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Simple Assay for Colorimetric Quantification of Unamplified Bacterial 16S rRNA in Activated Sludge using Gold Nanoprobes

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Title: Simple Assay for Colorimetric Quantification of Unamplified Bacterial 16S rRNA in Activated Sludge using Gold Nanoprobes
Authors: Nakajima, Meri Browse this author
Hirano, Reiko Browse this author
Okabe, Satoshi Browse this author →KAKEN DB
Satoh, Hisashi Browse this author →KAKEN DB
Keywords: Simple colorimetric assay
Bacterial 16S rRNA
Bacterial activity
Issue Date: Jan-2021
Publisher: Elsevier
Journal Title: Chemosphere
Volume: 263
Start Page: 128331
Publisher DOI: 10.1016/j.chemosphere.2020.128331
Abstract: Domestic and industrial wastewater treatment systems are vital in the protection of natural ecosystems and human health. Identification of microbial communities in the systems is essential to stable treatment performance. However, the current tools of microbial community analysis are labor intensive and time consuming, and require expensive equipment. Therefore, we developed a simple assay for colorimetric quantification of bacterial 16S rRNA extracted from environmental samples. The assay is based on RNA extraction with commercial kits, mixing the unamplified RNA sample with Au-nanoprobes and NaCl, and analyzing the absorbance spectra. Our experimental results confirmed that the assay format was valid. By analyzing the synthesized DNA, we optimized the operational parameters affecting the assay. We achieved adequate capture DNA density by setting the capture DNA probe concentration at 10 μM during the functionalization step. The required incubation time after NaCl addition was 30 min. The binding site of the target had negligible effect on DNA detection. Under the optimized condition, a calibration curve was created using 16S rRNA extracted from activated sludge. The curve was linear above 5.0 × 107 copies/μL of bacterial 16S rRNA concentration, and the limit of detection was 1.17 × 108 copies/μL. Using the calibration curve, the bacterial 16S rRNA concentration in activated sludge samples could be quantified with deviations between 48% and 208% against those determined by RT-qPCR. The findings of our study introduce an innovative tool for the quantification of 16S rRNA concentration as the activity of key bacteria in wastewater treatment processes, achieving stable treatment performance.
Rights: © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
Type: article (author version)
Appears in Collections:工学院・工学研究院 (Graduate School of Engineering / Faculty of Engineering) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 佐藤 久

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