Determination of fish origin by using 16S rDNA fingerprinting of bacterial communities by PCR-DGGE: An application on Pangasius fish from Viet Nam

Determination of citrus fruit origin by using 16S rDNA fingerprinting of bacterial communities by PCR- DGGE: an application to clementine from Morocco and Spain

“…DNA extraction of the bacterial community was done on the skin of the clementines using the method of Leesing [6] improved by Le Nguyen et al [7], which has been successfully applied to fish bacteria. We verified the extraction efficiency with a 2% (w/v) agarose gel.…”

“…The V3 variable region of bacterial 16S rDNA of the bacterial flora of the fruit was specifically amplified using the primers gc338f (5'CGCCCGCCGCGCGCGGCGGGCGGGG-CGGGGGCACGGGGGGACTCCTACGGGA-GGCAGCAG, Sigma, France) and 518r (5'-ATTACCGCGGCTGCTGG, Sigma, France) [6,7,11,12] was added to the forward primers in order to insure that the fragment of DNA would remain partially doublestranded and that the region screened was in the lowest melting domain [18]. Each mixture (final volume 50 µL) contained about 100 ng of template DNA, 0.2 µM for all the primers, 200 µM for all the deoxyribonucleotide triphosphate (dNTPs), 1.5 mM MgCl 2 , 5 µL of 10x of reaction Tag buffer (MgCl 2 free) (Promega, France) and 5U of Taq polymerase (Promega, France).…”

“…Each mixture (final volume 50 µL) contained about 100 ng of template DNA, 0.2 µM for all the primers, 200 µM for all the deoxyribonucleotide triphosphate (dNTPs), 1.5 mM MgCl 2 , 5 µL of 10x of reaction Tag buffer (MgCl 2 free) (Promega, France) and 5U of Taq polymerase (Promega, France). In order to increase the specificity of amplification and to reduce the formation of spurious byproducts, a "touchdown" PCR was performed [6,7,13]. An initial denaturation at 94°C for 1 min and 10 touchdown cycles of denaturation at 94°C for 1 min, then annealing at 65°C (with the temperature decreasing 1°C per cycle) for 1 min, and extension at 72°C for 3 min, followed 20 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 3 min.…”

“…The PCR products were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) by using a Bio-Rad DCode TM universal mutation detection system (Bio-Rad Laboratories, Hercules, USA) and the procedure first described by Muyzer et al [12] and improved by Leesing [6] and Le Nguyen et al [7]. Samples containing approximately equal amounts of PCR amplicons were loaded into 8% (w/v) polyacrylamide gels (acrylamide/NN'-methylene bisacrylamide, 37.5:1, Promega, France) in 1X TAE buffer (40 mM tris-HCl pH 7.4, 20 mM sodium acetate, 1.0 mM Na 2 -EDTA).…”

“…Our study relates to the determination of bacterial DNA present on clementines that come from different areas, by the method of PCR-DGGE adapted from the method developed by our team to determine the geographical origin of fish [6,7].…”