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Publication Number
US-9096884-B2
Publication Date
2015-08-04
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 a method for detecting NAD-dependent bacterial ligase activity in a sample comprising mammalian cell material. Bacterial cells in the sample are lysed under conditions sufficient to lyse bacterial cells, and the sample is treated under high pH conditions that inhibit the mammalian background from ATP-dependent ligase while not inhibiting bacterial NAD-dependent ligases released during the lysis. This enables selective ligase activity readout despite the presence of mammalian ATP-dependent ligase background.
After the high pH treatment, the sample or a portion of the sample is contacted with a nucleic acid molecule that acts as a substrate for NAD-dependent ligase activity. The contacted sample is incubated under conditions suitable for NAD-dependent ligase activity, and the presence, amount, or both of a ligated nucleic acid molecule resulting from the action of the NAD-dependent ligase on the substrate nucleic acid are determined to indicate the presence of NAD-dependent bacterial ligase activity.
The invention also provides a method of diagnosing the organism responsible for an infection, or a disease associated with the presence of a bacterial cell in a sample obtained from a mammalian subject. The same general workflow is used: lysis of bacterial cells, a subsequent high pH treatment that inhibits the mammalian ATP-dependent ligase background while preserving microbial NAD-dependent ligases, contacting with a substrate nucleic acid for NAD-dependent ligase activity, incubating under conditions suitable for NAD-dependent ligase activity, and determining the presence, amount, or both of a ligated nucleic acid molecule to indicate the presence of a bacterial cell causing the infection.
Claims Coverage
The partial claims provided include two independent claims. They share a common inventive approach centered on selectively suppressing mammalian ATP-dependent ligase background using high pH conditions while preserving microbial NAD-dependent ligase activity, followed by detection of a ligated nucleic acid product.
Selective suppression of mammalian ATP-dependent ligase background while preserving NAD-dependent bacterial ligases
Treating a sample under high pH conditions that inhibit the mammalian background from ATP-dependent ligase but which do not inhibit bacterial NAD-dependent ligases released during the lysis of the sample.
NAD-dependent ligation of a nucleic acid substrate
Contacting the sample with a nucleic acid molecule which acts as a substrate for NAD-dependent ligase activity in the sample, and incubating the thus contacted sample under conditions suitable for NAD-dependent ligase activity.
Detection of a ligated nucleic acid molecule to indicate NAD-dependent bacterial ligase activity
Determining the presence, amount, or both of a ligated nucleic acid molecule resulting from the action of the NAD-dependent ligase on the substrate nucleic acid molecule to indicate the presence of NAD-dependent bacterial ligase activity.
Diagnosis of infection organism based on NAD-dependent ligase-generated ligated nucleic acid
Determining the presence, amount, or both of a ligated nucleic acid molecule resulting from the action of the NAD-dependent ligase on the substrate nucleic acid molecule to indicate the presence of a bacterial cell causing the infection.
Across the independent claims, the inventive coverage is directed to high pH treatment that inhibits mammalian ATP-dependent ligase background while preserving microbial NAD-dependent ligases, followed by NAD-dependent ligation of a nucleic acid substrate and determining a ligated nucleic acid product to indicate bacterial NAD-dependent ligase activity and diagnose a bacterial cause of infection or a related disease.
Stated Advantages
Enables selective detection of NAD-dependent bacterial ligase activity by inhibiting mammalian ATP-dependent ligase background using high pH conditions while not inhibiting bacterial NAD-dependent ligases.
Provides a basis for diagnosing the organism responsible for an infection or a disease associated with the presence of a bacterial cell based on detection of a NAD-dependent-ligated nucleic acid product.
Documented Applications
Detecting NAD-dependent bacterial ligase activity in a sample comprising mammalian cell material.
Diagnosing the organism responsible for an infection, or a disease associated with the presence of a bacterial cell in a sample obtained from a mammalian subject.
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