Progress towards a rapid polymerase chain reaction diagnostic test for the identification of Mycobacterium avium...

Hybridization-capture polymerase chain reaction (HC-PCR), a nucleic acid sequence capture technique, was evaluated on faecal samples pooled from 50 sheep and individual faecal samples as a rapid diagnostic test for Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The status of each of the faecal samples was determined by radiometric culture. A simpler direct-PCR technique was evaluated on the same samples and was found to be more sensitive than HC-PCR. The lack of sensitivity of HC-PCR was neither due to location nor length of capture probe on IS 900 nor deterioration of the probe but was associated with inefficiencies in liquid phase hybridization and solid phase magnetic bead capture. Direct-PCR using primers from the 5' region of IS 900 was evaluated in a blind trial on 502 pooled faecal samples which were concurrently examined by culture. Twenty-one (64%) of the 33 culture positive pools were detected by direct PCR, representing 11 (79%) of the 14 farms with infected sheep. Direct-PCR was also more sensitive than immunomagnetic bead capture-PCR. Using individual faecal samples, 74% of culture positive samples were detected with direct-PCR compared to 44% with immunomagnetic bead capture-PCR. Direct-PCR from faeces can be used as a rapid means of screening pooled faecal samples for flock diagnosis of Johne's disease in sheep.