Design and development of an internal control plasmid for the detection of Mycobacterium avium subsp. paratuberculosis using real-time PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants. The hspX gene and insertion sequence IS900 can be used to diagnose Johne's with PCR. Generally, a single PCR tube containing the DNA sequence of interest is run as a positive control with each set of reactions. Single reactions within a PCR run can fail while the positive control does not. Thus, a single positive control tube does not determine if all PCR reactions worked properly. Our objective was to construct a plasmid to use as an internal control in each reaction. A plasmid containing an insert of M. bovis-hspX-M. bovis DNA was modified to remove a portion of the hspX insert used by the reverse hspX primer. The remaining insert was ligated back together and transformed into competent cells. Sequencing confirmed removal of 71 bp. PCR reactions using three primers (TB/M. bovis reverse, hspX forward and reverse) for hspX gene detection and four primers (IS900 forward and reverse, hspX forward, and TB/M. bovis reverse) for IS900 detection were optimized by titrating various amounts of plasmid against varied amounts of MAP genomic DNA. Plasmid insert amplification confirms a successful PCR reaction and identifies true positives and negatives within each individual reaction. The optimal plasmid amounts are 10 fg/reaction (hspX detection) and 1 fg/reaction (IS900 detection).