Patent EP0229701A2 successfully refreshed from Google Patents.
EP0229701A2
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Detection of viruses by amplification and hybridization
Assignee: F Hoffmann La Roche AG
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Filing: 1987-01-09
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Granted: —
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Expires: 2007-01-09
Abstract
Abstract The presence or absence of a nucleic acid sequence associated with one or more related viruses in a sample containing one or more nucleic acids and suspected of containing such sequence can be detected by amplifying the sequence using primers to form extension products as templates and detecting the amplified product if it is present. This may be accomplished by adding a labeled hybridization probe to the amplified product either free in solution or after immobilization on a solid support. Preferably the virus constitutes AIDs viruses and heapdnaviruses.
Inventors
- 👤 John Joseph Sninsky
- 👤 Shirley Lee Kwok
- 👤 David Henry Mack
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 19:26
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/575; C; C12; C12Q; C12Q1/00; C12Q1/70; C12Q1/701; C12Q1/706; C12Q1/702; C12Q1/703
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
227 words
A process for detecting or monitoring for the presence or absence of a nucleic acid sequence which is substantially conserved among the isolates of a virus and specific to the nucleic acids in said virus and which nucleic acid sequence is suspected of being contained in a sample, which process comprises: (a) treating the sample, together or separately, with an oligonucleotide primer for each strand of the nucleic acid sequence, four different nucleoside triphosphates, and an agent for polymerization, under hybridizing conditions, such that for each strand of the nucleic acid sequence an extension product of each primer is synthesized which is substantially complementary to each strand of the nucleic acid sequence being detected or monitored, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence to be detected is present; (c) treating the product of step (b) with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, resulting in amplification of the sequence to be detected if present; and (d) determining if the sequence to be detected is present in the sample.
Claim 2
Dependent Claim
227 words
A process for detecting or monitoring for the presence or absence of a nucleic acid sequence which is substantially conserved among the isolates of a virus and specific to the nucleic acids in said virus and which nucleic acid sequence is suspected of being contained in a sample, which process comprises: (a) treating the sample, together or separately, with an oligonucleotide primer for each strand of the nucleic acid sequence, four different nucleoside triphosphates, and an agent for polymerization, under hybridizing conditions, such that for each strand of the nucleic acid sequence an extension product of each primer is synthesized which is substantially complementary to each strand of the nucleic acid sequence being detected or monitored, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence to be detected is present; (c) treating the product of step (b) with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, resulting in amplification of the sequence to be detected if present; and (d) determining if the sequence to be detected is present in the sample.
Claim 3
Dependent Claim
50 words
A process according to claim 1 wherein step (d) comprises the steps of: (1) adding to the product of step (c) a labeled probe capable of hybridizing with the amplified nucleic acid sequence; and (2) determining whether the probe has hybridized to an amplified sequence in the nucleic acid sample.
Claim 4
Dependent Claim
24 words
A process according to claim 1 or 2 wherein in step (a) the sample is treated without first culturing the virus in the sample.
Claim 5
Dependent Claim
18 words
A process according to any one of claims 1-3 wherein the virus constitutes AIDS viruses, hepadnaviruses, or herpesvirus.
Claim 6
Dependent Claim
39 words
A process according to claim 4 wherein steps (b) and (c) are repeated at least once and the primers are selected from the gag region of the AIDS genome or from the polymerase or envelope genes of the hepadnaviruses.
Claim 7
Dependent Claim
15 words
A process according to any one of claims 1-5 wherein said nucleic acid is DNA.
Claim 8
Dependent Claim
15 words
A process according to any one of claims 1-5 wherein said nucleic acid is RNA.
Claim 9
Dependent Claim
72 words
A process according to any one of claims 1-7 wherein said agent for polymerization is an enzyme selected from the group consisting of E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, reverse transcriptase, or an enzyme that after being exposed to a temperature sufficient to denature nucleic acids retains its enzymatic activity to form said extension products at the temperature of reaction during steps (a) and (c).
Claim 10
Dependent Claim
56 words
A process according to claim 2 wherein step (2) comprises the steps of: (1) digesting the hybridized mixture from step (d) with a restriction enzyme recognizing a site within the sequences in the probe; and (2) detecting whether the restriction digest contains a restriction fragment correlated with the presence of the virus sequence to be detected.
