A novel real-time PCR assay for specific detection and quantification of Mycobacterium avium subsp. paratuberculosis in milk with the inherent possibility of differentiation between viable and dead cells

被引:21
作者
Dzieciol M. [1 ]
Volgger P. [1 ]
Khol J. [2 ]
Baumgartner W. [2 ]
Wagner M. [1 ]
Hein I. [1 ]
机构
[1] Department for Farm Animals and Veterinary Public Health, Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, A-1210 Vienna
[2] Department for Farm Animals and Veterinary Public Health, Clinic for Ruminants, University of Veterinary Medicine, A-1210 Vienna
关键词
Milk Sample; Internal Amplification Control; Mycobacterium Avium Subsp; IS900 Target; Pasteurize Milk Sample;
D O I
10.1186/1756-0500-3-251
中图分类号
学科分类号
摘要
Background. Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (Johne's disease) in ruminants and is suggested to be one of the etiologic factors in Crohn's disease in humans. Contaminated milk might expose humans to that pathogen. The aim of the present study was to develop a novel real-time PCR assay providing the additional possibility to detect viable Mycobacterium avium subsp. paratuberculosis (MAP) based on the MAP-specific Mptb52.16 target. The design included an internal amplification control to identify false negative results. Findings. Inclusivity and exclusivity tested on 10 MAP strains, 22 non-MAP mycobacteria, and 16 raw milk microflora strains achieved 100%. The detection limit in artificially contaminated raw milk was 2.42 × 101 MAP cells/ml milk. In a survey of naturally contaminated samples obtained from dairy herds with a known history of paratuberculosis, 47.8% pre-milk and 51.9% main milk samples tested positive. Real-time PCR-derived MAP-specific bacterial cell equivalents (bce) ranged from 1 × 100 to 5.1 × 102 bce/51 ml; the majority of samples had less than one bce per ml milk. Expression of the chosen target was detected in artificially contaminated raw milk as well as inoculated Dubos broth, thus confirming the real-time PCR assay's potential to detect viable MAP cells. Conclusions. Concentrating the DNA of a large sample volume in combination with the newly developed real-time PCR assay permitted quantification of low levels of MAP cells in raw milk and pasteurized milk. The selected target - Mptb52.16 - is promising with regard to the detection of viable MAP. Future studies integrating quantitative DNA- and RNA-based data might provide important information for risk assessment concerning the presence of MAP in raw milk and pasteurized milk. © 2010 Hein et al; licensee BioMed Central Ltd.
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