Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk

Development of a PCR method for detecting proteolytic psychrotrophic bacteria in raw milk

“…PCR was carried out using the primers FP aprI/RP aprII (Bach, Hartmann, Schloter, & Munch, 2001;Martins et al, 2005), SM2F/SM3R (Marchand et al, 2009b) and 16SPSEfluF/16SPSER (Scarpellini et al, 2004) Table 1 Polymerase chain reaction (PCR) primer sets used in this study. Table 1).…”

“…The detection of P. fluorescens using polymerase chain reaction (PCR) to amplify the aprX gene, which encodes a metalloprotease, is an alternative method for the assessment of raw milk (Martins, de Araújo, Mantovani, Moraes, & Vanetti, 2005;Vanetti, 2009). The PCR detection limit was reported to be 10 6 cfu mL À1 for the amplification of a 194 bp fragment of the aprX gene from P. fluorescens (Martins et al, 2005). However, the detection limit when amplifying an approximately 900 bp fragment from the same gene was reported to be 10 2 cfu mL À1 (Marchand et al, 2009b).…”

Characterising the genetic diversity of Pseudomonas syringae pv. syringae isolated from rice and wheat in Iran

“…DNA extraction. DNA was extracted from the strains that were grown overnight in nutrient broth (Merck, Darmstadt, Germany) at 26°C using the protocols described by Martins et al (2005). One tube of 1.5 ml was used to centrifuge the cells at 13 000× g for 5 min and the pellet was suspended in 200 ml Tris 0.1 mol/l and added 200 ml of lysis solution (NaOH 0.2N and 1% SDS), mixed and deproteinased with 700 ml of phenol/chloroform/ isoamyl alcohol (25:24:1 v/v/v), homogenised and centrifuged at 13 000× g for 10 minutes.…”

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“…For bacterial DNA extraction, 8 isolates were grown overnight in nutrient broth (Merck, Darmstadt, Germany) at 26 °C and the DNA was extracted as described 12 . The cells were pelleted by centrifugation at 13,000 g for 5 min and the pellet was resuspended in 200 µl each of Tris 0.1 mM and lysis solution (NaOH 0.2 N and 1% SDS).…”