Closed-system multi-stage nucleic acid amplification reactions

A method of detecting the presence or absence of one or more target polynucleotides in a sample, the method comprising the steps of:

• providing a fluidly closed system comprising a reaction chamber selectably in fluid communication with a waste reservoir, a sample reservoir containing a sample, a first reactant reservoir containing first-stage amplification reagents, and a second reactant reservoir containing second-stage amplification reagents;
• the reaction chamber comprising a retaining member positioned in the reaction chamber to retain a defined volume of the liquid in the reaction chamber whenever the remainder of the liquid is removed from the reaction chamber;
• fluidly transferring sample from the sample reservoir and first-stage amplification reagents from the first reactant reservoir to the reaction chamber so that the first-stage amplification reagents react with the sample in a first stage amplification reaction in the reaction chamber to produce a reaction product containing a first amplicon whenever a target polynucleotide is present in the sample;
• optically monitoring a fluorescent indicator in the first stage amplification reaction in the reaction chamber;
• fluidly transferring the reaction product to the waste reservoir, except for an effective portion withheld by the retaining member in the reaction chamber, when the optical signal reaches or exceeds a threshold level;
• fluidly transferring second-stage amplification reagents from the second reactant reservoir to the reaction chamber so that the second-stage amplification reagents react with the effective portion of the reaction product in a second amplification reaction in the reaction chamber to produce a second amplicon whenever the first amplicon is present in the reaction product;
• and
• detecting the second amplicon to determine whether the target polynucleotide is present in the sample.

A method of amplifying one or more RNA sequences, the method comprising the steps of:

• transcribing one or more RNA sequences in a fluidly closed reaction system to form one or more complementary single stranded DNA sequences using reverse transcriptase reagents in a first reaction mixture;
• optically monitoring a fluorescent indicator in the first stage amplification reaction
• isolating a first effective portion of the first reaction mixture in the fluidly closed reaction system, wherein said first reaction mixture is in a reaction chamber of said fluidly closed reaction system, the reaction chamber comprising a retaining member positioned in the reaction chamber to retain a defined volume of the liquid in the reaction chamber whenever the remainder of the liquid is removed from the reaction chamber, and wherein said step of isolating said first effective portion includes fluidly transferring said first reaction mixture to a waste reservoir, except for said first effective portion that is withheld by the retaining member in the reaction chamber, when the optical signal in the reaction chamber reaches or exceeds a threshold level;
• and
• amplifying in the reaction chamber the one or more complementary single stranded DNA sequences in the first effective portion using first-stage amplification reagents in a second reaction mixture to form one or more first amplicons, the first-stage amplification reagents including initial primers for each of the complementary single stranded DNA sequences.

A method for detecting the presence or absence of one or more target polynucleotides in a sample, the method comprising the steps of:

• providing a reaction chamber containing first-stage amplification reagents, the reaction chamber being selectably in fluid communication with a waste reservoir, a sample reservoir containing a sample, and a second reactant reservoir containing second-stage amplification reagents, each of said reservoirs being fluidly closed;
• the reaction chamber comprising a retaining member positioned in the reaction chamber to retain a defined volume of the liquid, as an effective portion, in the reaction chamber whenever the remainder of the liquid is removed from the reaction chamber;
• fluidly transferring sample from the sample reservoir to the reaction chamber so that the first-stage amplification reagents react with the sample in a first stage amplification reaction in the reaction chamber to produce a first reaction product containing a first amplicon if a target polynucleotide is present in the sample;
• optically monitoring a fluorescent indicator in the first stage amplification reaction in the reaction chamber;
• fluidly transferring the first reaction product to the waste reservoir, except for the an effective portion of the first reaction product withheld by the retaining member in the reaction chamber, when the optical signal reaches or exceeds a threshold level;
• fluidly transferring the second-stage amplification reagents from the second reactant reservoir to the reaction chamber so that the second-stage amplification reagents react with the effective portion of the first reaction product in a second stage amplification reaction in the reaction chamber to produce a second amplicon whenever the first amplicon is present in the first reaction product;
• and
• detecting the second amplicon to determine whether the target polynucleotide is present in the sample.