WO2022003723A1 - Procédé pour l'extraction d'acides nucléiques à partir d'un échantillon biologique - Google Patents

Procédé pour l'extraction d'acides nucléiques à partir d'un échantillon biologique Download PDF

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Publication number
WO2022003723A1
WO2022003723A1 PCT/IN2021/050637 IN2021050637W WO2022003723A1 WO 2022003723 A1 WO2022003723 A1 WO 2022003723A1 IN 2021050637 W IN2021050637 W IN 2021050637W WO 2022003723 A1 WO2022003723 A1 WO 2022003723A1
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Prior art keywords
biological sample
magnetic nanoparticles
nucleic acids
agent
biological
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PCT/IN2021/050637
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English (en)
Inventor
Sreedhar SANTHOSH
Aniruddha BHATI
Minu SARMA N
Sreepriya ARUN
Anu VARGHESE
Vandana S
Gayathri R
ArunPrasath SEKAR
Mohamed KASHITH M
Reenu JOSEPH
Yedhu MOHAN
Seethal BABU
Ajith ARUMUGAM
Tessy IYPE
Cn Ramchand
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Maggenome Technologies Pvt. Ltd.
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Publication of WO2022003723A1 publication Critical patent/WO2022003723A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present embodiment relates to a method for the extraction of nucleic acids from a biological sample of the subject using magnetic nanoparticles, and more particularly relates to the method for the extraction of DNA and RNA from a biological sample.
  • Nucleic acid is an important class of macromolecules found in all cells and viruses. The functions of nucleic acids have to do with the storage and expression of genetic information. Deoxyribonucleic acid (DNA) encodes the information the cell needs to make proteins. The extraction of nucleic acids from the biological samples plays a central role in molecular biology. The purified DNA is used in the diagnosis of genetic disorders and in the field of pharmacogenomics. The purified Ribonucleic Acid (RNA) also has a lot of importance in the diagnosis of diseases, especially those caused by RNA viruses.
  • DNA Deoxyribonucleic acid
  • RNA Ribonucleic Acid
  • nucleic acids are biological samples.
  • the extraction of nucleic acids from the blood sample requires stringent laboratory conditions vis-a-vis centrifuge, water bath, refrigerator and other sophisticated instruments.
  • nucleic acid sample is required in larger quantity. Therefore, the blood sample also has to be collected in large quantities.
  • the blood sample is collected through a needle and the process is invasive, the collection of blood sample makes the patient uncomfortable. Therefore, there is a need of developing a method for the extraction of nucleic acids from a non-invasive sample. Further, there is a need of the method for eliminating the laborious and time consuming centrifugation steps during the extraction of nucleic acids.
  • viral infections especially respiratory infections
  • nasal and throat swabs are used for the detection of viral infections. More recently several research groups have started recommending saliva, especially deep throat saliva as a preferred sample. Collection of saliva and urinary samples is non-invasive and poses absolutely no threat to the healthcare worker or technician since the patients themselves can collect the samples.
  • DNA extraction is laborious and time-consuming in traditional methods and later that can be achieved by solid phase extraction method which allow quick and efficient purification.
  • Solid phase extraction procedures are based on the preferential binding of DNA to a solid support followed by washing step to remove contaminants and elute pure DNA.
  • Silica spin column based technique is widely used across the world, introduction of magnetic solid phases rendered several advantages to DNA extraction protocols. It minimizes the need to use centrifugation, toxic solvents or filtration under vacuum/column.
  • Magnetite Fe304
  • Fe304 is the most widely used magnetisable solid phase support material with sizes ranging from micron to nanometers.
  • MNPs magnetic nanoparticles
  • the magnetic nanoparticles (MNPs) used for DNA extraction can be either coated or uncoated/bare Fe304.
  • coated magnetic beads/particles DNA binding is facilitated by surface functionalization.
  • the adsorption of DNA to these particles in such cases is dependent mainly on several physiochemical interactions like electrostatic, hydrophobic or hydrogen bonding.
