A simple and sensitive direct mRNA multiplexed detection strategy for amoA-targeted monitoring of ammonia-oxidizing activity in water environment
A simple and sensitive direct mRNA multiplexed detection strategy for amoA-targeted monitoring of ammonia-oxidizing activity in water environment
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DOI：
10.1016/j.microc.2020.105794
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发表时间：
2021-03-01
影响因子：
4.8
通讯作者：
Huang，Zhen
中科院分区：
文献类型：
--
作者：
Wang，Lan;Wang，Jun;Huang，Zhen
含氮污染物是导致水体富营养化的主要原因，严重干扰了水生态系统的平衡。细菌生物脱氮是目前控制含氮污染物的主要技术。好氧氨氧化细菌（AOB）氨单加氧酶（AMO）的α亚基（amoA）是脱氮过程中的一种脱氮生物标志物。目前，RT-PCR和元转录组是分析amoA基因表达的常用方法，但这两种方法存在扩增偏倚，且耗时较长。在此，我们报告了一种新的直接mRNA多重检测策略的基础上，xMAP阵列，用于分析基因表达没有逆转录和扩增步骤。通过对数据库中allamoA基因序列的分析，我们设计了一个通用的AOB探针，覆盖率为79.8%，并通过元转录组进一步证实了其高覆盖率。此外，我们将AOB分为10个簇，然后设计每个簇的特异性探针。该技术能够使用锁定核酸探针进行单核苷酸、宽线性动态范围（5- 50，000阿莫尔）和高灵敏度（5阿莫尔，检测体积为1 µL）检测。特别地，灵敏度是迄今为止在没有扩增的多重mRNA检测中的最高水平。我们发现mRNA片段化在我们的直接检测中是不必要的，简化了我们的策略。我们成功地半定量检测了污水处理厂污泥中的damoA mRNA，并确定了优势簇，与RT-PCR和元转录组结果一致。同时，污泥的氨氧化活性验证了theamoA mRNA检测结果。综上所述，我们建立了一种直接、简便、特异、灵敏的mRNA多重检测策略，在基因表达分析、环境微生物检测和污水处理厂活性诊断等方面具有巨大的应用潜力。
Nitrogenous pollutants are the main causes of water eutrophication, which greatly interfere with the balance of the aquatic ecosystem. The biological nitrogen removal using bacteria is the leading technology to control nitrogenous pollutants. The α-subunit (amoA) of ammonia monooxygenase (AMO) of aerobic ammonia-oxidizing bacteria (AOB) is an ammonia-depleting biomarker in nitrogen removal processes. Currently, RT-PCR and metatranscriptome are the general methods for analyzingamoA gene expression, however they can introduce amplification bias and are time consuming. Herein we report a new direct mRNA multiplexed detection strategy based on xMAP array, for analyzing gene expression without reverse transcription and amplification steps. After analyzing allamoA sequences in database, we design a universal probe for AOB with coverage of 79.8% whose high coverage is further confirmed by metatranscriptome. Moreover, we classify AOB into 10 clusters, and then design the specific probes of each cluster. This technique enables single-nucleotide, wide linear dynamic range (5–50,000 amol) and high sensitivity (5 amol with detection volume of 1 µL) detection with using locked-nucleic-acid probes. Particularly, the sensitivity is by far among the highest level for multiplex mRNA detection without amplification. We found that mRNA fragmentation is not necessary in our direct detection, simplifying our strategy. We have successfully detectedamoA mRNA semi-quantitatively and determined the predominant cluster in sludge from wastewater treatment plants, which was consistent with RT-PCR and metatranscriptome results. Meanwhile, the ammonia-oxidizing activities of sludge verified theamoA mRNA detection results. In conclusion, we have developed a mRNA direct, simple, specific and sensitive multiplexed detection strategy, which shows great potential for application in gene expression analysis, environmental microbial detection and activity diagnosis of wastewater treatment plants.