EP0145356A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Method of assay of DNA or RNA from virus particles
Assignee: National Research Development Corp UK
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Filing: 1984-11-20
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Granted: —
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Expires: 2004-11-20
Abstract
Abstract In the extraction of DNA or RNA from virus particles particularly for assay of viral DNA or RNA, e.g. for hepatitis B virus, it has been a problem that the normal procedure involves precipitating the virus by centrifuging the sample, followed by extracting into phenol, which is unpleasant to handle, and the addition of large amounts of expensive protease enzyme to remove the proteolysis products. In the invention, these problems are avoided by a method which comprises precipitating virus particles from the sample by incubating it with an insolubilised monospecific, preferably monoclonal antibody to a surface antigen of the virus, breaking open the capsid of the precipitated virus particles and releasing the viral DNA or RNA thereof, extracting the viral DNA or RNA into the liquid phase, and separating the solid and liquid phases. Very desirably the above method of extraction of viral DNA or RNA is combined with a two-site hybridisation assay to provide a very convenient and sensitive assay for viral DNA or RNA. A kit for use in connection with this method comprises the monospecific antibody and the two polynucleotides, not necessarily in double-stranded ds-form, and the first not necessarily insolubilised.
Inventors
- 👤 Peter Karayiannis
- 👤 Joao Pedro Pulido Valente Monjardino
- 👤 Howard Christopher Thomas
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 15:23
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/576; C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1003; C12N15/1006; C12Q; C12Q1/00; C12Q1/68; C12Q1/6804; C12Q1/70; C12Q1/701; C12Q1/705; C12Q1/706; G01N33/569; G01N33/56983
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
87 words
A method of extracting DNA or RNA from virus particles present in a clinical specimen which comprises the steps of precipitating virus particles from the specimen by incubating it with an insolubilised monospecific antibody to a viral surface antigen, thereby forming a precipitate comprising the virus particle, breaking open the capsid of the virus particle to release the viral DNA or RNA thereof from any proteins closely associated therewith, extracting the viral DNA or RNA into the liquid phase, and separating the liquid phase from solid phase.
Claim 2
Independent Claim
87 words
A method of extracting DNA or RNA from virus particles present in a clinical specimen which comprises the steps of precipitating virus particles from the specimen by incubating it with an insolubilised monospecific antibody to a viral surface antigen, thereby forming a precipitate comprising the virus particle, breaking open the capsid of the virus particle to release the viral DNA or RNA thereof from any proteins closely associated therewith, extracting the viral DNA or RNA into the liquid phase, and separating the liquid phase from solid phase.
Claim 3
Dependent Claim
20 words
A method according to Claim 1 wherein the capsid is broken open and the viral DNA or RNA released enzymically.
Claim 4
Dependent Claim
35 words
A method according to Claim 2 wherein the capsid is reacted with a protease in a proportion lower than that required for proteolysis of proteins present in the specimen, by the conventional method of extraction.
Claim 5
Dependent Claim
16 words
A method according to Claim 1 or 2 wherein the monospecific antibody is a monoclonal antibody.
Claim 6
Dependent Claim
21 words
A method according to Claim 1 or 2 wherein the virus is of the hepatitis B, hepatitis D or herpetoviridae families.
Claim 7
Independent Claim
154 words
A method of assay of a liquid sample produced by a method claimed in any one of Claims 1 to 5, for DNA or RNA suspected of being present therein, which comprises subjecting the sample to conditions effective to render the DNA or RNA single-stranded, contacting the sample under hybridisation conditions with a first polynucleotide which.is in a solid phase and unlabelled, and which is hybridisable under the assay conditions to a first region of the nucleic acid sequence of the suspected DNA or RNA and with a second polynucleotide which is in a liquid phase and labelled and which is hybridisable under the assay conditions to a second region of the nucleic acid sequence of the suspected DNA or RNA, the second region being separate from the first and mutually exclusive therefrom with respect to hybridisation, separating the solid and liquid phases and determining the amount of label present in the solid phase.
Claim 8
Dependent Claim
28 words
A method according to Claim 6 wherein the hybridisation conditions are the same for the first and second polynucleotides and they are contacted substantially simultaneously with the sample.
Claim 9
Dependent Claim
96 words
A kit for use in a method claimed in Claim 1 comprising: (1) a monospecific antibody to the surface antigen of a virus; (2) a first polynucleotide which is unlabelled and which, when in single-stranded form, is hybridisable to a first region of the nucleic acid sequence of the viral DNA or RNA; and (3) a second polynucleotide which is labelled and which, when in single-stranded form, is hybridisable to a second region of the nucleic acid sequence of the viral DNA or RNA, separate from the first and mutually exclusive therefrom with respect to hybridisation.
Claim 10
Dependent Claim
104 words
A kit according to Claim 8 which further comprises any one or more of the following: (4) a support material for insolubilising the monospecific antibody thereon; (5) a support material for insolubilising the first polynucleotide in single-stranded form; (6) a control DNA or RNA for testing the kit, the control DNA or RNA comprising a sequence which, when the control DNA or RNA is T.n single-stranded form, is hybridisable to each polynucleotide, when the polynucleotides are in single-stranded form; and (7) a reagent for breaking open the capsid of the virus and releasing the viral DNA or RNA from the proteins closely associated therewith.
Claim 11
Dependent Claim
21 words
A kit according to Claim 8 or 9 wherein the virus is of the hepatitis B, hepatitis D, or herpetoviridae family.
Claim 12
Dependent Claim
13 words
A kit according to Claim 9 wherein the reagent (7) is an enzyme.
Claim 13
Dependent Claim
56 words
A kit according to Claim 11 wherein the reagent (7) is a protease and is provided in the form of a number of units, each unit suitable for use in connection with one specimen and comprising an amount lower than that required for proteolysis of proteins present in the specimen, by the conventional method of extraction.
Claim 14
Dependent Claim
19 words
A kit according to any one of Claims 8 to 12 wherein the monospecific antibody is a monoclonal antibody.
References Cited (Backward Citations)
No citation records indexed.
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