Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay
文献类型： 外文期刊
作者： Zhang, Xuzhi 1 ; Yang, Qianqian 3 ; Zhang, Qingli 1 ; Jiang, Xiaoyu 1 ; Wang, Xiaochun 1 ; Li, Yang 1 ; Zhao, Jun 1 ; Qu, 1 ;
作者机构： 1.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China
2.Pilot Natl Lab Marine Sci & Technol Qingdao, Lab Marine Fisheries Sci & Food Prod Proc, Qingdao 266071, Peoples R China
3.Shanghai Ocean Univ, Coll Marine Sci, Shanghai 201306, Peoples R China
期刊名称：SCIENTIFIC REPORTS （ 影响因子：4.379； 五年影响因子：5.133 ）
ISSN： 2045-2322
年卷期： 2020 年 10 卷 1 期
页码：
收录情况： SCI
摘要： The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.
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