Controls for primers in multiplex amplification reactions

A method for detecting a control polynucleotide in a multiplex amplification reaction, the method comprising,

• providing:
• (1) a fluorescently-labeled nucleic acid probe;
• (2) 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;
• (3) a second primer pair and a third primer pair sufficient to amplify a second and third target sequence, respectively, by polymerase chain reaction;
• (4) 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 sequence and said second primer binding sequence:
• (i) the sequences of the primers of the second and third primer pairs or subsequences thereof that are at least 5 nucleotides long, and
• (ii) a probe binding sequence that is complementary to said fluorescently-labeled nucleic acid probe;
• amplifying the single-stranded control polynucleotide by polymerase chain reaction using said first primer pair, wherein said second primer is sufficiently complementary to said second primer binding sequence to allow for template-dependent extension of said second primer, and wherein said nucleic acid probe is sufficiently complementary to said probe binding site to allow for detection of said single-stranded polynucleotide sequence;
• and
• detecting the presence or absence of the amplified control polynucleotide by detecting hybridization of the nucleic acid probe to the probe-binding sequence in the amplified single-stranded control polynucleotide, thereby detecting the control polynucleotide in a multiplex amplification reaction.