Part:BBa_K5087001

GalR

Introduction

This reverse PCR primer is designed to selectively identify Prymnesium parvum genomic DNA by binding to the ITS2 region of its genome.

This part can be used for amplification of DNA both using the standard PCR method as well as isothermal amplification methods such as RPA (Recombinase Polymerase Amplification).

Biology & Usage

The ITS2 (Internal Transcribed Spacer 2) region is a non-coding segment of DNA found within the ribosomal RNA (rRNA) gene cluster. In the genome of Prymnesium parvum, the ITS2 region lies between the 5.8S and nuclear large rRNA genes [1].

The ITS regions, including ITS2, are commonly used for species identification because they tend to vary between species while being conserved within a species [2]. This variability makes the ITS2 region an effective target for designing species-specific primers, such as those used to identify Prymnesium parvum [2].

RPA Reaction

Recombinase Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technique that operates at a temperature range of 37–42°C, distinguishing it from traditional PCR methods that require thermal cycling for denaturation and annealing of DNA [4].

RPA relies on three essential types of proteins: a recombinase, single-stranded DNA binding proteins (SSBs), and a strand-displacing DNA polymerase.

The process begins when the recombinase protein binds to a primer (about 30–35 nucleotides long) that matches the target DNA sequence. This complex then searches for homologous sequences in double-stranded DNA and initiates strand invasion. The SSBs stabilize the displaced strand to prevent primer dissociation, while the strand-displacing DNA polymerase extends the primer, resulting in exponential amplification of the target sequence [5].

Figure 1. The RPA (Recombinase Polymerase Amplification) Mechanism

SHERLOCK Method

The SHERLOCK platform is a modern synthetic biology tool that utilizes the properties of the Cas13a protein, an enzyme from the Nobel Prize-winning CRISPR-Cas system. The Cas13a protein is guided with high specificity to the target sequence using crRNA.

The crRNA molecule is crucial for the assay's specificity. It consists of a direct repeat (DR) sequence and a spacer sequence that is complementary to the target. The crRNA molecule is designed to uniquely identify the organism by targeting the Internal Transcribed Spacer (ITS) sequence in its genome.

First, the Cas13a protein binds to the organism's genetic material, which was previously amplified using RPA and transcribed into RNA. Once bound, the Cas13a protein is activated and exhibits a "collateral" RNase activity, meaning it non-specifically cleaves nearby single-stranded RNA molecules [6].

This activity can be used in assays by including synthetic RNA probes tagged with a fluorescent reporter and a quencher in the reaction mixture. A fluorescent signal indicates that the reporters have been cleaved by Cas13, confirming the presence of the DNA target in the sample. The SHERLOCK method can also be used with Lateral Flow Assays (LFA).

Figure 2. The SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) Method Mechanism

Experimental Validation

Description The Gal R primer was used along with the Gal F primer (without the added T7 promoter) in a PCR experiment aimed at identifying the internal transcribed spacer 2 (ITS2) sequence of Prymnesium parvum in a culture sample. The PCR product resulting from this amplification should be 132 base pairs (bp) in length. PCR Conditions Pre-denaturation: 98°C for 30 seconds Denaturation: 98°C for 10 seconds Annealing: 67°C for 60 seconds Elongation: 72°C for 5 seconds Final Elongation: 72°C for 5 minutes Number of Cycles: 40

Experimental Details PCR Reaction Volume: 20 µl Starting Primer Concentration: 100 µM. The primers were first diluted 10x and then 25x, resulting in a final concentration of 400 nM. Then, 0.4 µl of each was used. PCR Master Mix (A&A Biotechnology): 10 µl Gel Electrophoresis of PCR Product Gel Composition: 2% agarose gel Electrophoresis Conditions: 85 V for 45 minutes Loading Volume: 10 µl of each PCR product Standards Used: GeneRuler 1kB DNA Ladder O'GeneRuler DNA Ladder Mix

Results Each tested sample displays the ITS2 sequence of 132 bp as six distinct bands between 200 and 100 bp, confirming the success of the experiment. Figure 3. Gel electrophoresis of the PCR product with Gal primers. Well 1: O'GeneRuler DNA Ladder Mix, Wells 2-7: PCR products, and Well 8: O'GeneRuler 1 kB DNA Ladder Mix.

Optimization

The objective of this experiment was to determine whether the ITS2 sequence is present in DNA samples isolated from Prymnesium parvum cultures NOW5, P, BOW2, and Gdańsk2 and to test which PCR conditions work best with the Gal primer set.

