The number of cases of Johne’s disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) is growing around the world. Detection of shed bacteria from faecal samples has been widely accepted as being an effective approach for the identification of infected cows, which, in turn, facilitates the implementation of quarantine procedures. A solid-phase nested PCR-enzyme-linked immunosorbent assay (PCR-ELISA) is reported here for detecting MAP. The primers and probes were designed to target the conserved region of insertion sequence 900 (IS900). The detection limit of this assay was ten copies of the IS900 gene in a standard plasmid, which is equivalent to one copy of bacterial genomic DNA. A good linearity was observed in the standard curve across 10⁵ orders of magnitude. No positive results were obtained when the test was carried out on E. coli, Staphylococcus sp., and Enterococcus sp., which are common pathogens isolated from cows in Taiwan. Thirty-one samples were tested by bacterial isolation, PCR, nested PCR, LAMP, nested PCR-ELISA, and serum ELISA in parallel. The results from the nested PCR-ELISA were consistent with those of a combination of bacterial culture and serum ELISA. In conclusion, this nested PCR-ELISA provides an option for laboratories with a thermocycler and an ELISA reader and can be used for both research and clinical purposes.

Key words: Mycobacterium paratuberculosis, Detection, Nested PCR-Linked Immunosorbent Assay