Simple assay for colorimetric quantification of unamplified bacterial 16S rRNA in activated sludge using gold nanoprobes

doi:10.1016/j.chemosphere.2020.128331

Chemosphere

Volume 263, January 2021, 128331

https://doi.org/10.1016/j.chemosphere.2020.128331 Get rights and content

Highlights

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A simple 16S rRNA quantification method with gold nanoprobes was developed.
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Reverse transcription and amplification of 16S rRNA were not necessary.
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The operational parameters affecting the assay were optimized.
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The detection limit was approximately 1% of 16S rRNA of total bacteria.
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16S rRNA in anammox could be determined from activated sludge analysis.

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.

Graphical abstract

Section snippets

Author contribution

Meri Nakajima: Data curation, Methodology, Writing - review & editing. Reiko Hirano: Data curation. Satoshi Okabe: Funding acquisition, Resources. Hisashi Satoh: Conceptualization, Methodology, Funding acquisition, Resources, Writing - original draft.

Chemicals and materials

Single-strand oligonucleotides were synthesized by Eurofins Genomics K.K. (Tokyo, Japan). The sequences of these oligonucleotides are provided in Table 1. As we targeted the 16S rRNA of total bacteria, we designed Au-nanoprobes, which are target-specific capture DNA probes conjugated to AuNPs, specific to total bacteria. Two types of Au-nanoprobes for total bacteria were synthesized (see below). One capture DNA probe was complementary to a PCR primer for the detection of total bacteria (341F),

DNA detection with Au-nanoprobes

The typical absorbance spectra of the test solutions of Au-nanoprobes designated as Probe, Au-nanoprobe solution in the presence of TGT (a positive sample), and the positive sample (abbreviated as POS sample) and Au-nanoprobe solution in the absence of Art Back (a negative sample) at 1 min after the addition of 5 M of NaCl solution were analyzed (Fig. 1). The maximum peak of the absorbance spectrum of an Au-nanoprobe solution provides the status of the Au-nanoprobes. The absorbance spectrum of

Confirmation of the sensing mechanism

To confirm the sensing mechanism, we performed detection primarily of DNA instead of 16S rRNA with the experimental setup shown in Scheme S1. The method comprises adding the sample to the Au-nanoprobe solution, followed by NaCl addition, and spectrophotometric comparison of the solutions before and after NaCl-induced Au-nanoprobes aggregation. It is well known that collective oscillation of electrons on the surface of AuNPs causes a phenomenon called surface plasmon resonance, which results in

Conclusions

We developed a simple assay to quantify bacterial 16S rRNA in biomass taken from wastewater treatment processes. After RNA extraction with commercial kits, bacterial 16S rRNA could be quantified by only adding Au-nanoprobes and NaCl to a sample and analyzing UV–visible absorbance spectra for 30 min without reverse transcription and qPCR. Our assay has potential for the monitoring of dominant bacteria (>1% total bacteria) that contribute to major processes (e.g., nitrification, denitrification,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported financially by JSPS KAKENHI [grant number 19K21979 and 26630243].

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View full text

Simple assay for colorimetric quantification of unamplified bacterial 16S rRNA in activated sludge using gold nanoprobes

Highlights

Abstract

Graphical abstract

Section snippets

Author contribution

Chemicals and materials

DNA detection with Au-nanoprobes

Confirmation of the sensing mechanism

Conclusions

Declaration of competing interest

Acknowledgements

Int. J. Hyg Environ. Health

Enzym. Microb. Technol.

Biosens. Bioelectron.

Biosens. Bioelectron.

Chemosphere

Biophys. J.

Sensor. Actuator. B Chem.

Water Res.

Water Res.

Water Res.

Sci. Total Environ.

Biosens. Bioelectron.

Biosens. Bioelectron.

Sci. Total Environ.

Chemosphere