Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's disease, a chronic enteritis of the gastrointestinal tract of ruminants. The insertion sequence IS900 and the hspX gene can be targeted for detection of MAP using PCR. Generally, each set of PCR reactions contains a positive and a negative control tube. Unfortunately, it is possible for a reaction to fail while the controls do not. Thus, a single positive and negative control tube cannot determine if all PCR reactions in a set worked properly. Our objective was to construct a plasmid to serve as a universal internal control in PCR reactions that test for Mycobacterium, including MAP and Mycobacterium bovis /tuberculosis-complex organisms. A previously constructed plasmid containing an insert of M. bovis-hspX-M. bovis DNA was enzymatically digested with BSG I and Sac II to remove 71 bp of the hspX portion (including the sequence we use for the reverse hspX primer). The remaining insert was blunt-ended and ligated back together. The new plasmid was transformed into competent cells and grown on LB/Amp/X-Gal/IPTG plates for screening. Both Eco RI digestion and sequencing confirmed the desired plasmid product. A reverse primer was designed to anneal to the M. bovis portion of the plasmid insert located farthest from the hspX forward primer. We performed PCR reactions, using various amounts of plasmid (1 ng to 100 ag) and MAP genomic (10 ng to 100 fg) DNA (ATCC 19698), to determine the optimal amount of plasmid to be used in both IS900 and hspX PCR reactions. The optimal amount of plasmid for IS900 and hspX detection was 1 fg and 10 fg, respectively. In conclusion, the plasmid construct can be included in MAP PCR reactions to confirm if a PCR reaction is successful and identify true positives and true negatives within each individual reaction tube.