Development, validation and application of specific primers for analyzing the clostridial diversity in dark fermentation pit mud by PCR-DGGE.
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.
State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology of Ministry of Education, Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiang Su 214122, China.
Xiao-Long
XL
Hu
State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology of Ministry of Education, Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiang Su 214122, China.
Hai-Yan
HY
Wang
State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology of Ministry of Education, Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiang Su 214122, China.
Qun
Q
Wu
State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology of Ministry of Education, Synergetic Innovation Center of Food Safety and Nutrition, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiang Su 214122, China. Electronic address: yanxu1688@gmail.com.
Yan
Y
Xu
D001483:Base Sequence; D003013:Clostridium / Q000145:classification* / Q000235:genetics; D017931:DNA Primers; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods*; D005285:Fermentation; D005798:Genes, Bacterial; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity
D017931:DNA Primers
Biodiversity; Clostridia-specific primers; Complex microbial ecosystem; Dark fermentation pit mud; PCR-DGGE