Method for distinguishing methicillin resistant S. aureus from methicillin sensitive S. aureus in a mixed culture
The present invention provides isolated oligonucleotides and methods for detecting a methicillin resistant Staphylococcus aureus in a sample, including a sample that comprises nucleic acid molecules of higher biological complexity than that of amplified nucleic acid molecules.
1 . An isolated oligonucleotide consisting of:
a. a nucleic acid sequence as set forth in SEQ ID NO: 1; SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; or
b. a nucleic acid sequence that hybridizes with the complement of the nucleic acid sequence as set forth in SEQ ID NO: 1; SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
2 . A vector comprising the nucleic acid molecule of claim 1 .
3 . A host cell comprising the vector of claim 3 .
4 . A kit comprising an isolated oligonucleotide of claim 1 .
5 . A method for detecting methicillin resistant Staphylococcus aureus in a sample, the method comprising the steps of:
a. providing an addressable substrate having a capture probes bound thereto, the capture probes comprising an oligonucleotide of claim 1;
b. providing a detection probe comprising detector oligonucleotides, wherein the detector oligonucleotides have sequences that are complementary to at least a portion of the MRSA nucleic acid sequence;
c. contacting the sample with the substrate and the detection probe under conditions that are effective for the hybridization of the capture oligonucleotide to the MRSA nucleic acid sequence and the hybridization of the detection probe to the MRSA nucleic acid sequence;
d. washing the substrate to remove non-specifically bound material; and
e. detecting whether the capture oligonucleotide and detection probe hybridized with the MRSA nucleic acid sequence.
6 . The method of claim 5 , wherein the capture oligonucleotide comprises a nucleic acid sequence as set forth in SEQ ID NO: 1; SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
7 . The method of claim 5 , wherein the detector oligonucleotides comprise a nucleic acid sequence as set forth in SEQ ID NO: 1; SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
8 . The method of claim 5 , wherein sample is contacted with the detector probe so that methicillin resistant Staphylococcus aureus nucleic acid present in the sample hybridizes with the detector oligonucleotides on the detector probe, and the methicillin resistant Staphylococcus aureus nucleic acid bound to the detector probe is then contacted with the substrate so that the methicillin resistant Staphylococcus aureus nucleic acid hybridizes with the capture oligonucleotide on the substrate.
9 . The method of claim 5 , wherein sample is contacted with the substrate so that a methicillin resistant Staphylococcus aureus nucleic acid present in the sample hybridizes with a capture oligonucleotide, and the methicillin resistant Staphylococcus aureus nucleic acid bound to the capture oligonucleotide is then contacted with the detector probe so that the methicillin resistant Staphylococcus aureus nucleic acid hybridizes with the detector oligonuclotides on the detector probe.
10 . The method of claim 5 , wherein the sample is contacted simultaneously with the detector probe and the substrate.
11 . The method of claim 5 , wherein the detector oligonucleotides comprise a detectable label.
12 . The method of claim 11 , wherein the detectable label allows detection by photonic, electronic, acoustic, opto-acoustic, gravity, electrochemical, electro-optic, mass-spectrometric, enzymatic, chemical, biochemical, or physical means.
13 . The method of claim 11 , wherein the label is fluorescent.
14 . The method of claim 11 , wherein the label is luminescent.
15 . The method of claim 11 , wherein the label is phosphorescent.
16 . The method of claim 11 , wherein the label is radioactive.
17 . The method of claim 11 , wherein the label is a nanoparticle.
18 . The method of claim 11 , wherein the label is a dendrimer.
19 . The method of claim 11 , wherein the label is a molecular aggregate.
20 . The method of claim 11 , wherein the label is a quantum dot.
21 . The method of claim 11 , wherein the label is a bead.
22 . The method of claim 5 , wherein the detector probe is a nanoparticle probe having detector oligonucleotides bound thereto.
23 . The method of claim 22 , wherein the nanoparticles are made of a noble metal.
24 . The method of claim 23 , wherein the nanoparticles are made of gold or silver.
