# US 11,993,812 B2 · Systems and methods of diagnosing and characterizing infections > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,993,812 B2 - Document ID: US11993812B2 - Status: Granted - Kind: B2 - Application number: 17/395,432 - Filed: 2021-08-05 - Published: 2021-12-09 - Granted: 2024-05-28 - Earliest claimed: 2015-11-04 - Assignees: The Translational Genomics Research Institute; Arizona Board of Regents acting for and on behalf of Northern Arizona University - Applicants: The Translational Genomics Research Institute; Arizona Board of Regents On Behalf of Northern Arizona University - Inventors: Elizabeth Driebe; Jolene Bowers; David Engelthaler; Paul Keim - Web page: https://patentgrep.com/patent/US11993812B2 ## CPC - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q 1/689: for bacteria - C12Q 2600/156: Polymorphic or mutational markers - C12Q 2600/16: Primer sets for multiplex assays - 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 ## Abstract Embodiments of the invention include methods of identifying microorganisms and/or diagnosing infections in subjects cause by microorganisms. Embodiments of the invention may also include further characterizing (e.g., determining the presence of one or more antibiotic resistance markers) the microorganisms and determining a strain identity of the microorganisms. ## Claims ### Claim 1 A method of detecting and characterizing one or more microorganisms within a sample, the method comprising the steps of: receiving the sample; extracting a template from the sample; performing a multiplex polymerase chain reaction assay comprising the steps of: amplifying a first marker from the template to create a first amplicon, comprising mixing the template with a first oligonucleotide comprising a sequence selected from SEQ ID Nos: 1-49, 54-109; and a second oligonucleotide comprising a sequence selected from SEQ ID Nos: 1-49, 54-109, wherein the first oligonucleotide and the second oligonucleotide independently comprise sequences selected from SEQ ID Nos: 1-49, 54-109 and wherein the first marker is specific for at least a genus of microorganism; amplifying a second marker from the template to create a second amplicon, comprising mixing the template with a third oligonucleotide and a fourth oligonucleotide, wherein the second marker is specific for at least one antibiotic resistance gene; amplifying a third marker from the template to create a third amplicon, comprising mixing the template with a fifth oligonucleotide and a sixth oligonucleotide comprising, wherein the third marker is specific for strain identity and/or lineage of the one or more microorganisms; wherein the multiplex polymerase chain reaction assay is performed within a single reaction vessel; and sequencing the first, second, and third amplicons to detect and characterize the one or more microorganisms. ### Claim 2 (depends on 1) The method of claim 1, wherein the third and fourth oligonucleotide independently comprise a sequence selected from SEQ ID NOs: 110-298. ### Claim 3 (depends on 1) The method of claim 1, wherein the fifth and sixth oligonucleotide independently comprise a sequence selected from SEQ ID NOs: 299-431. ### Claim 4 (depends on 1) The method of claim 1, further comprising adding an index to the first amplicon, second amplicon, and third amplicon using at least one indexing oligonucleotide. ### Claim 5 (depends on 1) The method of claim 1, wherein at least one of the one or more microorganisms is a pathogenic microorganism selected from the group consisting of: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. ### Claim 6 (depends on 1) The method of claim 1, wherein the template comprises nucleic acids from a subject and the one or more microorganisms and the template comprises DNA. ### Claim 7 (depends on 1) The method of claim 1, wherein the subject is an animal. ### Claim 8 (depends on 7) The method of claim 7, wherein the animal is a human. ### Claim 9 (depends on 1) The method of claim 1, wherein the sample is selected from at least one of a respiratory sample, a nasal swab sample, a urine sample, and a blood sample. ### Claim 10 A method of detecting and characterizing one or more microorganisms within a sample, the method comprising the steps of: receiving the sample; extracting a template from the sample; performing a multiplex polymerase chain reaction assay comprising the steps of: amplifying a first marker from the template to create a first amplicon, comprising mixing the template with a first oligonucleotide comprising a sequence selected from SEQ ID Nos: 54-57; and a second oligonucleotide comprising a sequence selected from SEQ ID Nos: 54-57, wherein the first oligonucleotide and the second oligonucleotide independently comprise sequences selected from SEQ ID Nos: 54-57 and wherein the first marker is specific for at least a genus of microorganism; amplifying a second marker from the template to create a second amplicon, comprising mixing the template with a third oligonucleotide and a fourth oligonucleotide, wherein the second marker is specific for at least one antibiotic resistance gene; amplifying a third marker from the template to create a third amplicon, comprising mixing the template with a fifth oligonucleotide and a sixth oligonucleotide, wherein the third marker is specific for strain identity and/or lineage of the one or more microorganisms; wherein the multiplex polymerase chain reaction assay is performed within a single reaction vessel; and sequencing the first, second, and third amplicons to detect and characterize the one or more microorganisms. ### Claim 11 (depends on 10) The method of claim 10, wherein the third and fourth oligonucleotide independently comprise a sequence selected from SEQ ID NOs: 110-298. ### Claim 12 (depends on 10) The method of claim 10, wherein the fifth and sixth oligonucleotides independently comprise a sequence selected from SEQ ID NOs:299-431. ### Claim 13 (depends on 10) The method of claim 10, further comprising adding an index to the first amplicon, second amplicon, and third amplicon using at least one indexing oligonucleotide. ### Claim 14 (depends on 10) The method of claim 10, wherein the template comprises nucleic acids from a subject and the one or more microorganisms and the template comprises DNA. ### Claim 15 (depends on 10) The method of claim 10, wherein the subject is an animal. ### Claim 16 (depends on 15) The method of claim 15, wherein the animal is a human. ### Claim 17 (depends on 10) The method of claim 10, wherein the sample is selected from at least one of a respiratory sample, a nasal swab sample, a urine sample, and a blood sample. ### Claim 18 A method of detecting and characterizing one or more microorganisms within a sample, the method comprising the steps of: extracting a template from the sample; performing a multiplex polymerase chain reaction assay comprising the steps of: amplifying a first marker from the template to create a first amplicon, comprising mixing the template with a first oligonucleotide comprising a sequence selected from SEQ ID Nos: 1-49, 54-109; and a second oligonucleotide comprising a sequence selected from SEQ ID Nos: 1-49, 54-109, wherein the first oligonucleotide and the second oligonucleotide independently comprise sequences selected from SEQ ID Nos: 1-49, 54-109 and wherein the first marker is specific for at least a genus of microorganism; amplifying a second marker from the template to create a second amplicon, comprising mixing the template with a third oligonucleotide and a fourth oligonucleotide, wherein the second marker is specific for at least one antibiotic resistance gene; and amplifying a third marker from the template to create a third amplicon, comprising mixing the template with a fifth oligonucleotide and a sixth oligonucleotide comprising, wherein the third marker is specific for strain identity and/or lineage of the one or more microorganisms; and sequencing the first, second, and third amplicons to detect and characterize the one or more microorganisms. ### Claim 19 (depends on 18) The method of claim 18, wherein the third and fourth oligonucleotide independently comprise a sequence selected from SEQ ID NOs: 110-298. ### Claim 20 (depends on 18) The method of claim 18, wherein the fifth and sixth oligonucleotide independently comprise a sequence selected from SEQ ID NOs: 299-431.