Method and kit of detecting the absence of micro-organisms
Inventors
Crow, Matthew A. • Bennett, Helen V. • Wratting, Daniel S. • Mullen, William H.
Assignees
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Publication Number
US-11746389-B2
Publication Date
2023-09-05
Expiration Date
Abstract
Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism. Corresponding kits are also provided.
Core Innovation
The invention provides methods of detecting a micro-organism in a sample by contacting the sample with a nuclease resistant nucleic acid molecule. The nuclease resistant nucleic acid molecule comprises a plurality of nuclease resistant nucleotides and is acted upon by the micro-organism in the sample such that it is extended by polymerase activity or ligated by ligase activity. After incubation under conditions suitable for polymerase activity or ligase activity, a nucleic acid molecule that has been extended or ligated is detected compared to a negative control.
A related method detects the absence of a micro-organism by using the same nuclease resistant nucleic acid molecule and incubation conditions, but detecting a lack of a nucleic acid molecule that has been extended by polymerase activity or ligated by ligase activity. The absence determination is made compared to a positive control. In both the presence and absence approaches, the detection outcome is based on the presence or lack of an extended or ligated nucleic acid product relative to the appropriate control.
The nucleic acid substrate is defined as nuclease resistant and, in refinements, includes methylated nucleotides and/or nucleotides protected at the 3′ and/or 5′ ends, and/or synthetic nucleotides. The nucleic acid modifying activity of the micro-organism is used as the mechanism that generates the extended or ligated nucleic acid product that is subsequently detected. Further refinements include probe-based detection, nucleic acid amplification for the detection step, and optional processing using Uracil DNA Glycosylase (UDG) to degrade uracil residues in a complementary strand of an at least partially double stranded substrate.
The document also recites use of an internal positive control (IPC) nucleic acid molecule and specifies that the IPC can be susceptible to nuclease activity or nuclease resistant and built from nucleotides resistant to nuclease activity. The IPC is included to detect or quantify contaminating nuclease activity, grounding interpretation of the extended or ligated product relative to expected control behavior. Detection can also be performed using a nucleic acid probe that binds to a target probe sequence within the nucleic acid molecule.
Claims Coverage
The document includes two independent methods: one for detecting the presence of a micro-organism and one for detecting the absence of a micro-organism. Across these, the inventive features are centered on a nuclease resistant nucleic acid substrate that is extended or ligated by micro-organism polymerase/ligase activity, followed by detection relative to a control.
Nuclease resistant nucleic acid substrate extended or ligated by micro-organism activity
Contacting the sample with a nuclease resistant nucleic acid molecule comprising a plurality of nuclease resistant nucleotides which is either extended by polymerase activity or is ligated by ligase activity of the micro-organism in the sample.
Incubation under conditions suitable for polymerase activity or ligase activity
Incubating the thus contacted sample under conditions suitable for polymerase activity or ligase activity.
Detection of extended or ligated nucleic acid product compared to a negative control
Detecting in the sample a nucleic acid molecule that has been extended by the polymerase activity or ligated by the ligase activity of the micro-organism as compared to a negative control, thereby indicating the presence of the micro-organism in the sample.
Nuclease resistant nucleic acid substrate absence readout relative to a positive control
Contacting the sample with a nuclease resistant nucleic acid molecule comprising a plurality of nuclease resistant nucleotides which is either extended by polymerase activity or is ligated by ligase activity of the micro-organism in the sample.
Incubation under conditions suitable for polymerase activity or ligase activity (absence method)
Incubating the thus contacted sample under conditions suitable for polymerase activity or ligase activity.
Detection of lack of extended or ligated nucleic acid product compared to a positive control
Detecting in the sample a lack of a nucleic acid molecule that has been extended by the polymerase activity or ligated by the ligase activity of the micro-organism as compared to a positive control, thereby indicating the absence of the micro-organism in the sample.
Overall claim coverage is directed to ETGA-style detection logic that uses a nuclease resistant nucleic acid molecule to be extended or ligated by micro-organism polymerase or ligase activity, then reads out the presence or lack of an extended/ligated nucleic acid product relative to a control (negative control for presence detection; positive control for absence detection).
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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