Lost in translation: the quest for Nitrosomonas cluster 7-specific amoA primers and TaqMan probes.,Microbial Biotechnology

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Lost in translation: the quest for Nitrosomonas cluster 7-specific amoA primers and TaqMan probes.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2020-07-19 , DOI: 10.1111/1751-7915.13627
Laura Orschler ₁ , Shelesh Agrawal ₁ , Susanne Lackner ₁

Affiliation

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.

中文翻译：

迷失在翻译中：寻找亚硝化单胞菌簇 7 特异性 amoA 引物和 TaqMan 探针。

选择引物和 TaqMan 探针来定量环境样品中的氨氧化细菌 (AOB) 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的重新评估揭示了 (1) 环境样本中 AOB 群体的显着错误表述，(2) 以及欧洲亚硝化单胞菌和富养亚硝化单胞菌缺乏完美匹配的引物对。我们设计了两个新的amoA簇 7 特异性引物对和 TaqMan 探针来定量欧洲猪笼草(nerF/nerR/nerTaq) 和富养猪笼草(netF/netR/netTaq)。使用来自各种 AOB 培养物的 DNA 作为单独模板以及 DNA 混合物和环境样本，将新设计的引物组的特异性和定量偏差与最流行的引物对 (amoA1f/amoA2r) 进行比较。根据 qPCR 结果，我们发现新设计的引物对和最常用的引物对对于单个模板的表现相似，但对于 DNA 混合物和环境样本的表现不同。使用流行的引物对导致环境样品中 AOB 的高度低估，特别是对于富养亚硝化单胞菌。因此，迫切需要更特异的引物和探针来了解不同环境中欧洲猪笼草和富养猪笼草之间的发生和竞争。

更新日期：2020-07-19

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