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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process
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Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process

机译：通过原位PCR直接检测脱氮过程中生物膜中的amoA基因

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摘要

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.
机译：氨氧化是从废水中生物脱氮的限速步骤。具有amoA基因的微生物群落的分析对于控制氮的去除很重要，该基因是编码氨单加氧酶的基因的一个小亚基。在这项研究中，通过原位PCR扩增了纯培养物和亚硝化反应器中生物膜中存在于欧洲亚硝化单胞菌细胞中的amoA基因。在该程序中，用溶菌酶使固定的细胞通透，并用洋地黄毒苷标记的引物进行半巢式PCR。然后，用碱性磷酸酶标记的抗洋地黄毒苷抗体和HNPP（2-羟基-3-萘甲酸-2'-苯胺磷酸酯）与Fast Red TR结合，并与Alexa Fluor 488标记的抗洋地黄毒苷一起检测扩增子。抗体。当使用前一种方法进行检测时，会以不可避免的非特异性信号检测生物膜中的amoA基因。另一方面，当使用Alexa Fluor 488标记的抗地高辛配基抗体进行检测时，在生物膜中检测到的amoA基因没有非特异性信号，并且在生物膜表面附近清楚地观察到了具有amoA基因的细胞。尽管在这项研究中未检测到功能性基因表达，但已证明可以根据其功能检测生物膜中的细胞。

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