A light cycler based real-time PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in food samples

摘要

Significant public health concerns have been raised that Mycobacterium avium subsp. paratuberculosis (MAP), the etiological for Johnes' disease in world dairy and beef herds, may also be involved in some cases of human Crohn's disease. Therefore animal food products are potential sources for human infection with MAP. PCR methods are potential tools for rapid monitoring animal food products for MAP contamination but a number of challenges remain. These include sample material associated PCR inhibition as well as MAP DNA template isolation difficulties in food matrices. Moreover, the IS900 element targeted by most of the current MAP PCR protocols may not be highly specific for MAP organisms. In order to address some of these challenges, we developed a real-time PCR protocol that amplifies the f57 sequence as an indicator for MAP presence. This PCR method incorporates an internal amplification control template to monitor for potential PCR inhibition or failures that would otherwise lead to false negatives in routine PCR screening of food samples. The developed assay's specificity was confirmed through analysis of 10 MAP isolates and 63 isolates of other bacterial species. This real-time PCR assay has a linear quantitative amplification range from 2x10~1 to 2 x10~6 copies of MAP f57 target on purified DNA. In parallel an optimal strategy for MAP DNA isolation from artificially contaminated milk samples was also developed. The combined protocols when applied to MAP spiked raw milk samples, a minimum detection limit of 10 MAP cells per ml was achieved starting from 10 ml samples.