SSCP技术分析活性污泥微生物群落结构的条件优化及检验

Optimal Conditions and Validation of Single-strand Conformation Polymorphism Technology for the Analysis of Microbial Communities in Activated Sludge

摘要:

单链构象多态性(SSCP) 技术是一种分析环境微生物遗传多态性的有效方法, 具有快速、简便、灵敏等特点。但是, 该技术用于分析复杂环境微生物群落结构时需要有针对性地优化操作条件。本研究针对聚丙烯酰胺凝胶电泳(PAGE)胶浓度、上样缓冲液中去离子甲酰胺浓度、电泳温度、电泳时间等确定了PCR-SSCP技术的优化条件, 并以自养脱硫反硝化反应器启动期的活性污泥样品对此优化条件进行了检验, 证明了16S rDNA V1~V3区为靶对象时, 丙烯酰胺与甲叉的质量比49:1, 质量分数12%的聚丙烯酰胺凝胶, 上样缓冲液中去离子甲酰胺的体积分数1/3, 在4℃, 300 V, 电泳18 h是最优的操作条件, 可以获较理想的SSCP图谱。

Abstract:

Single-Strand Conformation Polymorphism(SSCP) is an effect method for investigating environ-ment microbial genetic polymorphism, with its characterization of rapidness, simplicity, and sensitivity. However, many factors can influence the results of SSCP in the analysis of complex environment samples, and its optimization is highly needed. In this paper, optimal PCR-SSCP conditions were discussed based on PAGE concentration, formamide deionized in denaturing loading buffer, electrophoresis time and tempera-ture. The resluts showed that the optimal conditions were as follows: 16S rDNA V1~V3 was selected as the targeted gene, the ratio of acrylamide to N, N-dimethylacrylamide in 12% polyacrylamide gel electrophore-sis(PAGE)gel was 49:1, the ratio of formamide deionized in denaturing loading buffer was 1:3, running the SSCP gel at 300 V for 18 h (under 4℃). Aside from this, the validations using samples from a simultaneous desulfurification and denitrification bioreactor were conducted under this optimal conditions.