Abstract
To the authors’ knowledge, this is the first report of the use of emulsion-Polymerase chain reaction (e-PCR) coupled with denaturing gradient gel electrophoresis (DGGE) analysis. In the present work the effectiveness of ePCR in improving the power of the DGGE technique for microbial population studies was tested. Our results indicated that ePCR results in uniform amplification of several DNA molecules, overcoming the major limitations of conventional PCR, such as preferential amplification and DNA concentration dependence. Moreover, ePCR-DGGE resulted in higher sensitivity when compared to conventional PCR-DGGE methods used for studying microbial populations in a complex matrix. In fact, compared to conventional PCR, the DGGE profiles of ePCR products permitted the detection of a higher number of the species that were present in the tested sample.
Highlights
Conventional methods for microbial enumeration, identification, and characterization are insufficient for monitoring specific species in complex, mixed-species microbial communities
Three different experiments were performed to compare the capabilities of emulsion PCR (ePCR) and conventional PCR to amplify gene fragments from unique microbial DNA targets in the same sample by using universal 16S primers: (i) using a suspension of lactic acid bacteria (LAB) DNAs extracted from pure cultures, at various concentration ratios; (ii) using DNA extracted from bacterial cells collected in bulk from agar plates after traditional microbiological sampling of fermented sausages; (iii) using DNA directly extracted from sourdough, fermented sausages and food-grade starch
As Stolovitzky and Cecchi [35] demonstrated, longer molecules will be affected by a decrease in efficiency before the shorter one; the guanine-plus-cytosine (G + C) content conditions the efficiency of PCR, and this is important when PCR is applied to microbial populations, in which genomes vary widely, resulting in preferential amplification of templates with low G + C content [16,17,35,36]
Summary
Conventional methods for microbial enumeration, identification, and characterization are insufficient for monitoring specific species in complex, mixed-species microbial communities. Denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electophoresis (TGGE), introduced by Muyzer, et al [1], has been widely and successfully used to study microbial communities in food, making it a well-established tool for investigating microbial ecology [2,3,4,5,6,7,8,9,10,11,12] It has some disadvantages, many of which introduced by Polymerase Chain Reaction PCR itself, and it can carry bias. Data analysis and verification of sequencing data are a crucial point, which may generate artificial results on the base of imprecise data modulations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
