基于引物的湖泊沉积物氨氧化细菌PCR扩增策略比较

PCR扩增是检测环境中β-变形杆菌纲氨氧化细菌(β-AOB)群落的主要方法,但是引物的敏感性和特异性对结果具有关键影响.本研究首先比较了2组常用的β-AOB 16S rRNA基因引物,结果显示,在扩增一组不同性质湖泊沉积物样品时,βAMO引物均获得明亮的单一条带,但CTO引物则未能全部扩增.克隆及序列分析证实,βAMO引物扩增获得的序列均不属于β-AOB所在的Nitrosomonadales目,而CTO引物扩增获得的序列来自β-AOB中的Nitrosomonas europaea/"Nitrosococcus mobilis"分支.采用变性梯度凝胶电泳方法,对4种不同引物组合PCR策略扩增所得产物进行分析,发现以βAMO或16S rRNA通用引物结合CTO引物的巢式方案可以提高扩增的效率,且β-AOB的群落轮廓与CTO引物直接扩增方案高度相似.这些结果表明βAMO引物具有较高的敏感性但特异性较低,而CTO引物则相反.因此,特定巢式扩增方案既可提高扩增的效率,也能真实反映湖泊沉积物中β-AOB的群落轮廓,是较为理想的β-AOB研究方法.

Comparative Analysis of Primer-based PCR Strategies for Characterizing Ammonia-oxidizing Bacterial Communities in Lake Sediments

PCR-based techniques are commonly used to characterize ammonia-oxidizing bacterial (AOB) communities, but are subjected to the bias induced by PCR primers. In this study, the sensitivity and specificity of two pairs of primer were assessed based on a group of lake sediment samples. Compared to the low sensitivity of the CTO primer, the betaAMO primer produced intensive single bands in all samples tested. However, the sequences retrieved from one of the samples by betaAMO did not cluster with Nitrosomonadales, to which all beta-AOB affiliate. In contrast, all sequences amplified with CTO fell within the Nitrosomonas europaea/"Nitrosococcus mobilis" lineage. Furthermore, the products of four approaches were subjected to denaturing gradient gel electrophoresis (DGGE), and the patterns of the nested strategies with betaAMO followed CTO or the universal primer of 16S rRNA gene followed CTO were highly similar to that of CTO amplification. These findings suggest that betaAMO is not competent for charactering the beta-AOB communities due to the low specificity. The nested approaches with both selective and specific primers are the choice because they can amplify effectively and retrieve community composition of beta-AOB in lake sediments.