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A polymerase chain reaction (PCR)-based test was developed for the detection of Mycoplasma agalactiae in sheep milk samples. Two oligonucleotide primers were designed to amplify a 375 bp fragment of M. agalactiae chromosomal DNA. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization using a fluorescein labeled 528 bp probe. The primers allowed the amplification of fragment of M. agalactiae DNA and did not amplify any specific fragment of other mycoplasmal DNAs (M. capricolum, M. mycoides subsp. mycoides, M. mycoides subsp. capri, M. putrefaciens, M. arginini and M. bovis) or other bacterial DNAs (S. aureus, S. epidermidis, P. haemalytica, E. coli, S. agalactiae, S. dysgalactiae, S. uberis, B. cereus, P. aeruginosa, S. durans, L. lactis, L. lactis var. diacetilactis, L. mesenteroides, S. thermophilus, L. bulgaricus and L. casei). The limit of detection of PCR assay was between 2.5 and 25 fg of purified DNA and 10(2) CCU ml-1 on mycoplasma cultures. These results indicate that the PCR technique can be used as a rapid and specific diagnostic method for detection of M. agalactiae.
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