A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk.
A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. 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(4) M. avium subsp. paratuberculosis cells 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 subsp. paratuberculosis.
Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.
David
D
Rodríguez-Lázaro
Joy
J
Lloyd
Arnold
A
Herrewegh
John
J
Ikonomopoulos
Martin
M
D'Agostino
Maria
M
Pla
Nigel
N
Cook
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics; D005516:Food Microbiology; D005618:Fresh Water / Q000382:microbiology; D005798:Genes, Bacterial; D008892:Milk / Q000382:microbiology; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000302:isolation & purification*; D011786:Quality Control; D021161:Self-Sustained Sequence Replication; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria