A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis |
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| Authors: | Miguel Salgado Cristobal Verdugo Cord Heuer Pedro Castillo Patricia Zamorano |
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| Institution: | 1.Departments of Biochemistry and Microbiology, Faculty of Sciences, Austral University of Chile, Valdivia, Chile.;2.Graduate School, Faculty of Sciences, Austral University of Chile, Valdivia, Chile.;3.Epicentre, Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4474, New Zealand. |
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| 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. |
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| Keywords: | DNA extraction Map MGIT paratuberculosis PCR |
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