 qPCR machine (Bio-Rad Laboratories, Hercules, CA).

[0047] The diagnostic sensitivity and specificity along with Jeffrey's 95% CI were also estimated by testing positive clinical milk samples for gram-positive bacterial pathogens associated with bovine mastitis (S. aureus and coagulase-negative staphylococci) (Table 1 below). Rapid milk sample preparation, followed by real-time PCR testing, was performed as described above, and then amplicons were detected in real time by a CFX96 qPCR machine (Bio-Rad Laboratories, Hercules, CA) instrument.

[0048] Example 5 - Optimization and performance of milk sample preparation method The method of the present invention involves the use of magnetic beads to bind bacteria in milk samples, and in some cases also to free DNA and other host cells present in the milk sample. Different types of magnetic beads were evaluated, including simple magnetic beads (Dynabead™ MyOne™ silane), or other surface-derivatized beads (lectin magnetic beads, streptavidin and amine-derivatized magnetic beads), and immunomagnetic beads (magnetic beads conjugated with antibodies). Among the different magnetic beads tested, Dynabeads™ with silica-like surface chemistry, Dynabeads™ with streptavidin, lectin-derivatized, and amine-derivatized magnetic beads appeared promising. Lectin-derivatized magnetic beads may have specificity for sugars located on the bacterial surface, such as N-acetylglucosamine. Dynabeads™ with streptavidin bind to extracellular biotin on the surface of bacterial cells. Again, without wishing to be bound by any one theory, the hypothesis driving the inclusion of simple magnetic beads (Dynabeads™ with silica-like surface chemistry) is that binding to bacterial cells is random and may be due to electrostatic interactions (Vander Waals forces). Furthermore, the amine groups on the amine-derivatized magnetic beads interact with proteins localized on the surface of bacterial cells in the milk samples. Cultivation of magnetic bead suspensions prepared from clinical milk samples on selective agar plates revealed bacterial colonies, confirming binding to bacterial cells.

[0049] With regard to the selection of the aqueous solution used to resuspend the beads containing the bound bacterial cells, as described in the detailed description, the aqueous solution is preferably an aqueous buffer compatible with downstream PCR amplification, such as Tris-HCl, MOPS (3-(N-morpholino)propanesulfonic acid), or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), the pH of the aqueous buffer being 8.0-9.5. In one desired embodiment, the aqueous buffer is stabilized by Tris-HCl, e.g., present at about 20-30 mM and a pH of about 8-9. In a non-limiting example of the invention, the aqueous buffer is 25 mM Tris-HCl, pH 8.5, compatible with downstream qPCR. We also added detergents (Brij-L23, saponin, CHAPS, SDS), high salt (NaCl), and / or chelating agents (EDTA) to 25 mM or 50 mM Tris pH 8.5 buffers, as shown below, to evaluate whether these added components improved performance. 1.25 mM Tris (pH 8.5) + 0.5% Brij-L23 2.25 mM Tris (pH 8.5) + 1.0% Brij-L23 3.25 mM Tris (pH 8.5) + 1.5% Brij-L23 4.25mM Tris (pH 8.5) + 0.5% saponin 5.25 mM Tris (pH 8.5) + 1.0% saponin 6.25 mM Tris (pH 8.5) + 0.5% CHAPS 7.25 mM Tris (pH 8.5) + 50 mM NaCl 8.25 mM Tris (pH 8.5) + 50 mM NaCl + 5 mM EDTA + 1% SDS 9. 50 mM Tris (pH 8.5) + 100 mM NaCl + 5 mM EDTA

[0050] The above Tris buffer solutions 1-9 were found to perform similarly to 25 mM Tris (pH 8.5) without added detergents, salts, or chelating agents. However, no significant improvement in performance was observed, so 25 mM Tris buffer (pH 8.5) was chosen for its simplicity.

[0051] The real-time PCR program was also modified by the inventors to increase the time of the initial denaturation step at 95 °C, where bacterial cells can be lysed and DNA released, from 5 min to 7.5-10 min.

[0052] With regard to the choice of magnetic beads, it was found that there was no difference in the results obtained when testing the milk sample preparation method using Dynabeads™ with silica-like surface chemistry, Dynabeads™-streptavidin, lectin-derivatized or amine-derivatized magnetic beads. Therefore, Dynabeads™ with silica-like surface chemistry were used for subsequent testing due to their simplicity.

[0053] Another notable aspect of the milk sample preparation method of the present invention is the separation of bacterial cells from clinical milk, thereby eliminating the inhibitory effects of milk components on downstream real-time PCR assays.

[0054] During further optimization, different combinations of milk sample input and magnetic beads were evaluated and it was observed that the combination of 200 μL clinical milk sample and 10 μL magnetic beads appeared promising.

[0055] Example 6 - Detection of pathogens in milk samples For the testing of the rapid milk sample preparation method and downstream real-time PCR assay described herein, a total of 42 archived milk samples collected from mastitis quarters of cows were considered (Table 1). A combination of culture and MALDI-TOF analysis performed on these milk samples identified Gram-positive pathogens such as S. aureus and coagulase-negative staphylococci (CNS). To estimate specificity, a set of 26 milk samples collected from mastitis quarters of cows with E. coli (Gram-negative) growth was also included (Table 1). A preliminary evaluation of the milk sample preparation method according to the invention with a downstream real-time PCR assay was performed on these 68 clinical milk samples using culture and MALDI-TOF as reference methods.

