I used three primers Bcsp31(B4,B5), IS711 & B.abortus & IS7711 & B.Melitensis for the control positive and I got nice result with samples I got smear?

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Robert Adolf Brinzer

Are these individual colonies you are testing or are you using mixed samples that can contain other bacteria as well?

Kaadhia Al-Kharousi

NO, it DNA extracted from milk and buffy coat blood. only I am testing to see type of Brucella.

Then could be there is off target amplification of other bacteria species in the sample.