A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis
Journal of Veterinary Science
;
: 233-239, 2014.
Artigo
em Inglês
| WPRIM
| ID: wpr-191845
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Paratuberculose
/
DNA Bacteriano
/
Doenças dos Bovinos
/
Reprodutibilidade dos Testes
/
Técnicas Bacteriológicas
/
Reação em Cadeia da Polimerase em Tempo Real
Tipo de estudo:
Avaliação Econômica em Saúde
Limite:
Animais
Idioma:
Inglês
Revista:
Journal of Veterinary Science
Ano de publicação:
2014
Tipo de documento:
Artigo
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