The tubes were incubated at 37 ☌ for 2 h in water bath HAAKE SC 150 L (Thermo Scientific, Marietta, OH, USA). , as follows: 500 μL of 50 mg/mL of Lysozyme from chicken egg white (Sigma-Aldrich, Milan, Italy), 1 M of sorbitol (Sigma-Aldrich, Milan, Italy) and 0.1 M EDTA (Invitrogen Thermo Fisher, Milan, Italy), pH 7.4 were added to the cellular pellet. , modified as reported in Iacumin, et al. A sample (1 mL) of each culture was centrifuged at 14,000× g for 10 min at 4 ☌, and the pellet was subjected to DNA extraction according to Querol, et al. įor the first experiment (i) standard strains of lactic acid bacteria were cultured overnight at 30 ☌ in De Man, Rogosa e Sharpe broth (MRS, Oxoid, Milan, Italy). Although this innovative automation of the system allows efficient process management, which reduces the risk of laboratory errors or cross-contamination, it does not currently allow population studies, for which further processing of the generated amplicons are essential. After the ePCR reaction, obtained by microfluidics technology, the droplets stream individually pass through the reader for fluorescence analysis. This last evolution, also called droplet digital PCR (ddPCR), is considered to be the third generation of PCR. In fact, ePCR has been already used to increase the PCR sensitivity in many research fields, in which an absolute quantification of a single target nucleic acid was needed, such as detection of virus, genetically modified organisms, bacterial pathogens, adhesion studies in biofilm structures, and single mutation detection. This method is becoming a powerful technique for high-throughput assays in chemistry and biology. The principle is to disperse the template DNA molecules in a thermo-stable water-in-oil emulsion at a concentration where, statistically, a few droplets contain more than one gene amplified in situ by PCR to give >10 3 copies in each droplet. Nakano demonstrated that the water in oil ( w/ o) emulsion PCR (ePCR) method can solve many biases of traditional PCR, among all the compartmentalization of template DNA molecules reducing the competition between fragments of different lengths and in different concentrations, thus diminishing the bias for amplifying smaller fragments and increasing the concentration of the initial template DNA even if the number of templates is very limited and the segregation of template DNA molecules in each droplet of the emulsion prevents recombination between homologous or partially homologous gene fragments during PCR, thus eliminating the synthesis of short, chimeric products and other artefacts. In addition, data analysis and verification of sequencing data are a crucial point, which may generate artificial results on the base of imprecise data modulations. The costs for HTS may differ across countries or depend on direct access to a sequencing unit, which are expensive and require extensive expertise. However, the costs for HTS still remain high in comparison to ePCR/DGGE analysis. Over the last two decades, HTS technologies became ubiquitous in microbial ecology studies, in part due to the progressive reduction of costs. ![]() reported that mixed amplicons obtained from complex ecosystems can be sequenced and reveal novel microbial fingerprints of so far cryptic populations (identified as operational taxonomic units). More recently, high-throughput sequencing (HTS) technologies have been demonstrated to be a powerful approach to study microbial biodiversity in a wide range of environments, including food. ![]() When preferential PCR amplification occurs by using extracted metagenomic bacterial DNA from complex communities, certain species may remain undetected by DGGE. Differential or preferential amplification of rDNA genes by PCR was reported by Reysenbach and Suzuki and Giovannoni. Not all of the present species have similar sensitivities, and the different amounts of the species present in the sample can affect the concentration and detectability of the extracted DNA. However, it has some disadvantages, many of which introduced by Polymerase Chain Reaction PCR itself, and it can carry bias. ![]() , has been widely and successfully used to study microbial communities in food, making it a well-established tool for investigating microbial ecology. Denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electophoresis (TGGE), introduced by Muyzer, et al. Conventional methods for microbial enumeration, identification, and characterization are insufficient for monitoring specific species in complex, mixed-species microbial communities.
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