A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk.

National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden.
Journal of Microbiological Methods (Impact Factor: 2.16). 11/2008; 75(2):335-40. DOI: 10.1016/j.mimet.2008.07.009
Source: PubMed

ABSTRACT A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis (MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk. Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was assessed to 100 organisms per ml milk (corresponding to 1-10 CFU per ml) for samples of 10 ml. The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive by environmental culture, while 30% were positive by milk PCR. Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.

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    ABSTRACT: 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. 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. 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.
    BMC Research Notes 10/2010; 3:251.
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    Revista medica de Chile 06/2011; 139(6):794-801. · 0.36 Impact Factor
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    ABSTRACT: Molecular diagnostic tests are widely implemented in animal and human health microbiology. Efficient nucleic acid extraction methods are essential in diagnostic laboratories and automation is a valuable tool for those with high throughput activity. Nucleic acid extraction protocols present variable efficiency depending on the composition of the specimen and the chemical-physical characteristics of the target pathogen. In the present study, we compared the DNA extraction performances of four automated methods (kits I, M, P, Q) adapted on a Hamilton Robotics “Microlab Starlet” extraction unit, and one manual method (kit R). Ten-fold dilutions of Mycobacterium avium subsp. paratuberculosis (MAP) were used to contaminate bovine central nervous system (CNS) and lung-spleen-liver pools (LSL). In consideration of its chemical-physical characteristics, MAP was selected as a template for pathogen DNA detection. Analytical performance and repeatability were assessed through downstream real-time PCR amplification, hands-on time (HOT), total turnaround time (TAT) and costs of all kits. MAP was detected differently depending on extraction kit and type of matrix analysed. Kits M and I showed the highest analytical performance on CNS (1MAP/ml) and LSL (10MAP/ml), respectively. Besides analytical results, kits I and M displayed high repeatability, the same HOT, very similar TAT, and were inexpensive. In conclusions, different standardized automated systems have been established with high throughput, sensitivity and repeatability for CNS and LSL. Our results also demonstrated that is necessary to assess the effectiveness of extraction kits in matrices not previously tested to avoid the risk of unreliable diagnostic outcomes. KeywordsAutomated–DNA extraction–Evaluation–IS900– Mycobacterium avium subsp. paratuberculosis –Real-time PCR
    World Journal of Microbiology and Biotechnology 01/2011; 27(1):31-37. · 1.26 Impact Factor


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