[0056] The Gram-positive specific primers and probes used in the PCR assay are described in Example 4 above. Staphlylococcus aureus and coagulase-negative staphylococci (CNS) are Gram-positive bacteria and should be amplified, providing an indication of sensitivity. On the other hand, Escherichia coli is a Gram-negative bacteria, and the Gram-positive specific primers and probes used in this study should not amplify E. coli, providing an indication of specificity. Culture and MALDI-TOF are reference methods that provide the true state of the milk sample for comparison with the method of the present invention.

[0057] The results of this study showed superior performance of bead-free supernatant compared to bead suspension as template (Table 2). Additional molecular assay optimization and fresh clinical milk samples may improve the assay outcome. Typically, molecular methods detect both dead and viable cells. Culture methods, on the other hand, have the potential to demonstrate only viable cells. Thus, molecular methods appear to be less specific compared to culture methods. [Table 1] [Table 2]

[0058] In the present invention, a rapid milk sample preparation method compatible with downstream real-time PCR assays for the detection of bacterial pathogens in milk has been successfully developed. The key advantages compared to the prior art include the following features: 1. This rapid method is feasible for point-of-care applications to detect bacterial pathogens in milk. 2. No instrumentation (centrifuge and heating block) is required. 3. This method has the potential to reduce the inhibitory effects of milk components in downstream molecular assays. 4. No multiple washing or pipetting steps are required. 5. No special storage of the buffer solution (refrigeration or -20°C) is required. 6.It does not require the use of any dangerous chemicals.

Claims

1. 1. A method for preparing a milk sample for a downstream nucleic acid amplification process, comprising: a) contacting a milk sample containing at least one suspected bacterial pathogen involved in mastitis with uncoated magnetic beads; b) incubating the milk sample in the presence of the uncoated magnetic beads to allow bacterial cells in the milk sample to bind to the beads, thereby forming bead-bacteria complexes; c) subjecting the bead-bacteria complexes to a magnet such that the bead-bacteria complexes are separated from the milk sample supernatant; d) removing the milk sample supernatant; e) directly resuspending the separated bead-bacteria complexes in an aqueous solution to release the bead-bound bacteria into said solution; f) using an aliquot of the bead resuspension from e) as a template for primer-specific downstream nucleic acid amplification.

2. 2. The method of claim 1, further comprising subjecting the bead resuspension from f) to a magnet to separate the beads from the supernatant, and using an aliquot of the bead-free supernatant as a template for the primer-directed downstream nucleic acid amplification.

3. The method of claim 1 , wherein the milk sample is a raw milk sample.

4. The method of claim 3, wherein the raw milk sample is from a mastitis quarter of a dairy cow.

5. 2. The method of claim 1, wherein the bead-bacteria complex in e) is resuspended in a buffered aqueous solution that provides a suitable chemical environment for activity of a DNA polymerase.

6. The method according to claim 5, wherein the pH of the aqueous buffer solution is from 8.0 to 9.

5.

7. The buffer solution is Tris-HCl, Tris-H ３ P.O. ４ 8. The method of claim 6, wherein the cellulose is stabilized by MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or a combination thereof.

8. The aqueous buffer solution is Tris-HCl or Tris-H, present at about 10 to about 50 mM and at a pH of about 8.0 to about 9.

0. ３ P.O. ４ The method of claim 7, wherein the compound is stabilized by

9. 9. The method of claim 8, wherein the aqueous buffer solution is 25 mM Tris-HCl, pH 8.

5.

10. The method of claim 5 , wherein the buffered aqueous solution comprises a chelating agent, a salt, a detergent, or a combination thereof.

11. The method of claim 10, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), or sodium 4-aminosalicylate.

12. 11. The method of claim 10, wherein the detergent is tricosaethylene glycol dodecyl ether (Brij L23), octoxynol-1 (Triton X-100), polyoxyethylene sorbitan monolaurate (Tween-20), triethanolamine (TEA), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), sodium dodecyl sulfate (SDS), or a combination thereof.

13. The salt may be sodium chloride (NaCl), magnesium chloride (MgCl ２ 11. The method of claim 10, wherein the phosphate phosphatase is selected from the group consisting of potassium chloride (KCl), ... and combinations thereof.

14. The method of claim 1 , wherein the milk sample and the uncoated magnetic beads are mixed prior to incubation.

15. The method of claim 1, wherein the incubation is for about 5 to about 10 minutes at room temperature.

16. The method of claim 1 , wherein the step of preparing the milk sample is performed in a tube.

17. The method of claim 16, wherein the tube is placed in a magnetic stand to capture the magnetic beads.

18. The method of claim 1 , wherein the uncoated magnetic beads comprise a silica-like surface chemistry.

19. The method of claim 1 , wherein the uncoated magnetic beads are surface derivatized with lecithin, streptavidin, or amines.

20. The method of claim 1 , wherein the templates are analyzed by real-time PCR using primers specific for a bacterial pathogen involved in mastitis.

21. 21. The method of any one of claims 20, wherein the bacterial pathogen responsible for mastitis is selected from the group consisting of Staphylococcus aureus, Streptococcus sp., and coagulase-negative staphylococci (CNS).

22. A Point-of-Care (PoC) molecular test allowing the identification of an etiologic agent in a milk sample, comprising the use of a template for primer-directed nucleic acid amplification prepared according to the method of any one of claims 1 to 21.

23. 23. The method of claim 22, wherein the milk sample is from a mastitis quarter of a dairy cow.

24. 23. The method of claim 22, wherein the molecular test is real-time PCR.

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