  • a system and method to extract nucleic acid from the biological sample of the subject In view of the foregoing, a system and method to extract nucleic acid from the biological sample of the subject.
  • a method for extraction of nucleic acids from a biological sample using magnetic nanoparticles involves mixing the biological sample with a preservation buffer with or without an enzyme and a detergent to form a biological lysate, mixing the biological lysate with the magnetic nanoparticles, a salt and a binding agent, binding of the nucleic acids from the biological samples to the magnetic nanoparticles and washing to remove contaminants, and eluting the magnetic nanoparticles to obtain pure nucleic acids.
  • the biological sample is selected from blood, serum, plasma, urine, stool, semen, tears, saliva, amniotic fluids, oral fluids, sweat, paraffin fixed tissues, bacteria, viruses, fungi, soil or swabs.
  • the magnetic nanoparticles are coated or uncoated by a polymer or a protein.
  • the magnetic nanoparticles are prepared by a physical, chemical, biological and co-precipitation method.
  • the magnetic nanoparticles are a metal oxides comprising magnetite (Fe304), ulvospinel, hematite (a-Fe203), ilmenite, maghemite (y-Fe203), jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite, wairauite, and a combination thereof.
  • the magnetic nanoparticles has a size ranging from 1 nm to 500 nm.
  • the magnetic nanoparticles has a size ranging from 5 nm to 20 nm.
  • the detergent is an alkali metal alkylsulphate salt.
  • the salt is a monovalent and a divalent salt selected from sodium chloride, lithium chloride, Potassium chloride, magnesium chloride or a combination thereof.
  • the binding agent is a polymer selected from Polyethylene Glycol (PEG) 800 to 10,000.
  • the binding agent is a solvent selected from acetone, ethanol, methanol, acetonitrile or propanol.
  • the biological lysate is treated with a proteinase and DNase and RNase enzymes.
  • a preservation buffer for stabilization of cells, bacteria and virus consist of a buffering agent comprising phosphate, Tris, borate, citrate and glycine, wherein the buffering agent has a pH ranging from 4-9 and a concentration ranging from 0.1 M to 2 M, a monovalent and a divalent salt comprising sodium chloride, lithium chloride, potassium chloride, magnesium chloride and a combination thereof, wherein the salt has a concentration ranging from 0.01 M to 0.5 M, a chelating agent comprising ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis-tetraacetic acid, cyclohexane diaminotetraacetate (CDTA), diethylenetriaminepentaaceticacid (DTPA), tetraazacycylododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), deszferioximine and a chelatoranalog, wherein
  • a sample storage vial 200 for preservation of nucleic acids consist of a tubular body 202 for storing a biological sample, a cap 204 comprising a storage compartment for the storage of preservation buffer and a membrane for sealing the preservation buffer, and a narrow opening 206 comprising a teeth-like projection 208, wherein the teeth-like projections 208 ruptures the membrane of the cap 204.
  • the cap 204 is a flap like covering mounted on the tubular body 202.
  • the tubular body 202 is marked on the outer surface for indicating the volume of a biological sample.
  • the membrane is a plastic film, an aluminium film or a silver film.
  • the teeth-like projections pricks the membrane to release preservation buffer to the tubular body 202, which contains biological sample.
  • the tubular body 202 is attached with a detachable compartment which contains a swab for collecting the biological sample.
  • Figure 1 illustrates a method (100) for the extraction of nucleic acids, according to an embodiment herein;
  • Figure 2 illustrates a sample storing vial (200), according to an embodiment herein
  • Figure 3 illustrates a graph demonstrating a yield of nucleic acids, according to an embodiment herein;
  • Figure 4 illustrates a gel electrophoresis for determining the yield of DNA extracted from the present method and the yield of DNA extracted from the conventional methods, according to an embodiment herein;
  • Figure 5 illustrates a gel electrophoresis for determining the yield of extracted RNA, according to an embodiment herein;
  • Figure 6 illustrates the comparative yield and purity profiles of genomic DNA extracted from saliva sample stored in the SPB and the commercial buffer
  • Figure 7 depicts the comparative analysis of SPB and commercial buffer with respect to shelf life
  • Figure 8 shows the compatibility of SPB with commercially available kits
  • Figure 9 shows the yield and purity of DNA extracted using iron oxide magnetic nanoparticles under different lysis conditions; and Figure 10 shows the RT-PCR amplification of human and viral genes using the extracted viral RNA.
