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Miguel Salgado,Cristobal Verdugo,Cord Heuer,Pedro Castillo,Patricia Zamorano 대한수의학회 2014 Journal of Veterinary Science Vol.15 No.2
PCR is a highly accurate technique for confirming thepresence of Mycobacterium avium subsp. paratuberculosis(Map) in broth culture. In this study, a simple, efficient, andlow-cost method of harvesting DNA from Map cultured inliquid medium was developed. The proposed protocol(Universidad Austral de Chile [UACH]) was evaluated bycomparing its performance to that of two traditionaltechniques (a QIAamp DNA Stool Mini Kit andcethyltrimethylammonium bromide [CTAB] method). Theresults were statistically assessed by agreement analysis forwhich differences in the number of cycles to positive (CP)were compared by Student’s t-test for paired samples andregression analysis. Twelve out of 104 fecal pools culturedwere positive. The final PCR results for 11 samples analyzedwith the QIAamp and UACH methods or ones examinedwith the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between datafrom the CTAB and UACH methods. CP values for theUACH and CTAB techniques were not significantlydifferent, while the UACH method yielded significantlylower CP values compared to the QIAamp kit. The proposedextraction method combines reliability and efficiency withsimplicity and lower cost.