title: Real-time PCR-based methods for detection of Mycobacterium avium subsp. paratuberculosis in water and milk.
abstract: A real-time PCR assay for quantitative detection of Mycobacterium avium paratuberculosis has been developed. It targets and amplifies sequences from the IS900 insertion element which is specific for this bacterium, and includes an internal amplification control. The assay was tested against 18 isolates of M. avium paratuberculosis, 17 other mycobacterial strains, and 25 non-mycobacterial strains, and was fully selective. It is capable of detecting <3 genomic DNA copies with 99% probability or alternatively, using cells directly in the reaction, 12 cells can be detected with 99% probability. Using prior centrifugation, the assay was able to consistently and quantifiably detect 10(2) M. avium paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(2) M. avium paratuberculosis in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium paratuberculosis.
authors.affiliation: Institute of Food and Agricultural Technology (INTEA), University of Girona, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Rodríguez-Lázaro
authors.forename: Martin
authors.initials: M
authors.lastname: D'Agostino
authors.forename: Arnold
authors.initials: A
authors.lastname: Herrewegh
authors.forename: Maria
authors.initials: M
authors.lastname: Pla
authors.forename: Nigel
authors.initials: N
authors.lastname: Cook
authors.forename: John
authors.initials: J
authors.lastname: Ikonomopoulos
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D005618:Fresh Water / Q000382:microbiology*; D005784:Gene Amplification; D008892:Milk / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011786:Quality Control; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial