Detection of Mycobacterium avium subspecies paratuberculosis using nested polymerase chain reaction (nPCR)

Mycobacterium avium subspecies paratuberculosis, the cause of paratuberculosis (Johne’s disease) is a chronic debilitating infection in ruminants. The disease is one of the most widespread bacterial infections of ruminants throughout the world. Recently, the organism was reported to be associated with enteric infection in humans and hence the disease is of public health importance. In the present study, a nested Polymerase Chain Reaction (nPCR) was developed and evaluated for detection of Mycobacterium avium subspecies paratuberculosis. The insertion sequence (IS) 1311 was used as a target DNA for the nested PCR amplification. First, a pair of primers (P1 and P2) was designed from IS1311 nucleotide sequences to produce 549 bp PCR products. Next, a second pair of internal (nested) primers (P3 and P4) was used to produce a 260 bp PCR product. The nested amplification was necessary to increase the sensitivity of the PCR assay and to confirm the identity (specificity) of the first amplified PCR product. Because it is a highly sensitive technique the nPCR could be used for detection of Johne’s disease at an early stage of the infection. In addition, the described nPCR assay could be used as a supplement, or as an alternative, to conventional bacteriological procedures currently used for diagnosis of the disease in susceptible humans or animal populations.