Multiplex amplification detection assay

A method of analyzing a sample for multiple target nucleic acids in a PCR-flap assay, comprising:

• a) providing a sample comprising bisulfite-treated DNA prepared from a body fluid comprising plasma from a human subject, the DNA suspected of containing one or more of a plurality of at least 3 different target regions,
• b) treating said sample to an amplification reaction in a PCR-flap assay buffer under conditions wherein said at least 3 different target regions, if present in said sample, are amplified to form a pre-amplified mixture, wherein the PCR-flap assay buffer comprises 6 to 10 mM MgCl2,
• wherein said amplification reaction comprises PCR amplification reagents comprising at least 3 different primer pairs for amplifying said at least 3 different target regions, if present in said sample, from said bisulfite-treated DNA;
• c) partitioning said pre-amplified mixture into a plurality of different PCR-flap assay reaction mixtures comprising said PCR-flap assay buffer, each PCR-flap assay reaction mixture comprising an additional amount of a primer pair selected from said at least 3 different primer pairs;
• and
• d) conducting a plurality of PCR-flap assays with said PCR-flap assay reaction mixtures, wherein said different target regions, if present in said sample at step a), are amplified and detected in said PCR-flap assay reaction mixtures.