Methods for detection and discrimination of multiple analytes using fluorescent technology

Claim 1

A method for detecting and discriminating multiple target nucleic acid sequences, if present, within a mixture of multiple target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process, said method comprising the steps of: obtaining a mixture of different fluorescent dye-labeled target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process Using primer oligonucleotides labeled with multiple fluorescent dyes such that each different target nucleic acid sequence is labeled with a different fluorescent dye; obtaining multiple probe oligonucleotides, each said probe oligonucleotide being complementary to a portion of one strand of one target nucleic acid sequence such that said probe oligonucleotides can form hybrids with said fluorescent dye-labeled target nucleic acid sequences; obtaining a solid-phase system comprising a microtiter plate having microtiter wells and chemical linking means for efficiently binding said probe oligonucleotides to said microtiter wells; effecting chemical linking of said probe oligonucleotides to said microtiter wells to thereby immobilize said probe oligonucleotides in said microtiter wells: effecting hybridization of the probe oligonucleotides and the fluorescent dye-labeled target nucleic acid sequences, if present, within said mixture to thereby form and immobilize different fluorescent dye-labeled hybrids within said microtiter wells; removing non-hybridized fluorescent dye-labeled target nucleic acid sequences from within said microtiter wells; obtaining a specific combination filter set for spectrally distinguishing the fluorescence signal from each of the different fluorescent dye-labeled hybrids with a fluorescence reader instrument; and directly detecting and discriminating said different fluorescent dye-labeled hybrids within said microtiter wells with said fluorescence reader instrument.

Claim 2

A method for detecting and discriminating multiple target nucleic acid sequences, if present, within a mixture of multiple target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process, said method comprising the steps of: obtaining a mixture of different fluorescent dye-labeled target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process Using primer oligonucleotides labeled with multiple fluorescent dyes such that each different target nucleic acid sequence is labeled with a different fluorescent dye; obtaining multiple probe oligonucleotides, each said probe oligonucleotide being complementary to a portion of one strand of one target nucleic acid sequence such that said probe oligonucleotides can form hybrids with said fluorescent dye-labeled target nucleic acid sequences; obtaining a solid-phase system comprising a microtiter plate having microtiter wells and chemical linking means for efficiently binding said probe oligonucleotides to said microtiter wells; effecting chemical linking of said probe oligonucleotides to said microtiter wells to thereby immobilize said probe oligonucleotides in said microtiter wells: effecting hybridization of the probe oligonucleotides and the fluorescent dye-labeled target nucleic acid sequences, if present, within said mixture to thereby form and immobilize different fluorescent dye-labeled hybrids within said microtiter wells; removing non-hybridized fluorescent dye-labeled target nucleic acid sequences from within said microtiter wells; obtaining a specific combination filter set for spectrally distinguishing the fluorescence signal from each of the different fluorescent dye-labeled hybrids with a fluorescence reader instrument; and directly detecting and discriminating said different fluorescent dye-labeled hybrids within said microtiter wells with said fluorescence reader instrument.

Claim 11

A method for detecting and discriminating multiple target nucleic acid sequences, if present, within a mixture containing multiple target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process, said method comprising the steps of: obtaining multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence such that each said primer oligonucleotide can initiate amplification of a different target nucleic acid sequence in a polymerase chain reaction amplification process, said multiple target nucleic acid sequences including target nucleic acid sequences specific for microorganisms of the group consisting of Salmonella, Shigella, Campylobacter, Yersinia, and Escherichia coli; obtaining multiple probe oligonucleotides, each said probe oligonucleotide being complementary to a portion of one strand of one target nucleic acid sequence such that said probe oligonucleotides can form hybrids with said target nucleic acid sequences in a hybridization process; attaching multiple fluorescent dyes to either said primer oligonucleotides such that each said different target nucleic acid sequence resulting from said polymerase chain reaction amplification process is labeled with a different fluorescent dye or to said probe oligonucleotides such that each different target nucleic acid sequence/probe oligonucleotide hybrid is labeled with a different fluorescent dye; subjecting a sample to a polymerase chain reaction amplification process utilizing said primer oligonucleotides to thereby obtain a mixture of said multiple target nucleic acid sequences; obtaining a solid-phase system comprising a microtiter plate having microtiter wells and chemical linking means for efficiently binding said probe oligonucleotides to said microtiter wells; effecting chemical linking of said probe oligonucleotides to said microtiter wells to thereby immobilize said probe oligonucleotides in said microtiter wells; effecting hybridization of the probe oligonucleotides and the target nucleic acid sequences, if present, within said mixture to thereby form and immobilize different fluorescent dye-labeled hybrids within said microtiter wells; removing non-hybridized fluorescent dye-labeled components from within said microtiter wells; obtaining a specific combination filter set for spectrally distinguishing the fluorescence signal from each of the different fluorescent dye-labeled hybrids with a fluorescence reader instrument; and directly detecting and discriminating said different fluorescent dye-labeled hybrids within said microtiter wells with said fluorescence reader instrument.

