Method for rapid identification of microorganisms
The present invention relates, in general, to probes, methods, and kits used to determine the presence or absence of a microorganism in a sample. The probes, methods, and kits comprise at least one capture probe and/or at least one detector probe.
1. A method for detecting the presence of Staphylococcus aureus in a sample, the method comprising:
(a) releasing RNA or DNA from at least one microorganism in the sample;
(b) contacting the RNA or DNA with at least one capture probe capable of hybridizing to a first target sequence of the RNA or DNA, wherein the contacting is performed under conditions that permit hybridization between the first target sequence and the at least one capture probe to form a RNA or DNA-capture probe hybrid complex, and wherein the at least one capture probe comprises at least one sequence selected from the group consisting of SEQ ID NOs: 1, 2, 61, 62, and 73; and
(c) detecting the presence of the RNA or DNA-capture probe hybrid complex by
(i) contacting the RNA or DNA with at least one detector probe capable of hybridizing to a second target sequence of the RNA or DNA, wherein the detector probe comprises at least one reporter group and wherein the contacting is performed under conditions that permit hybridization between the second target sequence and the at least one detector probe to form a RNA or DNA-capture probe-detector probe hybrid complex, and wherein the at least one detector probe comprises at least one sequence selected from the group consisting of SEQ ID NOs: 61 and 62; and
(ii) detecting the RNA or DNA-capture probe-detector probe hybrid complex by detecting the at least one reporter group wherein the presence of the RNA or DNA-capture probe-detector probe hybrid complex indicates the presence of Staphylococcus aureus.
2. The method of claim 1 , wherein the first target sequence and the second target sequence comprise the same sequence.
3. The method of claim 1 , wherein the capture probe is immobilized on a solid support before hybridizing to the first target sequence.
4. The method of claim 1 , wherein the RNA or DNA-capture probe hybrid complex is immobilized on a solid support.
5. The method of claim 1 , wherein the RNA or DNA-capture probe-detector probe hybrid complex is immobilized on a solid support.
6. The method of any one of claims 3 , 4 , and 5 , wherein the solid support is selected from the group consisting of latex beads, agarose beads, paramagnetic beads, ferric oxide, microarray chips, filter paper, nitrocellulose filters, nylon membranes, glass slides and cellular membranes.
7. The method of claim 6 , wherein the solid support is a microarray chip.
8. The method of any one of claims 3 , 4 , and 5 , wherein two or more capture probes are immobilized on a single spot of the solid support.
9. The method of claim 3 , further comprising immobilizing the capture probe by hybridizing the capture probe to an immobilization probe on a solid support.
10. The method of claim 1 , wherein the reporter group is selected from the group consisting of a radioactive isotope, an enzyme, a fluorescent molecule and an amplification sequence.
11. The method of claim 10 , wherein the amplification sequence initiates an amplification reaction selected from the group consisting of strand displacement amplification (SDA), polymerase chain reaction (PCR), reverse transcriptase-strand displacement amplification (RT-SDA), reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification and QB replicase amplification.
12. A method of determining the efficacy of an antimicrobial patient therapy, comprising:
(a) detecting the presence of Staphylococcus aureus in a first sample obtained from a patient according to the method claim 1 ; and
(b) detecting the presence or absence of Staphylococcus aureus in a second sample obtained from the patient according to the method of claim 1 ;
wherein the first sample and the second sample are taken sequentially over time, and wherein the presence of Staphylococcus aureus in the first sample and the absence of Staphylococcus aureus in the second sample indicates a successful response to therapy; and the presence of Staphylococcus aureus in the first sample and in the second sample indicates a unsuccessful response to therapy.
13. A kit for detecting the presence or absence of Staphylococcus aureus in a sample, comprising:
(a) a solid support;
(b) at least one capture probe comprising at least one capture sequence that hybridizes to a first target sequence of RNA or DNA from Staphylococcus aureus to form a RNA or DNA-capture probe hybrid complex; wherein the at least one capture probe comprises at least one sequence selected from the group consisting of SEQ ID NOs: 1, 2, 61, 62 and 73;
(c) at least one detector probe that hybridizes to a second sequence of the RNA or DNA from Staphylococcus aureus , wherein the detector probe comprises at least one reporter group, and wherein the detector probe comprises at least one sequence selected from the group consisting of SEQ ID NOs: 61 and 62; and
(d) a vessel to collect, concentrate, amplify or isolate the RNA or DNA.
14. The kit of claim 13 , wherein the vessel is selected from the group consisting of evacuated blood collection tubes, eppendorf tubes and test tubes.
15. The kit of claim 14 , wherein the solid support is selected from the group consisting of latex beads, agarose beads, paramagnetic beads, ferric oxide, microarray chips, filter paper, nitrocellulose filters, nylon membranes, glass slides and cellular membranes.
16. An isolated oligonucleotide probe for use in detecting Staphylococcus aureus , wherein the probe is selected from the group consisting of SEQ ID NOs: 61 and 62.