Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers.
J Food Prot
; 67(12): 2833-8, 2004 Dec.
Article
em En
| MEDLINE
| ID: mdl-15633698
ABSTRACT
The likelihood that milk and milk products may act as a vehicle for antibiotic-resistant bacterial genes has become a concern to the food industry and a public health issue, and the demand for rapid tests has increased. The purity of DNA extracted from food samples is a key issue in the sensitivity and usefulness of biological analyses, such as PCR for pathogens and nonpathogens. A rapid, phenol-chloroform free method based on a modification of a sodium iodide DNA extraction, followed by a two-step PCR was developed for direct detection of the tet(M) gene in milk samples within a single working day. This study compares the proposed method with a traditional phenol solvent extraction method and with a commercial kit (QIAamp DNA blood mini kit, Qiagen). The three DNA extraction methods were used to ensure access to the tet(M) gene from 1 ml of raw milk, inoculated with a strain of Enterococcus faecalis, which carries the tet(M) gene. The proposed method, followed by a two-step PCR with nested primers specific for the tet(M) gene, was able to reach a detection limit below 10 CFU/ml in less than 4 h, including the two amplification cycles, thus outperforming in sensitivity and rapidity both the traditional and the commercial method.
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Base de dados:
MEDLINE
Assunto principal:
DNA Bacteriano
/
Reação em Cadeia da Polimerase
/
Enterococcus faecalis
/
Leite
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2004
Tipo de documento:
Article