Meinhardt Kelley A, Bertagnolli Anthony, Pannu Manmeet W, Strand Stuart E, Brown Sally L, Stahl David A
Civil and Environmental Engineering, University of Washington, Seattle, WA, 98105, USA.
Environ Microbiol Rep. 2015 Apr;7(2):354-63. doi: 10.1111/1758-2229.12259. Epub 2015 Feb 12.
Ammonia-oxidizing archaea (AOA) and bacteria (AOB) fill key roles in the nitrogen cycle. Thus, well-vetted methods for characterizing their distribution are essential for framing studies of their significance in natural and managed systems. Quantification of the gene coding for one subunit of the ammonia monooxygenase (amoA) by polymerase chain reaction is frequently employed to enumerate the two groups. However, variable amplification of sequence variants comprising this conserved genetic marker for ammonia oxidizers potentially compromises within- and between-system comparisons. We compared the performance of newly designed non-degenerate quantitative polymerase chain reaction primer sets to existing primer sets commonly used to quantify the amoA of AOA and AOB using a collection of plasmids and soil DNA samples. The new AOA primer set provided improved quantification of model mixtures of different amoA sequence variants and increased detection of amoA in DNA recovered from soils. Although both primer sets for the AOB provided similar results for many comparisons, the new primers demonstrated increased detection in environmental application. Thus, the new primer sets should provide a useful complement to primers now commonly used to characterize the environmental distribution of AOA and AOB.
氨氧化古菌（AOA）和细菌（AOB）在氮循环中发挥着关键作用。因此，经过充分验证的表征它们分布的方法对于开展有关其在自然和人工管理系统中重要性的研究至关重要。通过聚合酶链反应对编码氨单加氧酶（amoA）一个亚基的基因进行定量，常用于对这两类菌群进行计数。然而，包含氨氧化菌这一保守遗传标记的序列变体的可变扩增，可能会影响系统内部和系统之间的比较。我们使用一组质粒和土壤DNA样本，比较了新设计的非简并定量聚合酶链反应引物组与常用于定量AOA和AOB的amoA的现有引物组的性能。新的AOA引物组能更好地对不同amoA序列变体的模型混合物进行定量，并提高从土壤中回收的DNA中amoA的检测率。尽管AOB的两组引物在许多比较中都给出了相似的结果，但新引物在环境应用中的检测率更高。因此，新引物组应为目前常用于表征AOA和AOB环境分布的引物提供有益补充。
