关键词: 分子标记, 分子标记

Abstract:

The aim was to develop a method to isolate microbial DNA from pu-erh fermentated samples. Three assays for extraction of microorganisms from pu-erh fermentation samples were compared, the DNA isolation methods were improved on the basis of HP Plant DNA Kit (omega), and the DNA quality were measured by the quantity (OD260), purity (OD260/280), and the possibility for PCR amplification of both bacterial 16SrDNA and fungi ?-tubulin genes. The results of ultraviolet spectrophotometer analysis, PCR amplification, and agarose gel electrophoresis showed that microbes were accumulated by steeping fermented tea sample (5 g) in Tween-NaCl buffer (50 mL), washing with a sonicator for 10 min, and then centrifugation (10 min, 12000 r/min); DNA was extracted employed with cell lyses by grinding with liquid nitrogen, hydrolysis with lysomyme and lyticase, and then purification using the HP Plant DNA Kit (omega). Both bacterial 16S rDNA and fungi ?-tubulin genes could be amplified using the extracted DNA as templates. A simultaneous isolation of bacterial and fungal DNA from pu-erh tea fermentation samples was developed, and this provides a basement for the molecular biology research on the microbial communities and dynamics during pu-erh tea fermentation.