Compositions and methods for the simultaneous detection and quantification of multiple specific nucleic acid sequences

Claim 1

A composition for the assay of a plurality of nucleic acid analytes in a single sample comprising: (a) a plurality of different oligonucleotide hybridization assay probes, wherein each of said probes has a nucleotide sequence complementary to a specific different target nucleotide sequence of one or more nucleic acid analytes suspected of being in said test sample, (b) a plurality of different chemiluminescent labels, each of said labels coupled to one or more of said hybridization probes by a linker so that two or more nucleic acid analytes are each targeted by hybridization assay probes coupled to different of said chemiluminescent labels, wherein each of said hybridization probes will specifically hybridize to one or more analyte if present in said sample under conditions not favoring the hybridization of said probes to untargeted nucleic acids, said chemiluminescent labels being similarly protected from loss of chemiluminescent potential when coupled to a hybridization probe that is hybridized to a target nucleic acid, and said chemiluminescent labels being similarly susceptable to loss of their chemiluminescent potential when coupled to an unhybridized probe, and wherein after initiating a light-emitting reaction and selectively detecting labeled hybridized probes, each of said chemiluminescent labels emit light at one or more wavelengths sufficiently distinct from a wavelength of light emission of each other of said chemiluminescent labels that said chemiluminescent labels are independently detectable when the emitted light is simultaneously detected at said wavelengths as an indication of the presence of each of the nucleic acid analytes in said test sample.

Claim 26

A method for the assay of a plurality of nucleic acid analytes in a single sample comprising the steps: (a) providing in a medium, (i) a plurality of different oligonucleotide hybridization assay probes, each of said probes having a nucleotide sequence complementary to a target nucleotide sequence of a nucleic acid analyte suspected of being in said sample, (ii) a plurality of different chemiluminescent labels, each of said labels coupled to one or more of said hybridization probes, so that two or more nucleic acid analytes are each targeted by at least one hybridization assay probe coupled to a different chemiluminescent label, and (iii) said sample, and (b) establishing hybridization conditions under which the different hybridization assay probes will preferentially hybridize to said nucleic acid analytes, to form labeled double-stranded nucleic acid hybrids wherein said conditions do not favor the formation of double-stranded nucleic acid hybrids between said labeled probes and untargeted nucleic acids, (c) selectively destroying or inhibiting the chemiluminescent potential of said chemiluminescent labels coupled to unhybridized probe, (d) inducing the chemiluminescent labels associated with double-stranded nucleic acid hybrids to emit light, and (e) detecting each of said nucleic acid analytes, if present, by measuring the light emitted by the coupled chemiluminescent label hybridized thereto at a wavelength of light emission sufficiently distinct from a wavelength of light emission for each other of said chemiluminescent labels to allow said analytes to be independently detectable in a single test sample.

Claim 27

A method for the assay of a plurality of nucleic acid analytes in a single sample comprising the steps: (a) providing in a medium, (i) a plurality of different oligonucleotide hybridization assay probes, each of said probes having a nucleotide sequence complementary to a target nucleotide sequence of a nucleic acid analyte suspected of being in said test sample, (ii) a plurality of different chemiluminescent labels, each of said labels coupled to one or more of said hybridization probes, so that two or more nucleic acid analytes are each targeted by a hybridization assay probe coupled to a different chemiluminescent label, and (iii) said sample, and (b) establishing hybridization condition under which the hybridization assay probes will preferentially hybridize to said nucleic acid analytes, if present, to form labeled double-stranded nucleic acid hybrids wherein said conditions do not favor the formation of double-stranded nucleic acid hybrids between said labeled probes and untargeted nucleic acids, (c) selectively destroying or inhibiting the chemiluminescent potential of the chemiluminescent label coupled to unhybridized probe, (d) inducing the chemiluminescent labels associated with double-stranded nucleic acid hybrids to emit light, and (e) detecting each hybridized nucleic acid analyte by measuring, upon initiation of a light-emitting reaction, the time-to-peak and/or reaction duration values of the reacting chemiluminescent labels over predetermined time intervals after said initiation event.