  • Biological samples means any material, including without limitation, blood, serum, fluid and tissue biopsy samples, urine, sweat, tears, semen, plasma, stool, sweat, saliva, amniotic fluids, oral fluids, paraffin fixed tissues collected from subjects under study, or soil (containing microbes such as bacteria, fungi, virus) and other by-products, and any tangible material directly or indirectly derived there from.
  • the embodiment herein overcome the limitations of the prior art by eliminating the centrifugation steps during the extraction of nucleic acids and usage of toxic solvents.
  • Figure 1 illustrates the method for the extraction of nucleic acids from the biological sample.
  • the steps for the extraction of nucleic acids include:
  • the biological sample is collected in a sample storage vial.
  • the biological sample includes saliva, sputum, swab, toothpick, floss wire, toothbrush, blood, serum, semen, plasma, paraffin fixed tissue, saliva, faecal samples and urine sample.
  • the biological sample is mixed with a preservation buffer.
  • the biological sample is mixed with the preservation buffer by shaking a sample storage vial 200 for the time duration of 10-20 seconds.
  • the sample storage vial 200 contains the biological sample and the preservation buffer.
  • a preservation buffer for preserving a biological sample, and in turn for preserving nucleic acids.
  • the preservation buffer includes a salt, a detergent, an antimicrobial agent, a chelating agent, an osmotic agent, a denaturing agent and a buffering agent.
  • the preservation buffer preserves the nucleic acids for 12 months at room temperature and refrigerated conditions.
  • the buffering agent includes compounds such as, but not limited to, phosphate, Tris, borate, citrate and glycine.
  • the buffering agent has a pH ranging from 4 to 9. In a preferred embodiment, the pH of the buffering agent is 8. In an embodiment, the concentration of the buffering agent ranges from 0.1 M to 2 M. In a preferred embodiment, 1 M Tris is the buffering agent.
  • the salt is a monovalent or divalent salt.
  • the salt includes, but not limited to, sodium chloride, lithium chloride, potassium chloride, Magnesium chloride and a combination thereof.
  • the concentration of the salt ranges from 0.01 M to 0.5 M.
  • the chelating agent includes the reagents such as, but not limited to, ethylene di amine tetra acetic acid (EDTA), ethylene glycol-bis-tetra acetic acid, cyclohexane di amino tetra acetate (CDTA), di ethylene tri amine penta acetic acid (DTPA), tetra azacycylododecane tetraacetic acid (DOTA), tetra azacyclo tetradecane tetraacetic acid (TETA), desferioximine and a chelator analog.
  • the concentration of the chelating agent ranges from 0.01 M to 0.5 M
  • the denaturing agent includes, but not limited to, urea, dodecyl sulphate, guanidnium chloride, guanidinium thio-cyanate, perchlorate, an alcohol and a combination thereof.
  • urea is the denaturing agent.
  • the concentration of the denaturing agent ranges from 0.1% to 20%.
  • the detergent includes the reagents like, but not limited to, sodium dodecyl sulfate (SDS), sodium de oxy cholate, cetyl tri methyl ammonium bromide (CTAB), nonyl phenoxy poly ethoxyl ethanol (NP-40), octyl phenoxy poly ethoxy ethanol (Nonidet P-40), triton X-100 and polysorbate 20.
  • SDS sodium dodecyl sulfate
  • CTAB cetyl tri methyl ammonium bromide
  • NP-40 nonyl phenoxy poly ethoxyl ethanol
  • NP-40 nonyl phenoxy poly ethoxyl ethanol
  • Nonidet P-40 octyl phenoxy poly ethoxy ethanol
  • triton X-100 triton X-100 and polysorbate 20.
  • concentration of the detergent ranges from 0.1% to 5%.