Sample Information Prymnesium parvum genomic DNA isolated from liquid cultures was used, with the following concentrations for the samples: NOW5: 164.9 ng/µl P: 280.6 ng/µl BOW2: 154.3 ng/µl Gdańsk2: 127.2 ng/µl Primers were provided as stock solutions at 100 µM and were diluted 500x to achieve a final concentration of 200 nM. PCR Reaction Composition Total Reaction Volume: 20 µl DNA: 1 µl PCR Master Mix (A&A Biotechnology): 10 µl Primer Mix: 1 µl Water: 8 µl

PCR Conditions Two sets of PCR conditions were tested: Conditions A - Routine Conditions Pre-denaturation: 98°C for 30 seconds Denaturation: 98°C for 10 seconds Annealing: 67°C for 60 seconds Elongation: 72°C for 5 seconds Final Elongation: 72°C for 5 minutes Number of Cycles: 40 Condition B - Adjusted Conditions Pre-denaturation: 95°C for 60 seconds Denaturation: 95°C for 15 seconds Annealing: 67°C for 60 seconds Elongation: 72°C for 15 seconds Final Elongation: 72°C for 5 minutes Number of Cycles: 30 Conditions A correspond to routine conditions used in our lab for Gal primers, while Conditions B correspond to adjusted conditions optimized for the Taq polymerase used, based on the manufacturer’s instructions.

Results

The PCR products of the correct length were observed in samples 8 and 10, corresponding to Conditions B for samples P and NOW5, respectively. A product of the expected size was also present in well 3, but this result was confounded by the presence of a similar-sized product in the control sample.

Although the results are not entirely conclusive, the band observed in well 10 under Conditions B was absent in the corresponding well for Conditions A with the same DNA sample. This suggests that Conditions B may offer a more reliable outcome for these PCR experiments

Figure 4. Optimization results visualization. The numbers on the gel correspond to the following samples:

1: O'Gene Ruler DNA Ladder Mix, 2: Control Sample, 3: P, 4: BOW2, 5: NOW5, 6: Gdańsk2, 7: Control Sample, 8: P, 9: BOW2, 10: NOW5, 11: Gdańsk2, 12: Gene Ruler 1kB DNA Ladder Mix,

Part Performance

This part is an integral component of the PrymDetect Toolkit, which is designed for detecting Prymnesium parvum in water samples using the SHERLOCK method. In our tests we used the LwaCas13a protein.

The SHERLOCK reactions were carried out according to the protocol, which can be accessed here.

In this test, the GalR primer was used for the pre-amplification of the detection target using RPA. Our team assessed the performance of this part in conjunction with other components of the toolkit to identify the most reliable configurations, thereby simplifying its use for future teams. In our tests we used the LwaCas13a protein.

Figure 5: Fluorescence readout results demonstrating the performance of various part combinations from our toolkit, with the GalR primer serving as the connecting component. For simplicity, the negative controls for combinations that consistently showed no signal are not included in the graph, as they have been subtracted from the obtained results. The negative control which was included corresponds to the GalF-GalR-PrymCrRNA1 combination.

PrymFlow

Introduction to the Lateral Flow Assay

The SHERLOCK method, like other CRISPR/Cas-based detection techniques, is compatible with a Lateral Flow readout format. The Lateral Flow Test is known for its speed, simplicity, and ease of interpretation. We aimed to adapt the previously optimized SHERLOCK components for use with Lateral Flow dipsticks, resulting in the development of the PrymFlow test for detecting the presence of Prymnesium parvum.

LFA Result Interpretation

If the target is present, the T-line becomes visible, indicating a positive result. Meanwhile, the C-line serves as a control that is less visible when greater amounts of the target sequence are present. Gold nanoparticles (GNPs) with anti-FAM antibodies provide the visual indication of the test result.

WHAT HAPPENS WHEN Prymnesium parvum IS NOT PRESENT IN THE WATER SAMPLE?

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Streptavidin, immobilized on the C-line, captures the biotin-labeled ends of the intact reporters. The reporters are captured on the C-line, and the binding of gold nanoparticles (GNPs) conjugated with anti-FAM antibodies makes only the C (control) line visible.

AND WHEN IT IS PRESENT?

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The reporters are cleaved by the activated Cas protein. Consequently, gold nanoparticles (GNPs) with anti-FAM antibodies capture the FAM-labeled fragments, which then bind to the anti-anti-FAM antibody immobilized on the test line (T-line), producing a strong signal. The presence of the T-line indicates the presence of Prymnesium parvum. Some intact reporters might remain in the mix (the amount depends on how many target DNA molecules were present in the sample, influencing the ratio of activated to non-activated Cas13 protein and the number of cleaved reporter molecules). As a result, a weak control line (C-line) is visible.

Figure 6. Explanation of the lines visible on the Lateral Flow test during a negative and positive result.

The LFA tests were conducted following the protocol, which can be accessed here.

False positives

We observed false positives when using combinations that included GalF, GalR, and the PrymCrRNA1 design. However, this issue did not occur with the PrymCrRNA2 design.

