Selecting LAMP as the DNA amplification method

After multiple meetings with various stakeholder groups (see Human Practice wiki page), the iGEM Toronto wetlab team determined the most important requirements for their hypothetical diagnostic method: detecting the oak wilt pathogen quickly (in ~1 hour or less) and in the field with minimal lab equipment. To achieve these broad requirements, our diagnostic method must have strong diagnostic and analytical sensitivity and specificity. Additionally, it would need to be portable to be easily brought into field environments (such as forests without electricity) where oak wilt resides.

Top of mind in the realm of sensitive and specific diagnostics were methods based on the amplification of genomic material, which in our case was oak wilt DNA. While a variety of current amplification techniques existed, they each had drawbacks that would inhibit their success in the detection of oak wilt DNA in the field.

Some well-known techniques are listed below (Zanoli, 2013):

Given the need to create a fast, sensitive, specific, and portable diagnostic tool, the wetlab team decided that a newer DNA amplification method called loop-mediated isothermal amplification (LAMP) was a perfect candidate, since it overcame almost all of the drawbacks of the techniques above. Especially compared to PCR, thermal cycler is not necessary for LAMP.

This led to the team’s main project goal: develop a suitable LAMP reaction and process (with high viability and easy visualization) to amplify and detect oak wilt DNA. Ideally, our developed LAMP reaction would also meet the WHO’s ASSURED list of characteristics for diagnostic tests: high affordability, sensitivity, specificity, user friendliness, rapidity and robustness, being equipment-free, and deliverability.

Determining target DNA

The drylab and wetlab teams worked together to find suitable regions of oak wilt DNA to target for amplification; this region would be conserved for all individuals in the oak wilt species, but non-conserved between similar fungal species. This would ensure that if any DNA were to be amplified by our suitable LAMP reaction, it would solely belong to oak wilt and not another fungus.

After BLASTing a variety of oak wilt and other fungal genomic fragments, the drylab and wetlab teams found multiple target regions that were suitable for LAMP. The two most suitable regions selected for experimentation were BT and MCM7 (see Dry Lab wiki for in-depth selection and analysis of primers).

Finding suitable primers

Drylab used two online tools, Primer Explorer (PE) and GLAPD, to generate suitable primers for a LAMP reaction using the above target DNA regions as templates.

Drylab generated eight primer sets in total, with each set containing 4 primers: FIP (Forward inner primer), BIP (Backward inner primer), F3 (Forward primer), and B3 (Backward primer). The naming conventions of our candidate primer designs are as follows:

Note: Primer explorer design (PE). Whole Genome Based LAMP Primer Design (GLAPD)

Determining possible LAMP reaction reagents

Addressing requirement 1: Easy visualization

Because of the need to do in-field diagnostics of oak wilt, wetlab needed a way to visualize whether their LAMP reaction would be successful as the reaction proceeded. Colorimetric LAMP was the optimal solution for this requirement.

The binding of nucleotides by a DNA polymerase to template DNA releases hydrogen ions, thus reducing the pH of the reaction solution – this could be easily visualized via the addition of an indicator to the reaction mix, indicating that a successful amplification of DNA must cause the reaction to change color! (Tanner, 2015)

The New England Biolabs (NEB) WarmStart® Colorimetric LAMP 2X Master Mix was used.

• Key features of NEB WarmStart Master Mix:

1. This master mix provides a low-buffer reaction solution containing a visible pH indicator
2. Expect color change from pink to yellow in 30 minutes after incubating the LAMP reaction at 65C
3. According to Dr. Nathan Tanner, the WarmStart Master Mix contains glycerol, which makes it incompatible for freeze-drying.

If the mix turns from pink to yellow, wetlab could proceed with further testing (such as gel electrophoresis and specificity testing); if not, they would know that the reaction did not work (provided proper controls were in place) and should not proceed with further testing, saving us time, reagents, and money.

Addressing requirement 2: Ensuring LAMP remains equipment free and deliverable in field settings

Given the existing body of research on freeze-dried LAMP reactions and the recommendations by Dr. Pardee, the wetlab team decided that utilizing freeze-dried LAMP would be most suitable to using LAMP in the field. (Pardee, 2016) Because the colourimetric NEB LAMP master mix cannot be freeze-dried, iGEM Toronto experimented with

in addition to NEB master mix.

• Key features of LavaLAMP:

1. Increased sensitivity and specificity
2. Can communicate with the manufacturer to customize the master mix for better performance
3. Freeze-dry compatible

Hence, wetlab developed the following combinations of LAMP experiments they could conduct given their primer sets, target DNA regions, and master mixes available.

LAMP experimental workflows

Now that the dry lab had generated their candidate LAMP primers, the wet lab needed a way to experimentally determine the validity of these candidates as a diagnostic tool to succeed at amplifying oak wilt DNA.

Summary of Experimental Plan

Details about what wet lab did can be found in the Notebook section.

Positive control: verified stock template DNA and primer set that work with the NEB master mix (We used the positive control provided by Lucigen)

Negative controls: consists of all LAMP reactions in a designated group WITHOUT template DNA. 1uL of water is added to replace the reaction’s template DNA.

Step 1: Screen the primer sets through colorimetric LAMP reaction

Materials and Protocols

The materials and protocols of the experiments are documented on the Protocol section.

Combination of experiments

Each group represents a defined set of candidate LAMP experiments to be conducted together (to best compare results and adjust the protocols further). Because the color change of the reaction mix is not clear, the NEB protocol recommended users to run the reaction for an additional 10 minutes (NEB, 2016).

Table 1: Groups of candidate LAMP experiments to be conducted by wetlab team (FIRST ROUND)

(Details in the Results section): We hypothesize that primers designed for the MCM7 region are better at amplifying oak wilt DNA than the primers designed for the BT region because the primer set of PE2 of the MCM7 region had a slight color change.

Learning from our experimental results

After reviewing these results, the wetlab team members met to discuss how to optimize our experimental protocol to yield better LAMP reaction products. The team had a few important realizations.

Since the results with the NEB WarmStart Colorimetric master mix are not ideal, we experimented on Lucigen master mix to see if we can get more indicative results.

Lucigen master mix is colorless. Without access to fluorescence dye at the moment, we decided to visualize the results by gel electrophoresis.

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

Materials and Protocols

The materials and protocols of the experiments are documented on the Protocol section.

Combination of experiments

Table 2: Groups of candidate LAMP experiments to be conducted by wetlab team (SECOND ROUND).

Note: 71 °C was in the middle of the Lucigen master mix recommended reaction temperature range (Lucigen);

Quick Summary of Step 2

(Details in the Results section):

Learning from our experimental results

After reviewing these results, the wetlab team members met to discuss how to optimize our experimental protocol to yield better LAMP reaction products. The team had a few important realizations.

We performed group 1C and group 1D again with the proposed contamination controls in place. The controls could not fully eliminate the contamination issue.

We reached out to Jennifer and a graduate student in the Pardee lab, both had experience with LAMP. They shared that the only way we could avoid environmental contamination is by fully disinfecting the lab space or by changing the lab space. They also suggested that our lab reagents (master mix, primers, and/or DNA templates) could also be contaminated.

Therefore, iGEM Toronto immediately canceled any experimental plans related to gel electrophoresis and worked on finding another lab space that we could conduct our experiments in.

Step 3: Fluorescence LAMP experiment and melting curve analysis, combined with step 4: perform replicates to ensure experimental validity

Context and rationale:

In order to determine the most robust primer set from the candidates and to assess their amplification speed to determine which of them was best suited to in-field diagnostic use (this would ensure that diagnosis was rapid, which is a WHO ASSURED criteria), wetlab performed a fluorescence LAMP experiment using a qPCR machine; this allowed for a quantitative comparison of the degree of amplification with respect to time for each of the above primer sets.

Additionally, melting curve analysis was performed in tandem with the fluorescence LAMP experiment to verify if the amplification is targeted. When there is one single peak, it implies that there is only one single amplification product. By comparing the melting temperatures of the experimental sets, the wet lab can verify if the amplification is specific. When there is a shift in the melt peak of the same sample, it implies that there is off target amplification.

These data would aid wetlab in further decision-making regarding optimizations to the final LAMP reaction.

Reagents

Clean NEB WarmStart® Fluorescent LAMP/RT-LAMP Kit with UDG (provided by Jennifer’s lab) was used for the fluorescence LAMP experiment to prevent contamination.

Primer stocks are also used to perform this experiment to prevent contamination.

All of our samples were run under 65 degree celsius with SYBR-Green fluorescent dye. Each tube of the test plate is 25 microliter with 12.5 microliter of NEB-WarmStart Master Mix 2X, 9 micro-liter of ddH2O, 1 microliter of customized concentration of DNA solution, and 2.5 microliter of customized primer mix.

qPCR machine set-up:

The qPCR machine was programmed to undergo 60 cycles of amplification at 65 °C, each for 1 minute, followed by a series of heating and cooling for the melting curve analysis (see the figure below for details).

qPCR plate set-up:

The qPCR plate had 384 slots for reagents (see image above). To test all four primer sets for the MCM7 region (PE1, PE2, GLAPD1 and GLAPD2) and determine their limit of detection (LoD), wetlab employed the following dilution series of oak wilt target DNA (in ng/uL): 10, 5, 1, 0.5, 0.1. Three replicates for each dilution were performed (see Table 4 below).

The wetlab team’s experimental results indicated that the above primer sets displayed logistic amplification above threshold levels, confirming their viability, with GLAPD1 displaying the most rapid amplification (see wetlab’s results page on the wiki).