Fungal nucleic acid extraction

Abstract

The invention provides methods for extraction of fungal (e.g., yeast spp., filamentous fungal spp.) nucleic acid (e.g., DNA, RNA) from a sample (e.g., be human or veterinary clinical or research samples, agricultural samples, agricultural commodity samples, food products, or environmental samples). In some embodiments, the present invention provides enhanced nucleic acid extraction from samples comprising fungal cell(s) wherein enzymatic (e.g., lysostaphin treatment, lyticase treatment) sample treatment is performed in combination with mechanical (e.g., bead beating) sample treatment.

Claims

1. A method of extracting fungal nucleic acid from a sample, comprising: a) treating said sample with enzymatic treatment wherein said enzymatic treatment comprises treatment with lysostaphin and lyticase in a reaction mixture wherein said reaction mixture does not comprise a chelator; b) treating said reaction mixture with mechanical treatment: and c) extracting said nucleic acid from said reaction mixture wherein a combination of said enzymatic treatment comprising said lysostaphin and said lyticase with said mechanical treatment results in an increased yield of said extracted nucleic acid compared to enzymatic treatment or mechanical treatment performed separately.

2. The method of claim 1 wherein said mechanical treatment is selected from the group consisting of bead beating, grinding, sonication, extrusion, freezing, freeze-thawing, exposure to pressure exceeding 1 atm, exposure to pressure below 1 atm, exposure to temperature above 37 degrees C., exposure to temperature below 10 degrees C., treatment with a hammer mill, treatment with a knife mill, treatment with a ball mill, treatment with a homogenizer, treatment with a chipping machine, treatment with a grinding machine, treatment with a French press, treatment with an extrusion device, and irradiation.

3. The method of claim 1, wherein said mechanical treatment comprises bead beating.

4. The method of claim 1, further comprising treatment of said sample with protease.

5. The method of claim 1, further comprising osmotic shock treatment of said sample.

6. The method of claim 1, further comprising exposure of said sample to sodium hydroxide.

7. The treatment of claim 1, wherein said lysostaphin is added to said sample at a final concentration of 0.0125 U per l of reaction mixture.

8. The treatment of claim 1, wherein said lyticase is added to said sample at a final concentration of 0.22 U per l of reaction mixture.

9. The method of claim 1, further comprising addition of buffer to said sample.

10. The method of claim 9, wherein said buffer is MOPS.

11. The method of claim 1, wherein said extracting is performed using a robotic sample handling system.

12. The method of claim 1, wherein said nucleic acid is DNA.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an experimental timeline for Experiment 1.

(2) FIG. 2 shows quantitative PCR results for detection of Candida albicans DNA from whole blood samples, as described in Experiment 1. CA, Candida albicans; IC, internal control.

(3) FIG. 3 shows quantitative PCR results for detection of Staphylococcus aureus DNA from whole blood samples, as described in Experiment 1. SA, Staphylococcus aureus; IC, internal control.

DEFINITIONS

(4) To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

(5) As used herein, bead beater refers to a device that facilitates sample homogenization by mechanical disruption, wherein a vessel containing the sample is held by the device and subjected to agitation, whether by rotating, oscillating, shaking, etc. Typically, particles (e.g., inert particles; also referred to as microparticles or beads) are added to the sample vessel to aid in sample homogenization or maceration.

(6) As used herein, mechanical treatment, mechanical processing, mechanical handling or similar terms refer to methods of physically disrupting a sample comprising solid material (e.g., a solid sample; a suspension of solids in liquid; a semi-solid (e.g., viscous, gel) sample). Physical effects of mechanical treatment may include but are not limited to reduction of sample particle size, reduction of sample viscosity, liquification, maceration, homogenization, and the like. Examples of mechanical treatment include but are not limited to particle-based disruption (e.g., homogenization using beads, microparticles, and the like, also referred to as bead beating), grinding, sonication, extrusion, freezing, freeze-thawing, high pressure and/or temperature, low pressure and/or temperature, and/or use of hammer mills, knife mills, ball mills, homogenizers (e.g., Dounce homogenizers), chipping machines, grinding machines, extrusion devices, sonication, and/or irradiation.

(7) As used herein, enzymatic treatment, enzymatic lysis, enzymatic processing or similar terms refer to methods of enzymatic treatment of a sample to achieve physical disruption. Physical effects of enzymatic treatment may include but are not limited to reduction of sample particle size, reduction of sample viscosity, liquification, maceration, homogenization, and the like. In some embodiments, enzymatic treatment achieves disruption of solid biological material (e.g., cell wall). Such disruption may be partial (e.g., increased porosity of cell wall materials; reduced thickness of cell wall material; reduced size of cell wall fragments) or complete.

