Methods and compositions for rapid and high throughput diagnosis

A method of amplifying a target nucleic acid sequence in a sample, said method comprising:

• preparing a reaction pre-mixture comprising said sample and a polymerase, wherein said reaction pre-mixture does not comprise a primer;
• contacting said reaction pre-mixture with a microdroplet comprising a pair of primers to form a reaction mixture, wherein said microdroplet contains a volume of equal to or less than 250 nL;
• and
• amplifying said target nucleic acid sequence by thermal cycling in said reaction mixture,
• wherein said amplification has a lower limit of detection (LoD) as compared to an amplification by thermal cycling in a reaction mixture comprising a sample, a polymerase, and a pair of primers that are pre-mixed prior to amplification.

A method for simultaneously detecting two or more target nucleic acid sequences in a plurality of samples at a rate of at least 10000 samples per day in a single device, said method comprising:

• contacting said plurality of samples with two or more pairs of primers on a plate comprising a plurality of wells, wherein each well comprises one of said plurality of samples and one pair of said two or more pairs of primers,
• wherein each one pair of said two or more pairs of primers hybridizes with one of said two or more target nucleic acid sequences,
• wherein said plurality of wells comprise different pairs of said two or more pairs of primers, and amplifying one of said two or more target nucleic acid sequences in each well to a threshold level, thereby detecting said two or more target nucleic acid sequences in said plurality of samples,
• wherein said amplification of said detection has a sensitivity of at least 95% as calibrated with a positive control.