发酵床垫料中微生物16S rDNA PCR反应条件的建立与优化

为探讨发酵床垫料中微生物16S rDNA基因扩增实验中诸多因素对实验结果的影响,采用单因素法对16S rDNA基因扩增时PCR反应体系中的5个潜在因素（Mg^2＋、dNTP、引物、Taq酶、模板DNA）5水平上进行优化实验,并进一步优化了反应程序中的退火温度、循环次数。结果表明：25μL的最佳反应体系为：10×PCR buffer 2.5μL、MgCl22.0 mmol·L^-1、dNTP 0.2 mmol·L^-1、引物0.2μmol·L^-1、Taq DNA聚合酶0.5 U、模板DNA 50 ng;最佳反应程序为：在94℃下进行4 min预变性;随后循环扩增25个循环（包括94℃变性45 s,53.7℃退火30 s,72℃延伸90 s）;最后在72℃下延伸10 min。

Establishment and Optimization of Microbial 16S rDNA PCR Reaction Conditions in Fermentation Bed Padding Material

In order to study the influence of several potential factors on 16 S rDNA PCR reaction conditions in fermentation bed padding material, five factors（Mg^2＋, dNTP, primer, Taq DNA polymerase and DNA template）were optimized by single factor experiment at 5 concentration levels. Annealing temperatures and amplification cycle numbers were also optimized. The results showed that the optimum 25 μL reaction system comprised 10×PCR buffer 25 μL, MgCl22.0 mmol·L^-1, dNTP 0.2 mmol·L^-1, primer 0.2 μmol·L^-1, Taq DNA polymerase 0.5 U and DNA template 50 ng; and the optimum PCR procedure was as an initial pre-denaturing at 94 ℃ for 4 min, which preceded 25 cycles of denaturing at 94 ℃ for 45 s, annealing at 53.7 ℃ for 30 s, and extension at 72 ℃ for 90 s, with a final extension at 72 ℃ for 10 min.