  • the antimicrobial agent includes the compounds such as, but not limited to, sodium azide, sodium benzoate, halogen, phenol, quaternary ammonium compounds and anti-fungal agents.
  • the osmotic agent includes the compounds such as, but not limited to, glycerol, mannitol and urea.
  • the preservation buffer includes reagents like Tris, EDTA, NaCl, glycerol, azide, SDS and urea in combination with each other.
  • the preservation buffer includes 35mM-45mM Tris, 45mM-55mM EDTA, 250mM-350mM NaCl and 1.8-2.2% Glycerol.
  • the preservation buffer includes 35mM-45mM Tris, 45Mm-55mM EDTA, 250mM-350mM NaCl, 25-35% Glycerol and 0.08- 0.12% Azide.
  • the preservation buffer includes 35mM- 45mM Tris, 95mM-110mM EDTA, 0.8-1.2% SDS and 0.08-0.12%Azide. In yet another embodiment, the preservation buffer includes 35mM-45mM Tris, 95mM- llOmM EDTA and 0.8-1.2% SDS. In yet another embodiment, the preservation buffer includes 180mM-220mM Tris, 95mM-110mM EDTA, 1.8M-2.2M Urea, 0.8-1.2% SDS and 0.08-0.12% Azide. In yet another embodiment, the preservation buffer includes 180mM-220mM Tris, 95mM-l lOmM EDTA, 1.8M-2.2M Urea and 0.8- 1.2% SDS. In yet another embodiment, the preservation buffer includes 200mM Tris, 37Mm-47mM EDTA, 0.8M-1.2M Urea, 0.8- 1.2% SDS and 0.08-0.12%Azide.
  • the preservation buffer is capable of providing increased stability to the nucleic acids present inside the biological sample. In a preferred embodiment, the preservation buffer is capable of providing the stability of 12 months to the nucleic acids present inside the biological sample.
  • the sample storage vial 200 is provided (shown in figure 2).
  • the vial 200 includes a tubular body 202, a cap 204 and a narrow opening 206.
  • the vial 200 is capable of stabilizing the nucleic acids for 12 months.
  • the tubular body 202 is a long, slender compartment, capable of storing the biological sample.
  • the tubular body 202 has markings on the outer surface for indicating the volume of the biological sample.
  • the cap 204 is a flap like covering, capable of sealing the biological sample in the sample storage vial 200.
  • the cap 204 has a storage compartment for storing the preservation buffer on the inner surface.
  • the preservation buffer is stored and is sealed with a membrane.
  • the narrow opening 206 includes a teeth-like projection 208.
  • the teeth like projections 208 are capable of rupturing/pricking the membrane of the cap 204.
  • the membrane is a plastic film, an aluminium film or a silver film, The rupturing/pricking of the membrane allows the preservation buffer to mix with the biological sample in the tubular body 202.
  • the tubular body 202 is attached with a detachable compartment which contains a swab for collecting the biological sample.
  • the mixture of the biological sample and the preservation buffer is mixed with a lysate to form a biological lysate.
  • the lysate is capable of disrupting the cell membrane and release the contents such as, but not limited to, DNA, RNA and proteins.
  • the lysate is capable of degrading the unwanted contaminants such as, proteins, polysaccharides etc.
  • the lysate is mixed with magnetic nanoparticles, a salt and a binding agent.
  • the salt is a monovalent and a divalent salt including sodium chloride, lithium chloride, potassium chloride, magnesium chloride and a combination thereof.
  • the concentration of the salt is 1 M to 5 M.
  • binding agent is a polymer.
  • the polymer includes polyethylene glycol-800 to 10,000 (PEG).
  • the polymer is polyethylene glycol- 8000 (PEG).
  • the concentration of the polymer ranges from 1% to 25%.
  • the binding agent is a solvent.
  • solvent is selected from acetone, ethanol, methanol, acetonitrile or propanol.
  • the magnetic nanoparticles are prepared by physical, chemical, co-precipitation and biological method.
