Multiplex amplification detection assay

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

• a) providing a DNA sample prepared from a biological sample from a subject, the DNA sample suspected of comprising one or more of at least 3 different target regions, the DNA sample further comprising an endogenous reference DNA and an exogenous process control DNA;
• b) treating the DNA sample in an amplification reaction in a PCR-flap assay buffer under conditions wherein one or more of the at least 3 different target regions, if present in the DNA sample, the endogenous reference DNA, and the exogenous process control DNA are amplified to form a pre-amplified mixture, wherein the PCR-flap assay buffer comprises 3-(n-morpholino) propanesulfonic acid (MOPS) buffer and 6 to 10 mM MgCl2,
• wherein the amplification reaction comprises PCR amplification reagents comprising:
• i) at least 3 different primer pairs for amplifying the at least 3 different target regions, if present in the DNA sample;
• ii) a primer pair for amplifying the endogenous reference DNA;
• and
• iii) a primer pair for amplifying the exogenous process control DNA;
• c) partitioning the pre-amplified mixture into a plurality of different PCR-flap assay reaction mixtures comprising the PCR-flap assay buffer, each PCR-flap assay reaction mixture comprising an additional amount of one or more primer pairs selected from the at least 3 different primer pairs, an additional amount of the primer pair for amplifying the endogenous reference DNA, and an additional amount of the primer pair for amplifying the exogenous process control DNA;
• and
• d) conducting a plurality of different PCR-flap assays with the PCR-flap assay reaction mixtures, wherein a target region selected from the at least 3 different target regions, if present in the DNA sample at step a), the endogenous reference DNA, and the exogenous process control DNA are amplified and detected in each of the PCR-flap assays.