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Updated: Aug 14, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Design and development of an internal control plasmid for the detection of Mycobacterium avium subsp.
Becky J Brey1, Roy P Radcliff, Dorn L Clark
1Food Safety Services, Division of Laboratory Medicine, Marshfield Clinic Laboratories, 1000 North Oak Avenue, Marshfield, WI 54449, USA.
Abstract:
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).
Insights
A novel plasmid internal control was developed to validate PCR diagnostic tests for Johne's disease in ruminants. This method ensures the accuracy of each Mycobacterium avium subspecies paratuberculosis (MAP) test by confirming successful amplification within every reaction.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Bacterial Pathogenesis
Background:
- Johne's disease in ruminants is caused by Mycobacterium avium subspecies paratuberculosis (MAP).
- Current PCR diagnostics for MAP rely on external positive controls, which do not guarantee the success of individual reactions.
- A need exists for an internal control to validate each PCR test for MAP detection.
Purpose of the Study:
- To construct and validate a plasmid-based internal control for PCR assays detecting MAP.
- To improve the reliability and accuracy of Johne's disease diagnosis using molecular methods.
Main Methods:
- A plasmid containing M. bovis-hspX-M. bovis DNA was modified by removing a 71 bp segment of the hspX insert.
- The modified plasmid was used as an internal control in PCR reactions for detecting the hspX gene and IS900 insertion sequence.
- PCR conditions were optimized by titrating plasmid amounts against MAP genomic DNA.
Main Results:
- The modified plasmid successfully served as an internal control in PCR assays.
- Plasmid insert amplification confirmed the success of individual PCR reactions, distinguishing true positives from negatives.
- Optimal plasmid concentrations were determined as 10 fg/reaction for hspX detection and 1 fg/reaction for IS900 detection.
Conclusions:
- The developed plasmid internal control enhances the reliability of PCR diagnostics for Johne's disease.
- This internal control system validates each reaction, ensuring accurate identification of MAP.
- The optimized method provides a robust tool for veterinary diagnostics and research.
