초록 ▼

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 spe

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.

대표청구항 ▼

[ We claim:] [1.]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 in a dual chamber reaction vessel, wherein said dual chamber reaction vessel comprises tw

[ We claim:] [1.]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 in a dual chamber reaction vessel, wherein said dual chamber reaction vessel comprises two reaction chambers, a first and a second, which can be placed in fluid communication with each other, whereby said fluid communication can be controllably interrupted, said method comprising:a) combining in said first reaction chamber: a sample, said sample potentially containing said first nucleic acid, reaction buffer, a mixture of free nucleotides, a first and second specific oligonucleotide primer, and placing said reaction vessel in an automated apparatus such that;b) the automated apparatus heats the 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 such that a hybridization product can form, said hybridization product comprising said first nucleic acid and at least one of said first and second oligonucleotide primer;c) the automated apparatus then cools the first reaction chamber to a sufficient temperature such that said hybridization product forms, if said first nucleic acid is present;d) the automated apparatus then transfers the reaction mixture from the first reaction chamber to said second reaction chamber via said controllable fluid communication, such that the reaction mixture is brought into contact with nucleic acid polymerization enzyme;e) the automated apparatus maintains the temperature of the second reaction chamber at a sufficient temperature which allows for the specific oligonucleotide primer mediated amplification of said first nucleic acid, if present;f) the automated apparatus then contacts any amplicon and first nucleic acid in the second reaction chamber with a capture nucleic acid specific for said first nucleic acid such that a specifically-bound nucleic acid-capture probe hybridization complex can form;g) the automated apparatus optionally washes the hybidization complex mixture such that non-specifically bound nucleic acid is washed away from the specifically-bound nucleic acid-capture probe complex;h) the automated apparatus contacts the specifically-bound nucleic acid-capture probe complex with a labeled nucleic acid probe specific for said first nucleic acid such that a specifically-bound nucleic acid-capture probe-labeled probe complex can form;i) the automated apparatus optionally washes the specifically-bound nucleic acid-capture probe-labeled probe complex such that non-specifically bound labeled probe nucleic acid is washed away from the specifically-bound nucleic acid-capture probe-labeled probe complex;j) and the automated apparatus detects the presence or absence of said generated signal and optionally displays a value for the signal, and optionally records a value for the signal, wherein the automated apparatus contacts the specifically-bound nucleic acid-capture probe-labeled probe complex with a solution wherein a detectable signal is generated if said first nucleic acid is present, wherein the signal generated from the sample is proportional to the amount of said first nucleic acid in the sample;wherein each of steps h, i and j can be performed sequentially or concurrently.