Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR).
Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was constructed by insertion of rpoB fragments from Rhodococcus equi into CEM-1P, which is a recombinant plasmid that includes a T. equigenitalis-specific sequence region. In CEM-PCR, the size of the PCR product from CEM-POS was clearly different from the true positive PCR product. In addition, CEM-POS retained high stability under convenient storage conditions of 4 degrees C. These results suggest CEM-POS to be a useful tool as a reaction control in routine CEM-PCR examinations.
Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke-shi, Tochigi 329-0412, Japan. niwa@epizoo.equinst.go.jp
Hidekazu
H
Niwa
Toru
T
Anzai
Seiji
S
Hobo
D000818:Animals; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000662:veterinary*; D006734:Horse Diseases / Q000175:diagnosis* / Q000382:microbiology; D006736:Horses; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D015231:Sexually Transmitted Diseases, Bacterial / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D020625:Taylorella equigenitalis / Q000235:genetics* / Q000302:isolation & purification*