Open AccessA molecular beacon‐based real‐time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk
Author(s) -
RodríguezLázaro David,
Lloyd Joy,
Herrewegh Arnold,
Ikonomopoulos John,
D'Agostino Martin,
Pla Maria,
Cook Nigel
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2004.tb09686.x
Subject(s) - paratuberculosis , mycobacterium , microbiology and biotechnology , biology , nucleic acid , molecular beacon , centrifugation , bacteria , nasba , virology , chromatography , chemistry , dna , gene , biochemistry , rna , oligonucleotide , genetics
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 .