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Publication Number
US-9796997-B2
Publication Date
2017-10-24
Expiration Date
Abstract
A method of detecting a ligase expressing micro-organism in a sample comprises steps of treating the sample under conditions that inhibit the activity of ATP-dependent ligase from mammalian cells but which do not inhibit the activity of the microbial ligases, contacting the sample or a portion of the sample with a nucleic acid molecule which acts as a substrate for ligase activity in the sample, incubating the thus contacted sample under conditions suitable for ligase activity; and specifically determining the presence and/or the amount of a ligated nucleic acid molecule resulting from the action of the ligase on the substrate nucleic acid molecule to indicate the presence of the ligase expressing micro-organism. The micro-organism may be a fungus or a bacterium or both. High pH conditions may be employed to inactivate mammalian ligases. Related kits are described.
Core Innovation
The invention provides methods for detecting a ligase-expressing microorganism in a sample containing mammalian cells. The methods include selectively permeabilizing the cell membrane of the mammalian cells in the sample without lysing microorganisms, thereby generating a resultant sample that maintains intracellular microbial ligase activity. The resultant sample is treated under high pH conditions that inhibit the activity of ATP-dependent ligase from mammalian cells while not inhibiting the activity of intracellular microbial ligases.
After high pH treatment, microorganisms are lysed in the resultant sample to release ATP-dependent ligase and NAD-dependent ligase, as relevant. The resultant sample is then contacted with a nucleic acid molecule that is a substrate for the corresponding microbial ligase, and incubating under conditions suitable for microbial ligase activity produces a ligated nucleic acid molecule. Determining the presence and/or amount of the ligated nucleic acid indicates the presence of the ligase-expressing microorganism.
The invention further applies the same selective permeabilization and background-inhibition concept to distinguish fungi and/or bacteria. Separate assay portions are used to contact nucleic acid substrates specific for microbial ATP-dependent ligase and bacterial NAD-dependent ligase, followed by determining ligated nucleic acid products. In infection or disease contexts, determining the presence and/or amount of ligated nucleic acid products from the ATP-dependent and NAD-dependent microbial ligase reactions indicates fungi and/or bacteria causing the infection or disease.
Claims Coverage
Independent claims cover detecting a ligase-expressing microorganism in mammalian-cell-containing samples, fungal and/or bacterial cells with ATP-dependent and NAD-dependent ligase readouts, the microorganism responsible for an infection or disease, and determining whether fungi are responsible for infection or disease. Across the independent claims, the core inventive features are selective mammalian cell membrane permeabilization without lysing microorganisms, high pH inhibition of mammalian ATP-dependent ligase while preserving intracellular microbial ligase activity, microbial lysis to release ligases, ligase-specific nucleic acid substrate ligation, and determining ligated nucleic acids to indicate presence, amount, or responsibility.
Selective permeabilization without microbial lysis followed by high pH inhibition of mammalian ATP-dependent ligase
The method includes selectively permeabilizing the cell membrane of the mammalian cells in the sample without lysing microorganisms in the sample, and treating the resultant sample under high pH conditions that inhibit the activity of ATP-dependent ligase from mammalian cells but do not inhibit the activity of intracellular microbial ligases.
Microbial lysis to release ATP-dependent ligase and NAD-dependent ligase
The method includes lysing microorganisms in the resultant sample under the high pH conditions to release ATP-dependent ligase and NAD-dependent ligase.
Ligase-specific nucleic acid substrate and determination of ligated nucleic acid product
The method includes contacting the resultant sample with a nucleic acid molecule that is a substrate for microbial ligase in the sample, incubating under conditions suitable for microbial ligase activity, and determining the presence and/or the amount of a ligated nucleic acid molecule resulting from the action of the intracellular microbial ligase on the microbial substrate nucleic acid molecule to indicate the presence of the ligase-expressing microorganism.
Dual ligation readout distinguishing fungi and bacteria
The method includes treating the resultant sample under conditions that inhibit background from mammalian ATP-dependent ligase but do not affect intracellular microbial ATP-dependent and NAD-dependent ligases, lysing microorganisms to release microbial ATP-dependent and NAD-dependent ligases, contacting a first portion with a nucleic acid substrate for microbial ATP-dependent ligase and determining a ligated nucleic acid product to indicate the presence of fungi and/or bacteria, then contacting a second portion with a nucleic acid substrate for bacterial NAD-dependent ligase and determining a ligated nucleic acid product to indicate the presence of bacteria only.
Infection or disease microorganism determination using ATP-dependent and NAD-dependent microbial ligase reactions
The method includes determining the presence, amount, or both of a ligated nucleic acid molecule resulting from the action of the microbial ATP-dependent ligase on the substrate nucleic acid molecule to indicate the presence of fungi and/or bacteria causing the infection, and determining the presence, amount, or both of a ligated nucleic acid molecule resulting from the action of the bacterial NAD-dependent ligase on the substrate nucleic acid molecule to indicate the presence of bacteria causing the infection.
Fungal detection using high pH inhibition of mammalian ATP-dependent ligase and fungal ATP-dependent ligation
The method includes selectively permeabilizing the cell membrane of mammalian cells without lysing the fungi, treating under high pH conditions that inhibit the background signal from mammalian ATP-dependent ligase but do not affect intracellular fungal ATP-dependent ligases, lysing fungal cells to release fungal ATP-dependent ligases, contacting with a nucleic acid substrate for fungal ATP-dependent ligase, and determining the presence, amount, or both of a ligated nucleic acid molecule to indicate the presence of fungi.
Determining whether a fungus is responsible for infection or disease using fungal ATP-dependent ligation
The method includes selectively permeabilizing the cell membrane of mammalian cells without lysing the fungi, treating under conditions that inhibit the background from mammalian ATP-dependent ligase but do not affect fungal ATP-dependent ligases, lysing any fungal cells to release the fungal ATP-dependent ligase, contacting with a nucleic acid substrate for fungal ATP-dependent ligase, and determining the presence, amount, or both of a ligated nucleic acid molecule to indicate the presence of fungi causing the infection or disease.
Collectively, the independent claims define a detection workflow that selectively permeabilizes mammalian cells without lysing microorganisms, uses high pH to inhibit mammalian ATP-dependent ligase while preserving intracellular microbial ligase, lyses microorganisms to release microbial ligases, performs ligation on ligase-specific nucleic acid substrates, and determines the presence and/or amount of ligated nucleic acids to indicate presence, amount, and in some claims, the microorganism responsible for infection or disease. For fungi and bacteria differentiation, the claims further include separate detection portions for microbial ATP-dependent ligase activity and bacterial NAD-dependent ligase activity.
Stated Advantages
Reduces background from mammalian ATP-dependent ligase in samples containing mammalian cells.
Enables very high sensitivity for detection, including sensitivity down to femto/atto levels.
Links signal to viability-linked ligase activity by using intracellular microbial ligases and determining ligated nucleic acid products.
Documented Applications
Detecting fungal or bacterial cells or both in a sample comprising mammalian cells, including blood broth, blood, plasma, serum, platelets, and urine.
Detecting the microorganism responsible for an infection, or a disease associated with the presence of bacterial or fungal cell in a sample obtained from a mammalian subject.
Determining whether a fungus is responsible for an infection, or a disease associated with the presence of a fungal cell in a sample obtained from a subject.
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