Controls for primers in multiplex amplification reactions

A reagent kit, comprising,

• i. at least one fluorescently-labeled nucleic acid probe;
• ii. a first primer pair comprising a first and second primer, wherein the first primer pair is sufficient to amplify a first target sequence by polymerase chain reaction;
• iii. a second primer pair and a third primer pair sufficient to amplify a second and third target sequence, respectively, by polymerase chain reaction;
• iv. a single-stranded control polynucleotide comprising:
• (a) a nucleotide sequence of the first primer and a nucleotide sequence (designated a “second primer binding sequence”) that is complementary to the second primer;
• and
• (b) located between said nucleotide sequence of the first primer and said second primer binding sequence:
• (1) the sequences of the primers of the second and third primer pairs or subsequences thereof that are at least 5 nucleotides long, and
• (2) a probe binding sequence that is complementary to said fluorescently-labeled nucleic acid probe;
• wherein said nucleic acid probe is sufficiently complementary to said probe binding site to allow for detection of said single-stranded control polynucleotide sequence.