Claim 12

A method for detecting and discriminating multiple target nucleic acid sequences, if present, within a mixture containing multiple target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process, said method comprising the steps of: obtaining multiple primer oligonucleotides, each said primer oligonucleotide being complementary to a portion of one strand of a target nucleic acid sequence such that each said primer oligonucleotide can initiate amplification of a different target nucleic acid sequence in a polymerase chain reaction amplification process, said multiple target nucleic acid sequences including target nucleic acid sequences specific for microorganisms of the group consisting of Salmonella, Shigella, Campylobacter, Yersinia, and Escherichia coli; obtaining multiple probe oligonucleotides, each said probe oligonucleotide being complementary to a portion of one strand of one target nucleic acid sequence such that said probe oligonucleotides can form hybrids with said target nucleic acid sequences in a hybridization process; attaching multiple fluorescent dyes to either said primer oligonucleotides such that each said different target nucleic acid sequence resulting from said polymerase chain reaction amplification process is labeled with a different fluorescent dye or to said probe oligonucleotides such that each different target nucleic acid sequence/probe oligonucleotide hybrid is labeled with a different fluorescent dye; subjecting a sample to a polymerase chain reaction amplification process utilizing said primer oligonucleotides to thereby obtain a mixture of said multiple target nucleic acid sequences; obtaining a solid-phase system comprising a microtiter plate having microtiter wells and chemical linking means for efficiently binding said probe oligonucleotides to said microtiter wells; effecting chemical linking of said probe oligonucleotides to said microtiter wells to thereby immobilize said probe oligonucleotides in said microtiter wells; effecting hybridization of the probe oligonucleotides and the target nucleic acid sequences, if present, within said mixture to thereby form and immobilize different fluorescent dye-labeled hybrids within said microtiter wells; removing non-hybridized fluorescent dye-labeled components from within said microtiter wells; obtaining a specific combination filter set for spectrally distinguishing the fluorescence signal from each of the different fluorescent dye-labeled hybrids with a fluorescence reader instrument; and directly detecting and discriminating said different fluorescent dye-labeled hybrids within said microtiter wells with said fluorescence reader instrument.

Claim 13

A method for detecting and discriminating multiple target nucleic acid sequences, if present, within a mixture containing multiple target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process, said method comprising the steps of: obtaining a mixture of different fluorescent dye-labeled target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process using multiple oligonucleotide primers labeled with multiple fluorescent dyes such that each different target nucleic acid sequence is labeled with a different fluorescent dye, said multiple target nucleic acid sequences including target nucleic acid sequences specific for microorganisms of the group consisting of Salmonella, Shigella, Campylobacter, Yersinia, and Escherichia coli; obtaining multiple probe oligonucleotides, each said probe oligonucleotide being complementary to a portion of one strand of one target nucleic acid sequence such that said probe oligonucleotides can form hybrids with said fluorescent dye-labeled target nucleic acid sequences; obtaining a solid-phase system comprising a microtiter plate having microtiter wells and chemical linking means for efficiently binding said probe oligonucleotides to said microtiter wells; effecting chemical linking of said probe oligonucleotides to said microtiter wells to thereby immobilize said probe oligonucleotides in said microtiter wells; effecting hybridization of the probe oligonucleotides and the fluorescent dye-labeled target nucleic acid sequences, if present, within said mixture to thereby form and immobilize different fluorescent dye-labeled hybrids within said microtiter wells; removing non-hybridized fluorescent dye-labeled target nucleic acid sequences from within said microtiter wells; obtaining a specific combination filter set for spectrally distinguishing the fluorescence signal from each of the different fluorescent dye-labeled hybrids with a fluorescence reader instrument; and directly detecting and discriminating said different fluorescent dye-labeled hybrids within said microtiter wells with said fluorescence reader instrument.

Claim 14

A method for detecting and discriminating multiple target nucleic acid sequences, if present, within a mixture containing multiple target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process, said method comprising the steps of: obtaining a mixture of different fluorescent dye-labeled target nucleic acid sequences resulting from a selective polymerase chain reaction amplification process using multiple oligonucleotide primers labeled with multiple fluorescent dyes such that each different target nucleic acid sequence is labeled with a different fluorescent dye, said multiple target nucleic acid sequences including target nucleic acid sequences specific for microorganisms of the group consisting of Salmonella, Shigella, Campylobacter, Yersinia, and Escherichia coli; obtaining multiple probe oligonucleotides, each said probe oligonucleotide being complementary to a portion of one strand of one target nucleic acid sequence such that said probe oligonucleotides can form hybrids with said fluorescent dye-labeled target nucleic acid sequences; obtaining a solid-phase system comprising a microtiter plate having microtiter wells and chemical linking means for efficiently binding said probe oligonucleotides to said microtiter wells; effecting chemical linking of said probe oligonucleotides to said microtiter wells to thereby immobilize said probe oligonucleotides in said microtiter wells; effecting hybridization of the probe oligonucleotides and the fluorescent dye-labeled target nucleic acid sequences, if present, within said mixture to thereby form and immobilize different fluorescent dye-labeled hybrids within said microtiter wells; removing non-hybridized fluorescent dye-labeled target nucleic acid sequences from within said microtiter wells; obtaining a specific combination filter set for spectrally distinguishing the fluorescence signal from each of the different fluorescent dye-labeled hybrids with a fluorescence reader instrument; and directly detecting and discriminating said different fluorescent dye-labeled hybrids within said microtiter wells with said fluorescence reader instrument.