Development of a selective polymerase chain reaction assay for the detection of Mycoplasma mycoides subsp. Mycoides S.C. (contagious bovine pleuropneumonia agent).
A new selective assay for the detection of Mycoplasma mycoides subsp. mycoides SC (MmmSC) via the polymerase chain reaction (PCR) has been developed. This test used two PCR assays: a control-PCR (MYC-PCR) identifying the pathogen as a member of the mycoides cluster and the MSC-PCR which is specific for MmmSC. The MYC primers targeted a DNA sequence of about 460 bp from all the 59 mycoides cluster-strains tested. No amplification occurred with bovine genomic DNA or with the 11 other bacterial species assayed. The MSC primers selectively amplified a 275 bp sequence from the 27 MmmSC strains tested, with three specific internal restriction sites allowing confirmation of the identification. The sensitivity assessed by direct agarose gel analysis for both PCR assays was 100 CFU. The sensitivity of the MSC-PCR was increased to 1 CFU by a dot-blot hybridization step using, as a probe, the entire 275 bp sequence digoxigenin-labeled by PCR. These two PCR assays were successfully used to detect MmmSC in pleural fluids from naturally-infected cattle. We conclude that these two PCR assays may be valuable tools for the diagnosis of contagious bovine pleuropneumonia.
Dedieu
L
L
Laboratoire PATHOTROP, Département Elevage et Médecine Vétérinaire du Centre de Coopération Internationale en Recherche Agronomique pour le Dévelopement (CIRAD-EMVT), Maisons-Alfort, France.
Mady
V
V
Lefevre
P C
PC
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D008969:Molecular Sequence Data; D009176:Mycoplasma mycoides / Q000302:isolation & purification*; D011002:Pleuropneumonia, Contagious / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity