Pre-amplification assay

A method of detecting a condition in a first amplification reaction, comprising:

• assembling a first mixture, wherein the first mixture comprises a first nucleic acid template, a second nucleic acid template, a first set of primers specific for the first nucleic acid template, and a sample comprising sample nucleic acids;
• carrying out the first amplification reaction with the first mixture for a first number of cycles to generate a product mixture comprising the second nucleic acid template and an amplification product of the first nucleic acid template, wherein the second nucleic acid template is not amplified in the first amplification reaction;
• assembling a second mixture, wherein the second mixture comprises at least a portion of the product mixture, and reagents for carrying out a second nucleic acid assay, wherein the second nucleic acid assay is quantitative;
• carrying out the second nucleic acid assay with the second mixture to generate a signal indicative of the amount of the second nucleic acid template in the second mixture;
• and
• determining a signal indicative of the amount of the amplification product of the first nucleic acid template in the second mixture, and comparing the amount of the second nucleic acid template in the second mixture to the amount of the amplification product of the first nucleic acid template in the second mixture;
• wherein a condition in the first amplification reaction is detected when the difference between the amount of the amplification product of the first nucleic acid template and the amount of the second nucleic acid template in the second mixture is less than a threshold value.