Methods and devices for detecting and identifying microorganisms
Inventors
Singer, Alon • Prakash, Ranjit • Rapireddy, Srinivas • Nolling, Jork
Assignees
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Publication Number
US-11414712-B2
Publication Date
2022-08-16
Expiration Date
Abstract
Methods and devices for isolating microbial cells from a sample, extracting eukaryotic DNA from a sample, and identifying the microbial species in the sample are disclosed herein.
Core Innovation
A method identifies one or more specific microbial species in a sample obtained from a subject by selectively depleting eukaryotic DNA and then releasing and processing microbial genetic material. The method adds a eukaryotic cell lysis solution that selectively targets and predominantly lyses eukaryotic cells as opposed to microbial cells to release eukaryotic DNA for depletion.
The method then lyses one or more microbial cells to release a plurality of microbial genetic materials and isolates those microbial genetic materials. The method amplifies the plurality of microbial genetic materials and then contacts the amplified microbial genetic materials with one or more DNA Invading Artificial Nucleic Acids (DIANAs) capable of specifically invading duplex nucleic acids.
The DIANAs are gamma-modified PNA probes and comprise a sequence complementary to a bacterial 16S rDNA nucleic acid or a sequence complementary to a fungal 18S rDNA nucleic acid. Binding of one or more DIANAs to the amplified microbial genetic material of its respective single species or group of microbes is detected, and the detection indicates the presence of specific microbial species or groups.
The eukaryotic cell lysis solution includes Tween surfactant and Triton or IGEPAL, has a pH of about 6-9, and contains less than about 0.15 M monovalent salts. The documented workflow further includes device/cartridge concepts for automated, low-intervention multidirectional fluid handling and detection using DIANA-based optical signatures.
Claims Coverage
The partial content identifies one independent claim, with multiple dependent claims refining chemical conditions, nucleic-acid targets, and structural features of the DIANAs. Across the independent claim, the coverage centers on an integrated workflow that couples selective eukaryotic DNA depletion with microbial lysis, isolation, amplification, and DIANA binding detection using gamma-modified PNA probes complementary to 16S rDNA or 18S rDNA.
Selective eukaryotic DNA depletion with targeted microbial processing
Adding a eukaryotic cell lysis solution selectively targeting and predominantly lysing eukaryotic cells as opposed to microbial cells to release eukaryotic DNA, followed by lysing one or more microbial cells to release a plurality of microbial genetic materials and isolating the plurality of microbial genetic materials.
Gamma-modified PNA DIANA probes for invading duplex nucleic acids
Contacting amplified microbial genetic materials with one or more DNA Invading Artificial Nucleic Acids (DIANAs) capable of specifically invading duplex nucleic acids, wherein the DIANAs are gamma-modified PNA probes comprising a sequence complementary to a bacterial 16S rDNA nucleic acid or a sequence complementary to a fungal 18S rDNA nucleic acid.
DIANA binding detection as a species or group indicator
Detecting binding of the DIANAs to the amplified microbial genetic material of its respective single species or group of microbes, wherein the detection of binding indicates the presence of one or more specific microbial species or groups of microbes in the sample.
Low-load capability for microbial species detection
The method is capable of detecting a microbial species in the sample that has a load level as low as 1-10 CFU/ml.
Overall claim coverage ties together selective eukaryotic cell lysis/dna depletion, microbial cell lysis and genetic material isolation, amplification, and DIANA binding detection using gamma-modified PNA probes complementary to 16S rDNA or 18S rDNA, with an explicitly stated ability to detect microbial species at 1-10 CFU/ml.
Stated Advantages
Capable of detecting a microbial species at a load level as low as 1-10 CFU/ml.
Documented Applications
Detection and identification of microbial species from subject-derived samples such as whole blood.
Detection of multiple pathogens, including bacterial and fungal species listed in the partial content (e.g., Staphylococcus aureus and Candida albicans), including antibiotic resistance detection markers referenced in the partial content (e.g., MecA, VanA/VanB, OXA-48, NDM-1, blaKPC).
On-chip versus on-bench processing comparison and automated, low-intervention multidirectional fluid handling using a cartridge concept.
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