US 11,993,812 B2
Systems and methods of diagnosing and characterizing infections
Elizabeth Driebe, Flagstaff, AZ (US); Jolene Bowers, Flagstaff, AZ (US); David Engelthaler, Flagstaff, AZ (US); and Paul Keim, Flagstaff, AZ (US)
Assigned to The Translational Genomics Research Institute, Phoenix, AZ (US); and Arizona Board of Regents acting for and on behalf of Northern Arizona University, Flagstaff, AZ (US)
Filed by THE TRANSLATIONAL GENOMICS RESEARCH INSTITUTE, Phoenix, AZ (US); and ARIZONA BOARD OF REGENTS ON BEHALF OF NORTHERN ARIZONA UNIVERSITY, Flagstaff, AZ (US)
Filed on Aug. 5, 2021, as Appl. No. 17/395,432.
Application 17/395,432 is a continuation of application No. 15/773,270, granted, now 11,098,349, previously published as PCT/US2016/060369, filed on Nov. 3, 2016.
Claims priority of provisional application 62/250,565, filed on Nov. 4, 2015.
Prior Publication US 2021/0381030 A1, Dec. 9, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/686 (2018.01); C12Q 1/689 (2018.01)
CPC C12Q 1/686 (2013.01) [C12Q 1/689 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/16 (2013.01)] 20 Claims
 
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.
 
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.
 
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.