  • the magnetic nanoparticles are metal oxide. In an embodiment, the magnetic nanoparticles are coated by a polymer or a protein. In another embodiment, the magnetic nanoparticles are uncoated by a polymer or a protein. In an embodiment, the magnetic nanoparticles include materials, such as magnetite (Fe304), ulvospinel, hematite (a-Fe203), ilmenite, maghemite (y-Fe203), jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite, wairauite and a combination thereof.
  • magnetite Fe304
  • ulvospinel hematite
  • a-Fe203 ilmenite
  • maghemite y-Fe203
  • jacobsite trevorite
  • the magnetic nanoparticles lie in the range between 10 to 15 nanometres. In a preferred embodiment, the magnetic nanoparticles lie in the range between 1 to 500 nanometers. In a preferred embodiment, the magnetic nanoparticles has a size ranging from 5 nm to 20 nm.
  • the preparation of the biological lysate includes the mixing of a proteinase K with the mixture of the biological sample and the preservation buffer.
  • 20-30 pi of the proteinase K is mixed with the mixture of the biological sample and the preservation buffer.
  • the proteinase K is capable of degrading the proteins present inside the biological sample.
  • the mixture of the proteinase K, the biological sample and the preservation buffer is incubated at a temperature of 56°C for the time duration of 30 minutes.
  • 5 - 15 m ⁇ RNase A is added to the mixture of the proteinase K, the biological sample and the preservation buffer and is incubated at room temperature for the time duration of 15 minutes.
  • the RNase A is capable of degrading the RNA present inside the biological sample.
  • the mixture including the RNase A, the proteinase K, the biological sample and the preservation buffer is centrifuged at a speed of 14,000 rpm for the time duration of 5 minutes.
  • a pellet is discarded and a supernatant is stored in a tube for the further processing.
  • the supernatant is the biological lysate.
  • DNase enzyme will be added instead of RNase A, to remove the contaminating DNA.
  • the swab sample is collected from naso- pharygneal and oro -pharyngeal for RNA extraction.
  • the RNA extraction is achieved by adding 80-120m1 of viral lysis buffer, 3-8 m ⁇ of carrier RNA followed by incubation at 56°C for 10-15 minutes.
  • the incubated RNA sample is added with lysate and washed with 50-100% ethanol without dispersing the magnetic properties.
  • the obtained nanoparticles pellet is air dried, eluted in 20-40 m ⁇ RNase free water and incubated at 56°C for 10-15 minutes.
  • the nucleic acid is washed to remove the unwanted contaminants such as, but not limited to, cell debris, salts, buffer components and proteins.
  • 350-450pl of MagNa Mix is added inside the tube having the biological lysate.
  • the MagNa Mix includes a magnetic nanoparticle.
  • the MagNa mix includes a salt of Fe2+ and Fe3+.
  • the biological lysate is mixed with MagNa Mix and is incubated for the time duration of 5-10 minutes.
  • the tube is placed on a MagNa stand for the time duration of 5-10 minutes.
  • a supernatant is discarded and a pellet is not disturbed.
  • the nucleic acids in the biological lysate bind to the magnetic nanoparticles.
  • the magnetic pellets with the nucleic acids are immobilized through external magnetic field.
  • 0.8- 1.2 ml of wash buffer- 1 is added inside the tube.
  • the pellet is suspended in the wash buffer- 1 which contains Tris-HCl, EDTA, NaCl, ammonium chloride. Multivalent citrus salts and sterile water.
  • the wash buffer- 1 includes 1.8-1.2M Tris- HCl having a pH 7.5-8.5, 0.4-0.6M EDTA having a pH 7.5-8.5, NaCl, Ammonium Chloride, multivalent citrate salts and Autoclaved type III Water.
  • the multivalent citrate salts includes divalent and trivalent metallic salts.
  • the metallic salts includes salts of metals such as, but not limited to, sodium, potassium and lithium.
  • the tube is placed on MagNa stand for the time duration ranging between 30 seconds to 1 minute. In a preferred embodiment, the tube is placed on the MagNa stand, until the mixture becomes clear.
