副结核
分枝杆菌
微生物学
生物
核酸
分子信标
离心
细菌
纳斯巴
病毒学
分子生物学
色谱法
化学
DNA
基因
生物化学
核糖核酸
寡核苷酸
遗传学
作者
David Rodríguez‐Lázaro,Joy Lloyd,Arnold Herrewegh,John Ikonomopoulos,Martin D’Agostino,María Pla,Nigel Cook
出处
期刊:Fems Microbiology Letters
[Oxford University Press]
日期:2004-08-01
卷期号:237 (1): 119-126
被引量:44
标识
DOI:10.1111/j.1574-6968.2004.tb09686.x
摘要
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.
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