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.
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.
SourceAvailable from: Patricio RetamalRevista medica de Chile 06/2011; 139(6):794-801. DOI:10.4067/S0034-98872011000600015 · 0.37 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The causative agent of paratuberculosis in ruminants, Mycobacterium avium subsp. paratuberculosis (MAP), although still a matter of debate, has been linked with Crohn's and other human diseases. The availability of rapid methods for assessing the viability of MAP cells in food, in particular milk, could be of great use for risk management in food safety. MAP viability is generally assessed using culture techniques that require prolonged incubation periods for the growth of MAP. To differentiate between viable and non-viable MAP cells in milk samples, this study explores the combination of two already described techniques: peptide magnetic bead separation followed by Propidium Monoazide-qPCR. By using an Ordinal Multinomial Logistic Regression model to analyse the results obtained after spiking milk samples with mixtures containing different percentages of viable/dead cells, we were able to assess the probability of the viability status of MAP found in milk. This model was applied to contaminated pasteurized milk to ascertain the efficacy of heat treatment in MAP killing. The method reported herein can potentially be used for direct detection of MAP viability in milk. This article is protected by copyright. All rights reserved.FEMS Microbiology Letters 05/2014; 356(1). DOI:10.1111/1574-6968.12480 · 2.72 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Two mechanical lysis devices have been developed as compact, robust components to provide rapid sample preparation for nucleic acid diagnostic systems. One such component, known as the Micro Bead-Beater (TM) (mu BB (TM), BBTM, Claremont BioSolutions, Upland, CA), is a compact device that is capable of ultra-rapid lysis (>90% lysis in 30 s) of micro volumes (<80 mu L) of Bacillus spores in a continuous-flow format or in a disposable single-tube format. The mu BB is also capable of processing much larger volumes of solutions containing spores or vegetative cells using a continuous-flow mode. A second mechanical lysis device designed as a disposable component is the microfluidic bead blender, which uses a small electric motor to spin vanes within the bead-laden solution. DNA quantification results using dsDNA-binding fluorescence dyes and real-time PCR are presented, comparing the lysis of Bacillus subtilis spores using the mu BB (TM) with other well-known lysis techniques. Nanoscale imaging results obtained using scanning electron microscopy and transmission electron microscopy on B. subtilis spores lyzed using the mu BB (TM) are also presented. (JALA 2009;14:119-25)Journal of the Association for Laboratory Automation 06/2009; 14(3):119-125. DOI:10.1016/j.jala.2009.02.010 · 1.50 Impact Factor