(8) As used herein, protease refers to any enzyme that binds to and cleaves a proteinaceous (e.g., polypeptide, peptide) substrate.

(9) As used herein, the term subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms subject and patient are used interchangeably herein in reference to a human subject.

(10) As used herein, the term non-human animals refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc.

(11) As used herein, the term sample is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans), plants, fungi, microbes, etc. and encompass fluids, solids, tissues, and combinations thereof. Biological samples include blood products, such as plasma, serum and the like and tissue samples, such as biopsy samples and the like. Such examples are not however to be construed as limiting the sample types applicable to the present invention.

(12) As used herein, the term nucleic acid molecule refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

DETAILED DESCRIPTION OF THE INVENTION

(13) The invention provides methods for extraction of fungal (e.g., yeast spp., filamentous fungal spp.) nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention provides enhanced nucleic acid extraction from samples comprising fungal cell(s) wherein enzymatic (e.g., lysostaphin, lyticase, or a combination thereof) sample treatment is performed in combination with mechanical (e.g., bead beating) sample treatment.

(14) In experiments conducted during the course of developing some embodiments of the present invention, it was found that pre-treatment of samples (Candida albicans or Staphylococcus aureus diluted in whole blood) with lyticase and lysostaphin followed by mechanical disruption using bead beating resulted in an eight-fold increase in the amount of fungal DNA extracted, as measured by quantitative PCR.

(15) Accordingly, in some embodiments, the present invention provides methods for extraction of fungal DNA from samples wherein increased yield and/or increased efficiency of nucleic acid extraction is realized.

(16) Mechanical Disruption Methods

(17) The present invention provides methods for fungal nucleic acid extraction from a sample wherein the sample is subjected to mechanical disruption. Types of mechanical disruption include but are not limited to particle-based disruption (e.g., homogenization using beads, microparticles, and the like, also referred to as bead beating), grinding, sonication, extrusion, freezing, freeze-thawing, high pressure and/or temperature, low pressure and/or temperature, and/or use of hammer mills, knife mills, ball mills, homogenizers (e.g., Dounce homogenizers), chipping machines, grinding machines, extrusion devices, French press, and/or irradiation

(18) Bead Beating

(19) A laboratory-scale mechanical method for cell disruption uses small glass, ceramic, zirconium, or steel beads and a high level of agitation by stirring or shaking of the mix. The method, often referred to as bead beating, works well for all types of cellular materialfrom spores to animal and plant tissues.

(20) In some embodiments, beads are added to the cell or tissue suspension in a test tube and the sample is mixed on a common laboratory vortex mixer. While processing time is 3-10 times longer than that in specially machines described herein, it works for easily disrupted cells and is inexpensive.

(21) At a more sophisticated level, bead beating is done in closed vials, centrifuge tubes, or sealed titer plates. The sample and the beads are vigorously agitated (e.g., at about 2000 oscillations per minute) in a specially designed clamp driven by a high energy electric motor. In some machines hundreds of samples can be processed simultaneously. To prevent degradation of biological macromolecules (e.g., DNA, RNA, proteins), some form of cooling is often used because samples experience an increase in heat due to collisions of the beads. Cooling can be accomplished by placing titer plates or vials in chilled aluminum blocks. Another configuration suitable for larger sample volumes uses a rotor inside a sealed 15, 50 or 200 ml chamber to agitate the beads. The chamber can be surrounded by a cooling jacket. Using this same configuration, commercial machines capable of processing many liters of cell suspension are available.

(22) Sonication

(23) A method for cell disruption applies ultrasound (typically 20-50 kHz) to the sample (sonication). In principle, the high-frequency is generated electronically and the mechanical energy is transmitted to the sample via a metal probe that oscillates with high frequency. The probe is placed into the cell-containing sample and the high-frequency oscillation causes a localized low pressure region resulting in cavitation and impaction, ultimately breaking open the cells. Some systems permit cell disruption in smaller samples (including multiple samples under 200 L in microplate wells) and with an increased ability to control ultrasonication parameters.

(24) Valve-Type Processors

(25) Valve-type processors disrupt cells by forcing the media with the cells through a narrow valve under high pressure (20,000-30,000 psi or 140-210 MPa). As the fluid flows past the valve, high shear forces in the fluid pull the cells apart. By controlling the pressure and valve tension, the shear force can be regulated to optimize cell disruption. Due to the high energies involved, sample cooling is generally required, especially for samples requiring multiple passes through the system. Three major implementations of the technology exist: the French pressure cell press, constant cell disruption systems, and pumped-fluid processors.

(26) French press technology uses an external hydraulic pump to drive a piston within a larger cylinder that contains the sample. The pressurized solution is then squeezed past a needle valve. Once past the valve, the pressure drops to atmospheric pressure and generates shear forces that disrupt the cells.

