本研究以豉香型白酒酒饼中的细菌类群为对象，针对PCR-DGGE分析方法中DNA提取、PCR扩增、DGGE电泳时间和凝胶染色方法等重要环节开展了相关技术参数的比较和优化。结果显示：试剂盒法、CTAB法、SDS法和SDS-CTAB结合法等四种DNA提取方法中，SDS-CTAB结合法提取的DNA得率最高，蛋白质去除干净，完整性好，虽然步骤稍繁琐，但优于其他方法；经均匀设计法优化后，PCR反应中退火温度为50 ℃，引物浓度为0.4 μmol/L，模板2.5 μL（约34 ng）时所扩增出的条带最清晰、产物量最高，对应的DGGE分析中DNA条带的多样度和丰度最佳；以进程法比较了不同时长的DGGE电泳效果，发现在变性剂梯度范围为30%~60%、电压85 V、温度60 ℃条件下，电泳9 h DGGE胶中的DNA条带分离充分，分布位置适中；同时还发现，利用银染色法对胶进行染色，效果优于Goldview染色法。综合上述因素，初步建立了豉香型白酒酒饼微生物PCR-DGGE技术的分析方法。

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.

科技部农业科技成果转化资金项目（2012E0002005）；广东省高等教育教学改革项目（GDJG20142097）；广东省自然科学基金项目（2015A030313401）