Comparison of Multiplex and ordinary PCR for diagnosis of Paratuberculosis and Tuberculosis in blood samples (buffy coat) of cattle and buffaloes

Document Type: Short paper


Department of Veterinary Microbiology College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India


Paratuberculosis or Johne’s disease (JD) and tuberculosis (TB) caused by M. avium paratuberculosis (MAP) and M. Tuberculosis complex (MTC) respectively are endemic, chronic debilitating diseases affecting the dairy herds, causes huge economic loss to the dairy farmers in India and developing countries; and are also potential zoonotic threats. In this study, an in-house developed multiplex PCR targeting MAP, M. bovis and M. smegmatis was used in blood samples (buffy coat) in parallel to IS900 PCR and esxB PCR for early diagnosis of JD and TB respectively. A total of 202 blood samples of cattle and buffaloes were collected, buffy coat was obtained by centrifugation and used for DNA extraction and subsequently PCR. Out of 202 animals, 12 (5.9%) animals were positive for MAP by in-house multiplex PCR and 17 (8.4%) animals were positive by IS900 PCR. However, only 8 (4%) animals were positive by both the multiplex and IS900 PCR; whereas 4 and 9 animals were exclusively positive by multiplex PCR and IS900 PCR respectively. None of the animals were found to be positive for M. bovis and M. smegmatis by the in-house developed multiplex PCR. However, the esxB PCR detected 13 (6.4%) animals positive for TB, indicating its early diagnostic potential for TB from buffy coat. The in-house developed multiplex PCR detected MAP in buffy coat, though the IS900 PCR detected more number of animals to be positive for MAP, probably due to the presence of multiple copies of IS900 gene in a single MAP genome.


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