title: A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis.
abstract: PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.
authors.affiliation: Department of Biochemistry and Microbiology, Faculty of Sciences, Austral University of Chile, Valdivia, Chile. miguelsalgado@uach.cl.
authors.forename: Miguel
authors.initials: M
authors.lastname: Salgado
authors.forename: Cristobal
authors.initials: C
authors.lastname: Verdugo
authors.forename: Cord
authors.initials: C
authors.lastname: Heuer
authors.forename: Pedro
authors.initials: P
authors.lastname: Castillo
authors.forename: Patricia
authors.initials: P
authors.lastname: Zamorano
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000191:economics / Q000662:veterinary*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005260:Female; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics*; D010283:Paratuberculosis / Q000175:diagnosis / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D015203:Reproducibility of Results
chemical_list: D004269:DNA, Bacterial
keywords: DNA extraction; MGIT; Map; PCR; paratuberculosis