Diagnostic kit and method using a solid phase capture means for detecting nucleic acids

Claim 1

A method for the detection of a specific nucleic acid sequence of a predetermined nucleic acid, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing a predetermined nucleic acid with first and second oligonucleotide primers complementary to the strands of the specific nucleic acid sequence, at least the first primer being labeled with a specific binding ligand, so as to form a mixture of hybridized products of the first and second primers and the complementary acid strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with a detectably labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, and G. detecting the complementary product formed in step F as an indication of the presence of the predetermined nucleic acid in the specimen, provided that prior to detection step G, the specific binding ligand of the first primer is complexed with a receptor therefor which is bound to a solid support material.

Claim 2

A method for the detection of a specific nucleic acid sequence of a predetermined nucleic acid, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing a predetermined nucleic acid with first and second oligonucleotide primers complementary to the strands of the specific nucleic acid sequence, at least the first primer being labeled with a specific binding ligand, so as to form a mixture of hybridized products of the first and second primers and the complementary acid strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with a detectably labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, and G. detecting the complementary product formed in step F as an indication of the presence of the predetermined nucleic acid in the specimen, provided that prior to detection step G, the specific binding ligand of the first primer is complexed with a receptor therefor which is bound to a solid support material.

Claim 3

A method for the detection of a specific nucleic acid sequence of a predetermined nucleic acid, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing a predetermined nucleic acid with (1) first and second oligonucleotide primers complementary to the strands of the specific nucleic acid sequence, at least the first primer being labeled with a specific binding ligand, and (2) a solid support material having a receptor for the specific binding ligand bound thereto, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, at least the first primer being bound to the support material through a complex of the specific binding ligand and the receptor, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with a detectably labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F as an indication of the presence of the predetermined nucleic acid in the specimen.

Claim 4

A method for the detection of a specific nucleic acid sequence of a predetermined nucleic acid, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing a predetermined nucleic acid with (1) first and second oligonucleotide primers complementary to the strands of the specific nucleic acid sequence, at least the first primer being labeled with a specific binding ligand, and (2) a solid support material having a receptor for the specific binding ligand bound thereto, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, at least the first primer being bound to the support material through a complex of the specific binding ligand and the receptor, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with a detectably labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F as an indication of the presence of the predetermined nucleic acid in the specimen.

Claim 5

A method for the detection of a specific nucleic acid sequence of a predetermined nucleic acid, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing the predetermined nucleic acid with first and second oligonucleotide primers complementary to the specific nucleic acid sequence strands, at least the first primer being labeled with a specific binding ligand, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with a detectably labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with a solid support material to which is bound a receptor for the specific binding ligand of the first primer, thereby insolubilizing the probe-first primer complementary product through complexation of the specific binding ligand and receptor therefor, and H. detecting the insolubilized complementary product formed in step G as an indication of the presence of the predetermined nucleic acid in the specimen.

Claim 6

A method for the detection of a specific nucleic acid sequence of a predetermined nucleic acid, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing the predetermined nucleic acid with first and second oligonucleotide primers complementary to the specific nucleic acid sequence strands, at least the first primer being labeled with a specific binding ligand, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with a detectably labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with a solid support material to which is bound a receptor for the specific binding ligand of the first primer, thereby insolubilizing the probe-first primer complementary product through complexation of the specific binding ligand and receptor therefor, and H. detecting the insolubilized complementary product formed in step G as an indication of the presence of the predetermined nucleic acid in the specimen.

Claim 7

The method as claimed in any of claims 1 to 3 wherein the solid support material is a polymeric particle.

Claim 9

The method as claimed in any of claims 1 to 5 for the detection of HIV-I.

Claim 10

The method as claimed in any of claims 1 to 6 wherein the specific binding moiety is biotin or a derivative thereof, and the receptor is avidin or a derivative thereof.

Claim 11

The method as claimed in any of claims 1 to 6 wherein the specific binding moiety is an immunological species.

Claim 12

The method as claimed in any of claims 1 to 6 wherein the specific binding moiety is either a lectin or sugar, and the receptor is, respectively, a sugar or a lectin.

Claim 13

A method for the detection of a specific nucleic acid sequence in the gag region of HIV-I DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HIV-I DNA with (1) first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in the gag region of HIV-I DNA, at least the first primer being labeled with biotin, and (2) polymeric particles having an average particle size of from 0.1 to 10 µmeter and having avidin covalently bound thereto, so as to form a mixture of hybridized products of the first and second primers and HIV-I complementary strands, the first primer being bound to the particles through a biotin-avidin complex, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HIV-I nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of HIV-I in the specimen.

Claim 14

A method for the detection of a specific nucleic acid sequence in the gag region of HIV-I DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HIV-I DNA with (1) first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in the gag region of HIV-I DNA, at least the first primer being labeled with biotin, and (2) polymeric particles having an average particle size of from 0.1 to 10 µmeter and having avidin covalently bound thereto, so as to form a mixture of hybridized products of the first and second primers and HIV-I complementary strands, the first primer being bound to the particles through a biotin-avidin complex, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HIV-I nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of HIV-I in the specimen.

Claim 15

A method for the detection of a specific nucleic acid sequence in HLA DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HLA DNA with (1) first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in HLA DNA, at least the first primer being labeled with biotin, and (2) polymeric particles having an average particle size of from 0.1 to 10 µmeter and having avidin covalently bound thereto, so as to form a mixture of hybridized products of the first and second primers and HLA DNA complementary strands, the first primer being bound to the particles through a biotin avidin complex, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HLA DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of HLA DNA in the specimen.