25 . The method of claim 24 , wherein the nanoparticles are made of gold.
26 . The method of claim 23 , wherein the detecting comprises contacting the substrate with silver stain.
27 . The method of claim 23 , wherein the detecting comprises detecting light scattered by the nanoparticle.
28 . The method of claim 23 , wherein the detecting comprises observation with an optical scanner.
29 . The method of claim 28 , wherein the scanner is linked to a computer loaded with software capable of calculating grayscale measurements, and the grayscale measurements are calculated to provide a quantitative measure of the amount of nucleic acid detected.
30 . The method of claim 23 , wherein the detecting comprises observation with a flatbed scanner.
31 . The method of claim 30 , wherein the scanner is linked to a computer loaded with software capable of calculating grayscale measurements, and the grayscale measurements are calculated to provide a quantitative measure of the amount of nucleic acid detected.
32 . The method of claim 23 , wherein the oligonucleotides attached to the substrate are located between two electrodes, the nanoparticles are made of a material that is a conductor of electricity, and step (d) comprises detecting a change in conductivity.
33 . The method of claim 32 , wherein the electrodes are made of gold and the nanoparticles are made of gold.
34 . The method of claim 32 , wherein the substrate is contacted with silver stain to produce the change in conductivity.
35 . The method of claim 5 , wherein the sample comprises nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules.
36 . The method of claim 35 , wherein the higher biological complexity is greater than about 50,000.
37 . The method of claim 35 , wherein the higher biological complexity is between about 50,000 and about 3,000,000.
38 . The method of claim 35 , wherein the higher biological complexity is about 3,000,000.
39 . The method of claim 5 , wherein nucleic acid molecules in the sample are amplified.
40 . The method of claim 39 , wherein the nucleic acid molecules in the sample are amplified by polymerase chain reaction, rolling circle amplification, NASBA, or iCAN.
41 . A method for detecting methicillin resistant Staphylococcus aureus in a sample, the method comprising the steps of:
a. providing an addressable substrate having a capture oligonucleotide bound thereto, wherein the capture probe comprises an oligonucleotide having a sequence complementary to at least a portion of the MRSA nucleic acid sequence;
b. providing a detection probe comprising detector oligonucleotides, wherein the detector oligonucleotides is an oligonucleotide of claim 1;
c. contacting the sample with the substrate and the detection probe under conditions that are effective for the hybridization of the capture oligonucleotide to the MRSA nucleic acid sequence and the hybridization of the detection probe to the MRSA nucleic acid sequence;
d. washing to the substrate to remove non-specifically bound material; and
e. detecting whether the capture oligonucleotide and detection probe hybridized with the MRSA nucleic acid sequence.
42 . The method of claim 41 , wherein the detector oligonucleotides comprise a nucleic acid sequence as set forth in SEQ ID NO: 1; SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
43 . The method of claim 41 , wherein the capture oligonucleotides comprise a nucleic acid sequence as set forth in SEQ ID NO: 1; SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
44 . The method of claim 41 , wherein sample is contacted with the detector probe so that a methicillin resistant Staphylococcus aureus nucleic acid present in the sample hybridizes with the detector oligonucleotides on the detector probe, and the methicillin resistant Staphylococcus aureus nucleic acid bound to the detector probe is then contacted with the substrate so that the methicillin resistant Staphylococcus aureus nucleic acid hybridizes with the capture oligonucleotide on the substrate.
45 . The method of claim 41 , wherein sample is contacted with the substrate so that a methicillin resistant Staphylococcus aureus nucleic acid present in the sample hybridizes with a capture oligonucleotide, and the methicillin resistant Staphylococcus aureus nucleic acid bound to the capture oligonucleotide is then contacted with the detector probe so that the methicillin resistant Staphylococcus aureus nucleic acid hybridizes with the detector oligonuclotides on the detector probe.
46 . The method of claim 41 , wherein the sample is contacted simultaneously with the detector probe and the substrate.
47 . The method of claim 41 , wherein the detector oligonucleotides comprise a detectable label.
48 . The method of claim 47 , wherein the detectable label allows detection by photonic, electronic, acoustic, opto-acoustic, gravity, electrochemical, electro-optic, mass-spectrometric, enzymatic, chemical, biochemical, or physical means.
49 . The method of claim 47 , wherein the label is fluorescent.
50 . The method of claim 47 , wherein the label is luminescent.
51 . The method of claim 47 , wherein the label is phosphorescent.
52 . The method of claim 47 , wherein the label is radioactive.
53 . The method of claim 47 , wherein the label is a nanoparticle.
54 . The method of claim 47 , wherein the label is a dendrimer.
55 . The method of claim 47 , wherein the label is a molecular aggregate.
56 . The method of claim 47 , wherein the label is a quantum dot.
57 . The method of claim 47 , wherein the label is a bead.
58 . The method of claim 41 , wherein the detector probe is a nanoparticle probe having detector oligonucleotides bound thereto.
59 . The method of claim 58 , wherein the nanoparticles are made of a noble metal.
60 . The method of claim 59 , wherein the nanoparticles are made of gold or silver.
61 . The method of claim 60 , wherein the nanoparticles are made of gold.
62 . The method of claim 58 , wherein the detecting comprises contacting the substrate with silver stain.
63 . The method of claim 58 , wherein the detecting comprises detecting light scattered by the nanoparticle.
64 . The method of claim 58 , wherein the detecting comprises observation with an optical scanner.
65 . The method of claim 64 , wherein the scanner is linked to a computer loaded with software capable of calculating grayscale measurements, and the grayscale measurements are calculated to provide a quantitative measure of the amount of nucleic acid detected.
66 . The method of claim 58 , wherein the detecting comprises observation with a flatbed scanner.
67 . The method of claim 66 , wherein the scanner is linked to a computer loaded with software capable of calculating grayscale measurements, and the grayscale measurements are calculated to provide a quantitative measure of the amount of nucleic acid detected.
68 . The method of claim 58 , wherein the oligonucleotides attached to the substrate are located between two electrodes, the nanoparticles are made of a material that is a conductor of electricity, and step (d) comprises detecting a change in conductivity.
69 . The method of claim 68 , wherein the electrodes are made of gold and the nanoparticles are made of gold.
70 . The method of claim 68 , wherein the substrate is contacted with silver stain to produce the change in conductivity.
71 . The method of claim 41 , wherein the sample comprises nucleic acid molecules of higher biological complexity relative to amplified nucleic acid molecules.
72 . The method of claim 66 , wherein the higher biological complexity is greater than about 50,000.
73 . The method of claim 66 , wherein the higher biological complexity is between about 50,000 and about 3,000,000.
74 . The method of claim 66 , wherein the higher biological complexity is about 3,000,000.
75 . The method of claim 41 , wherein nucleic acid molecules in the sample are amplified.
76 . The method of claim 41 , wherein the nucleic acid molecules in the sample are amplified by polymerase chain reaction, rolling circle amplification, NASBA, or iCAN.
77 . The method of claims 1 or 41 , wherein the capture probe and substrate are bound by specific binding pair interactions.
78 . The method of claim 77 wherein the capture probe and substrate comprise complements of a specific binding pair.
79 . The method of claim 78 wherein complements of a specific binding pair comprise nucleic acid, oligonucleotide, peptide nucleic acid, polypeptide, antibody, antigen, carbohydrate, protein, peptide, amino acid, hormone, steroid, vitamin, drug, virus, polysaccharides, lipids, lipopolysaccharides, glycoproteins, lipoproteins, nucleoproteins, oligonucleotides, antibodies, immunoglobulins, albumin, hemoglobin, coagulation factors, peptide and protein hormones, non-peptide hormones, interleukins, interferons, cytokines, peptides comprising a tumor-specific epitope, cells, cell-surface molecules, microorganisms, fragments, portions, components or products of microorganisms, small organic molecules, nucleic acids and oligonucleotides, metabolites of or antibodies to any of the above substances.