Orschler Laura, Agrawal Shelesh, Lackner Susanne
Institute IWAR, Technical University of Darmstadt, Franziska-Braun-Straße 7, Darmstadt, 64287, Germany.
Microb Biotechnol. 2020 Nov;13(6):2069-2076. doi: 10.1111/1751-7915.13627. Epub 2020 Jul 19.
The choice of primer and TaqMan probes to quantify ammonia-oxidizing bacteria (AOB) in environmental samples is of crucial importance. The re-evaluation of primer pairs based on current genomic sequences used for quantification of the amoA gene revealed (1) significant misrepresentations of the AOB population in environmental samples, (2) and a lack of perfect match primer pairs for Nitrosomonas europaea and Nitrosomonas eutropha. We designed two new amoA cluster 7-specific primer pairs and TaqMan probes to quantify N. europaea (nerF/nerR/nerTaq) and N. eutropha (netF/netR/netTaq). Specificity and quantification biases of the newly designed primer sets were compared with the most popular primer pair (amoA1f/amoA2r) using DNA from various AOB cultures as individual templates as well as DNA mixtures and environmental samples. Based on the qPCR results, we found that the newly designed primer pairs and the most popular one performed similarly for individual templates but differed for the DNA mixtures and environmental samples. Using the popular primer pair introduced a high underestimation of AOB in environmental samples, especially for N. eutropha. Thus, there is a strong need for more specific primers and probes to understand the occurrence and competition between N. europaea and N. eutropha in different environments.
选择用于定量环境样品中氨氧化细菌（AOB）的引物和TaqMan探针至关重要。基于当前用于amoA基因定量的基因组序列对引物对进行的重新评估显示：（1）环境样品中AOB群体存在显著的错误表征；（2）缺乏与欧洲亚硝化单胞菌和嗜养亚硝化单胞菌完全匹配的引物对。我们设计了两个新的amoA簇7特异性引物对和TaqMan探针，用于定量欧洲亚硝化单胞菌（nerF/nerR/nerTaq）和嗜养亚硝化单胞菌（netF/netR/netTaq）。使用来自各种AOB培养物的DNA作为单独模板以及DNA混合物和环境样品，将新设计引物组的特异性和定量偏差与最常用的引物对（amoA1f/amoA2r）进行比较。基于qPCR结果，我们发现新设计的引物对和最常用的引物对在单独模板上表现相似，但在DNA混合物和环境样品上存在差异。使用常用引物对会导致环境样品中AOB的严重低估现象，尤其是对于嗜养亚硝化单胞菌。因此，迫切需要更具特异性的引物和探针，以了解欧洲亚硝化单胞菌和嗜养亚硝化单胞菌在不同环境中的存在情况和竞争关系。
