Improved template DNA preparation procedure for detection of Mycobacterium avium subsp. paratuberculosis in milk by PCR
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Date
2007Author
Gao, Anli
Mutharia, Lucy
Raymond, Melinda
Type
ArticleLanguage
enMetadata
Show full item recordAbstract
Factors affecting the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by PCR in raw milk and their interactions were investigated. Three day old bulk tank raw milk (50 ml) samples were seeded with MAP at a level of an estimated 30 CFU/ml. Heat-treatment of raw milk before centrifugation significantly affected the partitioning of MAP in the cream, whey and pellet fractions. Based on the IS900 PCR results, MAP preferentially partitioned into the cream fraction in unheated raw milk, and into the pellet fraction in the heat-treated milk. Treatment with 0.75% hexadecylpyridinium chloride (HPC) helped collect MAP in cream fraction. Heat treatment, use of pooled cream and pellet fractions and treatment with HPC improved the detection by PCR significantly, while washing of pellets prior to DNA extraction did not. The limit of detection using our optimized procedure was an estimated 15–50 CFU in 50 ml, or ≤ 1 CFU/ml.
URI
http://www.sciencedirect.com/science/article/pii/S0167701206003137http://erepository.uonbi.ac.ke:8080/xmlui/handle/123456789/43520
Citation
Journal of Microbiological Methods Volume 69, Issue 2, May 2007, Pages 417–420Publisher
Elsevier Department of Molecular and Cellular Biology (Mutharia, Raymond), University of Guelph, Guelph, ON, Canad
Collections
- Faculty of Health Sciences (FHS) [10378]