Non-replicative transduction particles and transduction particle-based reporter systems
AssigneeRoche Molecular Systems, Inc.
InventorsSlav Dugenny, Ellen H Fiss, Marc Rehfuss
Life of this patent
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Abstract
Methods and systems are provided for packaging reporter nucleic acid molecules into non-replicative transduction particles for use as reporter molecules. The non-replicative transduction particles can be constructed from viruses and use viral transduction and replication systems. The reporter nucleic acid molecules include a reporter gene, such as a reporter molecule or selectable marker, for detecting target genes or cells. Methods and systems are provided for detection of cells and target nucleic acid molecules using the non-replicative transduction particles as reporter molecules.
Claims11
A method for detecting S. aureus in a sample, the method comprising the steps of:
- (a) contacting the sample with a lysate comprised of a plurality of non-replicative transduction particles (NRTPs) such that the plurality of NRTPs transduces S. aureus, if present in the sample, wherein the plurality of NRTPs are produced by (i) inducing a lytic phase of a bacterial cell packaging system, wherein said bacterial cell packaging system comprises:
- a host bacteria cell;
- a φ80a bacteriophage genome having a disrupted terS packaging initiation site sequence existing as a lysogen within the host bacteria cell and comprises a sequence;
- a reporter nucleic acid molecule separate from the φ80a bacteriophage genome, having a luxAB reporter gene and a non-disrupted packaging initiation site sequence for facilitating packaging of a replicon of the reporter nucleic acid molecule into the NRTP, wherein the reporter nucleic acid molecule comprises a sequence selected from SEQ ID NO:
- 3, SEQ ID NO:
- 4, and SEQ ID NO:5;
- (ii) allowing the replicon of the reporter nucleic acid molecule to be packaged to produce the NRTPs;
- (b) providing conditions for expression of the luxAB reporter gene;
- and
- (c) detecting the presence or absence of light produced by the luxAB reporter gene, wherein the presence of light indicates the presence of S. aureus.
A method for producing and collecting a plurality of non-replicative transduction particles (NRTPs) for detecting S. aureus in a sample, comprising:
- (a) inducing a lytic phase of a bacterial cell packaging system, wherein said bacterial cell packaging system comprises:
- (i) a host bacteria cell;
- (ii) a φ80a bacteriophage genome having a disrupted packaging initiation site sequence existing as a lysogen within the host bacteria cell;
- (iii) a reporter nucleic acid molecule separate from the φ80a bacteriophage genome, having a luxAB reporter gene and a non-disrupted packaging initiation site sequence for facilitating packaging of a replicon of the reporter nucleic acid molecule into the NRTP, wherein the reporter nucleic acid molecule comprises a sequence selected from SEQ ID NO:
- 3, SEQ ID NO:
- 4, and SEQ ID NO:5;
- (b) allowing the replicon of the reporter nucleic acid molecule to be packaged to produce the NRTPs;
- and
- (c) collecting a lysate comprising the plurality of NRTPs.
Dependencies are inferred from the claim text.