  • supernatant is discarded and a pellet is washed with a wash buffer-2.
  • the wash buffer 2 includes 75% ethanol.
  • the supernatant is discarded and the pellet is air dried at a room temperature for the time duration of 10-15 minutes.
  • the pure nucleic acid is eluted out.
  • the pellet from step 106 is re-suspended in nuclease free water or Tris.
  • the pellet is re-suspended in 45-55 m ⁇ of nuclease free water or 8-12mM Tris having a pH 7.5- 8.5 and is incubated at a temperature of 56°C for the time duration of 10 minutes.
  • the tube is placed on the MagNa stand for the time duration of 5 minutes. In a preferred embodiment, the tube is placed on the MagNa stand, until the mixture becomes clear.
  • a supernatant is collected and a pellet is discarded.
  • the supernatant includes the pure nucleic acid.
  • the nucleic acids extracted from the present method gives higher yield than the nucleic acids extracted from the conventional methods (shown in figure 3). In a preferred embodiment, the yield of nucleic acid extracted from the present method is two to three times higher than the yield from the conventional methods.
  • Figure 2 illustrates a sample storing vial (200), according to an embodiment herein.
  • the sample storing vial 200 is provided and includes a tubular body 202, a cap 204, and a narrow opening 206.
  • the sample storing vial 200 is capable of stabilizing the nucleic acids for 12 months.
  • the sample storing vial 200 is capable for centrifuge.
  • the tubular body 202 is a long, slender compartment, capable of storing the biological sample.
  • the tubular body 202 has markings on the outer surface for indicating the volume of the biological sample.
  • the cap 204 is a flap like covering, capable of sealing the biological sample in the sample storage vial 200.
  • the cap 204 has a storage compartment for storing the preservation buffer on the inner surface.
  • the preservation buffer is stored and is sealed with a membrane.
  • the narrow opening 206 includes a teeth-like projection 208.
  • the teeth like projections 208 are capable of rupturing the membrane of the cap 204. The rupturing of the membrane allows the preservation buffer to mix with the biological sample in the tubular body 202.
  • Figure 3 illustrates a graph demonstrating a yield of nucleic acids, according to an embodiment herein.
  • Figure 4 illustrates a gel electrophoresis for determining the yield of DNA extracted from the present method and the yield of DNA extracted from the conventional methods.
  • the band of the purified DNA from the present method is brighter and sharper than the band of the purified DNA from the conventional methods.
  • the brighter band of the purified DNA depicts a higher concentration of the DNA.
  • the brighter band of the purified DNA depicts a higher yield than the DNA extracted from the conventional methods.
  • Figure 5 illustrates a gel electrophoresis for determining the yield of extracted RNA.
  • the yield of the RNA before the DNase treatment is compared with the yield of the RNA after the DNase treatment.
  • the 260/280 ratio for the RNA before the DNase treatment is 2.09 and for the RNA after the DNase treatment is 2.02.
  • the 260/230 ratio for the RNA before the DNase treatment is 1.82 and for the RNA after the DNase treatment is 1.62.
  • the yield of the RNA extracted from the present method is high over the traditional method.
  • Figure 6 illustrates the comparative yield and purity profiles of genomic DNA extracted from saliva sample stored in the (Saliva Preservation Buffers) SPB and the commercial buffer. Comparative analysis of genomic DNA extracted from the saliva sample stored in SPB and a commercial buffer (as shown in the figure 6) which depicts that the samples stored in SPB has significantly high purity profiles with respect to A260/280 and A260/230.
  • Figure 7 depicts the comparative analysis of SPB and commercial buffer with respect to shelf life.
  • the biological sample is stored in the SPB as well as the commercially available buffer for a period of time.
  • the DNA extracted from samples stored in SPB are loaded lane 1(702) and 2(704) of the agarose gel electrophoresis and DNA extracts from the sample stored in commercial buffers are loaded in lane 3 (706) and 4 (708) of the agarose gel electrophoresis to compare the integrity and size of DNA.
  • Figure 8 shows the compatibility of SPB with commercially available kits.
