Document Detail

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.
MedLine Citation:
PMID:  18694788     Owner:  NLM     Status:  MEDLINE    
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.
David Herthnek; Søren Saxmose Nielsen; Ann Lindberg; Göran Bölske
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-07-20
Journal Detail:
Title:  Journal of microbiological methods     Volume:  75     ISSN:  0167-7012     ISO Abbreviation:  J. Microbiol. Methods     Publication Date:  2008 Oct 
Date Detail:
Created Date:  2008-09-08     Completed Date:  2008-11-24     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8306883     Medline TA:  J Microbiol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  335-40     Citation Subset:  IM    
National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden.
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MeSH Terms
Culture Media
DNA, Bacterial / analysis,  isolation & purification*
Feces / microbiology
Immunomagnetic Separation / methods
Milk / chemistry,  microbiology*
Mycobacterium avium subsp. paratuberculosis / genetics,  isolation & purification*
Paratuberculosis / microbiology*
Polymerase Chain Reaction / methods*
Reg. No./Substance:
0/Culture Media; 0/DNA, Bacterial

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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