# US 12,215,376 B2 · Mitochondrial nucleic acid depletion and detection > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,215,376 B2 - Document ID: US12215376B2 - Status: Granted - Kind: B2 - Application number: 17/253,448 - Filed: 2019-06-20 - Published: 2021-08-19 - Granted: 2025-02-04 - Earliest claimed: 2018-06-21 - Assignees: Progenika Biopharma, S.A.; UEA Enterprises Limited - Applicants: Progenika Biopharma, S.A.; Uea Enterprises Limited - Inventors: Lourdes Palacios; Nerea Bartolome; Antonio Martinez; Justin Joseph O'Grady; Gemma Louise Kay - Web page: https://patentgrep.com/patent/US12215376B2 ## CPC - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition} - G01N 1/405: {by adsorption or absorption} - C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor} - C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention provides a method for depleting host nucleic acid in a biological sample, said sample having been previously obtained from an animal host and having been subjected to treatment to lyse host cells present in the sample, and having been subjected to a treatment to deplete nucleic acid released from host cells within said sample or otherwise to render such nucleic acid unidentifiable, the method comprising: (a) depleting host mitochondria or host mitochondrial DNA (mtDNA) present in the treated sample; and (b) extracting and/or analysing remaining nucleic acid from the treated sample. Also provided is a related kit comprising reagents for performing the method. Further claimed is a method for isolating mitochondrial DNA after host cells have been lysed and host cell nucleic acids have been removed from the sample. ## Claims ### Claim 1 A method for depleting host nucleic acid in a biological sample, said sample having been previously obtained from an animal host and having been subjected to treatment to lyse host cells present within the sample, said treatment to lyse host cells being a treatment with a cytolysin, and wherein said sample has been subjected to a treatment to deplete nucleic acid released from host cells within said sample or otherwise to render such nucleic acid unidentifiable, the method comprising: (a) depleting host mitochondria or host mitochondrial deoxyribonucleic acid (mtDNA) present in the treated sample; and (b) extracting and/or analysing remaining nucleic acid from the treated sample; wherein step (a) comprises: (i) adding a detergent to said treated sample; (ii) at least partial removal of host mitochondria by contacting the treated sample with an immobilised binding agent that selectively binds mitochondria; or (iii) extraction of whole nucleic acids and further capture of mitochondrial DNA using a panel of DNA and/or RNA and/or PNA baits that target the host mitochondrial genome; and wherein step (a) further comprises adding a nuclease to said treated sample. ### Claim 2 (depends on 1) The method according to claim 1, wherein said detergent comprises: saponin, digitonin, and/or filipin. ### Claim 3 (depends on 1) The method according to claim 1, wherein the binding agent comprises an antibody that selectively binds a protein present in the outer membrane of the mitochondria. ### Claim 4 (depends on 3) The method according to claim 3, wherein the antibody selectively binds Mitochondrial import receptor subunit TOM22 homolog (TOM22). ### Claim 5 (depends on 1) The method according to claim 1, wherein step (a) comprises extraction of whole nucleic acids and further RNA-guided DNA endonuclease enzyme-mediated recognition of host mitochondrial DNA using a sgRNA that targets said DNA endonuclease enzyme to a host mitochondrial DNA target sequence. ### Claim 6 (depends on 1) The method according to claim 1, wherein the treatment to which the sample has been subjected to lyse host cells is a treatment that is effective to lyse at least 3% of the host cells in the sample. ### Claim 7 (depends on 1) The method according to claim 1, wherein the cytolysin comprises a phospholipase C. ### Claim 8 A method for depleting host nucleic acid in a biological sample, said sample having been previously obtained from an animal host, said method comprising the steps of: (a) lysing host cells present within the sample, wherein said lysing comprises adding a cytolysin to the sample; (b) carrying-out a process to physically deplete at least genomic nucleic acid released from host cells within said sample or otherwise render such nucleic acid unidentifiable; (c) depleting host mitochondria or host mitochondrial deoxyribonucleic acid (mtDNA) present in the treated sample, wherein said depleting host mitochondria or mtDNA comprises adding a detergent and a nuclease to the sample; and (d) extracting and/or analysing remaining nucleic acid from the sample, wherein step (b) is performed before or after step (c). ### Claim 9 (depends on 8) The method according to claim 8, wherein step (a) comprises lysing at least 3% of the host cells in the sample. ### Claim 10 (depends on 8) The method according to claim 8, wherein the cytolysin comprises a phospholipase C. ### Claim 11 (depends on 8) The method according to claim 8, wherein the method results in at least a 5-fold depletion of host mitochondrial DNA originally contained within the sample. ### Claim 12 (depends on 8) The method according to claim 8, wherein the method further comprises amplifying and/or purifying the remaining nucleic acid. ### Claim 13 (depends on 8) The method according to claim 8, wherein the method further comprises sequencing the remaining nucleic acid or the amplified and/or extracted product thereof. ### Claim 14 (depends on 13) The method according to claim 13, wherein said sequencing identifies at least one drug resistance-conferring sequence in a pathogen present in the original sample. ### Claim 15 (depends on 8) The method according to claim 8, wherein the extracted remaining nucleic acid is bacterial, viral and/or fungal. ### Claim 16 (depends on 8) The method of claim 8, wherein the biological sample is blood. ### Claim 17 (depends on 8) The method of claim 8, wherein step (b) is performed before step (c), and the method further comprises performing a buffer exchange after the process to physically deplete genomic nucleic acid. ### Claim 18 (depends on 17) The method of claim 17, wherein the process to physically deplete genomic nucleic acid comprises adding a nuclease. ### Claim 19 (depends on 18) The method of claim 18, wherein the biological sample is blood.