1. Significant differences in target copy numbers in multiplex PCRs may result in incorrect identification at high Cq values.
Significant differences in target copy numbers in multiplex PCRs may result in incorrect identification at high Cq values. Cycle threshold (Cq) values range from 1 to 50 and are depicted on the x axis. The threshold (horizontal line) is arbitrary and just above the curve breakpoints. (A) Amplification curves for multiplex PCR targeting IS481 (purple) and IS1002 (red) in the presence of an internal control (phocine herpesvirus [green]) of a clinical sample positive for B. pertussis. At high concentrations of B. pertussis DNA, amplification of the internal control (green) is outcompeted for reaction components. (B) At low concentrations of DNA, a result interpretation error may arise because of the difference in the lower limits of detection for IS481 (high copy number) and IS1002 (low copy number). In this case, IS481 PCR shows a positive signal over the lowest concentrations, whereas IS1002 PCR is negative. A single amplification curve for IS481 can also indicate a B. holmesii infection (see the text). At high Cq values, this multiplex PCR may give a false B. holmesii result when B. pertussis is in fact the causative agent. (Courtesy of Lieuwe Roorda [reprinted with permission].)‏
Anneke van der Zee et al. Clin. Microbiol. Rev. 2015; doi: /CMR