Detection of Mycoplasma mycoides subspecies mycoides SC in clinical material by a rapid colorimetric PCR

A colorimetric polymerase chain reaction (PCR) test for the detection of Mycoplasma mycoides subsp. mycoides SC (MmmSC), the cause of contagious bovine pleuropneumonia (CBPP), was used to lest clinical samples from suspect and known cases of the disease. A commercially available detection kit, pleuroTRAP from AMRAD, Australia, which captures amplified double stranded DNA in the wells of a microtitre plate was used. Tissues tested from animals ranging from a variety of anatomical locations included lung, lymph node, kidney, spleen, pleural fluid and nasal swabs. Polymerase chain reaction was conducted on DNA extracted from tissues as well as on culture-derived mycoplasmas. Positive results could be seen by the development of a yellow colour in microtitre wells after the enzymatic detection procedure indicating the presence of specifically amplified products. Spectrophotometric values of OD450 for positive samples ranged from 0.211 to 1.409 compared to negative values of around 0.020 which reduced the subjectivity in the interpretation of results. MmmSC was successfully identified from cultures of reference and field strains, nasal swabs and excised tissue by amplification with primers supplied in the kit. The system is suitable for automation and may be a cost effective alternative to gel electrophoresis for the screening of moderately large sample numbers as is the case in surveillance programmes. (C) 1999 Academic Press.