Improved detection of Mycobacterium avium subsp paratuberculosis in milk by immunomagnetic PCR

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a ra pid method to screen milk samples for the presence of Mycobacterium avium s ubsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IM S) for M. ptb, developed at Queen's University, Belfast, was applied to mil k samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk Likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centr ifugation (2500g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatme nt. Following IMS, template DNA for IS900 PCR was obtained by heating the b ead-cell suspension in a thermal cycler at 100 degreesC for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3) CFU of M. ptb per 50 mi of milk (equivalent to 20 CFU/ml), whereas the minimum detec tion limit of direct IS900 PCR was estimated at 10(5) CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a to tal of 40 spiked (10(6) CFU M. ptb) and unspiked, raw and laboratory-pasteu rised milk samples were independently tested by IMS-PCR and conventional IS 900 PCR. IMS-PCR correctly identified 97.5% of milk samples (sensitivity 10 0%, specificity 95%), including spiked milk samples before and after labora tory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventio nal IS900 PCR correctly identified only 72.5% of the same 40 milk samples ( sensitivity 23%, specificity 100%). LMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commer cially pasteurised cows' milk. (C) 2000 Elsevier Science B.V. All rights re served.