Methods and devices for ultrasensitive direct detection of microorganisms
Inventors
Singer, Alon • Steinmiller, David • Goyal, Nadish • Nolling, Jork
Assignees
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Publication Number
US-11713330-B2
Publication Date
2023-08-01
Expiration Date
Abstract
The present disclosure generally relates to the field of ultrasensitive microbial pathogen detection and identification utilizing genomic sequence recognition.
Core Innovation
The invention provides an ultrasensitive method of detecting one or more species of microbial cells in a biological sample comprising mammalian cells. The method selectively lyses mammalian cells, including cells which comprise eukaryotic DNA, by contacting the biological sample with a composition consisting essentially of a magnesium salt, a buffering agent, water, and a compound of Formula 1. The method then amplifies a plurality of microbial genetic materials and detects the amplified microbial genetic material.
The detection is sequence- and binding-based using DNA Invading Artificial Nucleic Acids (DIANAs) having sequences complementary to genomic or plasmid sequences of specific microbial species, with DIANA binding indicating which microbial species are present. The composition is defined structurally by R1 and R2 selections within specified group options and by integer constraints for q, p, and n. The workflow supports microbial species identification in the presence of mammalian cells.
The document also describes removal or reduction of eukaryotic material and selective recovery of microbial genetic materials using anion-exchange selective capture. Free eukaryotic DNA is separated using anionic-exchange microparticles, and microbial genetic materials are isolated after selective lysis of mammalian cells. The resulting microbial genetic material is then amplified and detected using DIANAs.
Claims Coverage
Three independent claims are reflected across the partial content. The core inventive features are selective mammalian-cell lysis using a magnesium-salt-based composition consisting essentially of defined components and a compound of Formula 1, followed by amplification and detection of microbial genetic materials, with additional DIANA-based species identification and anion-exchange microparticle-based separation/isolation in the claim set.
Selective lysis and amplification-detection workflow using a magnesium salt composition and Formula 1 compound
An ultrasensitive method comprising selectively lysing mammalian cells, including eukaryotic DNA-containing cells, by contacting the sample with a composition consisting essentially of a magnesium salt, a buffering agent, water, and a compound of Formula 1; amplifying a plurality of microbial genetic materials; and detecting the amplified microbial genetic material.
Species-resolved DIANA detection of amplified microbial genetic material
Detecting amplified microbial genetic material by contacting it with a plurality of DIANAs having sequences complementary to genomic or plasmid sequences of specific microbial species, and detecting DIANA binding to indicate which microbial species are present in a biological sample.
Anion-exchange microparticle separation of free eukaryotic DNA and microbial genetic material isolation
Separating free eukaryotic DNA from a biological sample using anionic-exchange microparticles, removing the anionic-exchange microparticles, and isolating microbial genetic materials from microbial cells after selectively lysing mammalian cells.
Selective mammalian-cell lysis in a biological sample containing Borrelia cells
A method of selectively lysing mammalian cells in a biological sample comprising mammalian cells with eukaryotic DNA and Borrelia cells by contacting the biological sample with a composition consisting essentially of a magnesium salt, a buffering agent, water, and a compound of Formula 1.
Constrained Formula 1 compound concentration in the sample
Including the claimed composition so that the final concentration of the compound of Formula 1 is between 0.25 mM and 250 mM, inclusive.
Constrained pH window for selective mammalian cell lysis
Selectively lysing mammalian cells by adjusting the biological sample pH to within 8 to 11.5, inclusive.
Constrained Formula 1 substituent structures and integer subscript ranges
Defining substituent group R1 and R2 selections within specified group options and constraining q, p, and n to defined integer ranges.
Across the independent claims, the inventive coverage centers on a magnesium salt and Formula 1 compound composition for selective lysis of mammalian cells, followed by amplification and detection of microbial genetic materials. The claim set further includes DIANA-based species-specific detection and anion-exchange microparticle-based separation/isolation while reducing free eukaryotic DNA interference.
Stated Advantages
Ultrasensitive detection of one or more microbial cell species in a biological sample comprising mammalian cells.
Reduced interference by mammalian/eukaryotic genetic material via selective lysis and eukaryotic DNA reduction/removal, enabling amplification of microbial genetic materials.
Enrichment/capture of microbial genetic material through anion-exchange selective capture.
Reported detection limits and sensitivity improvements, including very low limits and improvements above cutoff.
Documented Applications
Detecting one or more species of microbial cells in a biological sample comprising mammalian cells using selective mammalian cell lysis and microbial genetic material amplification/detection.
DIANA-based species identification by contacting amplified microbial genetic material with DIANAs having sequences complementary to genomic or plasmid sequences of specific microbial species.
Sample processing that includes removal of free eukaryotic DNA using anionic-exchange microparticles and isolation of microbial genetic materials prior to amplification and DIANA detection.
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