Background Brucellosis is a zoonotic disease occurring worldwide. Brucella melitensis is the main cause of malta fever in humans and a major cause of abortion in sheep. In herd control and prevention programs, applying a suitable noninvasive method for accurate, rapid, and cost-effective monitoring of infected animals is a major concern. This study aimed to investigate lots of pooled sheep milk using qPCR to detect Brucella spp. infection in sheep. By calculating the limit of detection of Brucella in qPCR and microbial culture, the maximum number of pooled milk lots that retained the ability to be identified positively for Brucella was determined. A total of 144 milk samples were collected from the different seropositive sheep herds. The samples were randomly divided into six groups, each further divided into two subgroups, respectively. Then, DNA extraction was performed on 186 pooled and individual samples, followed by qPCR. Results The minimum detectable limits for qPCR and microbial culture per ml of milk were 100 and 300 CFU, respectively. Only 40% of the samples in microbial culture tested positive when the concentration decreased to 200 CFU. The results of qPCR indicated that four pools of 24 tested positive, whereas two pools tested negative. After examining the subgroups and individual samples within the two negative groups, it was revealed that all qPCR tests for these samples were negative. In the positive pools, at least one of the samples in the subgroups and corresponding individual samples tested positive. The two positive pools of 24, contained only one individual positive sample in each, indicating that the qPCR test could detect a positive Brucella sample in a pool of 24. Conclusions This noninvasive (milk instead of blood), rapid, and cost-effective method can be used to monitor suspected herds to identify infected animals with fewer tests.

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