Quantitative real-time polymerase chain reaction (PCR) and droplet digital PCR duplex assays for

detecting Zostera marina DNA in coastal sediments

Summary

This study developed a method using quantitative real-time polymerase chain reaction (qPCR) and
droplet digital PCR (ddPCR) to detect seagrass DNA in coastal sediments. The study found that
seagrass DNA can be preserved in sediments for a long time and that ddPCR is more accurate than
qPCR for detecting low concentrations of ancient seagrass DNA. The method can be used to explore
seagrass carbon sequestration and identify past seagrass meadows.

Latar belakang

The background of this study is focused on the detection of seagrass DNA in coastal sediments using
quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). Seagrass meadows are known to
be efficient blue carbon sinks, playing a crucial role in carbon storage and global climate change
mitigation. Understanding the current status of blue carbon storage and estimating organic carbon
accumulation rates in seagrass meadows are important for global actions related to climate change.

Previous studies have attempted to estimate the organic carbon stored in seagrass sediment and its
accumulation rate. However, accurate quantification of seagrass DNA in sediments is essential for
reliable estimation. This study aimed to develop and compare qPCR and ddPCR methods for
detecting seagrass DNA in sediment samples, providing a more accurate and sensitive approach for
quantification.

The study also investigated the efficiency of different DNA extraction methods and evaluated the
specificity of newly designed primers and probes for seagrass DNA detection. The results of this
study contribute to a better understanding of seagrass meadow dynamics and their role in carbon
storage.

Alasan menggunakan metode PCR:

PCR (Polymerase Chain Reaction) is a widely used method in molecular biology and genetics for
amplifying specific DNA sequences. There are several reasons for using the PCR method:

1. Amplification of specific DNA sequences: PCR allows for the amplification of a specific target DNA
sequence, even if it is present in very low quantities. This is useful for various applications such as
genetic testing, diagnostics, and DNA sequencing.

2. Detection and quantification of DNA: PCR can be used to detect and quantify the presence of
specific DNA sequences in a sample. This is particularly useful in environmental studies, where PCR
can be used to detect the presence of certain organisms or genetic markers in water or sediment
samples [4].

3. High sensitivity and specificity: PCR is highly sensitive and specific, meaning it can detect and
amplify very small amounts of DNA and discriminate between closely related DNA sequences. This
makes it a valuable tool in various fields, including forensics, medical research, and infectious disease
diagnostics.

4. Speed and efficiency: PCR is a rapid and efficient method for DNA amplification. It can amplify a
specific DNA sequence within a few hours, whereas traditional cloning methods can take days or
weeks [1].

5. Versatility: PCR can be used for a wide range of applications, including genotyping, gene
expression analysis, mutation detection, and DNA sequencing. It can also be combined with other
techniques, such as gel electrophoresis or DNA sequencing, to further analyze the amplified DNA [1].

Overall, PCR is a powerful and versatile technique that has revolutionized molecular biology and
genetics research. Its ability to amplify specific DNA sequences with high sensitivity and specificity
has made it an essential tool in various fields.

Komposisi dan program PCR

Materials and Methods:

1. Design of Primers and DLPs for Detecting Z. marina DNA:

- Specific primers and Dual-Labeled Probes (DLPs) were designed to detect Zostera marina (Z.
marina) DNA.
- Two DNA regions were used for primer and DLP design: the nuclear internally transcribed
spacer-2 (ITS-2) and the chloroplast gene for maturase K (matK).
- Sequences for these regions were obtained from GenBank (Accession numbers: AY553598 and
AB125354).
- Primer 3 software was employed for primer and DLP design.
- Design criteria included an optimal PCR product length of approximately 100 base pairs,
estimated melting temperatures of around 60°C for primers and 70°C for DLPs.
- Primers and DLPs were synthesized by Sigma Genosys, with DLPs labeled at the 5' end with 6-
carboxyfluorescein (6-FAM) and 6-carboxy-2',4',4,5',7',7,7'-hexachlorofluorescein (6-HEX), and
at the 3' end with Black Hole Quencher-1 (BHQ-1).
- The designed primer and DLP sets are detailed in Table 1.
2. Specificity Testing of Designed Primers and DLPs:

 To assess the specificity of the newly designed primers and DLPs (AmaITS and AmaMatK),
various plant species were tested, including Z. marina, Zostera japonica, Miscanthus sinensis,
Phragmites australis, Oryza sativa (Japanese rice), and Triticum aestivum (common wheat).
 To prevent cross-contamination, leaf sample treatments and DNA extractions were
conducted in separate laboratories.
 Leaf samples were freeze-dried and disrupted using TissueLyser, following modified
instructions based on Shimabukuro et al. (2012).
 DNA extraction was performed using the DNeasy Plant Mini kit (Qiagen) with 50 mg of dried
plant material.

3. Efficiency Testing of Designed Primers and DLPs:

 A duplex quantitative PCR (qPCR) was conducted with a final reaction volume of 10 µL.
 The reaction mixture included 5 µL of iQ multiplex powermix (Bio-Rad), 0.3 µL of each primer
(final concentration: 300 nM), 0.2 µL of each DLP (final concentration: 200 nM), 1 µL of
template DNA, and 3.5 µL of sterilized pure water.
 Amplification reactions were performed using the CFX96 Touch Real-Time PCR detection
system (Bio-Rad).
 The annealing temperature was determined using the thermal gradient function of the qPCR
equipment, ranging from 55°C to 65°C.
 The thermal cycling conditions comprised an initial 2-minute denaturation at 95°C, followed
by 50 cycles of 10 seconds at 95°C and 30 seconds at the annealing temperature (ranging
from 55°C to 65°C).
 Specificity of the newly designed primers and DLPs was assessed through qPCR with 50
cycles of 10 seconds at 95°C and 30 seconds at 58°C.

This protocol details the design, specificity testing, and efficiency assessment of primers and DLPs for
the detection of Z. marina DNA.

1. Plasmid Development for the Construction of the Standard Curve:

- Amplified PCR amplicons of AmaITS and AmaMatK using specific PCR primers (AmaITS forward,
AmaITS reverse, AmaMatK forward, AmaMatK reverse).
- Cloned both amplicons into TOPO PCR4 plasmid vectors.
- Transformed the plasmids into Escherichia coli TOP 10 competent cells.
- Selected positive colonies using colony direct-PCR.
- Cultured positive colonies in LB broth and extracted plasmids using the Qiaprep plasmid
purification Kit.
- Measured plasmid concentration with the GeneQuant II spectrophotometer.
 - Calculated the number of gene copies in the plasmid (molecules/µL) using a formula based on
plasmid concentration and length.
- Subcloned plasmid standards for each target sequence and diluted them 10 times.
- Amplified diluted plasmid standards using primers and DLPs designed in this study.

2. Quantitation of Genomic DNA in Z. marina Parts:

- Collected various parts of Z. marina (third leaf, youngest leaf, sheath, meristematic region,
rhizome, roots, and pollen).
- Freeze-dried the collected samples and extracted DNA from 50 mg of each sample and pollen
traces using the DNeasy Plant Mini Kit.
- Determined the gene copy number per ng of extracted DNA using qPCR and the plasmid
standard curve obtained earlier.

3. DNA Extraction from Coastal Sediments:

- Utilized different commercial DNA extraction kits (Qiamp stool Mini Kit, Isoil, Nucleospin from
Soil Kit with buffer systems SL-1 and SL-2, and DNeasy Plant Mini Kit) for extracting DNA from
sediment samples.
- Collected surface sediment samples from various sites and freeze-dried them.
- Disrupted the sediment samples using TissueLyser.
- Extracted DNA from approximately 100 mg of powdered sediment using the specified DNA
extraction kits.
- Determined the gene copy number per gram of sediment using qPCR and the plasmid standard
curve developed earlier.

4. Application of qPCR and ddPCR to Detect Z. marina DNA in Coastal Sediments:

- Used long-core sediment samples from seagrass meadows and non-vegetated tidal-flats.
- Followed strict contamination prevention protocols during sample manipulation.
- Prepared sediment subsamples by freeze-drying and disrupting with TissueLyser.
- Extracted DNA from 100 mg of powdered sediment samples using the DNeasy Plant Mini Kit.
- Determined the gene copy number per gram of sediment using both qPCR and ddPCR with the
primers and DLPs.
- Conducted ddPCR optimization for AmaITS and AmaMatK.
- Performed ddPCR in a QX100 ddPCR system and analyzed the results using QuantaSoft
software.

5. Statistical Analyses:

- Performed statistical analyses including Welch’s t-test, nonparametric Kruskal-Wallis, and Type I
regression in R 3.1.0 and SPSS 22.0.
- Considered p-values lower than 0.05 as statistically significant.

1. Assessment of AmaITS and AmaMatK Optimal Annealing Temperatures:

- Amplicon lengths of AmaITS and AmaMatK were 90 bp and 102 bp, respectively.
- Ct values of both amplicons increased gradually over 59.1°C.
- Annealing and extension temperature for qPCR was set at 58°C for both AmaITS and AmaMatK.

2. AmaITS and AmaMatK Specificity:

 - In silico verification of AmaITS and AmaMatK specificity using BLAST searches demonstrated
high specificity.
- Experimental evaluation using qPCR with DNA extracted from different Zostera species and
terrestrial plants from the Seto Inland Sea area confirmed the specificity of AmaITS and
AmaMatK for Z. marina.

3. Plasmid Standard Curves:

- The linear dynamic range for AmaITS and AmaMatK quantification was from 10^6 to 10^2 gene
copies per reaction.
- Standard curves exhibited a strong correlation between gene copy numbers and Ct values.
- No significant differences were observed between simplex and duplex qPCR for AmaITS and
AmaMatK.

4. Reproducibility of qPCR:

- Reproducibility was assessed by performing twenty replications using 10^6 to 10^2 gene copies
of plasmid standard curves.
- The percent coefficient of variation (CV) of Ct values ranged from 0.6% to 2.1% for AmaITS and
AmaMatK.
- Slight reductions in reproducibility were noted for gene copy numbers below 10^2 in AmaITS
and 10^1 in AmaMatK regions.

5. Quantitation of Genomic DNA in Z. marina Parts:

- AmaITS gene copy numbers were approximately ten times higher than AmaMatK in all Z.
marina parts.
- The lower part of the leaf sheath had the highest gene copy numbers for AmaITS.
- AmaMatK gene copy numbers were significantly lower in rhizomes and roots.
- Pollen had fewer gene copies compared to other parts.

6. DNA Extraction Methods for Detecting Z. marina DNA from Sediments:

- Four commercial DNA extraction kits and two buffer systems from the Nucleospin from Soil Kit
were compared for efficiency in extracting DNA from sediment and plants.
- The DNeasy Plant Mini Kit demonstrated the most efficient extraction from both sediments and
plants.
- Gene copy numbers differed significantly among samples extracted using different kits and
buffer systems.

7. Detecting Z. marina Genes in Coastal Sediments by qPCR and ddPCR:

- For ddPCR, the duplex reaction with AmaITS and AmaMatK showed decreased efficiency below
54°C, so this temperature was chosen for annealing and extension.
- Quantification using qPCR was cross-validated with absolute numbers obtained via ddPCR.
- A significant positive correlation was observed between gene copy numbers determined by
qPCR and ddPCR for AmaITS and AmaMatK.
- ddPCR demonstrated higher accuracy at low target DNA concentrations compared to qPCR.
- Sediment samples from Z. marina meadows contained significantly more gene copies of both
AmaITS and AmaMatK compared to samples from non-vegetated tidal flats.
- Gene copy numbers in Z3 sediments decreased with depth, while those below 1 meter
remained stable.
 - Both AmaITS and AmaMatK genes were detected in layers dated to approximately 5000
calibrated years before present (yr cal BP) using radiocarbon dating.
- A positive correlation was confirmed between gene copy numbers of AmaITS and AmaMatK
and the concentration of organic carbon (OC) derived from seagrass in Z3 samples, but not in
E1 samples.

For qPCR:

- Initial denaturation: 2 minutes at 95°C

- Amplification: 50 cycles of 10 seconds at 95°C, followed by 30 seconds at 55-65°C

- Specificity testing: 50 cycles of 10 seconds at 95°C, followed by 30 seconds at 58°C [5]

For ddPCR:

- Initial denaturation: 10 minutes at 95°C

- Amplification: 40 cycles of 30 seconds at 94°C, followed by 1 minute at 54°C

- Final enzyme deactivation: 10 minutes at 98°C [2]

Please note that the specific temperature ranges for the annealing step were not provided in the
article.

Komposisi pcr

The ddPCR mixtures contained 10 μL 2x ddPCR Supermix for Probes (Bio-Rad), 1 μM each primer,
0.25 μM each probe, 2 μL template DNA, and sterilized pure water up to a total volume of 20 μL [3].

Hasil PCR

Analisa hasil PCRnya gimana

Perbandingan qPCR dan ddPCR :

The advantages of using droplet digital PCR (ddPCR) over quantitative real-time PCR (qPCR) for
detecting seagrass DNA in sediment samples are as follows:

1. Higher sensitivity and accuracy: ddPCR is more sensitive than qPCR, especially when the sample
DNA quality is poor or the concentration of the target DNA is low. It performs better in reducing
background noise and is less susceptible to PCR inhibitors, resulting in higher accuracy at low
concentrations of target DNA [1].

2. Improved reproducibility: ddPCR provides better reproducibility compared to qPCR, particularly at

low gene copy numbers. It offers higher accuracy in quantification, especially in samples with low
gene copy densities [2].

3. Absolute quantification: ddPCR is an absolute quantification method, which means it does not
require a standard reference curve for quantification. This makes it more accurate and reliable than
qPCR, which relies on relative quantification using a standard curve [4].
4. Reduced false positives: ddPCR has a lower rate of false positives compared to qPCR, as it is less
affected by background noise and PCR inhibitors. This is particularly advantageous when detecting
low concentrations of target DNA [1].

5. Potential for multiplexing: While qPCR can detect multiple fluorophores, ddPCR is limited to two
fluorophores (FAM and HEX). However, duplex or multiplex assays can still be performed using
ddPCR, improving the accuracy of molecular investigations [3].

Overall, ddPCR offers higher sensitivity, accuracy, reproducibility, and reduced false positives
compared to qPCR, making it a more suitable method for detecting seagrass DNA in sediment
samples.