Detection of bordetella
First Claim
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1. A method for detecting the presence or absence of Bordetella pertussis in a biological sample from an individual, said method comprising:
- performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of IS481 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of IS481 probes, wherein the members of said pair of IS481 probes hybridize within no more than five nucleotides of each other, wherein a first IS481 probe of said pair of IS481 probes is labeled with a donor fluorescent moiety and a second IS481 probe of said pair of IS481 probes is labeled with a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first IS481 probe and said corresponding acceptor fluorescent moiety of said second IS481 probe, wherein the presence of FRET is indicative of the presence of B. pertussis in said biological sample, and wherein the absence of FRET is indicative of the absence of B. pertussis in said biological sample.
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Abstract
The invention provides methods to detect Bordetella pertussis and/or Bordetella parapertussis in a biological sample. Primers and probes for the differential detection of B. pertussis and B. parapertussis are provided by the invention. Articles of manufacture containing such primers and probes for detecting B. pertussis and/or B. parapertussis are further provided by the invention.
20 Citations
48 Claims
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1. A method for detecting the presence or absence of Bordetella pertussis in a biological sample from an individual, said method comprising:
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performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of IS481 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of IS481 probes, wherein the members of said pair of IS481 probes hybridize within no more than five nucleotides of each other, wherein a first IS481 probe of said pair of IS481 probes is labeled with a donor fluorescent moiety and a second IS481 probe of said pair of IS481 probes is labeled with a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first IS481 probe and said corresponding acceptor fluorescent moiety of said second IS481 probe, wherein the presence of FRET is indicative of the presence of B. pertussis in said biological sample, and wherein the absence of FRET is indicative of the absence of B. pertussis in said biological sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for detecting the presence or absence of Bordetella parapertussis in a biological sample from an individual, said method comprising:
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performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of IS1001 primer to produce an IS1001 amplification product if a B. parapertussis IS1001 nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said biological sample with a pair of IS1001 probes, wherein the members of said pair of IS1001 probes hybridize within no more than five nucleotides of each other, wherein a first IS1001 probe of said pair of IS1001 probes is labeled with a donor fluorescent moiety and a second IS1001 probe of said pair of IS1001 probes is labeled with a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of FRET between said donor fluorescent moiety of said first IS1001 probe and said corresponding acceptor fluorescent moiety of said second IS1001 probe, wherein the presence of FRET is indicative of the presence of B. parapertussis in said biological sample, and wherein the absence of FRET is indicative of the absence of B. parapertussis in said biological sample. - View Dependent Claims (23, 24)
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25. A method for detecting the presence or absence of Bordetella pertussis and/or B. parapertussis in a biological sample from an individual, said method comprising:
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performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of IS481 primers and a pair of IS1001 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample and an IS1001 amplification product if a B. parapertussis IS1001 nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of IS481 probes and a pair of IS1001 probes, wherein the members of said pair of IS481 probes hybridize within no more than five nucleotides of each other and wherein the members of said pair of IS1001 probes hybridize within no more than five nucleotides of each other, wherein a first IS481 probe of said pair of IS481 probes is labeled with a donor fluorescent moiety and wherein a second IS481 probe of said pair of IS481 probes is labeled with a corresponding acceptor fluorescent moiety, wherein a first IS1001 probe of said pair of IS1001 probes is labeled with a donor fluorescent moiety and wherein a second IS1001 probe of said pair of IS1001 probes is labeled with a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of FRET between said donor fluorescent moiety of said first IS481 probe and said corresponding acceptor fluorescent moiety of said second IS481 probe and/or between donor fluorescent moiety of said first IS1001 probe and said corresponding acceptor fluorescent moiety of said second IS1001 probe, wherein the presence of FRET is indicative of the presence of B. pertussis and/or B. parapertussis in said biological sample, and wherein the absence of FRET is indicative of the absence of B. pertussis or B. parapertussis in said biological sample. - View Dependent Claims (26, 27, 28)
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29. An article of manufacture, comprising:
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a pair of IS481 primers;
a pair of IS481 probes; and
a first donor fluorescent moiety and a corresponding first acceptor fluorescent moiety. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38)
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39. An article of manufacture, comprising:
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a pair of IS1001 primers;
a pair of IS1001 probes; and
a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.
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40. A method for detecting the presence or absence of B. pertussis in a biological sample from an individual, said method comprising:
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performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of IS481 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with an IS481 probe, wherein the IS481 probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety and said acceptor fluorescent moiety of said IS481 probe, wherein the presence or absence of FRET is indicative of the presence or absence of B. pertussis in said sample. - View Dependent Claims (41, 42, 43, 44, 45)
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46. A method for detecting the presence or absence of B. pertussis in a biological sample from an individual, said method comprising:
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performing at least one cycling step, wherein a cycling step comprises an amplifying step and a dye-binding step, wherein said amplifying step comprises contacting said sample with a pair of IS481 primers to produce an IS481 amplification product if a B. pertussis IS481 nucleic acid molecule is present in said sample, wherein said dye-binding step comprises contacting said IS481 amplification product with a nucleic acid binding dye; and
detecting the presence or absence of binding of said nucleic acid binding dye to said amplification product, wherein the presence of binding is indicative of the presence of B. pertussis in said sample, and wherein the absence of binding is indicative of the absence of B. pertussis in said sample. - View Dependent Claims (47, 48)
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Specification