Improved template DNA preparation procedure for detection of Mycobacterium avium subsp. paratuberculosis in milk by PCR. 2007

Anli Gao, and Lucy Mutharia, and Melinda Raymond, and Joseph Odumeru
Laboratory Services Division (Gao, Odumeru), University of Guelph, 95 Stone Rd W, Guelph, ON Canada N1H 8J7.

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 <or=1 CFU/ml.

UI MeSH Term Description Entries
D007091 Image Processing, Computer-Assisted A technique of inputting two-dimensional or three-dimensional images into a computer and then enhancing or analyzing the imagery into a form that is more useful to the human observer. Biomedical Image Processing,Computer-Assisted Image Processing,Digital Image Processing,Image Analysis, Computer-Assisted,Image Reconstruction,Medical Image Processing,Analysis, Computer-Assisted Image,Computer-Assisted Image Analysis,Computer Assisted Image Analysis,Computer Assisted Image Processing,Computer-Assisted Image Analyses,Image Analyses, Computer-Assisted,Image Analysis, Computer Assisted,Image Processing, Biomedical,Image Processing, Computer Assisted,Image Processing, Digital,Image Processing, Medical,Image Processings, Medical,Image Reconstructions,Medical Image Processings,Processing, Biomedical Image,Processing, Digital Image,Processing, Medical Image,Processings, Digital Image,Processings, Medical Image,Reconstruction, Image,Reconstructions, Image
D008892 Milk The off-white liquid secreted by the mammary glands of humans and other mammals. It contains proteins, sugar, lipids, vitamins, and minerals. Cow Milk,Cow's Milk,Milk, Cow,Milk, Cow's
D010283 Paratuberculosis A chronic GASTROENTERITIS in RUMINANTS caused by MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS. Johne's Disease,Johne Disease,Disease, Johne,Disease, Johne's,Johnes Disease,Paratuberculoses
D002417 Cattle Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor. Beef Cow,Bos grunniens,Bos indicus,Bos indicus Cattle,Bos taurus,Cow,Cow, Domestic,Dairy Cow,Holstein Cow,Indicine Cattle,Taurine Cattle,Taurus Cattle,Yak,Zebu,Beef Cows,Bos indicus Cattles,Cattle, Bos indicus,Cattle, Indicine,Cattle, Taurine,Cattle, Taurus,Cattles, Bos indicus,Cattles, Indicine,Cattles, Taurine,Cattles, Taurus,Cow, Beef,Cow, Dairy,Cow, Holstein,Cows,Dairy Cows,Domestic Cow,Domestic Cows,Indicine Cattles,Taurine Cattles,Taurus Cattles,Yaks,Zebus
D002418 Cattle Diseases Diseases of domestic cattle of the genus Bos. It includes diseases of cows, yaks, and zebus. Bovine Diseases,Bovine Disease,Cattle Disease,Disease, Bovine,Disease, Cattle,Diseases, Bovine,Diseases, Cattle
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016927 Mycobacterium avium subsp. paratuberculosis A subspecies of gram-positive, aerobic bacteria. It is the etiologic agent of Johne's disease (PARATUBERCULOSIS), a chronic GASTROENTERITIS in RUMINANTS. Mycobacterium paratuberculosis,Mycobacterium avium paratuberculosis,Mycobacterium avium subspecies paratuberculosis

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