Dataset Information

Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies.

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.

SUBMITTER: Iacumin L

PROVIDER: S-EPMC7465085 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies.

Iacumin Lucilla L Cecchini Francesca F Vendrame Marco M Comi Giuseppe G

Microorganisms 20200723 8

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 D ...[more]

PMID: 32717823

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Project description:Daqu made from raw wheat, barley or pea is used as an inoculum for the fermentation of Chinese Baijiu. In this study, the microbial communities of four different types of Daqus (sauce-flavor Wuling Daqu, sauce and strong-flavor Baisha Daqu, strong-flavor Deshan Daqu, and light-flavor Niulanshan Daqu) were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), phospholipid fatty acid (PLFA) analysis, and Biolog EcoPlates analysis (Biolog). Clear differences were seen between the microbial communities of the four Daqus. PCR-DGGE showed differences in the number and brightness of bands between the Daqus, indicating the presence of unique bacterial species in Deshan Daqu, Wuling Daqu, and Niulanshan Daqu. Lactobacillus sanfranciscensis, Bacillus thermoamylovorans, and some unclassified bacteria were unique to Wuling Daqu, Deshan Daqu, and Niulanshan Daqu, respectively. Moreover, some bacterial species were observed in all four Daqus. A total of 26 PLFAs between C12 to C20 were detected from the four Daqus by PLFA analysis. Wuling Daqu had the highest total and fungal biomasses, Baisha Daqu had the highest bacterial biomass, and Niulanshan Daqu had the highest ratio of fungal biomass to bacterial biomass. The Biolog results indicated differences in the carbon source use and mode of the four Daqus, and also demonstrated that each Daqu had varying abilities to utilize different types of carbon sources. The cluster analysis of the three methods showed that the microbial communities of the four Daqus were different. This study also demonstrates the applicability of the three analytical methods in the evaluating of the microbial communities of Daqus.Daqu made from raw wheat, barley or pea is used as an inoculum for the fermentation of Chinese Baijiu. In this study, the microbial communities of four different types of Daqus (sauce-flavor Wuling Daqu, sauce and strong-flavor Baisha Daqu, strong-flavor Deshan Daqu, and light-flavor Niulanshan Daqu) were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), phospholipid fatty acid (PLFA) analysis, and Biolog EcoPlates analysis (Biolog). Clear differences were seen between the microbial communities of the four Daqus. PCR-DGGE showed differences in the number and brightness of bands between the Daqus, indicating the presence of unique bacterial species in Deshan Daqu, Wuling Daqu, and Niulanshan Daqu. Lactobacillus sanfranciscensis, Bacillus thermoamylovorans, and some unclassified bacteria were unique to Wuling Daqu, Deshan Daqu, and Niulanshan Daqu, respectively. Moreover, some bacterial species were observed in all four Daqus. A total of 26 PLFAs between C12 to C20 were detected from the four Daqus by PLFA analysis. Wuling Daqu had the highest total and fungal biomasses, Baisha Daqu had the highest bacterial biomass, and Niulanshan Daqu had the highest ratio of fungal biomass to bacterial biomass. The Biolog results indicated differences in the carbon source use and mode of the four Daqus, and also demonstrated that each Daqu had varying abilities to utilize different types of carbon sources. The cluster analysis of the three methods showed that the microbial communities of the four Daqus were different. This study also demonstrates the applicability of the three analytical methods in the evaluating of the microbial communities of Daqus.

Project description:BACKGROUND: Contamination of grains with trichothecene mycotoxins, especially deoxynivalenol (DON), has been an ongoing problem for Canada and many other countries. Mycotoxin contamination creates food safety risks, reduces grain market values, threatens livestock industries, and limits agricultural produce exports. DON is a secondary metabolite produced by some Fusarium species of fungi. To date, there is a lack of effective and economical methods to significantly reduce the levels of trichothecene mycotoxins in food and feed, including the efforts to breed Fusarium pathogen-resistant crops and chemical/physical treatments to remove the mycotoxins. Biological approaches, such as the use of microorganisms to convert the toxins to non- or less toxic compounds, have become a preferred choice recently due to their high specificity, efficacy, and environmental soundness. However, such approaches are often limited by the availability of microbial agents with the ability to detoxify the mycotoxins. In the present study, an approach with PCR-DGGE guided microbial selection was developed and used to isolate DON -transforming bacteria from chicken intestines, which resulted in the successful isolation of several bacterial isolates that demonstrated the function to transform DON to its de-epoxy form, deepoxy-4-deoxynivalenol (DOM-1), a product much less toxic than DON. RESULTS: The use of conventional microbiological selection strategies guided by PCR-DGGE (denaturing gradient gel electrophoresis) bacterial profiles for isolating DON-transforming bacteria has significantly increased the efficiency of the bacterial selection. Ten isolates were identified and isolated from chicken intestines. They were all able to transform DON to DOM-1. Most isolates were potent in transforming DON and the activity was stable during subculturing. Sequence data of partial 16S rRNA genes indicate that the ten isolates belong to four different bacterial groups, Clostridiales, Anaerofilum, Collinsella, and Bacillus. CONCLUSIONS: The approach with PCR-DGGE guided microbial selection was effective in isolating DON-transforming bacteria and the obtained bacterial isolates were able to transform DON.

Dataset Information

Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies.

Publications

Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies.

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