松江湿地沉积物细菌T-RFLP分析中PCR体系的建立与优化

Songjiang Wetland Sediment Bacteria Established in the T-RFLP Analysis and Optimization of PCR System

细菌是微生物的重要组成部分,其对保持湿地生态系统平衡起着重要作用,在沉积物细菌T-RFLP(terminal restriction fragment length polymorphism)分析中建立与优化PCR体系对进一步研究沉积物细菌群落组成具有重要意义。此次研究以松江湿地沉积物细菌基因组DNA为模板,采用正交和单因素试验对PCR体系中各因素(模板DNA、Mg2+、dNTPs、引物、Taq酶)进行优化,系统分析各因素对PCR扩增结果的影响,建立最佳PCR反应体系。在20μL反应体系中,DNA模板30 ng、Mg2+2.0 mmol/L、dNTPs 0.2 mmol/L、引物0.2μmol/L、Taq酶0.5 U。该优化体系确保了沉积物细菌T-RFLP分析过程中PCR产物的质量与效果。

Bacteria are an important part of microorganisms, which play an important role to maintain the balance of wetland ecosystems. Establishment and optimization of PCR system in the T-RFLP analyzing of sediment bacteria is important for studying sediment bacterial community composition in the sediment. Taking Songjiang wetland sediment bacterial genomic DNA as template, orthogonal and single factor test was used to do the system optimization for PCR system factors（template DNA, Mg^2＋, dNTPs, primers, Taq enzyme）. Then we analyzed the influence of various factors on PCR amplification and established the best PCR reaction system. In 20 μL reaction system, DNA template, Mg^2＋, dNTPs, primers, Taq enzyme were 30 ng,2.0 mmol/L, 0.2 mmol/L, 0.2 μmol/L, and 0.5 U, respectively. The optimization system is able to ensure the quality and effectiveness of PCR products of the sediment bacterial T-RFLP analysis.