The invention provides a method that includes performing a pre-amplification of nucleic acid in a sample to generate amplicons that include a copy of a variant, annealing and extending variant-specific primers to create copies of the amplicon, and copying the copies with tailed primers to form tailed amplicons. The tailed amplicons are amplified with universal primers and detection probes that anneal to the tailed amplicons. Extension of the universal primers along the tailed amplicons through annealed detection probes generates a signal that shows the presence of the variant in the sample.

The invention provides a method that includes performing a pre-amplification of nucleic acid in a sample to generate amplicons that include a copy of a variant, annealing and extending variant-specific primers to create copies of the amplicon, and copying the copies with tailed primers to form tailed amplicons. The tailed amplicons are amplified with universal primers and detection probes that anneal to the tailed amplicons. Extension of the universal primers along the tailed amplicons through annealed detection probes generates a signal that shows the presence of the variant in the sample.

A two-step multiplex amplification reaction includes a first step which truncates the standard initial multiplex amplification round to boost the sample copy number by only a 100-1000 fold increase in the target. Following the first step the product is divided into optimized secondary single amplification reactions, each containing one of the primer sets that were used previously in the first or multiplexed booster step. The booster step can occur using an aqueous target nucleic acid or using a solid phase archived nucleic acid. In particular, nucleic acid sequences that uniquely identify E. Coli were identified using the multiplex amplification method.

A two-step multiplex amplification reaction includes a first step which truncates the standard initial multiplex amplification round to boost the sample copy number by only a 100-1000 fold increase in the target. Following the first step the product is divided into optimized secondary single amplification reactions, each containing one of the primer sets that were used previously in the first or multiplexed booster step. The booster step can occur using an aqueous target nucleic acid or using a solid phase archived nucleic acid. In particular, nucleic acid sequences that uniquely identify E. Coli were identified using the multiplex amplification method.