Establishment of a Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis (PCR-DGGE) Method to Analyze Bacteria from Xiaoqu of Soybean-flavor Liquor
Article
Figures
Metrics
Preview PDF
Reference
Related
Cited by
Materials
Abstract:
The bacterial populations in Xiaoqu of soybean-flavor liquor were used as the study object in this paper, and the technical parameters related to DNA extraction, polymerase chain reaction (PCR) amplification, denaturing gradient gel electrophoresis (DGGE) time, and staining protoco for PCR-DGGE analysis were compared and optimized. The results revealed that among four DNA extraction methods (kit, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and SDS-CTAB), the SDS-CTAB method produced the highest DNA yield with a low protein content and good integrity. Despite the slightly complicated steps, SDS-CTAB was better than other methods. After optimization was achieved using the uniform design method, the optimal PCR conditions were as follows: annealing temperature of 50℃, primer concentration of 0.4 μmol/L, and 2.5 μL (34 ng) of template DNA. Under these optimal conditions, the clearest bands were obtained, the yield of products was the highest, and the DNA bands in the corresponding DGGE analysis exhibited the best diversity and abundance. The performance of DGGE with different run times was compared using time-travel experiments, and the results showed that when the electrophoresis was conducted for nine hours using a denaturing gradient concentration range of 30%~60% at 85 V and a temperature of 60 ℃, the DNA bands on the DGGE gel were sufficiently separated with an appropriate distribution. Additionally, it was found that silver staining was better than Goldview staining. In summary, a PCR-DGGE method to analyze the microorganisms in Xiaoqu of soybean-flavor liquor was preliminarily established.
