高效液相色谱法提高红曲细菌分离与分析的效果

Increasing the Efficiency of the Separation and Analysis the Bacteria in Red Kojic Rice by Using High Performance Liquid Chromatography

为了探讨高效液相色谱技术(HPLC)在复杂微生态体系中微生物分离、分析的应用潜力,提高现代分子生物学技术检测的灵敏度和准确性,本文选取红曲作为模式体系,优化红曲中细菌样品的制备方法,应用HPLC技术对红曲中的生产菌株进行分离,并结合变性梯度凝胶电泳(DGGE)、细菌16SrDNA鉴定方法对色谱分离得到的菌株进行鉴定。结果显示:根据HPLC紫外检测图谱收集到5个明显的洗脱峰,其中有3个洗脱峰经DGGE分析为单一条带,16SrDNA鉴定洗脱峰分别包括环状芽孢杆菌、铜绿假单胞菌、人参土芽孢杆菌和脂肪酸芽孢杆菌。研究表明,高效液相色谱技术能够从复杂的红曲微生物样品中分离出单一的细菌,与传统PCR-DGGE技术相比,无需进行胶回收便可实现16SrDNA的直接测序,且具有准确性高,试验周期短,细菌细胞可回收等优点。色谱分离技术在下一代测序技术中的应用潜力也在探讨中。

Objectives: To eveluate the potential application of HPLC(High Performance Liquid Chromatography) to the seperation and analysis of microbe in micro-ecosystem, so that improves the sensitivity and accuracy of the techniques of modern molecular biology. Methods: Red kojic rice was selected as a model system, bacteria inside was separated and recovered by the optimized sample preparation procedure and chromatographic conditions of HPLC, then analyzed by PCR-DGGE(Denaturing Gradient Gel Electrophoresis) and identificated by 16S rDNA sequencing. Results: 5 fractions were recovered, and there were 3 fractions might be pure strains based on their electrophoretogram of PCR-DGGE. Direct sequencing of 16S rDNA of these 3 fractions contained Bacillus circulans, Bacillus ginsengihumi, Pseudomonas sp. or Alicyclobacillus respectively. Conclusions: This study highlights the HPLC technique could be a rapid, accurate and recoverable method for the separation of pure strain from Red Kojic Rice. Compared with the traditional PCR-DGGE technique, it can realize the direct sequencing of longer sequence of 16S rDNA without gel recovery. The potential application of the approach of chromatographic separation in the next generation sequencing is also discussed.