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.

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

SIDOTI, Francesca;BANCHE, Giuliana;ALLIZOND, VALERIA;CUFFINI, Annamaria;BERGALLO, Massimiliano
2011

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.
57
5
347
354
IS900 and F57 sequences; Mycobacterium avium subsp. paratuberculosis; Real-time qPCR assay
Sidoti F; Banche G; Astegiano S; Allizond V; Cuffini AM; Bergallo M
File in questo prodotto:
File Dimensione Formato  
CanJMicrobiol2011.pdf

Accesso aperto

Descrizione: Articolo principale
Tipo di file: POSTPRINT (VERSIONE FINALE DELL’AUTORE)
Dimensione 168.78 kB
Formato Adobe PDF
168.78 kB Adobe PDF Visualizza/Apri
Sidoti CanJ Microbiol 2011.pdf

Accesso riservato

Descrizione: Articolo principale
Tipo di file: PDF EDITORIALE
Dimensione 102.27 kB
Formato Adobe PDF
102.27 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/95976
Citazioni
  • ???jsp.display-item.citation.pmc??? 8
  • Scopus 20
  • ???jsp.display-item.citation.isi??? 19
social impact