(27) Cell Bomb

(28) Another system for cell disruption is rapid decompression or the cell bomb method. In this process, cells in question are placed under high pressure (usually nitrogen or other inert gas up to about 25,000 psi) and the pressure is rapidly released. The rapid pressure drop causes the dissolved gas to be released as bubbles that ultimately lyse the cell.

(29) Enzymatic Treatment

(30) Lysostaphin

(31) Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, is a family of enzymes (EC 3.2.1.17) which damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. Lysozyme is abundant in a number of secretions, such as tears, saliva, human milk and mucus. It is also present in cytoplasmic granules of the polymorphonuclear neutrophils (PMN). Large amounts of lysozyme can be found in egg white. C-type lysozymes are closely related to alpha-lactalbumin in sequence and structure making them part of the same family. Lysostaphin may be produced by recombinant expression (see, e.g., U.S. Pat. No. 4,931,390).

(32) Lyticase (Zymolase, Zymolyase)

(33) Zymolyase (zymolase), also referred to as lyticase, is a preparation of enzymes from Arthrobacter luteus. The main activities of the enzyme are -1,3 glucanase and -1,3-glucan laminaripentao-hydrolase, which hydrolyze glucose polymers at the -1,3-glucan linkages releasing laminaripentaose as the principal product. Optimal Zymolyase activity is at 30-37 C.; lytic activity ceases at higher temperatures. Susceptible fungal genera include but are not limited to Asbya, Candida, Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora, Kloekera, Kluyveromyces, Lipomyces, Metschikowia, Pichia, Pullularia, Saccharomyces, Saccharomycodes, Saccharomycopsis, Schizosaccahromyces, and Torulopsis.

Example

(34) The following example is provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and is not to be construed as limiting the scope thereof.

Fungal Nucleic Acid ExtractionMechanical and Enzymatic Processing

(35) The extraction of Candida albicans or Staphylococcus aureus DNA present in whole blood samples was performed. Six sets of conditions were used to compare extraction efficiency of mechanical processing (bead beating) alone or in combination with enzymatic treatment (lysostaphin and lyticase) under different osmotic or pH conditions. The combination of enzymatic pre-treatment followed by bead beating was approximately eight-fold more effective than either bead beating or enzymatic treatment performed separately.

(36) Methods and Reagents

(37) DNA was extracted using a DNA extraction kit formulated for the m2000 automated real-time PCR instrument (the mSample Preparation System (DNA) (Abbott Molecular, Abbott Park, Ill.) according to the manufacturer's instructions. Prior to starting, 35 ml of 95% ethanol was added to 70 ml lysis buffer to create wash buffer 2. Lysis buffer was used as wash buffer 1. Microparticles used for bead beating were Zirconia-Silica 9BioSpec). Stock solutions included 5N NaOH (Fisher brand), 1 M MOPS buffer (Sigma brand). Enzyme stock solutions were lysostaphin (2.8 U/l) which was diluted to 1.4 U/l with 10 l used per reaction for a final amount of 14 U per reaction); lyticase at 25 U/l (used at 250 U per reaction), and proteinase K (Abbott brand). Fungal stocks (S. aureus or C. albicans at 10.sup.5/ml) were diluted in whole blood to a final sample concentration of 300 ifu/ml.

(38) Six different extraction conditions were used, with 4 replicates of each condition. Two sets of extraction conditions included bead beating, while 4 did not. For bead beating, 900 mg zirconium/silica beads were used per sample. Extraction condition groups were:

(39) 1) standard bead beating extraction (tubes 1-4)

(40) 2) Lysostaphin and Lyticase pre-treatment followed by standard bead beating extraction (tubes 5-8)

(41) 3) Lysostaphin and Lyticase-NaOH-75 l water-MOPS (tubes 9-12)

(42) 4) Lysostaphin and Lyticase (tubes 13-16)

(43) 5) Lysostaphin and Lyticase-NaOH-775 pd water-MOPS (tubes 17-20)

(44) 6) Lysostaphin and Lyticase (tubes 21-24)

(45) DNA extraction plates were prepared for nucleic acid preparation using a KingFisher (Thermo Scientific) automated purification system. Extraction plates were prepared as follows:

(46) Loaded 1 ml of Lysis-ethanol into each well of extraction plate.

(47) Added 160 ul of microparticles into each well of extraction plate.

(48) Added extra lysis-ethanol into well that will have extra volume.

(49) For column 3, added 350 ul

(50) For column 4, added 600 ul

(51) For column 5, added 1.5 ml

(52) For column 6, added 1.8 ml

(53) Loaded 2 ml of lysis-ethanol into each well of Wash 1 plate.

(54) Loaded 2 ml of 70% ethanol into each well of Wash 2 plates (all 3)

(55) Loaded 250 ul water into elution plate.

(56) The extraction timeline is shown in FIG. 1. For enzyme treatment samples (groups 2-6), 20 l enzyme (lysostaphin and lyticase) stock was added to 720 l sample (groups 3-6) or 1100 l sample (group 2). Tubes were incubated at 37 C. for 30 min. Group 2 samples (enzyme treatment+bead beating) and Group 1 samples (bead beating only) were subjected to bead beating and incubated at 57 C. for 15 minutes in the presence of 325 l lysis buffer without ethanol in the presence of 110 l protease. For samples subjected to sodium hydroxide treatment (groups 3-6), 25 l of 5N sodium hydroxide was added along with 75 l water (groups 3 and 4) or 750 l water (groups 5 and 6). All sodium hydroxide-treated samples were incubated at 57 C. for 10 min, followed by adding 150 l MOPS buffer and 250 l lysis buffer. For group 3 and group 5 samples, 75 l protease was also added. All sodium hydroxide-treated samples (groups 3-6) were then incubated at 57 C. for an additional 15 minutes. Prior to extraction using a KingFisher purification system, all samples were centrifuged and supernatents were loaded on the extraction plate. Extractions were performed with 160 l magnetic microparticles.

(57) The KingFisher extraction program included a 25 minute drying step at the end to remove traces of ethanol that might otherwise inhibit quantitative PCR. Following extraction, eluates were stored at 20 C.

(58) To assess DNA extraction efficiency, quantitative PCR reactions were carried out:

(59) C. albicans assayfor 30 assays

(60) 1) Primer 1 0.1 ul/rx, 3 ul total

(61) 2) Primer 2 0.1 ul/rx, 3 ul total

(62) 3) Probe 0.1 ul/rx 3 ul

(63) 4) 2 Taqman Buffer AB #4324018 12.5 ul/rx, 375 ul total

(64) 5) 10IPC mix 2.5 ul/rx, 75 ul total

(65) 6) 50IPC template 0.5 ul/rx, 15 ul total

(66) 7) Water. 4.3 ul/rx, 129 ul total

(67) Master mix was made, and add 20 ul was added to each well in the plate. Following this, 5 l sample was added per reaction. Quantitative PCR was performed and samples were stored at 20 C. when complete.

(68) The S. aureus assay was performed identically using target-specific primers and probes.

(69) Results are shown below and in FIGS. 2 and 3.

(70) TABLE-US-00001 TABLE 1 C. albicans assay results. Standard C. albicans Standard Deviation, assay Internal Deviation, Internal Protocol results Control S. aureus Control Standard 30.5425 28.3625 0.50566 0.333504 bead beating (Group 1) Lysostaphin 27.245 28.27 0.169017 0.069761 and Lyticase + standard bead beating (Group 2) Lysostaphin 31.665 27.7475 1.31272 0.140089 and Lyticase, NaOH, MOPS (Group 3) Lystostaphin 1 27.47 0 0.100333 and Lyticase (Group 4) Lysostaphin 29.455 28.035 0.737496 0.106301 and Lyticase, NaOH, MOPS (Group 5) Lysostaphin 36.74 27.5375 1.24503 0.074554 and Lyticase (Group 6)

(71) TABLE-US-00002 TABLE 2 S. aureus assay results. Standard S. aureus Standard Deviation, assay Internal Deviation, Internal Protocol results Control S. aureus Control Standard 32.71 33.7625 0.280595 0.878725 bead beating (Group 1) Lysostaphin 31.6125 33.3 0.737626 0.402409 and Lyticase + standard bead beating (Group 2) Lysostaphin 33.7375 31.345 0.724724 0.635898 and Lyticase, NaOH, MOPS (Group 3) Lystostaphin 1 30.9675 0 0.581112 and Lyticase (Group 4) Lysostaphin 32.03 32.185 0.508789 0.814064 and Lyticase, NaOH, MOPS (Group 5) Lysostaphin 37.4675 30.6275 0.843421 0.263486 and Lyticase (Group 6)

(72) The lysticase-lysostaphin treatment appeared to help the bead beating and gave the best result. Over a 3 CT improvement for the C. albicans assay and over a 1 CT improvement for the S. aureus assay was observed. This extraction without the bead beating was different in that the NaOH treatment was done prior to the protease treatment. No sample clumping was observed when protease treatment was done before the NaOH treatment. The treatment with the osmotic shock and the protease (no bead beating) worked almost as well as the bead beating with the enzyme for S. aureus (0.7 CT improvement). The same protocol had over a 1 CT improvement for the C. albicans assay.

(73) All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or microbiology are intended to be within the scope of the following claims.