Comparison of DNA extraction methods to detect Salmonella spp. from pig faeces and pork

The quality of DNA extract significantly influences the outcome of PCR-based detection methods. Performances of four different DNA extraction methods were evaluated for their ability to recover Salmonella DNA from artificially contaminated specimens. Swine faecal and pork samples were spiked with known concentration of Salmonella Typhimurium, DNA was then extracted by each of the methods considered and finally tested by a commercial Salmonella Real Time kit. The QIAgen and Adiafood kits were the most suitable methods to detect Salmonella in pig faeces. For pork samples the best performances were obtained using the Invitrogen kit. The use of appropriate DNA extraction methods is a critical issue for successful and valid PCR results and it is advisable that DNA extraction techniques are carefully selected with particular regard to the type of samples they should be used for. Introduction Salmonella is an important pathogen causing food-borne diseases and pork is recognised as one of the main sources of human infection (1,2). Salmonella Typhimurium, that is one of the main serovars involved in human illness, is frequently associated with pigs (3). Contamination of pig carcasses can occur in the slaughtering plants as a result inaccurate procedures or environmental contamination, and subsequently Salmonella may be present in pork. Benefits of having rapid, sensitive, and specific tests for the detection of Salmonella in matrices such as faeces and foodstuffs are clear, for the possibility of timely identification of infection or contamination even at very low doses. However, some challenges associated with the usage of rapid methods based on PCR still remain and a crucial one is the removal of inhibitory compounds from target DNA (4). To date, only a limited number of studies comparing the efficacy of extraction methods to perform Real Time PCR, removing reaction inhibitors from different matrices, have been carried out. Thus, this study was aimed at identifying the most effective DNA extraction methods suitable for molecular protocols specifically for faecal and pork samples. Therefore different DNA isolation kits were tested to extract Salmonella DNA from artificially contaminated specimens and their performances were evaluated by applying a Real Time PCR kit. Preliminary results allow to identify the commercial kits yielding the best DNA products useful for further molecular analysis. Material and methods Spiked faecal and pork samples Forty-four samples of pig faeces and 44 samples of pork were spiked with 3 different concentrations of S. Typhimurium (from 2 to 14 CFU/g). Each sample was prepared in triplicate. For all methods the first step was the pre-enrichment of 10 g of matrix (faeces or pork) into 90 ml Buffered Peptone Water (BPW). DNA extraction The following four extraction methods were used to extract DNA from faecal specimens, according to the manufacturers’ instructions: InviMag Stool DNA kit (Invitek, GmbH), combining the Invisorb® technology with the use of magnetic particles for isolation of nucleic acids at high purity level [1]; QIAamp DNA Stool kit (Qiagen), based on an initial step with a fast spin-column, that specifically binds DNA to the silica-gel membrane, then a secondary step where the lysis using proteinase K ensures high yields of DNA in stool [2]; Lysis reagent (iCheck-Bio-Rad), that uses a lysing solution and beads [3]; Extraction DNA mix (AES Chemunex, AdiaFood), that uses a lysing solution combined with boiling method [4]. Similarly, DNA was extracted from pork samples using four different methods: Charge Switch gDNA mini Bacteria Kit (Invitrogen, Life technologies), that allows bacterial DNA purification by using the magnetic bead-based technology and cellular lysis with proteinase K and lysozime [1], the boiling method [4], (5).Then two methods that were tested also