A PCR-based test for the specific identification of Mycoplasma mycoides subspecies mycoides SC

Commonwealth Scientific and Industrial Research Organisation, Australian Animal Health Laboratory, Geelong, Victoria.

The polymerase chain reaction (PCR) was used to develop a test for the detection of Mycoplasma mycoides subspecies mycoides SC in the tissues of animals infected with contagious bovine pleuropneumonia (CBPP). Two sets of primers were designed; one set (MC323/MC358) to amplify a approximately 1.5-kbp DNA fragment from all the members of the M. mycoides 'Cluster' and the other set (MM450/MM451) specifically amplified a 574-bp DNA fragment from M. mycoides subspecies. The PCR products could be differentiated further by digestion with the restriction enzyme AsnI. Enzyme digestion of amplification products from M. m. mycoides SC produced 2 fragments, whereas the other 2 M. mycoides subspecies, M. m. mycoides LC and M. m. capri, produced 3 fragments. This test was shown to be very sensitive, being able to detect between 10 and 100 organisms. Cattle were experimentally infected with the Gladysdale strain of M. m. mycoides SC, and samples of serum and mucus were taken periodically, as were postmortem samples of lung, lymph node, pleural fluid, synovial fluid, and tracheal swabs. Complement fixation test on serum samples, culture of postmortem tissues, and histopathologic examination confirmed disease. DNA was extracted from postmortem samples and amplified by PCR using primers MM450 and MM451. Digestion of products using AsnI allowed the specific identification of M. m. mycoides SC. This test could confirm CBPP in 48 hours and was thus capable of giving a more rapid result than the traditional methods of culture, isolation, and identification using biochemical and serological techniques.