Claim 28

A method for the assay of a plurality of nucleic acid analytes in a single sample comprising the steps: (a) providing in a medium, (i) a plurality of different oligonucleotide hybridization assay probes, each of said probes having a nucleotide sequence complementary to a target nucleotide sequence of a nucleic acid analyte suspected of being in said sample, (ii) a plurality of different chemiluminescent labels, each of said labels coupled to one or more of said hybridization probes, so that two or more nucleic acid analytes are each targeted by a hybridization assay probe coupled to a different chemiluminescent label, and (iii) said sample, and (b) establishing hybridization conditions under which the hybridization assay probes will hybridize to said nucleic acid analytes, if present, to specifically form labeled double-stranded nucleic acid hybrids wherein said conditions do not favor the formation of double-stranded nucleic acid hybrids between said labeled probes and untargeted nucleic acids, (c) selectively destroying or inhibiting the chemiluminescent potential of the chemiluminescent label coupled to unhybridized probe, (d) inducing at least one of the coupled chemiluminescent labels associated with said double-stranded nucleic acid hybrids to participate in a light-emitting reaction at a first pH value, and measuring the light emitted by said label thereby, (e) adjusting the assay solution to at least one different pH value, (f) inducing at least one different coupled chemiluminescent label associated with one or more different double-stranded nucleic acid hybrid to participate in a light emitting reaction at said different pH value(s), and (g) measuring the light emitted by said different labels during a predetermined time period, thereby permitting the separate identification of different coupled chemiluminescent label as a measure of each of different nucleic acid analytes hybridized to labeled hybridization assay probes in a single test sample.

Claim 35

A method for simultaneously detecting the presence or amount of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids in a single sample comprising: (a) contacting said sample with at least one oligonucleotide hybridization assay probe which will preferentially hybridize to Chlamydia trachomatis nucleic acids over non- Chlamydia trachomatis nucleic acids and at least one oligonucleotide hybridization assay probe which will preferentially hybridize to Neisseria gonorrhoeae nucleic acids over non- Neisseria gonorrhoeae nucleic acids, wherein said Chlamydia trachomatis -specific hybridization assay probes are coupled to a first chemiluminescent labeling reagent and said Neisseria gonorrhoeae -specific hybridization assay probes are coupled to a second chemiluminescent labeling reagent, (b) establishing hybridization conditions sufficient to allow hybridization between said Chlamydia trachomatis -specific hybridization assay probes and Chlamydia trachomatis nucleic acids, if present, and Neisseria gonorrhoeae -specific hybridization assay probes and Neisseria gonorrhoeae nucleic acids, if present, wherein said hybridization conditions will not promote non-specific hybridization of said hybridization assay probes to non-target nucleic acids, (c) selectively destroying or inhibiting the chemiluminescent potential of the chemiluminescent label coupled to unhybridized probe, and (d) detecting said first chemiluminescent reagent coupled to hybridized Chlamydia trachomatis -specific probes as an indication of the presence of Chlamydia trachomatis nucleic acids in said sample, and said second chemiluminescent reagent coupled to hybridized Neisseria gonorrhoeae -specific probes as an indication of the presence of Neisseria gonorrhoeae nucleic acids in said sample.

Claim 36

A method for the assay of a plurality of analytes in a target nucleic acid in a sample comprising: (a) contacting said sample with a plurality of different oligonucleotide hybridization assay probes, wherein each of said different probes specifically hybridizes to a different target analyte region of said nucleic acid and wherein each of said different probes is coupled to a different chemiluminescent label, (b) establishing hybridization conditions in said sample sufficient to allow the specific hybridization between each said hybridization assay probe and its analyte, wherein said hybridization conditions do not promote hybridization of said hybridization assay probes to non-target nucleotide sequence regions of said nucleic acid, (c) selectively destroying or inhibiting the chemiluminescent potential of the chemiluminescent label coupled to unhybridized probe, (d) detecting said chemiluminescent labels coupled to said different probes as an indication of the presence of said nucleic acid analytes in said sample.