The design and optimisation of the Multiplex PCR for detection of Mycoplasma agalactiae, Yersinia enterocolitica and Aeromonas hydrophila in raw milk

Single-locus (sl) and multiplex (m) polymerase chain reaction (PCR) procedu res were developed for the detection of M. agalactiae, Y. enterocolitica an d A. hydrophila in raw milk samples. Three oligonucleotide primers for each pathogen were used in PCR, to detect a 375 base-pair (bp) fragment of M. a galactiae chromosomal DNA, the yst gene (145 bp) of Y. enterocolitica and a er gene (209 bp) of A. hydrophila. In optimisation studies, the sensitivity limits of sIPCR were found 10.0, 5.0 and 1.0 pg for M. agalactiae, Y. ente rocolitica and A. hydrophila, respectively. 2.0 mM MgCl2 concentration was found as superior for mPCR. When studied with naturally contaminated raw mi lk samples, detection rates obtained by PCR were 1.8%, 53% and 23% for M. a galactiae, Y. enterocolitica and A. hydrophila, respectively. Results indic ate the ability to detect these th ree pathogens effectively on the basis o f direct PCR analysis of raw milk.