  • the biological sample is stored in the SPB is extracted with commercially available DNA extraction kits such as spin column kit and magnetic bead based kit.
  • DNA extraction kits such as spin column kit and magnetic bead based kit.
  • the integrity and size of DNA is high in magnetic spin based 804 than spin column kit 802.
  • Figure 9 shows the yield and purity of DNA extracted using iron oxide magnetic nanoparticles under different lysis conditions.
  • the high purity of DNA extracted from saliva samples is attributed to (i) effective denaturation and removal of proteins by the use of chaotropic salt (guanidine hydrochloride) and proteinase K and (ii) removal of RNA contamination by treating with RNase A. Absence of any of these components decreases the purity and/or the yield of DNA.
  • the sample from the subject is lysed for protein and RNA digestion and DNA is selectively precipitated from the lysate after digestion of proteins and RNA followed by binding to the magnetic nanoparticles.
  • the high specificity of this method may be due to the presence of polyethylene glycol (PEG) and high salt concentration in the MNP formulation, which prevent the binding of denatured proteins or other contaminants to magnetic nanoparticles where, the presence of chaotropic salts and high salt concentration thermodynamically favored the binding of only double stranded DNA on solid support (magnetic nanoparticles) barring proteins and RNA.
  • PEG polyethylene glycol
  • Figure 10 shows the RT-PCR amplification of human and viral genes using the extracted viral RNA.
  • the extracted viral RNA is quantified using RT-PCR with primer specific for N gene, S gene and ORF 1 of viral or bacterial genome along with internal control gene MS2 by Taq path RT PCR kit.
  • the viruses are selected from SARS, MERS, or SARS COV-2.
  • the nucleic acid is eluted from DNA, RNA (genomic), cfDNA, mRNA, rRNA, tRNA, siRNA, MiRNA, or hnRNA.
  • viruses are selected from SARS, MERS, or SARS COV-2.
  • Nano particles were synthesized by freshly prepared solutions of 1 M FeC12.4H20 and 2 M FeC13.6H20 were used as precursors by co-precipitation of Fe2+ and Fe3+ ions, in the molar ratio of 1:2. Alkalinity was maintained to form ions precipitate of Fe 3 0 4, by adding 8 M NaOH drop wise along with constant stirring at 80 °C for maturation. Impurities were removed by washing several times under a strong magnetic field with hot distilled water and the suspension was dried overnight at 60 °C. Bare nanoparticles were obtained by dispersing magnetic nanoparticles in 100 mM Tris EDTA, pH 8 to a final stock concentration of 10 mg/ml. The mean size of magnetic nanoparticles was 10-12 nm as determined by TEM analysis. The particles at a concentration of 40pl/ml were dispersed in a formulation buffer contains a final concentration of 10% PEG and 2 M NaCl in assay.
  • the buffers was prepared by mixing Tris, EDTA, urea and sodium azide was used as an anti-bacterial agent.
  • Buffer 1 was prepared by mixing 40mM Tris, 50mM EDTA, 300 mM NaCl, and 2% glycerol.
  • Buffer 2 was prepared by mixing 40mM Tris, 50mM EDTA, 300 mM NaCl, 30% glycerol, and 0.1% azide.
  • Buffer 3 was prepared by mixing 40mM Tris, lOOmM EDTA, 1% SDS, and 0.1% azide.
  • Buffer 4 was prepared by mixing 40mM Tris, lOOmM EDTA, and 1% SDS.
  • Buffer 5 was prepared by mixing 200mM Tris, lOOmM EDTA, 2M urea, 1% SDS, and 0.1% azide.
  • Buffer 6 was prepared by mixing 200mM Tris, lOOmM EDTA, 2M urea, and 1% SDS.
