title: Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.
abstract: 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.
authors.affiliation: Department of Public Health and Microbiology, Virology Unit, Turin University, Italy. francesca.sidoti@unito.it
authors.forename: Francesca
authors.initials: F
authors.lastname: Sidoti
authors.forename: Giuliana
authors.initials: G
authors.lastname: Banche
authors.forename: Sara
authors.initials: S
authors.lastname: Astegiano
authors.forename: Valeria
authors.initials: V
authors.lastname: Allizond
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Cuffini
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bergallo
mesh_terms: D000328:Adult; D000368:Aged; D003424:Crohn Disease / Q000382:microbiology; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial