A simple and sensitive direct mRNA multiplexed detection strategy for amoA-targeted monitoring of ammonia-oxidizing activity in water environment

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 alpha-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 analyzing amoA 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 all amoA 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 mu L) 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 detected amoA 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 the amoA 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.

Automated summary

A new direct mRNA multiplexed detection strategy based on xMAP array has been developed to analyze gene expression without reverse transcription and amplification steps, showing great potential in environmental microbial detection and activity diagnosis of wastewater treatment plants. The technology enables high sensitivity and wide dynamic range detection, simplifying the process and providing accurate results.