Methods and devices for identifying microbial infections
Inventors
Singer, Alon • Prakash, Ranjit • Nolling, Jork
Assignees
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Publication Number
US-11840721-B2
Publication Date
2023-12-12
Expiration Date
Abstract
The present disclosure generally relates to the field of microbial pathogen detection and identification utilizing genomic sequence recognition.
Core Innovation
The invention provides a method of identifying one or more specific microbial species in a sample from a subject by depleting eukaryotic DNA, lysing microbial cells to release microbial genetic materials, isolating the microbial genetic materials, and amplifying the microbial genetic materials. The amplified microbial genetic materials are incubated with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs) for less than 10 minutes at a temperature below 65° C.
The amplified microbial genetic materials are invaded by at least one of the DIANAs during the incubation. Detection is based on detecting binding of one or more of the DIANAs to the amplified microbial genetic material of its respective single species or group of microbes, where detection of binding indicates the presence of one or more specific microbial species or groups of microbes in the sample.
One or more of the plurality of DIANAs is a peptide nucleic acid, a locked nucleic acid, and/or a bridged nucleic acid. The peptide nucleic acid, locked nucleic acid, and/or bridged nucleic acid comprises a sequence that shares at least 60% identity with a sequence selected from the group consisting of SEQ ID NOS: 20-571, including the complement, reverse, or reverse complement of such sequences, and sequences that lack six or fewer bases at either or both ends.
Claims Coverage
The independent claim is clm-00001. It includes eukaryotic DNA depletion and microbial genetic material preparation, together with DIANA incubation and binding-based detection, with inventive features centered on rapid DIANA invasion and sequence definitions tied to SEQ ID NOS: 20-571.
Rapid DIANA invasion and binding detection of amplified microbial genetic material
Incubating amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs) for less than 10 minutes at a temperature below 65° C, wherein the amplified microbial genetic materials are invaded by at least one of the DIANAs during the incubation and detecting binding indicates the presence of one or more specific microbial species or groups of microbes in the sample.
Eukaryotic DNA depletion and microbial genetic material preparation
Depleting eukaryotic DNA from the sample, lysing one or more microbial cells to release a plurality of microbial genetic materials, and isolating and amplifying the plurality of microbial genetic materials prior to DIANA incubation.
DIANAs as peptide, locked, and/or bridged nucleic acids with SEQ ID targeting
Using one or more DIANAs that is a peptide nucleic acid, a locked nucleic acid, and/or a bridged nucleic acid, wherein the peptide/locked/bridged nucleic acid comprises a sequence that shares at least 60% identity with a sequence selected from SEQ ID NOS: 20-571, including the complement, reverse, or reverse complement, and includes sequences that lack six or fewer bases at either or both ends.
Across the independent claim, microbial species detection is enabled by rapidly incubating amplified microbial genetic materials with a plurality of DIANAs for less than 10 minutes below 65° C, followed by detecting DIANA binding as the presence indicator, with DIANA identity-defined nucleic-acid targeting and upstream eukaryotic DNA depletion and microbial genetic material isolation/amplification.
Stated Advantages
Rapid detection using DIANA binding, performed for less than 10 minutes at a temperature below 65° C.
Documented Applications
Detection of bloodstream infections (BSIs) and sepsis contexts, including endocarditis and neonatal sepsis, using DIANA-driven microbial detection from subject samples.
Detection of antimicrobial resistance genes and antimicrobial resistance via targeted DIANAs.
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