Claim 11
Dependent Claim
55 words
A process according to claim 9 wherein the virus constitutes the AIDS viruses and steps (1) and (2) utilize a positive control containing one or more nucleic acids with a sequence of the AIDS viral genome and/or a negative control which does not contain any nucleic acid(s) with a sequence from among the AIDS isolates.
Claim 12
Dependent Claim
32 words
A process according to claim 2 wherein step (1) comprises the steps of: (1) spotting the product of step (c) on a membrane; and (2) adding the probe to the spotted membrane.
Claim 13
Dependent Claim
32 words
A process according to claim 2 wherein step (1) comprises the steps of: (1) spotting the probe on a membrane; and (2) adding the product of step (c) to the spotted membrane.
Claim 14
Dependent Claim
191 words
A composition containing an amplified nucleic acid sequence which is substantially conserved among the isolates of a virus and specific to the nucleic acid in a virus produced by: (a) treating a sample containing said nucleic acid sequence in unamplified form, together or separately, with an oligonucleotide primer for each strand of the nucleic acid sequence, four different nucleoside triphosphates, and an agent for polymerization, under hybridizing conditions, such that for each strand of the nucleic acid sequence an extension product of each primer is synthesized which is substantially complementary to each strand of the nucleic acid sequence being detected or monitored, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (c) treating the product of step (b) with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, resulting in amplification of the nucleic acid sequence.
Claim 15
Dependent Claim
191 words
A composition containing an amplified nucleic acid sequence which is substantially conserved among the isolates of a virus and specific to the nucleic acid in a virus produced by: (a) treating a sample containing said nucleic acid sequence in unamplified form, together or separately, with an oligonucleotide primer for each strand of the nucleic acid sequence, four different nucleoside triphosphates, and an agent for polymerization, under hybridizing conditions, such that for each strand of the nucleic acid sequence an extension product of each primer is synthesized which is substantially complementary to each strand of the nucleic acid sequence being detected or monitored, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (c) treating the product of step (b) with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, resulting in amplification of the nucleic acid sequence.
Claim 16
Dependent Claim
16 words
A composition according to claim 13 wherein said virus constitutes the AIDS viruses or the hepadnaviruses.
Claim 17
Dependent Claim
128 words
A kit for detecting or monitoring for the presence or absence of a nucleic acid sequence which is substantially conserved among the nucleic acids in a virus and specific to the nucleic acids in a virus and which nucleic acid sequence is suspected of being contained in a sample, which kit comprises: (a) an oligonucleotide primer for each strand of the nucleic acid sequence to be detected, which primer or primers are substantially complementary to each strand of each specific nucleic acid sequence such that an extension product synthesized from one primer, when it is separated from its complement, can serve as a template for the synthesis of the extension product of the other primer; and (b) a labeled probe capable of hybridizing with the nucleic acid sequence.
Claim 18
Dependent Claim
128 words
A kit for detecting or monitoring for the presence or absence of a nucleic acid sequence which is substantially conserved among the nucleic acids in a virus and specific to the nucleic acids in a virus and which nucleic acid sequence is suspected of being contained in a sample, which kit comprises: (a) an oligonucleotide primer for each strand of the nucleic acid sequence to be detected, which primer or primers are substantially complementary to each strand of each specific nucleic acid sequence such that an extension product synthesized from one primer, when it is separated from its complement, can serve as a template for the synthesis of the extension product of the other primer; and (b) a labeled probe capable of hybridizing with the nucleic acid sequence.
Claim 19
Dependent Claim
16 words
A kit according to claim 15 wherein said virus constitutes the AIDS viruses or the hepadnaviruses.
Claim 20
Dependent Claim
32 words
A kit according to claim 15 or 16 further comprising an agent for polymerization, each of four different nucleoside triphosphates, and a means for detecting hybrids of said probe and said sequence.
Claim 21
Dependent Claim
82 words
A kit according to any one of claims 15-17 wherein the virus constitutes AIDS viruses and the kit further comprises a positive control containing one or more nucleic acids with a sequence of the AIDS viral genome and/or a negative control which does not contain any nucleic acid(s) with a sequence from among the AIDS viruses, and one restriction enzyme capable of cleaving a nucleic acid containing the suspected sequence at a specific restriction site contained in a sequence in the probe.
References Cited (Backward Citations)
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