Claim 16

A method for the detection of a specific nucleic acid sequence in HLA DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HLA DNA with (1) first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in HLA DNA, at least the first primer being labeled with biotin, and (2) polymeric particles having an average particle size of from 0.1 to 10 µmeter and having avidin covalently bound thereto, so as to form a mixture of hybridized products of the first and second primers and HLA DNA complementary strands, the first primer being bound to the particles through a biotin avidin complex, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HLA DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of HLA DNA in the specimen.

Claim 17

A method for the detection of a specific nucleic acid sequence in human β-globin DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing human β-globin DNA with (1) first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in human β-globin DNA, at least the first primer being labeled with biotin, and (2) polymeric particles having an average particle size of from 0.1 to 10 µmeter and having avidin covalently bound thereto, so as to form a mixture of hybridized products of the first and second primers and the human β-globin DNA complementary strands, the first primer bound to the particles through a biotin-avidin complex, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific human β-globin DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of human β-globin DNA in the specimen.

Claim 18

A method for the detection of a specific nucleic acid sequence in human β-globin DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing human β-globin DNA with (1) first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in human β-globin DNA, at least the first primer being labeled with biotin, and (2) polymeric particles having an average particle size of from 0.1 to 10 µmeter and having avidin covalently bound thereto, so as to form a mixture of hybridized products of the first and second primers and the human β-globin DNA complementary strands, the first primer bound to the particles through a biotin-avidin complex, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, the first primer extension product being insoluble, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific human β-globin DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the insoluble first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and insoluble first primer extension product, and G. detecting the insoluble complementary product formed in step F by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of human β-globin DNA in the specimen.

Claim 19

A method for the detection of a specific nucleic acid sequence in the gag region of HIV-I DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HIV-I DNA with first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in the gag region of HIV-I DNA, at least the first primer being labeled with biotin, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HIV-I DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with polymeric particles having an average particle size of from 0.1 to 10 µmeters, and having avidin covalently bound thereto, thereby insolubilizing the probe-first primer complementary product through complexation of biotin and avidin, and H. detecting the insolubilized complementary product formed in step G by contacting it with a composition which will provide a dye in the presence of the enzyme as an indication of the presence of HIV-I in the specimen.

Claim 20

A method for the detection of a specific nucleic acid sequence in the gag region of HIV-I DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HIV-I DNA with first and second oligonucleotide primers complementary to the strands of a specific nucleic acid sequence in the gag region of HIV-I DNA, at least the first primer being labeled with biotin, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HIV-I DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with polymeric particles having an average particle size of from 0.1 to 10 µmeters, and having avidin covalently bound thereto, thereby insolubilizing the probe-first primer complementary product through complexation of biotin and avidin, and H. detecting the insolubilized complementary product formed in step G by contacting it with a composition which will provide a dye in the presence of the enzyme as an indication of the presence of HIV-I in the specimen.

Claim 21

A method for the detection of a specific nucleic acid sequence in HLA DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HLA DNA with first and second oligonucleotide primers complementary to the strands of a specific HLA DNA nucleic acid sequence, at least the first primer being labeled with biotin, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HLA DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with polymeric particles having an average particle size of from 0.1 to 10 µmeters, and having avidin covalently bound thereto, thereby insolubilizing the probe-first primer complementary product through complexation of biotin and avidin, and H. detecting the insolubilized complementary product formed in step G by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of HLA DNA in the specimen.

Claim 22

A method for the detection of a specific nucleic acid sequence in HLA DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing HLA DNA with first and second oligonucleotide primers complementary to the strands of a specific HLA DNA nucleic acid sequence, at least the first primer being labeled with biotin, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific HLA DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with polymeric particles having an average particle size of from 0.1 to 10 µmeters, and having avidin covalently bound thereto, thereby insolubilizing the probe-first primer complementary product through complexation of biotin and avidin, and H. detecting the insolubilized complementary product formed in step G by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of HLA DNA in the specimen.

Claim 23

A method for the detection of a specific nucleic acid sequence in human β-globin DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing human β-globin DNA with first and second oligonucleotide primers complementary to the strands of the specific nucleic acid sequence in human β-globin DNA, at least the first primer being labeled with biotin, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific human β-globin DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with polymeric particles having an average particle size of from 0.1 to 10 µmeters, and having avidin covalently bound thereto, thereby insolubilizing the probe-first primer complementary product through complexation of biotin and avidin, and H. detecting the insolubilized complementary product formed in step G by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of human β-globin DNA in the specimen.

Claim 24

A method for the detection of a specific nucleic acid sequence in human β-globin DNA, the sequence having two complementary strands, the method comprising: A. contacting a specimen suspected of containing human β-globin DNA with first and second oligonucleotide primers complementary to the strands of the specific nucleic acid sequence in human β-globin DNA, at least the first primer being labeled with biotin, so as to form a mixture of hybridized products of the first and second primers and the complementary strands, B. forming first and second extension products of the primers in the hybridized products, which extension products, when separated from their complements, can serve as templates for synthesis of the extension products of the primers, C. separating the primer extension products from the templates on which they were synthesized, D. contacting the separated primer extension products and the specimen with additional first and second primers, resulting in amplification of the specific human β-globin DNA nucleic acid sequence to form complementary products, E. separating the primer extension products from the complementary products formed in step D, F. contacting the first primer extension product separated in step E with an enzyme-labeled oligonucleotide probe which is complementary thereto to form a product of the labeled probe and first primer extension product, G. contacting the complementary product formed in step F with polymeric particles having an average particle size of from 0.1 to 10 µmeters, and having avidin covalently bound thereto, thereby insolubilizing the probe-first primer complementary product through complexation of biotin and avidin, and H. detecting the insolubilized complementary product formed in step G by contacting it with a composition which provides a dye in the presence of the enzyme as an indication of the presence of human β-globin DNA in the specimen.