Method and kit of detecting the absence of micro-organisms

Inventors

Crow, Matthew A. • Bennett, Helen V. • Wratting, Daniel S. • Mullen, William H.

Assignees

Momentum Bioscience Ltd

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 relates to a method of detecting the presence of a micro-organism in a sample by enzymatically generating a detectable nucleic acid product from a nuclease resistant nucleic acid molecule. The nuclease resistant nucleic acid molecule comprises a plurality of nuclease resistant nucleotides, wherein the nuclease resistant nucleotides comprise phosphorothioate nucleotides. The micro-organism nucleic-acid modifying activity extends the nuclease resistant nucleic acid molecule by polymerase activity or ligates it by ligase activity.

After contacting and incubating the sample under conditions suitable for polymerase activity or ligase activity, the method detects a nucleic acid molecule that has been extended by the polymerase activity or ligated by the ligase activity as compared to a negative control. The described processing provides diagnostic discrimination between micro-organism-dependent nucleic-acid modification and negative-control outcomes.

The invention also provides methods for detecting the absence of a micro-organism by detecting a lack of an extended or ligated nucleic acid product as compared to a positive control. In refined embodiments, an internal positive control (IPC) nucleic acid molecule is included, and the positive control may be configured as a nuclease-susceptible or nuclease-resistant nucleic acid. Further refinements include using nucleic acid structures that include uracil in a complementary strand and degrade uracil residues by Uracil DNA Glycosylase (UDG), and preventing extension by modifying a 3′ end of a complementary strand.

Claims Coverage

The independent claims cover two complementary detection modalities: detecting the presence of a micro-organism versus detecting the absence of a micro-organism. Across the independent claims, the core inventive concept is the enzymatic generation of a detectable extended or ligated nucleic acid product from a nuclease resistant phosphorothioate-containing nucleic acid, followed by detection relative to a control.

Overall, the claim coverage centers on using a nuclease resistant nucleic acid molecule comprising phosphorothioate nucleotides as an enzymatic substrate for polymerase extension or ligase ligation by a micro-organism, then determining presence by detection of the extended/ligated product relative to a negative control, or determining absence by detection of lack of that product relative to a positive control. Dependent claims further specify control configurations (including internal positive control), probe-based detection, uracil/UDG-based degradation features, and constraints to prevent extension.

Stated Advantages

Documented Applications