  • Buffer 7 was prepared by mixing 200mM Tris, lOOmM EDTA, 2M urea, 1% SDS, and 0.1% azide
  • Extraction of DNA was achieved by adding 1 ml of saliva mixed with the saliva preservation 0.5ml buffer, 25 pi of proteinase K, and incubated at 56 °C for 30 minutes. RNA contamination was removed by adding RNase. The sample was then centrifuged to remove the debris, 400 m ⁇ of magnetic nanoparticles formulation was added to the supernatant and incubated for 5 minutes. The DNA-bound magnetic nanoparticles were washed with 75 % ethanol without dispersing the magnetic particles. The magnetic Nano-particle pellet was air dried, the bound DNA was eluted in 50 m ⁇ nuclease free water and incubated at 56 °C for 10 minutes. The extracted DNA were resolved using 1% agarose gel.
  • Naso-pharyngeal and oro -pharyngeal swab sample were collected in 2-3ml of VTM / MTM as per CDC guidelines and RNA extraction from these sample was achieved by adding 100 m ⁇ of viral lysis buffer, 5 m ⁇ of Carrier RNA and incubated at 56 °C for 10 minutes. After lysis 125 m ⁇ of magnetic nanoparticles formulation was added to the lysate and incubated for 5 minutes at room temperature. The RNA-bound magnetic nanoparticles were washed with 75 % ethanol without dispersing the magnetic particles. The magnetic nanoparticles pellet was air dried, the bound RNA was eluted in 30 m ⁇ RNase free water and incubated at 56 °C for 2 minutes. The extracted RNA was used for downstream applications like Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) based detection using specific genes.
  • RT-PCR Reverse Transcriptase Polymerase Chain Reaction
  • Example 5 PCR amplification of human and bacterial genes from the extracted DNA
  • the PCR amplification was carried with human specific primer LCO-HCO (Light and Heavy chain cytochrome oxidase) and bacterial specific primers (16s rRNA) by thermal cycler.
  • the human specific primer LCO-HCO amplify a gene fragment length of 700bp (HCO 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA- 3',LCO 5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and was visualized on ethidium bromide stained 2 % agarose gel.
  • the bacteria-specific primers (16s rRNA) amplify a gene fragment length of 1.5Kb (FP 5'-AGA GTT TGA TCC TGG CTC AG-3', RP5'-GGT TAC CTT GTT ACG ACT T-3') and was visualized on ethidium bromide stained 1% agarose gel.
  • Example 6 RT-PCR amplification of human and SARS-COVID genes using the extracted viral RNA
  • the extracted viral RNA was quantified (as shown in the figure 10) using RT-PCR with primer specific for N gene, S gene and ORF 1 of SARS-COVID along with internal control gene MS2 by Taq path RT PCR kit.

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Abstract

Procédé d'extraction d'acides nucléiques à partir d'un échantillon biologique utilisant des nanoparticules magnétiques, le procédé implique le mélange de l'échantillon biologique avec un tampon de conservation avec ou sans enzyme et un détergent pour former un lysat biologique, le mélange du lysat biologique avec les nanoparticules magnétiques, un sel et un agent de liaison, la liaison des acides nucléiques de l'échantillon biologique aux nanoparticules magnétiques et le lavage pour éliminer les contaminants et l'élution des nanoparticules magnétiques pour obtenir des acides nucléiques purs.
PCT/IN2021/050637 2020-06-30 2021-06-30 Procédé pour l'extraction d'acides nucléiques à partir d'un échantillon biologique WO2022003723A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5346999A (en) * 1985-01-18 1994-09-13 Applied Biosystems, Inc. Method of nucleic acid extraction
WO1995011083A2 (fr) * 1993-10-22 1995-04-27 Abbott Laboratories Tube de reaction et methode d'utilisation de celui-ci minimisant les contaminations
WO2016075701A2 (fr) * 2014-11-11 2016-05-19 Scigenom Labs Pvt. Ltd. Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5346999A (en) * 1985-01-18 1994-09-13 Applied Biosystems, Inc. Method of nucleic acid extraction
WO1995011083A2 (fr) * 1993-10-22 1995-04-27 Abbott Laboratories Tube de reaction et methode d'utilisation de celui-ci minimisant les contaminations
WO2016075701A2 (fr) * 2014-11-11 2016-05-19 Scigenom Labs Pvt. Ltd. Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues

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