Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers.
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.
Cenci-Goga
B T
BT
Department of Pathology and Microbiology, School of Medical Sciences, Bristol BS8 1TD, UK. cencigoga@unipb.it
Crotti
S
S
Costarelli
S
S
Rondini
C
C
Karama
M
M
Bennett
P
P
D000818:Animals; D004269:DNA, Bacterial / Q000302:isolation & purification*; D013293:Enterococcus faecalis / Q000235:genetics*; D005506:Food Contamination; D005784:Gene Amplification; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial