Results: Engineering Success of LAMP Primers for oak wilt

Step 1: Screen the primer sets through colorimetric LAMP reaction

We first run the LAMP reaction for all sets with the NEB WarmStart Master Mix, the amplifications are shown by the color change from pink to yellow.

Expected Results

Negative reactions are shown as pink around RGB (245,66,141)

Positive reactions are shown as yellow around RGB (245, 215, 66

IGEM Toronto results

Fig 1. NEB LAMP kit on target region BT The primer ratio used is FIP/BIP:F3/B3 = 8:1, and diluted to 1.6uM and 0.2uM in the mix. The reaction also included two different target DNA concentrations, 2ng/ul, and 10 ng/ul. The picture was taken at the 40 min mark.

Fig 2. NEB LAMP kit on target region MCM7 The primer ratio used is FIP/BIP:F3/B3 = 8:1, and diluted to 1.6uM and 0.2uM in the mix. The reaction also includes two different target DNA concentrations, 2ng/ul, and 10 ng/ul. The left image was taken at the 30 min mark. The right image was taken at the 40 min mark.

Fig 3. NEB LAMP kit on target region MCM7 amplified by the primer set PE2. The negative control, positive control, and the experimental result are placed side by side to reflect the slight color change.

Using Google Colour Picker, the negative control RGB is (189,9,6). This pink is darker than the expected result due to shadows. The positive control RGB is (230,137,44). The yellow is not as bright yellow as the expected result. The graduate student from Pardee's lab suggested that is due to the heater we used in the lab does not have a heated lid to keep the temperature even throughout the reaction tube. The experimental result RGB is (235, 90, 12).

This experimental step does not give a clear result, although we could see a slight color change on the PrimerExplorer 2 design for MCM7 (Fig 3). We believe this is not a strong evidence of success. It serves as a direction indicator. We learnt that the pH dye changes color from pink to yellow if and only if amplification takes place within the mix.

We hypothesize that primers designed for the MCM7 region are better at amplifying oak wilt DNA than the primers designed for the BT region.

Step 2: Perform gel electrophoresis to better validate if the reaction results are false positive or false negative

Fig 4A. Gel eletrophoresis result of the primer sets designed for the BT region. Lucigen Master Mix was used. This experiment was performed on 20220822

Fig 4B. MCM7, Lucigen, 20220829

To visualize the effectiveness of each primer set, we run gel electrophoresis on each of the LAMP products for the two target regions BT and MCM7, respectively (Fig 4. A, B). From Fig 4. A, primers designed for BT show no amplification at all, which is contrary to our expectation. In comparison, there are amplifications for MCM7 (Fig 4. B), especially for primer designer PE1. There are visible bands at 200 bp that match the target amplicon (215bp). This suggests that PE1 design for MCM7 is successful, while it could not be concluded only by the note, because:

gel electrophoresis with LAMP products inevitably produces smear and multiple off-target bands.
The gel electrophoresis does not validates quantitatively.
Reiteration of the experiment does not guarantee in replication of the same result.

We repeat in the gel electrophoresis experiment with changes in LAMP parameters. There are contamination issues found in the negative control group, and the experiment is set-backed by those unexplainable results. After consulting with specialists in the LAMP, Jennifer Doucet, we decided to use qPCR to quantitatively visualize our experimental result to gain a better understanding.

Step 3: Quantitatively compare the degree of amplification for the better primer sets, combined with step 4: replicate the above results to ensure experimental validity

After a general test on each primer set and two different DNA regions, we focus on amplifying the MCM7 DNA region because these primer sets showed signs of success in previous experiments.

The context and rationale, reagents, qPCR machine set up and qPCR plate set up are explained in the Experiment page.

Successful amplification of Oak Wilt MCM7 DNA region with all the primer sets

** Please note that the background noise of the amplification plot goes up to delta Rn = 5.00e+4

Fig 5. Amplification plot of Oak Wilt PE1 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates.	Each primer set shows positive LAMP amplification on the MCM7 region for the following reasons: The fluorescence signals of all samples pass the threshold in less than 60 minutes. Issue: The no template negative control also showed amplifications. Since we used a new master mix from the Pardee lab and stock primers, we can confirm that our stock DNA template has been contaminated.
Fig 6. Amplification plot of Oak Wilt PE2 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates.
Fig 7. Amplification plot of Oak Wilt GLAPD1 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates.
Fig 8. Amplification plot of Oak Wilt GLAPD2 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates.

Comparing the Cq of each primer set

Conc. of DNA template	10ng/uL	5ng/uL	1ng/uL	0.5ng/uL	0.1ng/uL
Primer set	Mean Cq (cycles) and StDev (Note: 1 cycle = 1 minute)					Average
PE1	32.84+/-2.86	40.19+/-7.78	32.79+/-2.95	31.98+/-3.50	29.22+/-8.50	35 min
PE2	37.74+/-5.43	40.20+/-5.79	33.61+/-2.91	37.36+/-5.19	35.43+/-5.88	36 min
GLAPD1	24.65+/-2.06	25.64+/-1.62	25.02+/-1.91	29.60+/-4.07	35.17+/-0.41	27 min
GLAPD2	33.65+/-4.52	32.97+/-2.87	32.78+/-1.75	35.66+/-3.23	32.45+/-8.65	34 min

Fig 9. Amplification plot of Oak Wilt LAMP Primers with 10ng/uL of DNA template in the reaction. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates. The amplification curves of GLAPD1 look more similar to each other, compared to other primer sets.

Learnings

The GLAPD1 primer set took 27 cycles (approximate to 27 min based on the qPCR machine set up) to reach the threshold line. The reaction efficiency is higher than other primer sets. Three replicates were performed. The standard deviations of the mean Cq for the GLAPD1 replicates are smaller than the other primer sets. It also demonstrates consistency.

Comparing the Cq of the dilution series of each primer set

Fig 10. Amplification plot of a single dilution series.

Fig 11. Melting curve plot of a single dilution series.

Learnings

This dilution series is within the detection limits because the fluorescence signal increases rapidly in the linear phase and reaches a plateau within the time frame. The amplification plots and melt curve plots of all concentrations look the same. There is no sign of approaching the detection limit. We expect a slower increase of fluorescence signal and a larger standard deviation between the dataset when the dilution series is approaching the detection limit.

All primer sets demonstrated a targeted amplification

Fig 12. Melting curves of Oak Wilt PE1 LAMP Primers.	Each primer set shows specific LAMP amplification on the MCM7 region for the following reasons: Single melting peak Small standard deviation Note: PE1 has a generally higher peak height compared with other sets, indicating more fluorescence signals are observed. (Higher polymerase reaction.)
Fig 13. Melting curves of Oak Wilt PE2 LAMP Primers.
Fig 14. Melting curves of Oak Wilt GLAPD1 LAMP Primers.
Fig 15. Melting curves of Oak Wilt GLAPD2 LAMP Primers.

Observations and Learning

Conc. of DNA template	10ng/uL	5ng/uL	1ng/uL	0.5ng/uL	0.1ng/uL
Primer set	Melting temperature (degreeC) and StDev					Average
PE1	88.93+/-0.17	88.88+/-0.23	89.04+/-0.30	89.49+/-0.19	89.15+/-0.17	89.10
PE2	89.34+/-0.43	89.41+/-0.11	89.37+/-0.51	89.60+/-0.58	89.83+/-0.64	89.51
GLAPD1	88.97+/-0.42	89.05+/-0.49	88.63+/-0.36	89.11+/-0.17	89.15+/-0.34	88.98
GLAPD2	86.35+/-0.11	85.94+/-0.47	86.06+/-0.45	86.46+/-0.39	86.49+/-0.63	86.26

We observed that there are two different peaks, where PE1, PE2, and GLAPD1 primer sets have identical curves forming one peak on the right-hand side, and the melting curve of the GLAPD2 primer set forms another peak on the left-hand side.

Through the mean melting temperature of each primer set, we also observed that GLAPD2 has a lower melting temperature.

This shows that they have different amplification products. PE1,PE2, and GLAPD1 have one similar amplification product. GLAPD2 has another amplification product.
From a statistical perspective, it suggests that the PE1, PE2, GLAPD1 binding site is more common or universal. It implies that it can be more stable and have higher probability of binding success in on-site detection.

Comparing primer sets

PE1	[Pros] The melting curve peaks of the 15 experiments for PE1 primers are more concentrated together. Through calculating the mean melting temperature, it shows that PE1 has the smallest standard deviation. It implies that the LAMP reaction is stable and the result is repeatable. [Pros] The height of the overall melting curve peak of PE1 sample is higher thanother primer sets. It implies that more fluorescent signals were observed.
PE2	No outstanding pros or cons
GLAPD1	[Pros] The GLAPD1 primer set took 27 cycles (approximate to 27 min based on the qPCR machine set up) to reach the threshold line. The reaction efficiency is higher than other primer sets.
GLAPD2	[Cons] Through figure 16, it suggests that the GLAPD2 binding site is less common or universal. It implies that it can be less stable and have lower probability of binding success in on-site detection.

Summary:

For on site detection, stronger amplification is preferred. Therefore, we decided to move forward to our freeze-dry experiment with PE1

Step 5: Test if the selected LAMP reaction still works well when it is freeze-dried

Fig 9. The freeze-dried LAMP reaction

Fig 9. Amplification plot and melting curve analysis of freeze-dried LAMP reaction with PE1 primer set, targeting MCM7 region of the oak wilt DNA. The amplification passes the threshold at 32 cycles (32 minutes), indicating a positive amplification. There is one peak in the melting curve plot, indicating a single amplicon.

Learnings

Looking at figure 9 melting curve plot, it shows that the freeze-dried version of LAMP reaction emitted less fluorescence signals than the non-freeze dried version. The difference could come from: (1) using different master mixes as the NEB master mix cannot be freeze-dried. (2) Degradation of the fluorophores when the reagent was stored.