内参基因加标法定量土壤微生物目标基因绝对拷贝数

【目的】通过荧光定量PCR技术对土壤微生物目标基因进行绝对定量,其定量结果的准确性容易受到DNA提取得率以及腐殖酸抑制性的影响。【方法】采用内参基因加标法,利用构建的突变质粒DNA,对供试水稻土壤样品中的微生物16S r RNA目标基因的绝对拷贝数进行荧光定量PCR检测,用来表征该样品中细菌群落总体丰度。在定量前通过双向引物扩增方法验证突变质粒中的内参基因对供试土壤的特异性。【结果】不同水稻土壤样品的DNA提取量在样品间差异较大。通过内参基因加标法对DNA提取量进行校正,显著提高了16S r RNA基因绝对定量的精确度。不同水稻土壤样品间的变异系数为17.8,与未加标处理相比降低了66.7%。在此基础上,进一步通过内参基因加标法对土壤有机质和含水率均呈现典型空间特征差异的6处亚热带湿地土壤样品中的16S r RNA基因进行绝对定量。16S r RNA基因绝对拷贝数与土壤微生物生物量碳具有显著的线性相关性(R2=0.694,P0.001),表明内参校正后的16S r RNA基因绝对拷贝数可以准确反映单位质量土壤中微生物的丰度。【结论】内参基因加标法可以对DNA提取得率以及腐殖酸对PCR扩增的抑制性进行校正,从而提高绝对定量的准确性。基于内参基因加标法的目标基因绝对定量PCR检测,可作为土壤微生物生物量测量,以及微生物功能基因绝对丰度定量的一种核酸检测方法。

The absolute PCR quantification of microbial target genes in soils using internal gene as standards

Objective] Fluorescence quantitative PCR can absolutely or relatively quantify the target genes in soil samples. However, the accuracies of absolute quantification of target genes are influenced by DNA yields during DNA extraction and inhibition caused by co-extracted humic acids. Methods] Using prepared mutated plasmid DNA, this paper quantified the 16S rRNA gene from one paddy soil by the methods of adding such plasmid containing mutated internal gene before DNA extraction. Two-way primers amplification showed that the added internal gene inserted in this mutated plasmid DNA was specific to studied soils when used as internal standards. Results] The results showed that DNA yields varied greatly among these paddy soil samples, leading to incorrect results from absolute quantification. Using the mutated plasmid DNA, the precision of resulted data points has been improved significantly. It showed that the coefficient of variation for absolute 16S rRNA gene copies of these samples was 17.8, which was 66.7% lower than the values calculated for soil samples without internal gene correction. It indicates that added internal gene can correct the effects of varied DNA yields. Such tested methods were further used to quantify 16S rRNA gene from 6 wetland soils with great spatial and physic-chemical differences in soil organic matter contents and water moisture. The total bacterial abundance indicated by absolute 16S rRNA gene copies was linearly correlated (R2=0.694, p<0.001) with soil microbial biomass carbon, demonstrating that after addition of internal standards, the quantified 16S rRNA gene copies can accurately reflect the absolute microbial abundance in soil samples. Conclusion] The added internal gene can correct for DNA yields and the effects of inhibition of humic acids on PCR amplification. Using plasmid containing mutated internal gene as internal standards, this method, as a nucleic acid assay can be applied in soil ecology to quantify soil microbial biomass and absolute abundance of microbial functional genes.