Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

Abstract

Aims: To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). Methods and Results: Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10 1 -10 6 CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. Conclusions: When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10 3 and 10 1 CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. Significance and Impact of the Study: This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.

Laser-Assisted Synthesis of Superparamagnetic Fe@Au Core−Shell Nanoparticles

“…The magnetic Fe core of the Fe@Au nanoparticles is a single domain; its average size is significantly larger (see below) than that of the original Fe nanoparticles produced through thermal decomposition of Fe(CO) 5 , which was determined by electron microscopy to be equal to 7.8 nm (Figure 2). Panels a and b of Figure 3 show TEM micrographs and corresponding particle diameter histograms for the magnetically extracted Fe-containing nanoparticles before and after acid treatment, respectively.…”

“…Monodispersed Fe nanoparticles were prepared under nitrogen atmosphere via thermal decomposition of iron pentacarbonyl, Fe(CO) 5 . 17 In a typical homogeneous nucleation synthesis of ∼8-nm-diameter particles, 2.28 g of oleic acid (OA) was stirred in 20 mL of octyl ether, the solution was heated to 40°C, and 0.3 mL of Fe(CO) 5 was injected in a 1:3 molar ratio to the OA. Following the injection, the reaction mixture became orangeyellow.…”

“…3,4 Magnetic-core gold-shell nanoparticles are of particular importance because of their potential biodiagnostic applications, such as rapid magnetic separation and concentration of biomaterials. 5 Recently, gold-coated Fe 3 O 4 magnetic core nanoparticles (MNP) have been synthesized through wet chemical methods. 6 However, pure Fe core nanoparticles are expected to be superior for such applications because of the high saturation magnetization of iron, M s ) 218 emu/g at 300 K, which is a factor of >2 larger than that of Fe 3 O 4 (93 emu/g).…”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…Caseins may also be eliminated by pronase digestion before recovery of the cells by centrifugation (Allmann et al, 1995;Furet et al, 2004;Ogier et al, 2004;Flórez and Mayo, 2006). It has been reported that the recovery of the bacterial cells may be improved by addition of polyethylene glycol during the homogenisation step (Stevens and Jaykus, 2004). A matrix lysis buffer containing urea and SDS combined with an homogenisation in a Stomacher laboratory blender has been used by Rossmanith et al (2007) to recover Gram-positive cells from various food samples, including cheeses.…”

“…Baruzzi et al (2005), Trmcic et al (2008), and Furet et al (2004) used a commercial kit in which proteins are eliminated by the use of a protein precipitation solution. Column-based or DNA-binding matrix purification methods have also been used (Rudi et al, 2005;Parayre et al, 2007;Zago et al, 2009;Le Dréan et al, 2010), sometimes as a final purification step after phenol/chloroform extraction (Stevens and Jaykus, 2004;LopezEnriquez et al, 2007). Separation of cells from the food matrix simplifies the subsequent steps of DNA extraction because most undesirable compounds such as matrix-associated reaction inhibitors are eliminated at the first step of extraction.…”