A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of

National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden.
Journal of Microbiological Methods (Impact Factor: 2.03). 11/2008; 75(2):335-40. DOI: 10.1016/j.mimet.2008.07.009
Source: PubMed


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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    • "appear to be useful diagnostics that function well not only for fecal samples (Alinovi et al., 2009) but also for bulk-tank milk samples. Successful optimization of the template preparation methods and beadbeating durations relied on the observation that MAP fractionates to both the pellet and cream layers (Gao et al., 2007;Herthnek et al., 2008). Following optimization, we were able to use the Tetracore kits to reliably detect MAP concentrations >10 −4.5 (corresponding to 8.23 cfu/mL) in bulktank samples, with the caveat that optimization was conducted using spiked milk rather than with positive farm samples. "
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    ABSTRACT: Mycobacterium avium ssp. paratuberculosis (MAP), the etiologic agent of Johne's disease in dairy cattle, may enter the bulk tank via environmental contamination or direct excretion into milk. Traditionally, diagnostics to identify MAP in milk target either MAP antibodies (by ELISA) or the organism itself (by culture or PCR). High ELISA titers may be directly associated with excretion of MAP into milk but only indirectly linked to environmental contamination of the bulk tank. Patterns of bulk-milk ELISA and bulk-milk PCR results could therefore provide insight into the routes of contamination and level of infection or environmental burden. Coupled with questionnaire responses pertaining to management, the results of these diagnostic tests could reveal correlations with herd characteristics or on-farm practices that distinguish herds with high and low environmental bulk-tank MAP contamination. A questionnaire on hygiene, management, and Johne's specific parameters was administered to 292 dairy farms in New York, Oregon, and Wisconsin. Bulk-tank samples were collected from each farm for evaluation by real-time PCR and ELISA. Before DNA extraction and testing of the unknown samples, bulk-milk template preparation was optimized with respect to parameters such as MAP fractionation patterns and lysis. Two regression models were developed to explore the relationships among bulk-tank PCR, ELISA, environmental predictors, and herd characteristics. First, ELISA optical density (OD) was designated as the outcome in a linear regression model. Second, the log odds of being PCR positive in the bulk tank were modeled using binary logistic regression with penalized maximum likelihood. The proportion of PCR-positive bulk tanks was highest for New York and for organic farms, providing a clue as to the geographical patterns of MAP-positive bulk-tank samples and relationship to production type. Bulk-milk PCR positivity was also higher for large relative to small herds. The models revealed that bulk-milk PCR result could predict ELISA OD, with PCR-positive results corresponding to high bulk-milk ELISA titers. Similarly, ELISA was a predictor of PCR result, although the association was stronger for organic farms. Despite agreement between high bulk-milk ELISA titers and positive PCR results, a large proportion of high ELISA farms had PCR-negative bulk tanks, suggesting that farms are able to maintain satisfactory hygiene and management despite a presence of MAP in these herds.
    Full-text · Article · Dec 2015 · Journal of Dairy Science
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    • "MAP has been identified in many body locations, such as lymph nodes, spleen, liver and animal hide (Wells et al., 2009; Wu et al., 2007) as well as in semen (Sharifzadeh et al., 2010), drinking water (Gill et al., 2011), milk and milk products (Corti and Stephan, 2002; Djønne et al., 2003; Donaghy et al., 2004; Millar et al., 1996). With the progression of the disease, the animal begins shedding MAP in the feces (Herthnek et al., 2008). Therefore, milk could easily get contaminated with high numbers of MAP during teat preparation (Dundee et al., 2001). "
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    ABSTRACT: Prevalence of Mycobacterium avium paratuberculosis in commercially pasteurized milk was studied. A total of 300 commercially pasteurized milk samples were purchased from various parts of Eastern-Azerbaijan province of Iran. Two 50 ml from each sample were centrifuged. DNA extraction was performed on one of the pellets and extracted DNA was evaluated for the presence of Mycobacterium avium paratuberculosis specific IS900 by PCR assay. In order to detect viable cells of the bacterium, the second related pellet was treated with 0.75% HPC and decontaminated samples cultured on two Herrold's egg yolk medium (supplemented with mycobactin J and amphotericin B, nalidixic acid, and vancomycin). Isolated colonies by culture method were confirmed by IS900-based PCR. Although M. avium paratuberculosis DNA was detected in 32 (10.7%) samples by PCR assay, viable bacterium was not isolated by culture method in any sample.
    Full-text · Article · Feb 2012 · African journal of microbiology research
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    • "For the present study we used a non-mechanical method of cell lysis which was successfully applied for real-time PCR-based detection of the Gram-positive bacterial species Listeria monocytogenes in milk [15]. This protocol includes multiple incubation and washing steps at 45°C in combination with solvents and detergents, and maybe permits the recovery of MAP from milk fat as well [25]. "
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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.
    Full-text · Article · Oct 2010 · BMC Research Notes
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