Mar 20, 2026
Quantification of Phosphorus Cycling Functional Genes in Marine Sediments by SYBR Green qPCR
- DALING WANG1,2
- 1The Ocean University of China, Qingdao, China;
- 2The University of New South Wales, Kensington, NSW, Australia
Protocol Citation: DALING WANG 2026. Quantification of Phosphorus Cycling Functional Genes in Marine Sediments by SYBR Green qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrbwozlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 17, 2026
Last Modified: March 20, 2026
Protocol Integer ID: 313410
Keywords: quantification of phosphorus cycling functional gene, abundance of phosphorus cycling gene, phosphorus cycling gene, phosphorus cycling functional gene, marine sediments by sybr green qpcr, quantification of other environmental functional gene, other environmental functional gene, quantitative pcr, marine sediment, gene copy number, time quantitative pcr, sybr green qpcr, pcr products during amplification, fluorescence proportional to the accumulation, fluorescence signal, phod, target gene
Abstract
The abundance of phosphorus cycling genes (phoD, phoX, gcd and pqqC) were quantified using SYBR Green I-based real-time quantitative PCR (qPCR). SYBR Green I binds specifically to double-stranded DNA and emits fluorescence proportional to the accumulation of PCR products during amplification. Fluorescence signals were monitored in real time using an ABI PRISM 7500 Detection System. Absolute quantification was performed by generating standard curves from tenfold serial dilutions of plasmids containing the target genes. Gene copy numbers were calculated based on the linear regression between Cycle threshold (Ct) values and the logarithm of known copy numbers. This method can be applied to the quantification of other environmental functional genes.
Materials
Total genomic DNA was extracted from marine sediments using the PowerSoil®DNA Isolation kit (MoBio, Germany), and quantified prior to qPCR analysis. DNA purity and concentration were measured using a NanoDrop spectrophotometer (Thermo Scientific, USA) to ensure suitability for downstream amplification. The integrity of the DNA was assessed by 1% agarose gel electrophoresis.
Troubleshooting
Before start
Notes: prepare an ice box before starting the experiment. Keep all reaction mixes and reagents on ice throughout the procedure. PCR mix should be protected from light until loading onto the thermocycler.
Principle
The abundance of phosphorus cycling genes (phoD, phoX, gcd and pqqC) were quantified using SYBR Green I-based real-time quantitative PCR (qPCR). SYBR Green I is a fluorescent dye that intercalates into double-stranded DNA (dsDNA). In solution, the unbound dye exhibits minimal fluorescence; however, fluorescence increases markedly when the dye binds to dsDNA. During PCR amplification, the accumulation of newly synthesized dsDNA leads to a proportional increase in fluorescence intensity, which is monitored in real time. The emitted fluorescence signal is detected at approximately 530 nm using the SYBR Green (FAM) channel of the instrument.
Real-time fluorescence signals were recorded using an ABI PRISM 7500 Detection System. Absolute quantification was performed by generating standard curves from tenfold serial dilutions of plasmids containing the target genes. Gene copy numbers were calculated based on the linear regression between Cycle threshold (Ct) values and the logarithm of known copy numbers.
FastStart Universal SYBR Green Master (Rox).pdf238KB Plamids and Environmental DNA Extraction
Construct plamids and extract environmental DNA.
A clone library of plasmids harboring P-cycle functional genes was constructed following the general procedure outlined in the attached protocol. Optimized Cloning Protocol for Standard Curve Generation in qPCR of Environmental Functional Genes.pdf632KB
Optimized Cloning Protocol for Standard Curve Generation in qPCR of Environmental Functional Genes.pdf632KB Total genomic DNA was extracted from marine sediments using the PowerSoil®DNA Isolation kit (MoBio, Germany), and quantified prior to qPCR analysis. DNA purity and concentration were measured using a NanoDrop spectrophotometer (Thermo Scientific, USA) to ensure suitability for downstream amplification. The integrity of the DNA was assessed by 1% agarose gel electrophoresis.
Notes:
①. A260/A280 ratio: The ideal range is between 1.8 and 2.0. A260/A230 ratio: The ideal value is approximately 2.0.
②. Integrity: When checking DNA integrity through agarose gel electrophoresis, ideally, the DNA should appear as a clear high molecular weight band, indicating that the sample has not degraded.
qPCR Reaction System (20 µL )
preparation of qPCR master mix, primer, and DNA. All the reagent are prepared on ice and keep protected from light prior to amplification.
In a 1.5 or 2.0 mL brown reaction tube kept on ice, prepare the PCR mix(except for DNA) for a 20 µL reaction by adding the following components listed in Table 2. For multiple samples, prepare the PCR mix by multiplying the reagent volumes by (number of samples + 1) to compensate for pipetting losses during preparation.
The primer information
| pathway | target gene | primer | primer sequence (5'-3') | size(bp) | references | |
| organic phosphorus mineralization | phoD Alkaline phosphatase D | phoD_F | CAG TGG GAC GAC CAC GAG GT | 362 | Sakurai et al., 2008 | |
| phoD_R | GAG GCC GAT CGG CAT GTC G | |||||
| phoX Alkaline phosphatase X | phoX_F | GGC AAA ACG CCN TGG GGN AC | 278 | Zipeng Liao，2017 | ||
| phoX_R | GGG TCG ACC TCG ACN AYV YAG CC | |||||
| inorganic phosphorus solubilization | gcd Glucose dehydrogenase | gcd_F | CGG CGT CAT CCG GGS ITI YRA YRT | 360 | Bergkemper et al., 2016 | |
| gcd_R | GGG CAT GTC CAT GTC CCA IAD RTC RTG | |||||
| pqqC Pyrroloquinoline quinone synthase C | pqqC_F | AAC CGC TTC TAC TAC CAG | 304 | Zheng et al., 2017 | ||
| pqqC_R | GCG AAC AGC TCG GTC AG |
Table 2 the qPCR reaction system
| reagent names | volume（µL） | |
| 10x FastStart Universal SYBR Green Master (ROX) | 10 | |
| forward primer (10 μM) | 0.6 | |
| reverse primer (10 μM) | 0.6 | |
| bovine serum albumin (BSA,Takara, 20 mg·mL−1) | 0.2 | |
| double-distilled water (dd H2O) | 6.6 | |
| PCR mix (prepared from the above components, 18 µL) | ||
| DNA | 2 | |
| Total | 20 | |
BSA was added to alleviate the inhibitory effects of humic substances and other PCR inhibitors commonly present in environmental DNA extracts, to stabilize DNA polymerase activity, and to improve amplification efficiency and reproducibility.
Notes:
①. Keep the qPCR reaction away from light !!!
②. All samples were analyzed in triplicate. A no-template control (NTC) was included in each run, using DEPC-treated water (or dd H2O) instead of DNA template.
Mix the solution carefully by pipetting up and down
Add 2.0 µL DNA, and mix carefully by pipetting up and down. Prepare the tubes or microplates for PCR (e.g., seal tubes with optical tube caps or the plate with self-adhesive foil.)
qPCR thermocycler procedure
The amplification program was as following (eg. pqqC):
- 95°C for 10 min (initial denaturation)
- 35 cycles of:
- 95°C for 15 s
- 60°C for 30 s
- 72°C for 1 min
- 72°C for 5 min (final extension)
phoD：95°C for 10 min，35 cycles at 95℃ for 15 s, 60℃ for 30 s, 72℃ for
60 s, 72℃ for 8 min.
phoX: 95°C for 10 min，35 cycles at 95℃ for 15 s, 63℃ for 30 s, 72℃ for
1.5 min, 72℃ for 10 min.
gcd: 95°C for 10 min，33 cycles at 95℃ for 15 s, 60℃ for 1 min, 72℃ for
45 min, 72℃ for 7 min.
A melting curve analysis was performed at the end of amplification to confirm specificity. A single peak indicated specific amplification.
Figure 1. Combined qPCR analysis of target genes (e.g., pqqC), including amplification curves (a), melting curves (b), agarose gel electrophoresis showing specific amplification and the expected product size (c), and the qPCR standard curve used for quantification (d).
Standard Curve Construction
Plasmids containing nirS gene fragments were serially diluted tenfold from 101 to 107 copies µL⁻¹. Standard curves were generated by plotting Ct values against log10(copy number). The linear regression equation was expressed as: Ct = a × log10 (copies) + b. Amplification efficiency was calculated using the formula:
Efficiency (%) = (10^(-1/slope) − 1) × 100. Only assays with efficiencies between 80% and 110% and R² ≥ 0.95 were accepted.
| target gene | regression curve | R2 | efficiency | |
| phoD | y=-3.2732X+37.574 | 0.9974 | 102.07% | |
| phoX | y=-3.8991X+47.672 | 0.9993 | 80.50% | |
| gcd | y=-3.8972X+44.082 | 0.9892 | 80.55% | |
| pqqC | y=-3.7864X+40.866 | 0.9974 | 83.70% |
Calculation of Gene Abundance
Gene copy numbers in marine sediments were calculated from the standard regression curve and normalized to sample dry weight. Final results were expressed as copies g⁻¹ (dry weight).
Notes: This method is also suitable for quantifying the abundance of functional genes in cDNA. In this case, RNA must first undergo reverse transcription to synthesize cDNA. The advantage of using Real-time Reverse Transcription PCR (RT-qPCR) lies in its capacity to accurately reflect gene transcription activity, rather than merely quantifying gene copy number. By measuring the abundance of target genes in cDNA, people can evaluate gene expression levels, thereby gaining insights into gene function and activity under specific environmental conditions or contexts.
References
Bergkemper, F.; Kublik, S.; Lang, F.; Krüger, J.; Vestergaard, G.; Schloter, M.; Schulz, S. (2016). Novel oligonucleotide primers reveal a high diversity of microbes which drive phosphorus turnover in soil. Journal of Microbiological Methods, 125, 91–97. https://doi.org/10.1016/j.mimet.2016.04.011.
Liao, Z. (2017). Diversity of microbial alkaline phosphatase gene in farmland soil and its response to phosphorus. Master's thesis, South China University of Technology, Guangzhou, China.
Sakurai, M.; Wasaki, J.; Tomizawa, Y.; Shinano, T.; Osaki, M. (2008). Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter. Soil Science and Plant Nutrition, 54, 62-71. https://doi.org/10.1111/j.1747-0765.2007.00210.x.
Zheng, B. X.; Hao, X. L.; Ding, K.; Zhou, G. W.; Chen, Q. L.; Zhang, J. B.; Zhu, Y. G. (2017). Long-term nitrogen fertilization decreased the abundance of inorganic phosphate-solubilizing bacteria in an alkaline soil. Scientific Reports, 7, 42284. https://doi.org/10.1038/srep42284.
Protocol references
Bergkemper, F.; Kublik, S.; Lang, F.; Krüger, J.; Vestergaard, G.; Schloter, M.; Schulz, S. (2016). Novel oligonucleotide primers reveal a high diversity of microbes which drive phosphorus turnover in soil. Journal of Microbiological Methods, 125, 91–97. https://doi.org/10.1016/j.mimet.2016.04.011.
Liao, Z. (2017). Diversity of microbial alkaline phosphatase gene in farmland soil and its response to phosphorus. Master's thesis, South China University of Technology, Guangzhou, China.
Sakurai, M.; Wasaki, J.; Tomizawa, Y.; Shinano, T.; Osaki, M. (2008). Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter. Soil Science and Plant Nutrition, 54, 62-71. https://doi.org/10.1111/j.1747-0765.2007.00210.x.
Zheng, B. X.; Hao, X. L.; Ding, K.; Zhou, G. W.; Chen, Q. L.; Zhang, J. B.; Zhu, Y. G. (2017). Long-term nitrogen fertilization decreased the abundance of inorganic phosphate-solubilizing bacteria in an alkaline soil. Scientific Reports, 7, 42284. https://doi.org/10.1038/srep42284.