Cloning and development of synthetic internal amplification control for Bacillus anthracis real-time polymerase chain reaction assays

Abstract

An internal amplification control (IAC) was developed for Bacillus anthracis rpoB gene detection using TaqMan assay. Synthetic IAC oligonucleotides were subcloned using vector pDG1730 for ectopic integration into host Bacillus subtilis strain 1A772 genome. Differentially labeled target and IAC probes were used in real-time polymerase chain reaction (PCR) assays. There was no nonspecific cross-detection in single-well reactions. Limit of detection for both target and IAC DNA was 5 fg corresponding to a single gene copy. The IAC, in conjunction with target system, should decrease the rate of false-positive and false-negative results in real-time PCR assays.