A multiplex-PCR for the diagnosis of contagious agalactia of sheep and goats

A multiplex polymerase chain reaction (m-PCR) assay was optimized for the s imultaneous detection of several species of small ruminant mycoplasmas. Two sets of oligonucleotide primers specific for Mycoplasma agalactiae (Ma) an d Mycoplasma 'mycoides' cluster (M.m. cluster) were used in the test. The m -PCR was able to amplify a 375-bp fragment of Ma chromosomal DNA and a 257- 260-bp fragment of M.m. cluster chromosomal DNA. Four reference strains (M. agalactiae, M. mycoides subsp. mycoides LC, M. capricolum subsp. capricolu m, M. mycoides subsp, capri) and 56 samples (44 milk samples, two nasal swa bs, six ocular swabs, three vaginal swabs and one sample of fibrinous exuda te from carpal joint), from sheep and gears with clinical signs of contagio us agalactia (CA), were examined. The m-PCR confirmed the identification of reference strains and allowed to identify, of the 43 positive samples exam ined, 35 Ma strains, 12 M. 'mycoides' cluster strains; in four samples both Ma and mycoplasmas of M. 'mycoides' cluster were revealed. The m-PCR was a ble to detect 7 pg of mycoplasma DNA. The specificity and sensitivity of th e m-PCR suggest its use for the 'routine' diagnosis of CA. (C) 2001 Academi c Press.