Figure 7. Test 1: False positives obtained with the GalF-GalR-PrymCrRNA1 pair. 1. Negative control for GalF-GalR + PrymCrRNA1, 9. Positive control.

Figure 8. Test 2: 2. Prymnesium parvum DNA ModF-GalR + PrymCrRNA1, 3. Negative control for ModF-GalR + PrymCrRNA1, 7. Negative control, 8. Positive control.

The negative control for GalF-GalR with PrymCrRNA1 (Sample 1, Test 1) produced a false positive result, likely due to nonspecific interactions between the GalF-GalR primers and PrymCrRNA1. These false positives were consistently observed, which is why we advise against using the GalF-GalR primer pair with PrymCrRNA1.

However, GalR can be successfully used in combination with other forward primers, such as the ModF primer, as demonstrated by the positive test result (two visible bands) obtained for the sample containing Prymnesium parvum DNA (Sample 2, Test 2) and the negative result from the corresponding negative control (Sample 3, Test 2).

Parts Compatible with GalR

The GalR primer shows optimal performance when paired with the ModF primer and PrymCrRNA1 molecules, yielding the highest fluorescence readout. It can also be effectively used in combination with the AltF primer and PrymCrRNA1. While other pairings are possible, they consistently result in significantly lower outputs.

Detection of Prymnesium Types through Genomic Analysis

Our genomic analysis has identified which types of Prymnesium can be detected with our primer and crRNA combinations. This determination is based on the ITS sequence, corresponding to the specific type of prymnesin the algae produces [7].

Classification of Prymnesins

Prymnesins are classified into three distinct types based on the structure of their carbon backbone [8]:

• A-type Prymnesins: Have the largest carbon backbone with 91 carbon atoms. They are the most potent, exhibiting significant ichthyotoxic (fish-killing) properties.
• B-type Prymnesins: Feature a carbon backbone of 85 carbon atoms. They lack certain ring structures found in A-types and are generally less potent, though still toxic.
• C-type Prymnesins: Possess the shortest carbon backbone with 83 carbon atoms and show significant structural diversity. Their toxicity is variable and less well-characterized compared to A- and B-types.

Table 1: Primer Combinations Including GalR and Their Detection Capabilities

Primer Combination	Target Regions	Detected Types
Mod_F & Gal_R	5.8S & ITS2	All types
Alt_F & Gal_R	5.8S & ITS2	All types
Gal_F & Gal_R	ITS2 & ITS2	Type B

Table 2: crRNA Combinations and Their Detection Capabilities

crRNA Combination	Target Region	Detected Types
PrymCrRNA1	ITS2	Type A and B
PrymCrRNA2	ITS2	Type A, B, and some C

Sequence

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Primer and crRNA Collection Binding Sites

Here, we illustrate the positioning of all primers and crRNAs in our PrymDetect Toolkit on the ribosomal cistron of Prymnesium parvum genomic DNA.

Figure 9. Positioning of primers and crRNAs from the PrymDetect Toolkit on the ribosomal cistron of Prymnesium parvum genomic DNA.

Biosafety

We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.

Sequence Source and Design

This sequence was designed by Galluzzi et al [3]. The primer was specifically created to target the ITS2 region of the rRNA gene cluster using Primer Express software, with their specificity confirmed through a BLAST search in the GenBank and EMBL databases.

Resources

• [1] White, T.J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications. Academic Press.
• [2] Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet, A., Ravel, C., Marty, P., Delaunay, P., Kasbari, M., Granouillac, B., Gradoni, L., & Sereno, D. (2017). Leishmania infections: Molecular targets and diagnosis. *Molecular Aspects of Medicine, 57*, 1-29. https://doi.org/10.1016/j.mam.2016.11.012
• [3] Galluzzi, L., Bertozzini, E., Penna, A., et al. (2008). Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. *Letters in Applied Microbiology, 46*(2), 261-266. https://doi.org/10.1111/j.1472-765X.2007.02294.x
• [4] Lobato, I. M., & O'Sullivan, C. K. (2018). Recombinase polymerase amplification: Basics, applications and recent advances. Trends in Analytical Chemistry: TRAC, 98, 19–35. https://doi.org/10.1016/j.trac.2017.10.015
• [5] Ruichen Lv, Nianhong Lu and Junhu Wang et al. Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens: An Overview. Zoonoses, 2(1). DOI: 10.15212/ZOONOSES-2022-0002
• [6] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
• [7] Jian J, Wu Z, Silva-Núñez A, et al. Long-read genome sequencing provides novel insights into the harmful algal bloom species Prymnesium parvum. Sci Total Environ. 2024;908:168042. doi:10.1016/j.scitotenv.2023.168042
• [8] Binzer SB, Svenssen DK, Daugbjerg N, et al. A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum. Harmful Algae. 2019;81:10-17. doi:10.1016/j.hal.2018.11.010