Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions
Abstract
:1. Introduction
2. Importance of Absolute Quantification for Biological Questions
3. Brief Description of Advanced Absolute Quantification Methods
3.1. Fluorescence Spectroscopy
3.2. Flow Cytometry
3.3. Spike-In with Reference Markers
3.4. 16S qPCR and qRT-PCR Quantification
3.5. Droplet Digital PCR (ddPCR)
4. Decision-Making Regarding Different Biological Questions
4.1. Differentiation between Active and Dead Cells
4.2. Absolute Quantification of Specific Taxa of Interest
4.3. Absolute Quantification of Low Biomass Bacterial Samples
4.4. Rapid Quantification for a Large Number of Samples
4.5. Absolute Quantification of Bacteria Based on Other Features
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Absolute Quantification Method | Major Applications (Published) | Advantages | Limitations/Concerns | References |
---|---|---|---|---|
Fluorescence spectroscopy | Aquatic, soil, food and beverage, and air | High affinity; multiple dye selection to distinguish both live and dead cells | Fail to stain dead cells with complete DNA degradation; some dyes bind both DNA and RNA | Gordon et al., 2017, Guzaev et al., 2017, Saint-Ruf et al., 2010, Sieracki et al., 1999, Auty et al., 2001, |
CARD-FISH + flow cytometry/qPCR | Aquatic | Direct quantification of specific taxa; detects both live and dead cells; provides insights for function, morphology, and ecology among taxa | Large population of cells are required for rare taxa detection; possibility of unspecific probe binding; Sample fixation may cause operation and efficiency biases; background noise | Hinzke et al., 2021, Kuo et al., 2021, Piwosz et al., 2021, Priest et al., 2021, Neuenschwander et al., 2015, Kubota et al., 2013 |
Flow cytometry | Feces, aquatic, and soil | Rapid; single cell enumeration; flexible parameters based on physiological characteristics; capability to differentiate live and dead cells | Background noise exclusion may be required; gating strategy; dilution may be required; not ideal for complex systems/heterogeneous samples | Luhung et al., 2021, Heinrichs et al., 2021, Xu et al., 2021, Zhu et al., 2019, Deng et a., 2019, Vandeputte et al., 2017, Prest et al., 2013, Berney et al., 2007, Longnecker et al., 2005, Salcher et al., 2011 |
Spike-in with internal reference | Soil, sludge, and feces | Rapid; easy incorporation into high throughput sequencing; high sensitivity; easy handling | Internal reference, spiking amount, and spiking time point can greatly affect the accuracy; 16S rRNA copy number calibration possibly needed. | Yang et al., 2018, Tourlousse et al., 2017, Smets et al., 2016, Lou et al., 2018, Stämmler et al., 2016 |
16S qPCR | Feces, clinical (lung), soil, plant, air, and aquatic | Directly quantifies specific taxa; cost-effective and easy handling; high sensitivity; compatible with low biomass samples | 16S rRNA copy number calibration may be needed; PCR-related biases exist; standard curves are required | Luhung et al., 2021, Callegari et al., 2021, Blaud et al., 2021, Lei et al., 2021, Jian et al., 2020, Vandeputte et al., 2017, Stoddard et al., 2015, Sze et al., 2014, Brankatschk et al., 2012 |
16S qRT-PCR | Clinical (joint infection), food safety, feces, sludge, water remediation, and soil | High resolution and sensitivity; directly quantifies specific taxa; detects active cells; compatible with low biomass samples | More of an approximation for protein synthesis than overall cell count; unstable RNA/RNA degradation; 16S rRNA copy number calibration may be needed | Ma et al., 2018, Johnston and Behrens, 2020, Bui et al., 2012, Boyer and Combrisson, 2013, Kim et al., 2014, Stoddard et al., 2015, Matsuda et al., 2009 |
ddPCR | Clinical (lung, bloodstream infection), air, feces, and soil | Applicable to low concentrations of DNA; directly quantify specific taxa; high throughput capabilities, and no standard curve needed; compatible with low biomass samples | Dilutions are required for high concentrated template; may require a large number of replicates | Luhung et al., 2021, Ahn et al., 2020, Zeng et al., 2020, Sze et al., 2014, Kim et al., 2014, Ziegler et al., 2019, Gobert et al., 2018 |
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Wang, X.; Howe, S.; Deng, F.; Zhao, J. Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions. Microorganisms 2021, 9, 1797. https://doi.org/10.3390/microorganisms9091797
Wang X, Howe S, Deng F, Zhao J. Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions. Microorganisms. 2021; 9(9):1797. https://doi.org/10.3390/microorganisms9091797
Chicago/Turabian StyleWang, Xiaofan, Samantha Howe, Feilong Deng, and Jiangchao Zhao. 2021. "Current Applications of Absolute Bacterial Quantification in Microbiome Studies and Decision-Making Regarding Different Biological Questions" Microorganisms 9, no. 9: 1797. https://doi.org/10.3390/microorganisms9091797