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. It has been recognized that HTS data, despite providing information at high taxonomic resolution from a wide range of samples, poorly reflect the actual relative abundance of phylotypes, casting doubt on the reliability of data interpretation. Another key point is the detection and identification of species with cell numbers of less than 10 3 cfu/g. Ultimately, methods are needed which can span from 5 up to 60 days, in particular for when ecological studies are made for the duration of food fermentation processes. For the above reasons, HTS is limited in its general application. Moreover, HTS technologies, in addition to being subject to the same bias of PCR, can be affected by some intrinsic limitations: (a) DNA is a recalcitrant molecule, which remains long time after cell death and (b) only a relative quantification of the genera and/or species is possible.
#Emulsion pcr compared verification
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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