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Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis

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Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne’s disease and may contribute to the onset and development of Crohn’s disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.

Keywords: IS900 and F57 sequences; Mycobacterium avium sous-espèce paratuberculosis; Mycobacterium avium subsp. paratuberculosis; dosage par qPCR en temps réel; real-time qPCR assay; séquences IS900 et F57

Document Type: Research Article

DOI: http://dx.doi.org/10.1139/w11-022

Affiliations: 1: Department of Public Health and Microbiology, Virology Unit, Turin University, 10126 Turin, Italy. 2: Department of Public Health and Microbiology, Bacteriology and Mycology Unit, Turin University, 10126 Turin, Italy.

Publication date: May 4, 2011

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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