Nucleic acid assays
First Claim
1. A method for the detection of the presence or absence of a single stranded or double stranded first nucleic acid in a sample, by automated isothermal amplification of said first nucleic acid, said method performed in at least two reaction vessels which can be placed in fluid communication with each other, said method comprising:
- a) combining in a first reaction vessel;
a test sample and reagents suitable for carrying out a nucleic acid amplification reaction such that a reaction mixture can form and placing said reaction vessel in an automated apparatus such that;
b) the automated apparatus heats said first reaction vessel to a sufficient temperature, and for a sufficient time to render any double stranded first nucleic acid in the sample to be tested into sufficient single stranded nucleic acid available for hybridization, andc) the automated apparatus cools said first reaction vessel to a sufficient temperature to form a hybridization product, said hybridization product comprising at least one oligonucleotide primer and a first nucleic acid if said first nucleic acid is present in said test sample,d) contacting said product from said first reaction vessel with a nucleic acid amplification enzyme to provide a nucleic acid amplification mixture,e) amplifying said first nucleic acid in a second reaction vessel wherein the automated apparatus maintains the temperature of said second reaction vessel at a sufficient temperature which allows for a specific oligonucleotide primer mediated amplification of said first nucleic acid to produce amplicons, andf) detecting the presence of amplicons.
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Accused Products
Abstract
The present invention relates to the detection of specific nucleic acid sequences after an amplification process, or directly without amplification. In particular, the invention provides for the automation of the amplification and detection process, the amplification and detection of one or more specific nucleic acid sequences, the use of internal controls, reduced potential for contamination caused by the manual manipulation of reagents, and improved reagent compositions to better control assay performance and provide for further protection against contamination.
62 Citations
27 Claims
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1. A method for the detection of the presence or absence of a single stranded or double stranded first nucleic acid in a sample, by automated isothermal amplification of said first nucleic acid, said method performed in at least two reaction vessels which can be placed in fluid communication with each other, said method comprising:
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a) combining in a first reaction vessel;
a test sample and reagents suitable for carrying out a nucleic acid amplification reaction such that a reaction mixture can form and placing said reaction vessel in an automated apparatus such that;b) the automated apparatus heats said first reaction vessel to a sufficient temperature, and for a sufficient time to render any double stranded first nucleic acid in the sample to be tested into sufficient single stranded nucleic acid available for hybridization, and c) the automated apparatus cools said first reaction vessel to a sufficient temperature to form a hybridization product, said hybridization product comprising at least one oligonucleotide primer and a first nucleic acid if said first nucleic acid is present in said test sample, d) contacting said product from said first reaction vessel with a nucleic acid amplification enzyme to provide a nucleic acid amplification mixture, e) amplifying said first nucleic acid in a second reaction vessel wherein the automated apparatus maintains the temperature of said second reaction vessel at a sufficient temperature which allows for a specific oligonucleotide primer mediated amplification of said first nucleic acid to produce amplicons, and f) detecting the presence of amplicons. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method for the detection of the presence or absence of a single stranded or double stranded first nucleic acid in a sample, by automated isothermal amplification of said first nucleic acid, said method performed in a reaction vessel comprising at least two reaction chambers which can be placed in fluid communication with each other, said method comprising:
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a) combining in a first reaction chamber;
a test sample and reagents suitable for carrying out a nucleic acid amplification reaction such that a reaction mixture can form and placing said reaction vessel in an automated apparatus such that;(i) the automated apparatus heats said first reaction chamber to a sufficient temperature, and for a sufficient time to render any double stranded first nucleic acid in the sample to be tested into sufficient single stranded nucleic acid available for hybridization, and (ii) the automated apparatus cools said first reaction chamber to a sufficient temperature to form a hybridization product, said hybridization product comprising at least one oligonucleotide primer and a first nucleic acid if said first nucleic acid is present in said test sample, b) contacting said product from said first reaction chamber with a nucleic acid amplification enzyme to provide a nucleic acid amplification mixture, c) amplifying said first nucleic acid in a second reaction chamber wherein the automated apparatus maintains the temperature of said second reaction chamber at a sufficient temperature which allows for a specific oligonucleotide primer mediated amplification of said first nucleic acid to produce amplicons, and d) detecting the presence of amplicons. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification