Development, validation and application of specific primers for analyzing the clostridial diversity in dark fermentation pit mud by PCR-DGGE

Bioresour Technol. 2014 Jul;163:40-7. doi: 10.1016/j.biortech.2014.04.008. Epub 2014 Apr 16.


In this study, a Clostridia-specific primer set SJ-F and SJ-R, based on the available 16S rRNA genes sequences from database, was successfully designed and authenticated by theoretical and experimental evaluations. It targeted 19 clostridial families and unclassified_Clostridia with different coverage rates. The specificity and universality of novel primer set was tested again using the dark fermentation pit mud (FPM). It was demonstrated that a total of 13 closest relatives including 12 species were affiliated with 7 clostridial genera, respectively. Compared to the well-accepted bacterial universal primer pair P2/P3, five unexpected clostridial genera including Roseburia, Tissierella, Sporanaerobacter, Alkalibacter and Halothermothrix present in the FPM were also revealed. Therefore, this study could provide a good alternative to investigate the clostridial diversity and monitor their population dynamics rapidly and efficiently in various anaerobic environments and dark fermentation systems in future.

Keywords: Biodiversity; Clostridia-specific primers; Complex microbial ecosystem; Dark fermentation pit mud; PCR-DGGE.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Base Sequence
  • Clostridium / classification*
  • Clostridium / genetics
  • DNA Primers
  • Denaturing Gradient Gel Electrophoresis / methods*
  • Fermentation*
  • Genes, Bacterial
  • Phylogeny
  • Polymerase Chain Reaction / methods*
  • Species Specificity


  • DNA Primers