External control reagents for nucleic acid amplification are provided that
verify the absence or presence of specific target sequences, and correct primers
and probes. A single-stranded, external control polynucleotide is amplified with
primers of the same sequence as target primers. Probes with detectable labels and
sequences specific for target and external control polynucleotides allow for detection
and measurement. The primers and the detectable probe are adjacent or substantially
adjacent when hybridized to the external control polynucleotide. Target and control
amplicons may be detected by increased fluorescence induced by polymerase-mediated
5 nuclease cleavage or hybridization of a self-quenching probe complementary
to both target and external control polynucleotides. A kit of PCR reagents can
be dispensed into vessels for rapid and accurate nucleic acid amplification assay,
with real-time or end-point measurements. The amplification control reagents, kits,
and methods of the present invention provide positive and negative control tests
which can be conducted concurrently with target amplification. Allelic differences
at genetic loci can be detected, including single nucleotide polymorphisms (SNP).
