Method for nucleic acid amplification and detection using adhered probes
First Claim
1. A method for the detection of a target nucleic acid comprising:
- A. contacting a specimen suspected of containing a target nucleic acid with a nucleic acid test element comprising a sealable support having disposed thereon a nucleic acid reagent composition comprising a mixture of;
a. a nucleic acid reagent comprising;
particles composed of a first polymer having a glass transition temperature of at least about 70°
C., said particles having an average diameter of from about 0.1 to about 3 micrometers, andan oligonucleotide covalently attached to said particles which is complementary to said target nucleic acid, andb. a water insoluble adhesive comprising a second polymer which has a glass transition temperature which is at least about 30°
C. less than the glass transition temperature of said first polymer,said polymeric adhesive being present in said composition at from about 1 to about 20 weight percent,wherein said sealable support has hydrophilic surface groups provided by a treatment selected from the group consisting of corona discharge treatment, chromic acid treatment, and treatment with a radio frequency electromagnetic field in the presence of a reactive gas, or said hydrophilic surface groups are provided by a hydrophilic subbing layer on which said composition is disposed,so as to form a water insoluble hybridized product of said target nucleic acid and said oligonucleotide, andB. detecting said hybridized product as an indication of the presence of said target nucleic acid in said specimen.
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Abstract
Nucleic acids can be amplified and detected using an element which has a sealable support on which is disposed a nucleic acid reagent composition. The composition is a mixture of a nucleic acid reagent composed of polymeric particles to which an oligonucleotide is covalently attached. The particles are prepared from a first polymer having a glass transition temperature of at least about 70° C. and have an average diameter of from about 0.1 to about 3 micrometers. The reagent is adhered to the support using a water insoluble adhesive comprising a second polymer which has a glass transition temperature which is at least about 30° C. less than the glass transition temperature of the first polymer. The adhesive is present in the composition at from about 1 to about 20 dry weight percent. The method provides high sensitivity and low background in the assay of nucleic acids, preferably using polymerase chain reaction.
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Citations
24 Claims
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1. A method for the detection of a target nucleic acid comprising:
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A. contacting a specimen suspected of containing a target nucleic acid with a nucleic acid test element comprising a sealable support having disposed thereon a nucleic acid reagent composition comprising a mixture of; a. a nucleic acid reagent comprising;
particles composed of a first polymer having a glass transition temperature of at least about 70°
C., said particles having an average diameter of from about 0.1 to about 3 micrometers, andan oligonucleotide covalently attached to said particles which is complementary to said target nucleic acid, and b. a water insoluble adhesive comprising a second polymer which has a glass transition temperature which is at least about 30°
C. less than the glass transition temperature of said first polymer,said polymeric adhesive being present in said composition at from about 1 to about 20 weight percent, wherein said sealable support has hydrophilic surface groups provided by a treatment selected from the group consisting of corona discharge treatment, chromic acid treatment, and treatment with a radio frequency electromagnetic field in the presence of a reactive gas, or said hydrophilic surface groups are provided by a hydrophilic subbing layer on which said composition is disposed, so as to form a water insoluble hybridized product of said target nucleic acid and said oligonucleotide, and B. detecting said hybridized product as an indication of the presence of said target nucleic acid in said specimen. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for the amplification and detection of a target nucleic acid comprising:
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A. amplifying a target nucleic acid using at least one biotinylated primer to form at least one amplified biotinylated target nucleic acid, B. contacting said amplified biotinylated target nucleic acid with a nucleic acid test element comprising a sealable support having disposed thereon a nucleic acid reagent composition comprising a mixture of; a. a nucleic acid reagent comprising;
particles composed of a first polymer having a glass transition temperature of at least about 70°
C., said particles having an average diameter of from about 0.1 to about 3 micrometers, andan oligonucleotide covalently attached to said particles which is complementary to said target nucleic acid, and b. a water insoluble adhesive comprising a second polymer which has a glass transition temperature which is at least about 30°
C. less than the glass transition temperature of said first polymer,said polymeric adhesive being present in said composition at from about 1 to about 20 weight percent, wherein said sealable support has hydrophilic surface groups provided by a treatment selected from the group consisting of corona discharge treatment, chromic acid treatment and treatment with a radio frequency electromagnetic field in the presence of a reactive gas, and or said hydrophilic surface groups are provided by a hydrophilic subbing layer on which said composition is disposed, to form a water insoluble immobilized hybridized product of said amplified biotinylated target nucleic acid and said nucleic acid reagent on said element, and C. detecting the presence of said immobilized hybridized product by complexation of said amplified biotinylated target nucleic acid with a labeled conjugate of avidin as an indication of the presence of said target nucleic acid. - View Dependent Claims (23, 24)
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Specification