Design and development of an internal control plasmid for the detection of Mycobacterium avium subsp. paratuberculosis using real-time PCR. Mol Cell Probes 2006 Feb;20(1):51-9
Date
12/13/2005Pubmed ID
16337359DOI
10.1016/j.mcp.2005.09.005Scopus ID
2-s2.0-32844474682 (requires institutional sign-in at Scopus site) 11 CitationsAbstract
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).
Author List
Brey BJ, Radcliff RP, Clark DL Jr, Ellingson JLAuthor
Becky J. Buelow MD Associate Professor in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBase Sequence
Cattle
Molecular Sequence Data
Mycobacterium avium subsp. paratuberculosis
Paratuberculosis
Plasmids
Polymerase Chain Reaction
